ABSTRACT
Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities. However, their high molecular weight limits their bioavailability for otherwise-treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor, 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease spinal muscular atrophy (SMA). SMA, like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs that modulate splicing of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in elevated levels of bioavailability to the brain. Additionally, 8D3130-ASO yielded therapeutic levels of SMN2 splicing in the central nervous system of adult human SMN2-transgenic (hSMN2-transgenic) mice, which resulted in extended survival of a severely affected SMA mouse model. Systemic delivery of nucleic acid therapies with brain-targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.
Subject(s)
Muscular Atrophy, Spinal , Neurodegenerative Diseases , Mice , Animals , Humans , Oligonucleotides/pharmacology , Oligonucleotides/therapeutic use , Neurodegenerative Diseases/drug therapy , Muscular Atrophy, Spinal/drug therapy , Muscular Atrophy, Spinal/genetics , Central Nervous System , Oligonucleotides, Antisense/therapeutic use , Mice, Transgenic , Disease Models, AnimalABSTRACT
Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antibody Affinity/immunology , Brain/metabolism , Microdialysis/methods , Receptors, Transferrin/immunology , Animals , Antibodies, Monoclonal/cerebrospinal fluid , Antibodies, Monoclonal/immunology , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/blood , Area Under Curve , Biological Transport , Blood-Brain Barrier/metabolism , Brain/cytology , CHO Cells , Cricetinae , Cricetulus , Endothelial Cells/metabolism , Humans , Male , Rats, Sprague-Dawley , Transcytosis , Trastuzumab/administration & dosage , Trastuzumab/bloodABSTRACT
Delivery of biologic drugs across the blood-brain barrier is becoming a reality. However, the solutions often involve the assembly of complex multi-specific antibody molecules. Here we utilize a simple 12 amino-acid peptide originating from the melanotransferrin (MTf) protein that has shown improved brain delivery properties. 3D confocal fluorescence microscopic analysis demonstrated brain parenchymal localisation of a fluorescently labelled antibody (NIP228) when chemically conjugated to either the MTf peptide or full-length MTf protein. Measurement of plasma kinetics demonstrated the MTf peptide fusions had very similar kinetics to an unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system.
Subject(s)
Drug Carriers/metabolism , Interleukin 1 Receptor Antagonist Protein/therapeutic use , Membrane Glycoproteins/metabolism , Neuralgia/drug therapy , Peptides/metabolism , Animals , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Drug Carriers/chemistry , Humans , Interleukin 1 Receptor Antagonist Protein/administration & dosage , Interleukin 1 Receptor Antagonist Protein/pharmacokinetics , Male , Membrane Glycoproteins/chemistry , Mice, Inbred C57BL , Neuralgia/metabolism , Peptides/chemistryABSTRACT
The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.
Subject(s)
Antibodies/metabolism , Blood-Brain Barrier/metabolism , Receptors, Transferrin/immunology , Receptors, Transferrin/metabolism , Transcytosis/physiology , Animals , Antibodies/pharmacology , Antibody Affinity/physiology , Brain/cytology , Endosomes/drug effects , Endosomes/physiology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , In Vitro Techniques , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Mass Spectrometry , Protein Binding/physiology , Rats , Subcellular Fractions/metabolism , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins , Red Fluorescent ProteinABSTRACT
The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having KDs of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.
Subject(s)
Antibodies, Monoclonal/chemistry , Brain/drug effects , Galanin/chemistry , Receptors, Transferrin/chemistry , Receptors, Transferrin/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antibody Affinity/physiology , Bioengineering/methods , Blood-Brain Barrier/metabolism , Brain/metabolism , Cerebrospinal Fluid/metabolism , Galanin/metabolism , Male , Protein Transport/physiology , Rats , Rats, Sprague-DawleyABSTRACT
With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Neuralgia/drug therapy , Neuralgia/metabolism , Analgesics/therapeutic use , Animals , Antibodies/therapeutic use , Brain/cytology , CD11b Antigen/metabolism , Calcium-Binding Proteins/metabolism , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glial Fibrillary Acidic Protein/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Neuralgia/pathology , Neuroglia/drug effects , Signal Transduction/drug effectsABSTRACT
Neuropathic pain is a major unmet medical need, with only 30% to 35% of patients responding to the current standard of care. The discovery and development of novel therapeutics to address this unmet need have been hampered by poor target engagement, the selectivity of novel molecules, and limited access to the relevant compartments. Biological therapeutics, either monoclonal antibodies (mAbs) or peptides, offer a solution to the challenge of specificity as the intrinsic selectivity of these kinds of molecules is significantly higher than traditional medicinal chemistry-derived approaches. The interleukin-1 receptor system within the spinal cord has been implicated in the amplification of pain signals, and its central antagonism provides relief of neuropathic pain. Targeting the IL-1 system in the spinal cord with biological drugs, however, raises the even greater challenge of delivery to the central compartment. Targeting the transferrin receptor with monoclonal antibodies has proved successful in traversing the endothelial cell-derived blood-brain barrier and delivering proteins to the central nervous system. In this study, we describe a novel construct exemplifying an engineered solution to overcome these challenges. We have generated a novel anti-transferrin receptor-interleukin-1 receptor antagonist fusion that transports to the central nervous system and delivers efficacy in a model of nerve ligation-induced hypersensitivity. Approaches such as these provide promise for novel and selective analgesics that target the central compartment.
Subject(s)
Antibodies/therapeutic use , Central Nervous System/drug effects , Hyperalgesia/etiology , Interleukin 1 Receptor Antagonist Protein/therapeutic use , Receptors, Transferrin/immunology , Sciatica/complications , Animals , Antibodies/pharmacology , Central Nervous System/physiology , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Recombinant Fusion Proteins/pharmacology , Time FactorsABSTRACT
Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aß), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bß-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.
Subject(s)
Alzheimer Disease/drug therapy , Cerebral Amyloid Angiopathy/drug therapy , Neprilysin/administration & dosage , Serum Albumin/genetics , Alzheimer Disease/blood , Alzheimer Disease/complications , Amyloid beta-Peptides/metabolism , Animals , Blood Coagulation/drug effects , Cerebral Amyloid Angiopathy/blood , Cerebral Amyloid Angiopathy/genetics , Fibrin/drug effects , Fibrin/metabolism , Fibrinogen/antagonists & inhibitors , Humans , Macaca fascicularis , Neprilysin/adverse effects , Neprilysin/genetics , Partial Thromboplastin Time , Proteolysis/drug effects , Prothrombin Time , Rats , Serum Albumin/administration & dosage , Serum Albumin/adverse effects , Thromboplastin/geneticsABSTRACT
BACKGROUND/AIM: An in vitro chemoresponse assay may aid effective therapy selection in epithelial ovarian cancer (EOC). This study explores changes in chemoresponse between paired primary and recurrent EOC tumors. PATIENTS AND METHODS: RESULTS from metachronous tumors were examined in 242 patients. Changes in in vitro chemoresponse, measured by the area under the dose response curve (AUC) between paired tumors were assessed. RESULTS: A significant increase in AUC was identified in most first-line therapies over time. No significant difference was observed in most recurrent therapies. When the elapsed time between occurrences was <17 months, no difference was observed for any recurrent therapies, and half of first-line therapies exhibited significant increases in AUC. When ≥17 months, all 7 therapies showed significant increases. CONCLUSION: These results suggest an increase in chemoresistance over time, which is more pronounced for first-line therapies. This is consistent with clinical observations and suggests the biologic concordance between assay results and response to chemotherapy.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm , Neoplasm Recurrence, Local/drug therapy , Neoplasms, Glandular and Epithelial/drug therapy , Neoplasms, Second Primary/drug therapy , Ovarian Neoplasms/drug therapy , Carcinoma, Ovarian Epithelial , Disease-Free Survival , Female , Humans , Neoplasms, Glandular and Epithelial/mortality , Neoplasms, Second Primary/mortality , Ovarian Neoplasms/mortalityABSTRACT
Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/chemistry , Neprilysin/chemistry , Peptide Fragments/chemistry , Peptides/chemistry , Protein Engineering , Recombinant Proteins/chemistry , Alzheimer Disease/drug therapy , Amino Acid Sequence , Amino Acid Substitution , Gene Expression , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Neprilysin/genetics , Peptides/genetics , Protein Structure, Tertiary , Proteolysis , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Structure-Activity RelationshipABSTRACT
BACKGROUND: The feasibility of robotic staging for high-risk endometrial cancer is unclear. METHODS: Retrospective review of papillary serous and clear cell endometrial cancer open staging (OS) and robotic staging (RS) cases (2009-2011) by two gynaecological oncologists. RESULTS: There were 15 OS and 17 RS cases (no conversions). Age, uterine weight and body mass index were comparable, with more stage I RS cases. Operative time (172.5 vs 124.2 min, p = 0.0005), blood loss (71.9 vs 310.0 ml, p = 0.0002), hospital stay (5.4 vs 1.2 days, p = 0.0016) and lymphadenectomy yield (16.8 vs 10.2 nodes, p = 0.0041) were decreased for RS. Optimal cytoreduction rates (100% vs 93%, p = 0.2794), follow-up (19.9 vs 27.1 months, p = 0.2283) and recurrences (three vs five, p = 0.5395) were equivalent. Disease-free survival (54.5% vs 66.7%, p = 0.5302) and overall survival rates (81.8% vs 80.0%, p = 0.9075) were equivalent. CONCLUSIONS: Robotic staging is feasible with minimal blood loss, a short operative time and recovery and good optimal cytoreduction rates.
Subject(s)
Carcinoma, Papillary/surgery , Carcinoma/surgery , Endometrial Neoplasms/surgery , Neoplasm Staging/methods , Robotic Surgical Procedures/methods , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Neoplasm Recurrence, Local , Retrospective Studies , Treatment OutcomeABSTRACT
Alzheimer's disease is characterized by the accumulation of amyloid deposits in the brain and the progressive loss of cognitive functions. Although the precise role of amyloid-ß in disease progression remains somewhat controversial, many efforts to halt or reverse disease progression have focussed on reducing its synthesis or enhancing its removal. It is believed that brain and peripheral soluble amyloid-ß are in equilibrium and it has previously been hypothesized that a reduction in peripheral amyloid-ß can lower brain amyloid-ß, thereby reducing formation of plaques predominantly composed of insoluble amyloid-ß; the so-called peripheral sink hypothesis. Here we describe the use of an amyloid-ß degrading enzyme, the endogenous metallopeptidase neprilysin, which is fused to albumin to extend plasma half-life and has been engineered to confer increased amyloid-ß degradation activity. We used this molecule to investigate the effect of degradation of peripheral amyloid-ß on amyloid-ß levels in the brain and cerebrospinal fluid after repeated intravenous dosing for up to 4 months in Tg2576 transgenic mice, and 1 month in rats and monkeys. This molecule proved highly effective at degradation of amyloid-ß in the periphery but did not alter brain or cerebrospinal fluid amyloid-ß levels, suggesting that the peripheral sink hypothesis is not valid and is the first time that this has been demonstrated in non-human primates.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Brain/drug effects , Brain/metabolism , Neprilysin/administration & dosage , Animals , Female , Humans , Injections, Intravenous , Macaca fascicularis , Male , Mice , Mice, Transgenic , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: While most gynecologic cancers respond to first-line cytotoxic chemotherapy, treatment of recurrent disease is frequently associated with acquired drug resistance. In order to find an in vitro surrogate of this clinical phenomenon, a tumor chemoresponse assay was studied. METHODS/MATERIALS: Patients who had tissue submitted for repeated chemoresponse testing were identified through a retrospective search. Sixty-three patients met inclusion criteria (chemoresponse testing completed at primary diagnosis and upon recurrence of disease and assays completed ≥90 days apart). The Wilcoxon signed-rank test was used to compare chemoresponse, represented as a response index (RI), between primary and recurrent measurements. In a secondary analysis, response was categorized and coded as Responsive = 3, Intermediately Responsive = 2 and Non-Responsive = 1, and the paired t-test was used to compare chemoresponse between primary and recurrent measurement. RESULTS: Median time between primary and recurrent tumor testing was 309 days (IQR 208-422). Drugs tested included carboplatin, cisplatin, docetaxel, doxorubicin, gemcitabine, paclitaxel, topotecan, and combination carboplatin/gemcitabine and carboplatin/paclitaxel. There were no differences in chemoresponse between primary and recurrent measurement when chemoresponse was represented by RI scores; although a trend toward increased resistance to paclitaxel upon recurrence was noted. When chemoresponse was analyzed as a continuous variable corresponding to categorized response, a significant shift toward increased resistance to paclitaxel at recurrence, and a marginally significant trend toward increased resistance to carboplatin at recurrence, were observed. CONCLUSIONS: We observed a trend toward increased chemoresistance at recurrence for paclitaxel, and a marginally significant trend toward increased chemoresistance to carboplatin, but no change in chemoresponsiveness between primary diagnosis and recurrence of disease for other common chemotherapy drugs, including common second-line agents such as doxorubicin, gemcitabine, and topotecan.
ABSTRACT
Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments.
Subject(s)
Histocompatibility Antigens Class I/genetics , Immunoglobulin G/genetics , Mutation/genetics , Receptors, Fc/genetics , Recombinant Fusion Proteins/genetics , Single-Chain Antibodies/genetics , Animals , Half-Life , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Humans , Immunoglobulin G/administration & dosage , Immunoglobulin G/chemistry , Mice , Mice, Inbred BALB C , Protein Binding/drug effects , Protein Engineering , Protein Stability/drug effects , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Receptors, Fc/chemistry , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Single-Chain Antibodies/administration & dosage , Single-Chain Antibodies/chemistryABSTRACT
A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Drug Screening Assays, Antitumor/methods , Neoplasms/drug therapy , Antineoplastic Agents/chemistry , Cell Line, Tumor , Chronic Disease , Drug Discovery/methods , HL-60 Cells , Humans , Leukemia/drug therapy , Leukemia/pathology , Neoplasms/pathologyABSTRACT
Structural backbones of iron-scavenging siderophore molecules include polyamines 1,3-diaminopropane and 1,5-diaminopentane (cadaverine). For the cadaverine-based desferroxiamine E siderophore in Streptomyces coelicolor, the corresponding biosynthetic gene cluster contains an ORF encoded by desA that was suspected of producing the cadaverine (decarboxylated lysine) backbone. However, desA encodes an l-2,4-diaminobutyrate decarboxylase (DABA DC) homologue and not any known form of lysine decarboxylase (LDC). The only known function of DABA DC is, together with l-2,4-aminobutyrate aminotransferase (DABA AT), to synthesize 1,3-diaminopropane. We show here that S. coelicolor desA encodes a novel LDC and we hypothesized that DABA DC homologues present in siderophore biosynthetic clusters in the absence of DABA AT ORFs would be novel LDCs. We confirmed this by correctly predicting the LDC activity of a DABA DC homologue from a Yersinia pestis siderophore biosynthetic pathway. The corollary was confirmed for a DABA DC homologue, adjacent to a DABA AT ORF in a siderophore pathway in the cyanobacterium Anabaena variabilis, which was shown to be a bona fide DABA DC. These findings enable prediction of whether a siderophore pathway will utilize 1,3-diaminopropane or cadaverine, and suggest that the majority of bacteria use DABA AT and DABA DC for siderophore, rather than norspermidine/polyamine biosynthesis.
Subject(s)
Bacterial Proteins/metabolism , Carboxy-Lyases/metabolism , Evolution, Molecular , Siderophores/biosynthesis , Streptomyces coelicolor/enzymology , Anabaena variabilis/chemistry , Anabaena variabilis/enzymology , Anabaena variabilis/genetics , Bacteria/chemistry , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/genetics , Carboxy-Lyases/genetics , Molecular Sequence Data , Phylogeny , Polyamines/metabolism , Streptomyces coelicolor/geneticsABSTRACT
BACKGROUND: This study was performed to determine if a chemotherapy-induced apoptosis assay (MiCK) could predict the best therapy for patients with ovarian cancer. METHODS: A prospective, multi-institutional and blinded trial of the assay was conducted in 104 evaluable ovarian cancer patients treated with chemotherapy. The MiCK assay was performed prior to therapy, but treating physicians were not told of the results and selected treatment only on clinical criteria. Outcomes (response, time to relapse, and survival) were compared to the drug-induced apoptosis observed in the assay. RESULTS: Overall survival in primary therapy, chemotherapy naïve patients with Stage III or IV disease was longer if patients received a chemotherapy which was best in the MiCK assay, compared to shorter survival in patients who received a chemotherapy that was not the best. (p < 0.01, hazard ratio HR 0.23). Multivariate model risk ratio showed use of the best chemotherapy in the MiCK assay was the strongest predictor of overall survival (p < 0.01) in stage III or IV patients. Standard therapy with carboplatin plus paclitaxel (C + P) was not the best chemotherapy in the MiCK assay in 44% of patients. If patients received C + P and it was the best chemotherapy in the MiCK assay, they had longer survival than those patients receiving C + P when it was not the best chemotherapy in the assay (p = 0.03). Relapse-free interval in primary therapy patients was longer if patients received the best chemotherapy from the MiCK assay (p = 0.03, HR 0.52). Response rates (CR + PR) were higher if physicians used an active chemotherapy based on the MiCK assay (p = 0.03). CONCLUSION: The MiCK assay can predict the chemotherapy associated with better outcomes in ovarian cancer patients. This study quantifies outcome benefits on which a prospective randomized trial can be developed.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Ovarian Neoplasms/drug therapy , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Female , Humans , Ovarian Neoplasms/pathology , Survival Rate , Treatment OutcomeABSTRACT
Arginine decarboxylases (ADCs; EC 4.1.1.19) from four different protein fold families are important for polyamine biosynthesis in bacteria, archaea, and plants. Biosynthetic alanine racemase fold (AR-fold) ADC is widespread in bacteria and plants. We report the discovery and characterization of an ancestral form of the AR-fold ADC in the bacterial Chloroflexi and Bacteroidetes phyla. The ancestral AR-fold ADC lacks a large insertion found in Escherichia coli and plant AR-fold ADC and is more similar to the lysine biosynthetic enzyme meso-diaminopimelate decarboxylase, from which it has evolved. An E. coli acid-inducible ADC belonging to the aspartate aminotransferase fold (AAT-fold) is involved in acid resistance but not polyamine biosynthesis. We report here that the acid-inducible AAT-fold ADC has evolved from a shorter, ancestral biosynthetic AAT-fold ADC by fusion of a response regulator receiver domain protein to the N terminus. Ancestral biosynthetic AAT-fold ADC appears to be limited to firmicute bacteria. The phylogenetic distribution of different forms of ADC distinguishes bacteria from archaea, euryarchaeota from crenarchaeota, double-membraned from single-membraned bacteria, and firmicutes from actinobacteria. Our findings extend to eight the different enzyme forms carrying out the activity described by EC 4.1.1.19. ADC gene clustering reveals that polyamine biosynthesis employs diverse and exchangeable synthetic modules. We show that in Bacillus subtilis, ADC and polyamines are essential for biofilm formation, and this appears to be an ancient, evolutionarily conserved function of polyamines in bacteria. Also of relevance to human health, we found that arginine decarboxylation is the dominant pathway for polyamine biosynthesis in human gut microbiota.
Subject(s)
Bacillus subtilis/metabolism , Biofilms , Carboxy-Lyases/metabolism , Polyamines/chemistry , Alanine Racemase/metabolism , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/metabolism , Genomics , Humans , Intestines/microbiology , Molecular Conformation , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Homology, Amino AcidABSTRACT
We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Enzyme Assays/methods , Animals , Base Sequence , Cattle , DNA/genetics , DNA Gyrase/metabolism , DNA, Superhelical/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Topoisomerase II InhibitorsABSTRACT
A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.