Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.692
Filtrar
1.
Se Pu ; 37(10): 1090-1097, 2019 Oct 08.
Artigo em Chinês | MEDLINE | ID: mdl-31642288

RESUMO

A partially filled monolith was prepared by in situ polymerization, and then carrier ampholytes (CAs, pH 3-10) were immobilized on its surface. For effective utilization of capillary isoelectric focusing (cIEF) with the monolithic immobilized pH gradient (M-IPG), a new online platform was established by the introducing of an eight-way injection valve, a three-way valve and a cross-shaped unit. Besides, a capillary coated with hydroxypropyl cellulose (HPC capillary) was prepared and used to determine the isoelectric points (pI) of trastuzumab and etanercept. In parallel, using the newly built capillary isoelectric focusing platform, the pI values of trastuzumab and etanercept were measured with the M-IPG column, and compared with the results obtained using the HPC capillary. It was found that these two cIEF columns can be effectively used to separate proteins and determine the pI values of monoclonal antibodies and fusion proteins in protein drugs. Moreover, the measured pI values were consistent with those estimated using the HPC capillary.


Assuntos
Focalização Isoelétrica , Proteínas/química , Força Próton-Motriz , Anticorpos Monoclonais/química , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Polimerização
2.
Adv Exp Med Biol ; 1172: 97-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628653

RESUMO

The IL-17 family in humans consists of six distinct cytokines (IL-17A-F) that can interact with five IL-17 receptors (IL-17RA-E). The interaction between these cytokines and their receptors are critical in mediating host defenses while also making major contributions to inflammatory and autoimmune responses as demonstrated through both in vitro and in vivo experiments as well as human clinical trials. Inhibition of the IL-17A/IL-17RA interaction by monoclonal antibodies has also displayed remarkable efficacies in clinical trials against psoriasis and other autoimmune diseases. Recently, we and others reported the identification and characterization of both small-molecule and peptide IL-17A antagonists. These non-antibody IL-17A antagonists can effectively and selectively disrupt the IL-17A/IL-17RA complex and may provide alternative modalities to treat IL-17-related autoimmune and inflammatory diseases. This chapter summarizes the reported crystal structures of the IL-17 cytokines, their complexes with IL-17RA, and their complexes with both monoclonal antibodies as well as small-molecule and peptide antagonists.


Assuntos
Interleucina-17 , Receptores de Interleucina-17 , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Doenças Autoimunes/imunologia , Cristalização , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Interleucina-17/imunologia , Receptores de Interleucina-17/antagonistas & inibidores , Receptores de Interleucina-17/química , Receptores de Interleucina-17/imunologia
3.
Soft Matter ; 15(33): 6660-6676, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389467

RESUMO

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.


Assuntos
Anticorpos Monoclonais/química , Fenômenos Biofísicos , Difusão , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência , Modelos Químicos , Multimerização Proteica , Estabilidade Proteica , Soluções , Viscosidade
4.
Chem Commun (Camb) ; 55(69): 10312-10315, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397446

RESUMO

Herein, we report a novel magnet-mediated antibody-boronate sandwich-typed assay (ABSTA) strategy for the ultrasensitive, specific, rapid, and enzyme-free detection of glycoproteins in complex samples. The proposed ABSTA method exhibited ultrahigh sensitivity for HCG with a detection limit of 0.19 mIU mL-1, which is approximately 40-fold lower than that of conventional sandwich enzyme immunoassay.


Assuntos
Anticorpos Imobilizados/química , Ácidos Borônicos/química , Gonadotropina Coriônica/sangue , Corantes Fluorescentes/química , Imãs/química , Anticorpos Monoclonais/química , Gonadotropina Coriônica/análise , Fluorescência , Humanos , Imunoensaio/métodos , Limite de Detecção
5.
Pharm Res ; 36(9): 130, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31264003

RESUMO

PURPOSE: An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest. METHODS: To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency. RESULTS: The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged. CONCLUSION: This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida de Alta Pressão , Imagem por Ressonância Magnética , Ligação Proteica , Conformação Proteica , Espectrometria de Massas em Tandem
6.
Chem Soc Rev ; 48(16): 4361-4374, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31294429

RESUMO

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Ácidos/química , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Catepsina B/metabolismo , Dissulfetos/química , Estabilidade de Medicamentos , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo
7.
Adv Exp Med Biol ; 1140: 225-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347050

RESUMO

Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30-40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.


Assuntos
Anticorpos Monoclonais/química , Sequenciamento de Nucleotídeos em Larga Escala , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional , Animais , Células CHO , Cricetinae , Cricetulus , Espectrometria de Massas
8.
Analyst ; 144(13): 3999-4005, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31172144

RESUMO

Micrococcal nuclease (MNase) is an extracellular endonuclease of Staphylococcus aureus (S. aureus). It digests single stranded nucleic acid. The presence of MNase is the gold standard to identify S. aureus and its content. The present study reports the ultrahigh sensitive and selective fluorescence platform for MNase detection, designed and developed based on the surface energy transfer mechanism. A "proof of concept" is being developed based on monoclonal antibody-conjugated quantum dots (mAb-QDs), wherein mAb-QDs act as donors and graphene oxide (GO) acts as an acceptor. mAb-QDs in close proximity to GO undergo adsorption due to weak affinity between them and this results in fluorescence quenching by the transfer of surface energy from mAb-QDs to GO. During sensing, a much stronger affinity of mAb-QDs towards MNase inhibits the energy transfer to GO and this allows the regaining of fluorescence. Immobilized mAb-QDs on nitrocellulose membrane strips were fabricated and tested for "ON-OFF-ON" sensing of MNase. The limit of detection for fluorescence based assay and strips is found to be 0.3 ng mL-1 and 0.5 ng mL-1, respectively. The developed strips were applied on real samples for the detection of S. aureus.


Assuntos
Corantes Fluorescentes/química , Grafite/química , Nuclease do Micrococo/análise , Pontos Quânticos/química , Staphylococcus aureus/química , Anticorpos Monoclonais/química , Técnicas Biossensoriais/métodos , Colódio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção , Membranas Artificiais , Nanocompostos , Estudo de Prova de Conceito , Sensibilidade e Especificidade , Staphylococcus aureus/enzimologia
9.
Pharm Res ; 36(9): 129, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31254106

RESUMO

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/análise , Animais , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/química , Conformação Proteica , Ratos Wistar , Soroalbumina Bovina/química
10.
Anal Bioanal Chem ; 411(21): 5617-5629, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214752

RESUMO

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.


Assuntos
Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio/química , Cromatografia Líquida/métodos , Imunoglobulina G/química , Espectrometria de Massas em Tandem/métodos
11.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 871-879, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223005

RESUMO

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.


Assuntos
Proteínas de Escherichia coli , Peptídeos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
J Chromatogr A ; 1602: 317-325, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248584

RESUMO

This study demonstrates how multimodal cation exchange chromatographic systems can be successfully employed to purify an IgG4 monomer from three distinct aggregate species. In addition, the steric mass action model is shown to be capable of facilitating the development of effective bind/elute processes for aggregate removal. A variety of multimodal anion and cation exchange resin materials and conditions were initially screened for both selectivity and recovery of the product. While the multimodal anion exchangers exhibited significant recovery issues, the Capto MMC and MMC ImpRes resin systems were observed to have good recoveries and some selectivity for these challenging separations under linear gradient conditions. Mechanistic modeling was then explored as a means to expedite the development of a bind/elute process for decreasing the aggregate content of this challenging monoclonal antibody mixture. The retention behavior of the monomer and the higher molecular weight species under different linear gradient conditions were used to estimate the SMA parameters of the proteins on both the Capto MMC and MMC ImpRes systems. A range of simulations were then carried out to determine an efficient bind/elute process for the removal of higher molecular weight species while also obtaining a good yield of the monomer in both resin systems. Finally, bind/elute experiments were carried out under the suggested simulation conditions for each resin system and were shown to be in good agreement with the theoretical predictions, with purities and yields of 99% and 78.6% for Capto MMC and 99.3% and 87.9% for Capto MMC ImpRes, respectively. The simple approach described in this paper presents a rapid and useful method for model-based process development of antibody monomer-aggregate separations with multimodal cation exchange chromatography.


Assuntos
Anticorpos Monoclonais/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica/métodos , Modelos Moleculares , Simulação por Computador , Peso Molecular , Temperatura Ambiente
13.
Ann Hematol ; 98(10): 2339-2346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250082

RESUMO

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Medula Óssea , Calreticulina , Mutação , Trombocitemia Essencial , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Calreticulina/genética , Calreticulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/patologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-31128526

RESUMO

Activated carbon (AC) is a porous solid with a larger surface area and lower cost than chromatography resins. AC has been used in the field of biopharmaceutical manufacturing for plasma-derived products and recombinant monoclonal antibodies (mAb). In our previous study, AC was employed in the purification process of therapeutic mAb as a replacement for Protein A affinity chromatography (PrA). In addition, we designed an all flow-through purification process using AC. In these investigations, greater effective clearance of high-molecular-weight species (HMW), low-molecular-weight species (LMW), host cell proteins (HCP), and DNA was observed compared to that of the conventional Protein A platform purification process. However, it was revealed that mAb recovery from the AC step was lower than that from the PrA step. In this work, to improve mAb recovery from the AC step while maintaining the effective removal of impurities, a pretreatment procedure conducted prior to the AC treatment was investigated. We found that both an ultrafiltration/dilution and reduction in the conductivity of the filtered cell culture supernatant after acid precipitation could improve both the impurity clearance and mAb recovery from the AC treatment. From the obtained results, we designed a two-step purification process in which AC treatment is followed by either cation exchange column chromatography or anion exchange column chromatography, and we compared this against the Protein A platform purification process. Excellent impurity clearance was achieved, even in the one-column process. Furthermore, we designed an innovative column-free flow-through purification process based on acid precipitation, clarification, ultrafiltration/dilution, and the implementation of an AC filter membrane and an anion exchange chromatography membrane. With this process in the pilot-scale, HCP level can be reduced to below 10 ng/mg, and HMW and LMW can be removed to below 1% while improving mAb recovery. From these results, it is strongly expected that AC is a promising candidate for the next generation of mAb purification processes to improve the economy and efficiency of the process.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Carvão Vegetal/química , Cromatografia por Troca Iônica/métodos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 1-17, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31128359

RESUMO

Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.


Assuntos
Anticorpos Monoclonais , Eletroforese Capilar , Imunoconjugados , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Humanos , Imunoconjugados/análise , Imunoconjugados/química , Imunoconjugados/isolamento & purificação , Focalização Isoelétrica , Espectrometria de Massas
16.
Mol Imaging ; 18: 1536012119829986, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044647

RESUMO

OBJECTIVE: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation. METHODS: [89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg). RESULTS: [89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses. CONCLUSIONS: [89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígeno B7-H1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Desferroxamina/química , Radioisótopos/química , Zircônio/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados , Camundongos , Tomografia por Emissão de Pósitrons , Distribuição Tecidual , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Biotechnol ; 37(7): 761-765, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31133742

RESUMO

The use of monoclonal antibodies in cancer therapy is limited by their cross-reactivity to healthy tissue. Tumor targeting has been improved by generating masked antibodies that are selectively activated in the tumor microenvironment, but each such antibody necessitates a custom design. Here, we present a generalizable approach for masking the binding domains of antibodies with a heterodimeric coiled-coil domain that sterically occludes the complementarity-determining regions. On exposure to tumor-associated proteases, such as matrix metalloproteinases 2 and 9, the coiled-coil peptides are cleaved and antigen binding is restored. We test multiple coiled-coil formats and show that the optimized masking domain is broadly applicable to antibodies of interest. Our approach prevents anti-CD3-associated cytokine release in mice and substantially improves circulation half-life by protecting the antibody from an antigen sink. When applied to antibody-drug conjugates, our masked antibodies are preferentially unmasked at the tumor site and have increased anti-tumor efficacy compared with unmasked antibodies in mouse models of cancer.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias/terapia , Animais , Anticorpos Monoclonais/química , Sobrevivência Celular , Citocinas/metabolismo , Células HEK293 , Humanos , Imunoconjugados , Integrinas/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
18.
PLoS Negl Trop Dis ; 13(5): e0007373, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31120889

RESUMO

Infections of humans and livestock with African trypanosomes are treated with drugs introduced decades ago that are not always fully effective and often have severe side effects. Here, the trypanosome haptoglobin-haemoglobin receptor (HpHbR) has been exploited as a route of uptake for an antibody-drug conjugate (ADC) that is completely effective against Trypanosoma brucei in the standard mouse model of infection. Recombinant human anti-HpHbR monoclonal antibodies were isolated and shown to be internalised in a receptor-dependent manner. Antibodies were conjugated to a pyrrolobenzodiazepine (PBD) toxin and killed T. brucei in vitro at picomolar concentrations. A single therapeutic dose (0.25 mg/kg) of a HpHbR antibody-PBD conjugate completely cured a T. brucei mouse infection within 2 days with no re-emergence of infection over a subsequent time course of 77 days. These experiments provide a demonstration of how ADCs can be exploited to treat protozoal diseases that desperately require new therapeutics.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antiprotozoários/administração & dosagem , Benzodiazepinas/administração & dosagem , Pirróis/administração & dosagem , Tripanossomíase Africana/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Antiprotozoários/química , Benzodiazepinas/química , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pirróis/química , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/parasitologia
19.
Nat Chem Biol ; 15(7): 730-736, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31110306

RESUMO

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and ß-1,4-galactosyltransferase (ß4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and ß4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.


Assuntos
Anticorpos Monoclonais/biossíntese , Engenharia Celular , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetulus , Glicosilação/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química
20.
Pharm Res ; 36(8): 109, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127417

RESUMO

PURPOSE: To investigate differences in the preferential exclusion of trehalose, sucrose, sorbitol and mannitol from the surface of three IgG1 monoclonal antibodies (mAbs) and understand its effect on the aggregation and reversible self-association of mAbs at high-concentrations. METHODS: Preferential exclusion was measured using vapor pressure osmometry. Effect of excipient addition on accelerated aggregation kinetics was quantified using size exclusion chromatography and on reversible self-association was quantified using dynamic light scattering. RESULTS: The doubling of excipient concentration in the 0 to 0.5 m range resulted in a doubling of the mAb transfer free energy for all excipients and antibodies tested in this study. Solution pH and choice of buffering agent did not significantly affect the magnitude of preferential exclusion. We find that aggregation suppression for trehalose, sucrose and sorbitol (but not mannitol) correlates with the magnitude of their preferential exclusion from the native state of the three IgG1 mAbs. We also find that addition of sugars and polyols reduced the tendency for reversible self-association in two mAbs that had weakly repulsive or neutral self-interactions in the presence of buffer alone. CONCLUSIONS: The magnitude of preferential exclusion for trehalose, sucrose and sorbitol correlates well with their partial molar volumes in solution. Mannitol is excluded to a greater extent than that expected from its partial molar volume, suggesting specific interactions of mannitol that might be different than the other sugars and polyols tested in this study. Local interactions play a role in the effect of excipient addition on the reversible self-association of mAbs. These results provide further insights into the stabilization of high-concentration mAb formulations by sugars and polyols.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Polímeros/química , Agregados Proteicos , Sacarose/química , Álcoois Açúcares/química , Trealose/química , Excipientes/química , Cinética , Simulação de Dinâmica Molecular , Conformação Proteica , Propriedades de Superfície
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA