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1.
Bioconjug Chem ; 24(5): 772-9, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23578050

RESUMO

Antibody-drug conjugates (ADCs) are target-specific anticancer agents consisting of cytotoxic drugs covalently linked to a monoclonal antibody. The number of ADCs in the clinic is growing, and therefore thorough characterization of the quantitative assays used to measure ADC concentrations in support of pharmacokinetic, efficacy, and safety studies is of increasing importance. Cytotoxic drugs such as the tubulin polymerization inhibiting auristatin, monomethyl auristatin E, have been conjugated to antibodies via cleavable linkers (MC-vc-PAB) through internal cysteines. This results in a heterogeneous mixture of antibody species with drug-to-antibody ratios (DAR) ranging from 0 to 8. In order to characterize the assays used to quantitate total MC-vc-PAB-MMAE ADCs (conjugated and unconjugated antibody), we used purified fractions with defined DARs from 6 therapeutic antibodies to evaluate different assay formats and reagents. Our investigations revealed that for quantitation of total antibody, including all unconjugated and conjugated antibody species, sandwich ELISA formats did not always allow for recovery of all purified DAR fractions (DAR 0-8) to within ±20% of the expected values at the reagent concentrations tested. In evaluating alternative approaches, we found that the recovery of DAR fractions with semihomogeneous assay (SHA) formats, in which sample, capture, and detection reagents are preincubated in solution, were less affected by the antibody's MMAE drug load as compared to traditional stepwise sandwich ELISAs. Thus, choosing the optimal assay format and reagents for total antibody assays is valuable for developing accurate quantitative assays.


Assuntos
Antineoplásicos/farmacocinética , Imunotoxinas/farmacocinética , Oligopeptídeos/farmacocinética , Moduladores de Tubulina/farmacocinética , Animais , Antineoplásicos/química , Ensaio de Imunoadsorção Enzimática , Imunotoxinas/química , Camundongos , Camundongos SCID , Oligopeptídeos/química , Moduladores de Tubulina/química
2.
Lab Chip ; 13(7): 1342-50, 2013 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-23380873

RESUMO

Miniaturization of immunoassays has numerous potential advantages over traditional ELISAs. Here we present a novel approach using patterned planar plates (PPPs). These 'wall-less' plates consist of a 16 × 24 array of 2 mm diameter hydrophilic regions surrounded by a hydrophobic polytetrafluoroethylene (PTFE) coating. Assays are performed by adding 2 µL droplets to the hydrophilic areas. These droplets are overlaid with an immiscible mixture of perfluorocarbon liquid (PFCL) that essentially eliminates evaporation. During wash steps, a thin film of PFCL covers the hydrophobic coating and prevents its wetting by wash buffer; as a result, the hydrophilic wells remain intact and inter-well cross-contamination is prevented. We compared the performance of three immunoassays using PPPs versus traditional 384-well ELISA plates. These included assays for soluble FcRH5 in human serum, SDF-1 in mouse serum, and human IgG in mouse plasma. The results show that the PPP assays were closely comparable to the ELISAs in terms of sensitivity, linearity of dilution, and sample quantitation. Moreover, the PPP assays were rapid to perform, easily adapted from ELISA protocols, and used 10- to 50-fold less sample and reagent volume as compared to 384- or 96-well plate ELISAs. As an additional advantage, PPPs conform to established microplate dimensional standards making them compatible with pre-existing equipment and workflows. PPPs therefore represent an attractive and broadly applicable approach to flexible miniaturization of plate-based immunochemical assays.


Assuntos
Imunoensaio/instrumentação , Análise em Microsséries/instrumentação , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Quimiocina CXCL12/análise , Quimiocina CXCL12/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/imunologia , Camundongos , Politetrafluoretileno/química , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/imunologia , Receptores Fc
3.
Bioanalysis ; 3(6): 677-700, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21417735

RESUMO

With more than 34 targets being investigated and nearly 20 clinical trials at various phases of development, antibody-drug conjugates (ADCs) hold a lot of promise for improving oncological malignancy therapy. This therapeutic strategy designed to specifically or preferentially deliver a cytotoxic agent to tumor cells through conjugation to a monoclonal antibody is not new. Although this approach is relatively simple conceptually, the history of ADCs clearly attests to the high degree of complexity in their development. Each component of an ADC is important to achieve efficacy with minimal toxicity, and the ability to monitor this multicomponent therapeutic entity is deemed to be critical for their successful optimization. In this article we review the different bioanalytical strategies that have been implemented to characterize various ADCs and discuss the challenges and issues associated with these approaches.


Assuntos
Anticorpos/análise , Imunoconjugados/análise , Preparações Farmacêuticas/análise , Animais , Anticorpos/imunologia , Técnicas de Química Analítica/métodos , Técnicas Citológicas/métodos , Humanos , Imunoensaio/métodos , Imunoconjugados/imunologia
4.
J Immunol Methods ; 362(1-2): 70-81, 2010 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-20833179

RESUMO

IL-17AA, IL-17FF, and IL-17AF are proinflammatory cytokines that have been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). In order to measure the levels of these cytokines in synovial fluid and serum samples from RA patients, immunoassays specific for IL-17AA, FF, and AF were developed. Although these assays could tolerate up to 50% pooled normal human serum, false positive reactivity was problematic in patient samples suggesting interference from heterophilic antibodies. We therefore evaluated the ability of several commercially available heterophilic antibody blocking agents to reduce false positive reactivity by testing them against samples that were confirmed as false positives in the IL-17AA, FF, and AF assays. Several of the blockers performed well, including HBR-1, HBR-9, HBR-11, HBR-Plus, Serum Cytokine Assay Diluent, and IIR. We chose to move forward using IIR blocker for sample analysis and verified that IIR had no effect on the assay standard curves and did not affect IL-17 quantitation in plasma from ex vivo stimulated human whole blood. IL-17FF and IL-17AF were below the limits of quantitation of the assays (12.3 and 10.5pg/ml, respectively) in synovial fluid and serum samples from patients with RA and osteoarthritis (OA). For the more sensitive IL-17AA assay (1.6pg/ml limit of quantitation), low levels of IL-17AA were measurable in 48% of RA synovial fluid samples (mean, 7.9pg/ml; median, <1.6pg/ml; range, <1.6-29.7pg/ml; n=23) but not in synovial fluid from patients with OA (n=33). For serum samples, however, IL-17AA was below the limit of detection for both RA and OA patients. When these same serum samples were analyzed in the absence of a heterophilic antibody blocker, false positive reactivity yielded apparent mean IL-17AA levels of 43.3pg/ml (28% positive; n=50) and 14.8pg/ml (12% positive; n=50) for RA and OA patients, respectively, results that could potentially be interpreted as consistent with disease biology. These studies demonstrate the importance of ensuring that HAb interference is well controlled, particularly when measuring low concentrations of cytokines in samples from patients with autoimmune disease.


Assuntos
Artrite Reumatoide/sangue , Interleucina-17/sangue , Osteoartrite/sangue , Líquido Sinovial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Anticorpos/química , Anticorpos/imunologia , Artrite Reumatoide/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Reações Falso-Positivas , Feminino , Humanos , Interleucina-17/imunologia , Masculino , Pessoa de Meia-Idade , Osteoartrite/imunologia , Sensibilidade e Especificidade , Líquido Sinovial/imunologia
5.
Cancer Res ; 68(22): 9280-90, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19010901

RESUMO

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Imunotoxinas/uso terapêutico , Maitansina/análogos & derivados , Receptor ErbB-2/análise , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/toxicidade , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunotoxinas/farmacocinética , Imunotoxinas/toxicidade , Maitansina/farmacocinética , Maitansina/uso terapêutico , Maitansina/toxicidade , Camundongos , Ratos , Ratos Sprague-Dawley , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Bioconjug Chem ; 19(8): 1673-83, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18637680

RESUMO

CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Maleimidas/metabolismo , Maitansina/análogos & derivados , Maitansina/metabolismo , Oligopeptídeos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Humanos , Imunoconjugados/análise , Imunoconjugados/imunologia , Camundongos , Sensibilidade e Especificidade
7.
J Immunol Methods ; 320(1-2): 58-69, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17280683

RESUMO

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.


Assuntos
Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Soro/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Animais , Anticorpos Monoclonais/imunologia , Relação Dose-Resposta a Droga , Macaca fascicularis , Camundongos , Pan troglodytes , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
8.
Protein Eng Des Sel ; 19(7): 291-7, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16621915

RESUMO

An AB.Fab (albumin-binding Fab) consists of a Fab and a phage-derived albumin-binding peptide. This molecule is capable of binding both antigen and albumin simultaneously. Using a Fab derived from Herceptin we generated a panel of AB.Fab variants with wide-ranging affinities for albumin. An assay that measured AB.Fab binding to albumin in solution was developed to most accurately reflect the binding affinity for albumin in vivo. Affinity varied depending upon the species of albumin tested. For rat and rabbit albumin, affinities ranged from 0.04 to 2.5 microM. Reduced affinity for albumin correlated with a reduced half-life and higher clearance rates in both species; the beta half-life ranged 6-fold while clearance ranged over 50-fold in rats and 20-fold in rabbits. To estimate the pharmacokinetic properties of an AB.Fab in humans, AB.Fab variants with similar affinities for rat and rabbit albumin were selected. Using their pharmacokinetic parameters and the principles of allometric scaling for albumin, we estimate an approximate beta half-life for an AB.Fab with 0.5 microM affinity for albumin of up to 4 days in humans with a clearance of 76 ml/h. These variants demonstrate the ability to modulate the clearance of a Fab fragment in vivo and help to establish guidelines for pharmacokinetic engineering of molecules through albumin binding.


Assuntos
Albuminas/metabolismo , Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Peso Molecular , Ligação Proteica , Coelhos , Ratos , Especificidade da Espécie , Trastuzumab
9.
Assay Drug Dev Technol ; 2(2): 131-40, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15165509

RESUMO

In developing a screening assay for a serine/threonine kinase, we evaluated various formats of an in-plate enzyme-linked immunosorbent assay (ELISA), as well as solution-phase kinase assays using either ELISA or AlphaScreen detection. Substrate was available both as a biotinylated 15-residue peptide and as a 25-residue peptide containing the same sequence expressed as a glutathione S-transferase fusion protein. When increasing concentrations of either of these substrates were coated directly onto ELISA plates, the rates of the kinase reactions progressively increased. In contrast, when the biotin-peptide was captured onto NeutrAvidin-coated plates, the finite peptide binding capacity of the plates limited the amount of substrate that could be incorporated into the assay system and thereby limited the rate of the reaction at a given kinase concentration. Solution-phase kinase reactions can tolerate high substrate concentrations; however, analysis of kinase reaction samples containing biotin-peptide concentrations higher than the binding capacity of NeutrAvidin-coated plates resulted in an inability to detect differences between reactions run at different substrate concentrations. For AlphaScreen detection following solution-phase kinase reactions, limitations in the binding capacity of the donor and acceptor beads caused loss of signal for substrate concentrations above the maximum binding capacity. Overall, the solution-phase assays required significantly more kinase than the in-plate assays (1-4 microg/ml versus <100 ng/ml, respectively). These studies demonstrate that the amount of substrate that can be incorporated into an assay system substantially affects the rate of the kinase reaction and therefore the amount of kinase required for the assay.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Tecnologia Farmacêutica/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/química , Especificidade por Substrato
10.
Nucleic Acids Res ; 31(6): e25, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12626724

RESUMO

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3'-ethylene glycol scaffolding with the incorporation of 2'-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.


Assuntos
Técnicas de Química Analítica/métodos , Ácidos Nucleicos/análise , Hemoglobina Fetal/genética , Expressão Gênica , Humanos , Células K562 , Ácidos Nucleicos/genética , RNA/genética , RNA/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
11.
Am J Pathol ; 161(3): 787-97, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12213706

RESUMO

Recent advances in molecular biology, human genetics, and functional genomics tremendously increase the number of molecular targets available for potential therapeutic and diagnostic use. To complement DNA array data, cost-efficient high-throughput technologies providing reliable information at the protein level need to be developed. Here we describe the generation of a frozen cell array that required the use of single cell suspensions and could serve various applications such as the analysis of specific antibody or ligand binding to a large panel of different cell types. As an example, binding of an anti-human epithelial cell adhesion molecule monoclonal antibody to 24 different cell lines has been analyzed using the cell array and compared to the data generated by fluorescence-activated cell sorting. The reliability and flexibility of our frozen cell array technology is compatible with the needs of high-throughput screening for drug discovery and target validation.


Assuntos
Criopreservação , Técnicas Citológicas , Anticorpos/análise , Antígenos de Neoplasias , Moléculas de Adesão Celular/análise , Linhagem Celular , Molécula de Adesão da Célula Epitelial , Citometria de Fluxo/métodos , Humanos , Sensibilidade e Especificidade
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