RESUMO
In Alzheimer's disease (AD) and other tauopathies, the cytosolic protein Tau misfolds and forms intracellular aggregates which accumulate within the brain leading to neurodegeneration. Clinical progression is tightly linked to the progressive spread of Tau pathology throughout the brain, and several lines of evidence suggest that Tau aggregates or "seeds" may propagate pathology by spreading from cell to cell in a "prion like" manner. Accordingly, blocking the spread of extracellular seeds with an antibody could be a viable therapeutic approach. However, as the structure of Tau seeds is unknown, it is only possible to rationally design therapeutic Tau antibodies by making a priori assumptions. To avoid this, we developed a robust and quantitative cell based assay and employed an unbiased screening approach to identify the antibody with the highest activity against human Tau seeds. The selected antibody (D), directed to the mid-region of Tau (amino acids 235-250), potently blocked the seeding of human AD Tau and was also fully efficacious against seeds from progressive supranuclear palsy. When we compared this antibody with previously described reference antibodies, we were surprised to find that none of these antibodies showed comparable efficacy against human pathological seeds. Our data highlight the difficulty of predicting antibody accessible epitopes on pathological Tau seeds and question the potential efficacy of some of the Tau antibodies that are currently in clinical development.
Assuntos
Anticorpos/metabolismo , Epitopos/imunologia , Proteínas tau/química , Proteínas tau/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Mapeamento de Epitopos , Epitopos/química , Células HEK293 , Humanos , Agregados Proteicos , Conformação Proteica , Ressonância de Plasmônio de Superfície , Transfecção , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.
Assuntos
Anticorpos , Animais , Humanos , Terminologia como AssuntoRESUMO
Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at "Site 3", blocking the interaction with the signaling co-receptor gp130.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Complexo Antígeno-Anticorpo/química , Artrite Experimental/imunologia , Artrite Experimental/terapia , Sítios de Ligação , Cristalografia por Raios X , Receptor gp130 de Citocina/química , Feminino , Humanos , Hibridomas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/química , Macaca fascicularis , Modelos Moleculares , Dados de Sequência Molecular , Ratos , Homologia de Sequência de AminoácidosRESUMO
The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Células CHO , Cricetinae , Dissulfetos/química , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , TemperaturaRESUMO
The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin-neutralizing monoclonal antibody (Scl-AbIV) to gonad-intact female cynomolgus monkeys. Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl-AbIV treatment had clear anabolic effects, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle-treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group. Taken together, the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Densidade Óssea/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/imunologia , Osso e Ossos/metabolismo , Marcadores Genéticos/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Osso e Ossos/patologia , Feminino , Macaca fascicularis , OsteogêneseRESUMO
A kappa-light chain from a Fab expression system was truncated by the insertion of a stop codon in the gene sequence to produce a variable light (VL) single domain antibody (dAb). Here, we describe the expression of dAb in the periplasm of Escherichia coli through fermentation in a defined media. Immunoglobulin binding domains from peptostreptococcal protein L (PpL) have been shown to bind specifically to kappa-light chains. We have produced recombinant PpL, at high yield, and this was used to custom-produce PpL-Sepharose affinity columns. Here, we show that the affinity purification of VL dAb by this method is simple and efficient with no apparent loss in protein at any stage. The truncated dAb protein product was confirmed by electrospray mass spectrometry and N-terminal sequencing. When analyzed by SDS-PAGE it was shown to be over 95% pure and produced at yields of 35-65 mg/L of culture medium. The dAb protein produced was shown by NMR and CD to be a folded beta-sheet domain. This domain is bound by PpL with a Kd of approximately 50 nM as determined by stopped-flow fluorimetry.
Assuntos
Anticorpos/imunologia , Proteínas de Bactérias/imunologia , Região Variável de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos/isolamento & purificação , Sítios de Ligação de Anticorpos/imunologia , Cromatografia de Afinidade , Dicroísmo Circular , Escherichia coli/metabolismo , Fluorometria , Região Variável de Imunoglobulina/metabolismo , Cadeias kappa de Imunoglobulina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Terciária de ProteínaAssuntos
Anticorpos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Sequência de Bases , Escherichia coli/citologia , Fermentação , Expressão Gênica , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Periplasma/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.
Assuntos
Aminoglicosídeos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Antineoplásicos/uso terapêutico , Moléculas de Adesão Celular/imunologia , Imunoconjugados/uso terapêutico , Lectinas/imunologia , Linfoma de Células B/terapia , Sequência de Aminoácidos , Aminoglicosídeos/química , Aminoglicosídeos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antineoplásicos/imunologia , Ligação Competitiva , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Humanos , Imunoconjugados/imunologia , Linfoma de Células B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Antibody-targeted chemotherapy with gemtuzumab ozogamicin (CMA-676, a CD33-targeted immunoconjugate of N-acetyl-gamma-calicheamicin dimethyl hydrazide [CalichDMH], a potent DNA-binding cytotoxic antitumor antibiotic) is a clinically validated therapeutic option for patients with acute myeloid leukemia (AML). Here, we describe the preclinical profile of another immunoconjugate of CalichDMH, CMC-544, targeted to CD22 expressed by B-lymphoid malignancies. CMC-544 comprises a humanized IgG4 anti-CD22 monoclonal antibody (mAb), G5/44, covalently linked to CalichDMH via an acid-labile 4-(4'-acetylphenoxy) butanoic acid (AcBut) linker. Both CMC-544 and unconjugated G5/44 bound human CD22 with subnanomolar affinity. CMC-544, but not unconjugated G5/44, exerted potent cytotoxicity against CD22+ B-cell lymphoma (BCL) cell lines (inhibitory concentration of 50%: 6-600 pM CalichDMH). CMC-544 caused a potent inhibition of growth of small but established BCL xenografts leading to cures (therapeutic index > 10). CMC-544 prevented the establishment of BCL xenografts and also caused regression of large BCLs (> 1.5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignancies.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos B/biossíntese , Moléculas de Adesão Celular , Imunoconjugados/uso terapêutico , Lectinas/biossíntese , Linfoma de Células B/terapia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Concentração Inibidora 50 , Inotuzumab Ozogamicina , Lectinas/metabolismo , Linfoma de Células B/metabolismo , Linfoma não Hodgkin/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Químicos , Transplante de Neoplasias , Ligação Proteica , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Fatores de TempoRESUMO
We demonstrate the importance of optimizing the balance of light chain (LC) and heavy chain (HC) expression to achieve high level production of Fab' fragments in the Escherichia coli periplasm. The LC:HC balance has been controlled by varying the codon usage of the signal peptide (SP) and 5' mature domain coding regions. Different SP coding regions have been identified from a codon wobble-based library using alkaline phosphatase (AP) as a reporter gene. A plasmid system that enables random combination of these variant SP coding regions is used to construct optimized Fab' expression plasmids. These small plasmid libraries facilitated selection of optimal Fab' expression plasmids and resulted in increases of periplasmic yield, up to 580 mgL(-1) from E. coli fermentations and will enable rapid variable region subcloning and selection of future Fab(') expression plasmids.