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1.
Proc Natl Acad Sci U S A ; 117(29): 16949-16960, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616569

RESUMO

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.


Assuntos
Anticorpos/química , Afinidade de Anticorpos , Arginase/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Humanos
2.
PLoS Comput Biol ; 15(5): e1006980, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31042706

RESUMO

Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.


Assuntos
Afinidade de Anticorpos/fisiologia , Biologia Computacional/métodos , Sequência de Aminoácidos , Animais , Anticorpos/química , Quimiocina CCL20 , Simulação por Computador , Desenho de Fármacos , Região Variável de Imunoglobulina , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
3.
J Immunol ; 198(1): 528-537, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881707

RESUMO

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.


Assuntos
Abatacepte/farmacologia , Desenho de Fármacos , Imunossupressores/farmacologia , Abatacepte/química , Animais , Antígeno B7-1/imunologia , Antígeno B7-2 , Estabilidade de Medicamentos , Humanos , Imunossupressores/química , Ligação Proteica/imunologia
4.
Anal Chem ; 89(4): 2361-2368, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28194941

RESUMO

Antibodies are an important class of drugs, comprising more than half of all new FDA approvals. Therapeutic antibodies must be chemically stable both in storage and in vivo, following administration to patients. Deamidation is a major degradation pathway for all natural and therapeutic proteins circulating in blood. Here, the linkage between deamidation propensity and structural dynamics is investigated by examining two antibodies with differing specificities. While both antibodies share a canonical asparagine-glycine (NG) motif in a structural loop, this is prone to deamidation in one of the antibodies but not the other. We found that the hydrogen-exchange rate at the adjacent two amides, often the autocatalytic nucleophiles in deamidation, correlated with the rate of degradation. This previously unreported observation was confirmed upon mutation to stabilize the deamidation lability via a generally applicable orthogonal engineering strategy presented here. We anticipate that the structural insight into chemical degradation in full-length monoclonal antibodies and the high-resolution hydrogen-exchange methodology used will have broad application across biochemical study and drug discovery and development.


Assuntos
Amidas/metabolismo , Anticorpos Monoclonais/metabolismo , Asparagina/metabolismo , Espectrometria de Massas/métodos , Amidas/química , Anticorpos Monoclonais/química , Asparagina/química , Catálise , Medição da Troca de Deutério , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo
5.
Blood ; 125(22): 3484-90, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25788700

RESUMO

Ticagrelor is a direct-acting reversibly binding P2Y12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y12. Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 5'-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures.


Assuntos
Adenosina/análogos & derivados , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Antídotos/química , Antídotos/farmacologia , Adenosina/antagonistas & inibidores , Adenosina/imunologia , Animais , Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Especificidade de Anticorpos , Anticorpos Amplamente Neutralizantes , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Hemorragia/prevenção & controle , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Camundongos , Modelos Moleculares , Agregação Plaquetária/efeitos dos fármacos , Engenharia de Proteínas , Ticagrelor
6.
Biotechnol Bioeng ; 112(7): 1472-7, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25619171

RESUMO

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab.


Assuntos
Expressão Gênica , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/isolamento & purificação , Dimerização , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
8.
Nat Chem ; 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755312

RESUMO

Several peptide dual agonists of the human glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R) are in development for the treatment of type 2 diabetes, obesity and their associated complications. Candidates must have high potency at both receptors, but it is unclear whether the limited experimental data available can be used to train models that accurately predict the activity at both receptors of new peptide variants. Here we use peptide sequence data labelled with in vitro potency at human GCGR and GLP-1R to train several models, including a deep multi-task neural-network model using multiple loss optimization. Model-guided sequence optimization was used to design three groups of peptide variants, with distinct ranges of predicted dual activity. We found that three of the model-designed sequences are potent dual agonists with superior biological activity. With our designs we were able to achieve up to sevenfold potency improvement at both receptors simultaneously compared to the best dual-agonist in the training set.

9.
Nat Biomed Eng ; 8(3): 214-232, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37814006

RESUMO

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody-antigen interactions. The method, which we named 'deep screening', involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument's flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.


Assuntos
Anticorpos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anticorpos/genética , Anticorpos/metabolismo , Biblioteca Gênica , Fragmentos de Imunoglobulinas , Ribossomos/genética , Ribossomos/metabolismo
10.
Sci Rep ; 13(1): 9825, 2023 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-37330528

RESUMO

Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33red) and oxidized IL-33 (IL-33ox) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products/epidermal growth factor receptor (RAGE/EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 107 M-1 s-1, to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33red and a fast association rate (8.5 × 107 M-1 s-1), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33red and IL-33ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.


Assuntos
Inflamação , Proteína 1 Semelhante a Receptor de Interleucina-1 , Camundongos , Humanos , Animais , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Inflamação/metabolismo , Interleucina-33/metabolismo , Citocinas/metabolismo , Receptores ErbB/metabolismo , Transdução de Sinais
11.
MAbs ; 13(1): 1992068, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34781832

RESUMO

Bioconjugates are an important class of therapeutic molecules. To date, O-glycan-based metabolic glycoengineering has had limited use in this field, due to the complexities of the endogenous O-glycosylation pathway and the lack of an O-glycosylation consensus sequence. Here, we describe the development of a versatile on-demand O-glycosylation system that uses a novel, widely applicable 5 amino acid O-glycosylation tag, and a metabolically engineered UDP-galactose-4-eperimase (GALE) knock-out cell line. Optimization of the primary sequence of the tag enables the production of Fc-based proteins with either single or multiple O-glycans with complexity fully controlled by media supplementation. We demonstrate how the uniformly labeled proteins containing exclusively N-azido-acetylgalactosamine are used for CLICK chemistry-based bioconjugation to generate site-specifically fluorochrome-labeled antibodies, dual-payload molecules, and bioactive Fc-peptides for applications in basic research and drug discovery. To our knowledge, this is the first description of generating a site-specific O-glycosylation system by combining an O-glycosylation tag and a metabolically engineered cell line.


Assuntos
Química Click , Polissacarídeos , Glicosilação , Polissacarídeos/química
12.
J Cell Biol ; 219(4)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32328641

RESUMO

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.


Assuntos
Pseudópodes/metabolismo , Nexinas de Classificação/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Feminino , Células HeLa , Humanos , Masculino , Nexinas de Classificação/genética , Proteínas de Xenopus/genética , Xenopus laevis
13.
Nat Commun ; 11(1): 1326, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165615

RESUMO

Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface. Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.


Assuntos
Fator H do Complemento/metabolismo , Trypanosoma/fisiologia , Moscas Tsé-Tsé/parasitologia , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Bovinos , Membrana Celular/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/química , Cricetinae , Cricetulus , Camundongos Endogâmicos BALB C , Parasitemia/sangue , Ligação Proteica , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Regulação para Cima
14.
MAbs ; 12(1): 1801230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32880207

RESUMO

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.


Assuntos
Afinidade de Anticorpos , Arginase/química , Anticorpos de Cadeia Única/química , Regulação Alostérica , Cristalografia por Raios X , Humanos
15.
PLoS One ; 14(3): e0214545, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925190

RESUMO

Doxorubicin is a chemotherapeutic agent that is commonly used to treat a broad range of cancers. However, significant cardiotoxicity, associated with prolonged exposure to doxorubicin, limits its continued therapeutic use. One strategy to prevent the uptake of doxorubicin into cardiac cells is the encapsulation of the drug to prevent non-specific uptake and also to improve the drugs' pharmacokinetic properties. Although encapsulated forms of doxorubicin limit the cardiotoxicity observed, they are not without their own liabilities as an increased amount of drug is deposited in the skin where liposomal doxorubicin can cause palmar-plantar erythrodysesthesia. Exosomes are small endogenous extracellular vesicles, that transfer bioactive material from one cell to another, and are considered attractive drug delivery vehicles due to their natural origin. In this study, we generated doxorubicin-loaded exosomes and demonstrate their rapid cellular uptake and re-distribution of doxorubicin from endosomes to the cytoplasm and nucleus resulting in enhanced potency in a number of cultured and primary cell lines when compared to free doxorubicin and liposomal formulations of doxorubicin. In contrast to other delivery methods for doxorubicin, exosomes do not accumulate in the heart, thereby providing potential for limiting the cardiac side effects and improved therapeutic index.


Assuntos
Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Exossomos/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico , Linhagem Celular , Exossomos/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Cinética
16.
Nat Microbiol ; 4(12): 2074-2081, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31636418

RESUMO

To maintain prolonged infection of mammals, African trypanosomes have evolved remarkable surface coats and a system of antigenic variation1. Within these coats are receptors for macromolecular nutrients such as transferrin2,3. These must be accessible to their ligands but must not confer susceptibility to immunoglobulin-mediated attack. Trypanosomes have a wide host range and their receptors must also bind ligands from diverse species. To understand how these requirements are achieved, in the context of transferrin uptake, we determined the structure of a Trypanosoma brucei transferrin receptor in complex with human transferrin, showing how this heterodimeric receptor presents a large asymmetric ligand-binding platform. The trypanosome genome contains a family of around 14 transferrin receptors4, which has been proposed to allow binding to transferrin from different mammalian hosts5,6. However, we find that a single receptor can bind transferrin from a broad range of mammals, indicating that receptor variation is unlikely to be necessary for promiscuity of host infection. In contrast, polymorphic sites and N-linked glycans are preferentially found in exposed positions on the receptor surface, not contacting transferrin, suggesting that transferrin receptor diversification is driven by a need for antigenic variation in the receptor to prolong survival in a host.


Assuntos
Interações Hospedeiro-Parasita/imunologia , Evasão da Resposta Imune , Receptores da Transferrina/química , Receptores da Transferrina/imunologia , Transferrina/metabolismo , Trypanosoma brucei brucei/imunologia , Variação Antigênica , Variação Genética , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Tripanossomíase Africana/imunologia
17.
Endocrinology ; 160(5): 1057-1064, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30888399

RESUMO

Both fibroblast growth factors (FGFs), by binding to FGF receptors (FGFRs), and activation of the gravitostat, by artificial loading, decrease the body weight (BW). Previous studies demonstrate that both the FGF system and loading have the capacity to regulate BW independently of leptin. The aim of the current study was to determine the possible interactions between the effect of increased loading and the FGF system for the regulation of BW. We observed that the BW-reducing effect of increased loading was abolished in mice treated with a monoclonal antibody directed against FGFR1c, suggesting interactions between the two systems. As serum levels of endocrine FGF21 and hepatic FGF21 mRNA were increased in the loaded mice compared with the control mice, we first evaluated the loading response in FGF21 over expressing mice with constant high FGF21 levels. Leptin treatment, but not increased loading, decreased the BW in the FGF21-overexpressing mice, demonstrating that specifically the loading effect is attenuated in the presence of high activity in the FGF system. However, as FGF21 knockout mice displayed a normal loading response on BW, FGF21 is neither mediating nor essential for the loading response. In conclusion, the BW-reducing effect of increased loading but not of leptin treatment is blocked by high activity in the FGF system. We propose that both the gravitostat and the FGF system regulate BW independently of leptin and that pharmacologically enhanced activity in the FGF system reduces the sensitivity of the gravitostat.


Assuntos
Peso Corporal/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica/efeitos dos fármacos , Leptina/farmacologia , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
18.
Ann Clin Transl Neurol ; 6(3): 554-574, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30911579

RESUMO

Objective: Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aßo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods: Potency of AZ59 was evaluated by binding to PrPC, blockade of Aßo interaction and interruption of Aßo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results: AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Interpretation: Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Anticorpos Monoclonais/uso terapêutico , Proteínas PrPC/antagonistas & inibidores , Proteínas PrPC/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Sítios de Ligação , Encéfalo/patologia , Células COS , Chlorocebus aethiops , Cognição , Modelos Animais de Doenças , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Sinapses/patologia
19.
Pain ; 160(9): 1989-2003, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31045747

RESUMO

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Neuralgia/metabolismo , Neuralgia/prevenção & controle , Receptores Purinérgicos P2X4/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/fisiologia , Antagonistas do Receptor Purinérgico P2X/administração & dosagem , Antagonistas do Receptor Purinérgico P2X/metabolismo , Ratos , Ratos Sprague-Dawley
20.
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
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