RESUMO
Convergence across B-cell receptor (BCR) and antibody repertoires has become instrumental in prioritizing candidates in recent rapid therapeutic antibody discovery campaigns. It has also increased our understanding of the immune system, providing evidence for the preferential selection of BCRs to particular (immunodominant) epitopes post vaccination/infection. These important implications for both drug discovery and immunology mean that it is essential to consider the optimal way to combine experimental and computational technology when probing BCR repertoires for convergence signatures. Here, we discuss the theoretical basis for observing BCR repertoire functional convergence and explore factors of study design that can impact functional signal. We also review the computational arsenal available to detect antibodies with similar functional properties, highlighting opportunities enabled by recent clustering algorithms that exploit structural similarities between BCRs. Finally, we suggest future areas of development that should increase the power of BCR repertoire functional clustering.
Assuntos
Anticorpos , Receptores de Antígenos de Linfócitos B , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
The origins of the various elements in the human antibody repertoire have been and still are subject to considerable uncertainty. Uncertainty in respect of whether the various elements have always served a specific defense function or whether they were co-opted from other organismal roles to form a crude naïve repertoire that then became more complex as combinatorial mechanisms were added. Estimates of the current size of the human antibody naïve repertoire are also widely debated with numbers anywhere from 10 million members, based on experimentally derived numbers, to in excess of one thousand trillion members or more, based on the different sequences derived from theoretical combinatorial calculations. There are questions that are relevant at both ends of this number spectrum. At the lower bound it could be questioned whether this is an insufficient repertoire size to counter all the potential antigen-bearing pathogens. At the upper bound the question is rather simpler: How can any individual interrogate such an astronomical number of antibody-bearing B cells in a timeframe that is meaningful? This review evaluates the evolutionary aspects of the adaptive immune system, the calculations that lead to the large repertoire estimates, some of the experimental evidence pointing to a more restricted repertoire whose variation appears to derive from convergent 'structure and specificity features', and includes a theoretical model that seems to support it. Finally, a solution that may reconcile the size difference anomaly, which is still a hot subject of debate, is suggested.
Assuntos
Formação de Anticorpos/imunologia , HumanosRESUMO
Molecularly imprinted polymers (MIPs) are tailor-made materials that can specifically rebind desired target molecules. Usually, the template is in solution with monomers that form a specific polymeric cavity around the template. Here, a model system with an amino acid (Fmoc-L-Histidine) is presented. This new approach entails the imprinting of templates that are bound to support materials. It is shown that the new approach leads to materials that exhibit symmetric peak shapes for both enantiomers under isocratic conditions.
Assuntos
Impressão Molecular , Polímeros/química , Cromatografia Líquida de Alta Pressão , Histidina/química , Estrutura Molecular , Estereoisomerismo , Propriedades de SuperfícieRESUMO
In antibody humanization, complementarity determining regions from a 'donor' antibody are often grafted onto a human framework selected by high sequence identity with the donor. In our own humanization experiments, we have found that species information is often incorrect. Here we take three mouse antibodies and perform BLAST searches against sequences annotated as being human. We find that the first genuine human hits for the six chains appear at Positions 30, 4, 11, 24, 18 and 29 in the hit lists. This illustrates both the need for caution in performing humanization and for improvements in annotation.
Assuntos
Anticorpos Monoclonais Humanizados/química , Biologia Computacional , Bases de Dados de Proteínas , Projetos de Pesquisa , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Anotação de Sequência Molecular , Especificidade da EspécieRESUMO
The crossreactivity of molecularly imprinted polymers (MIPs) and its practical implications are discussed. Screening of MIP libraries is presented as a fasttrack route to discovery of resins selective towards new targets, exploiting the fact that MIPs imprinted with one type of template molecule also show recognition to related and sometimes also to apparently unrelated molecules. Several examples from our own and others' studies are presented that illustrate this crossreactivity and the pattern of recognition is discussed for selected examples.
Assuntos
Impressão Molecular/métodos , Polímeros/química , Polímeros/síntese químicaRESUMO
The mitochondrial permeability transition (mPT) is considered to be an important mediator of apoptosis and necrosis, and is specifically blocked by cyclosporin A (CsA). CsA has been shown to exert a potent neuroprotective action in vivo when allowed to cross the blood-brain barrier in various animal models of acute neurological insults and neurodegenerative disease. The neuroprotective effect of CsA is considered to be mediated through specific inhibition of the mitochondrial permeability transition pore (mPTP) and through inhibition of neuronal calcineurin activity. Characterization of mPT has mainly been performed in liver and heart mitochondria, and some brain studies have reported a decreased inhibitory effect of CsA and questioned the importance of mPT in brain-derived mitochondria. We have used the de-energized model of swelling to examine the mPT in brain-derived non-synaptosomal mitochondria. Ca(2+)-induced swelling was evaluated by electron microscopy and by measurement of spectrophotometric alterations in light scattering. Permeability transition was readily induced in a majority of the mitochondria at a wide range of Ca(2+) levels and was powerfully inhibited by CsA with a half-maximal effect at approximately 23 nM CsA. The swelling kinetics and CsA effects were comparable to previous findings in de-energized liver and heart mitochondria. Careful characterization of mPT and CsA effects in brain-derived mitochondria is the first step in evaluating newly developed CsA analogues capable of crossing the blood-brain barrier and preferentially entering the brain. The importance of CsA causing a shift of the mitochondrial sensitivity to Ca(2+) in neurological disorders is discussed.
Assuntos
Química Encefálica/efeitos dos fármacos , Cálcio/antagonistas & inibidores , Cálcio/farmacologia , Ciclosporina/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/ultraestrutura , Dilatação Mitocondrial/efeitos dos fármacos , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Biological membranes normally restrict the passage of hydrophilic molecules. This impairs the use of a wide variety of drugs for biomedical applications. To overcome this problem, researchers have developed strategies that involve conjugating the molecule of interest to one of a number of peptide entities that are efficiently transported across the cell membranes. In the past decade, a number of different peptide families with the ability to cross the cell membranes have been identified. Certain of these families enter the cells by a receptor-independent mechanism, are short (10-27 amino acid residues), and can deliver successfully various cargoes across the cell membrane into the cytoplasm or nucleus. Surprisingly, some of these vectors, the SynB vectors, have also shown the ability to deliver hydrophilic molecules across the blood-brain barrier, one of the major obstacles to the development of drugs to combat diseases affecting the CNS.
Assuntos
Encéfalo/metabolismo , Endocitose/fisiologia , Animais , Transporte Biológico , Sistemas de Liberação de Medicamentos , Humanos , Peptídeos/farmacocinéticaRESUMO
This study describes the identification and evaluation of molecularly imprinted polymers (MIPs) for the selective removal of potentially genotoxic aminopyridine impurities from pharmaceuticals. Screening experiments were performed using existing MIP resin libraries to identify resins selective towards those impurities in the presence of model pharmaceutical compounds. A hit resin with a considerable imprinting effect was found in the screening and upon further investigation, the resin was found to show a broad selectivity towards five different aminopyridines in the presence of the two model active pharmaceutical ingredients (APIs) piroxicam and tenoxicam.
Assuntos
Aminopiridinas/isolamento & purificação , Carcinógenos/isolamento & purificação , Contaminação de Medicamentos , Nicotina/análogos & derivados , Preparações Farmacêuticas/química , Polímeros/química , Impressão Molecular , Nicotina/química , Piroxicam/análogos & derivados , Piroxicam/químicaRESUMO
A direct aqueous SFE system designed to extract water samples contained in vials has been coupled on-line with a reverse phase LC-MS-MS system using a single 10-port valve. An SFE trap system using C(1) stationary phase connected to a C(18) analytical HPLC column enabled the SFE-LC-MS-MS analysis of three polyether ionophore antibiotics in water using a step gradient. A quantitative SFE-LC-MS-MS method has been developed whereby the progress of SFE can be monitored directly on-line such that ionophore recovery profile data from a single water sample can be obtained. Using a continuous direct aqueous SFE period of 75 min, the SFE-LC-MS-MS recoveries of the ionophores were: monensin 76.2% with RSD 4.1%, lasalocid 84.6% with RSD 3.8% and narasin 91.2% with RSD 3.2%. With positive ion electrospray ionization, the SFE-LC-MS-MS system using a 4 mL water sample provided multiple reaction monitoring (MRM) limits of detection for monensin and lasalocid each equivalent to 90 ng/L whereas 30 ng/L for narasin. A two-way valve controlling carbon dioxide distribution to the SFE vessel has provided a means for the initial investigation of the recovery of ionophore sodium salts from water using static SFE.
Assuntos
Antibacterianos/análise , Cromatografia Líquida/métodos , Cromatografia com Fluido Supercrítico/métodos , Ionóforos/análise , Espectrometria de Massas em Tandem/métodos , Poluentes Químicos da Água/análise , Antibacterianos/química , Ionóforos/química , Estrutura Molecular , Poluentes Químicos da Água/químicaRESUMO
Fab 35PA83 is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part of the "self" proteins, are preferable. Accordingly, the proportion of FR residues in 35PA83 that were encoded by human V and J germline genes, i.e. the germinality index (GI) of 35PA83, was increased in a multistep cumulative approach. In a first step, the FR1 and FR4 residues of 35PA83 were changed simultaneously into their counterparts coded by 35PA83's closest human germline genes, without prior modelling. The resulting derivative of 35PA83 had the same affinity as its parental Fab. In a second step, the 3D structures of this first 35PA83 derivative, carrying the same type of residue changes but in the FR2 and FR3 regions, were modelled in silico from sequences. Some of the changes in FR2 or FR3 modified the predicted peptide backbone. The changes that did not seem to alter the structure were introduced simultaneously in the Fab by an in vitro method and resulted in a loss of reactivity, which could however be fully restored by a single point mutation. The final 35PA83 derivative had a GI higher than that of a fully human Fab, which had neutralization properties similar to 35PA83 and which was used as a benchmark in this study.
Assuntos
Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Biologia Computacional , Células Germinativas/imunologia , Primatas/imunologia , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos/química , Afinidade de Anticorpos/imunologia , Linhagem Celular , Humanos , Região Variável de Imunoglobulina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Mutação Puntual/genética , Estrutura Secundária de Proteína , Alinhamento de SequênciaRESUMO
Morphine-6-glucuronide (M6G), an active metabolite of morphine, has been shown to have significantly attenuated brain penetration relative to that of morphine. Recently, we have demonstrated that conjugation of various drugs to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated morphine-6-glucuronide to a peptide vector SynB3 to enhance its brain uptake and its analgesic potency after systemic administration. We show by in situ brain perfusion that vectorization of M6G (Syn1001) markedly enhances the brain uptake of M6G. This enhancement results in a significant improvement in the pharmacological activity of M6G in several models of nociception. Syn1001 was about 4 times more potent than free M6G (ED(50) of 1.87 versus 8.74 micromol/kg). Syn1001 showed also a prolonged duration of action compared with free M6G (300 and 120 min, respectively). Furthermore, the conjugation of M6G results in a lowered respiratory depression, as measured in a rat model. Taken together, these data strongly support the utility of peptide-mediated strategies for improving the efficacy of drugs such as M6G for the treatment of pain.
Assuntos
Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Derivados da Morfina/metabolismo , Oligopeptídeos/administração & dosagem , Oligopeptídeos/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Camundongos , Derivados da Morfina/administração & dosagem , Oligopeptídeos/síntese química , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodosRESUMO
A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein, which is capable of lowering intracellular drug concentrations. In the present study, we tested the capability of 2-pyrrolinodoxorubicin (p-DOX), a highly potent derivative of DOX, to bypass multidrug resistance. The accumulation, intracellular distribution and cytotoxicity of p-DOX were tested in two cell lines (K562 and A2780) and their DOX-resistant counterparts (K562/ADR and A2780/ADR). Cellular accumulation and cytotoxicity were dramatically lowered for DOX in resistant cell lines, in comparison with non-resistant cells. In contrast, cellular accumulation, intracellular distribution and cytotoxicity of p-DOX were independent of the nature of the cell lines. The p-DOX showed potent dose-dependent inhibition of cell growth against resistant cells as compared with DOX. After treatment of resistant cells with verapamil, the intracellular levels of DOX were markedly increased and consequent cytotoxicity improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement of cell uptake or an increase in the cytotoxic effect of the derivative p-DOX, indicating that the compound bypasses the P-glycoprotein. Finally, we show that vectorization of p-DOX by a peptide vector (SynB3) which has been shown to enhance the brain uptake of DOX and to decrease its heart accumulation does not affect this property. These results indicate that p-DOX and its vectorized form are potent and effective in overcoming multidrug resistance.
Assuntos
Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/fisiologia , Adjuvantes Farmacêuticos/administração & dosagem , Adjuvantes Farmacêuticos/síntese química , Adjuvantes Farmacêuticos/farmacologia , Transporte Biológico , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/síntese química , Proteínas de Transporte/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Expressão Gênica/genética , Genes MDR/genética , Humanos , Líquido Intracelular/química , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/fisiologia , Células K562 , Pirróis/metabolismo , Pirróis/farmacologia , Células Tumorais Cultivadas , Verapamil/farmacologiaRESUMO
A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.
Assuntos
Endocitose/fisiologia , Proteínas de Homeodomínio/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas Nucleares , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Proteína do Homeodomínio de Antennapedia , Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Cátions/metabolismo , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Fixadores/farmacologia , Proteínas de Homeodomínio/química , Humanos , Concentração de Íons de Hidrogênio , Células K562 , Dados de Sequência Molecular , Nocodazol/farmacologia , Oligopeptídeos/fisiologia , Fragmentos de Peptídeos/química , Temperatura , Vimblastina/farmacologiaRESUMO
The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.
Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Leucina Encefalina-2-Alanina/análogos & derivados , Leucina Encefalina-2-Alanina/administração & dosagem , Analgesia , Animais , Transporte Biológico , Leucina Encefalina-2-Alanina/química , Leucina Encefalina-2-Alanina/farmacocinética , Camundongos , Peptídeos/administração & dosagem , Peptídeos/químicaRESUMO
Linear peptides (SynB vectors) with specific sequence motifs have been identified that are capable of enhancing the transport of a wide range of molecules into cells. These peptide vectors have been used to deliver exogenous peptides and protein Ags across the cell membrane and into the cytoplasm of cells. Specifically, in vitro analysis indicated that these SynB peptides enhanced the uptake of two 9-mer peptide Ags, NP(147-155) and Mtb(250-258) (T cell epitopes of influenza nucleoprotein and Mycobacterium tuberculosis, respectively) and the M. tuberculosis Ag Mtb8.4 protein, into K562 cells when covalently linked to the respective Ags. Furthermore, selected SynB vectors, when conjugated to these same Ags and used as immunogens, resulted in considerably enhanced Ag-specific CTL responses. Several SynB vectors were tested and resulted in varying levels of cellular uptake. The efficiency of uptake correlated with the ability of the SynB construct to deliver each epitope in vivo and induce specific CTL responses in mice. These data suggest that peptide vectors, such as SynB that transport target Ags across the cell membrane in a highly efficient manner, have significant potential for vaccine delivery.
Assuntos
Antígenos de Bactérias/administração & dosagem , Citotoxicidade Imunológica , Epitopos de Linfócito T/administração & dosagem , Ativação Linfocitária , Nucleoproteínas/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Proteínas de Ligação a RNA , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/administração & dosagem , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Citotoxicidade Imunológica/genética , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Células K562 , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Mycobacterium tuberculosis/imunologia , Proteínas do Nucleocapsídeo , Nucleoproteínas/imunologia , Nucleoproteínas/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/metabolismoRESUMO
The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD4/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Humanos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Ligação ProteicaRESUMO
Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.