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1.
Artigo em Inglês | MEDLINE | ID: mdl-32062365

RESUMO

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.


Assuntos
Fragmentos Fab das Imunoglobulinas/análise , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/análise , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulinas/metabolismo , Sulfato de Amônio/química , Animais , Western Blotting , Galinhas , Cromatografia por Troca Iônica , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulinas/química , Papaína/metabolismo
2.
Acta Crystallogr D Struct Biol ; 75(Pt 11): 1003-1014, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31692474

RESUMO

Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.


Assuntos
Descoberta de Drogas , Fragmentos Fab das Imunoglobulinas/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Anticorpos de Cadeia Única/química , Animais , Apoptose , Células CHO , Clonagem Molecular , Cricetulus , Escherichia coli/genética , Humanos
3.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 10): 634-639, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31584011

RESUMO

An increased level of granulocyte-macrophage colony-stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J. 447, 205-215], except at the hinge regions between the immunoglobulin domains and the H3 loop region. This paper presents evidence for the flexibility of the hinge and H3 loop regions of the antibody based on the comparison of two independently solved crystal structures.


Assuntos
Anticorpos Neutralizantes/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fragmentos Fab das Imunoglobulinas/química , Animais , Anticorpos Neutralizantes/imunologia , Células CHO , Cricetulus , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Leucócitos Mononucleares , Modelos Moleculares , Conformação Proteica
4.
BMC Mol Cell Biol ; 20(1): 29, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382872

RESUMO

BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Antígenos CD40/agonistas , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/química , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Transdução de Sinais , Eletricidade Estática
5.
Adv Exp Med Biol ; 1162: 39-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332733

RESUMO

Cannabinoids have been widely used for recreational and medicinal purposes. The increasing legalization of cannabinoid use and the growing success in Medicinal Chemistry of cannabinoids have fueled recent interest in cannabinoid-sensing sites in receptor proteins. Here, we review structural data from high-resolution cryo-EM and crystallography studies that depict phytocannabinoid, endocannabinoid, and synthetic cannabinoid molecules bound to various proteins. The latter include antigen-binding fragment (Fab), cellular retinol binding protein 2 (CRBP2), fatty acid-binding protein 5 (FABP5), peroxisome proliferator-activated receptor γ (PPAR γ), and cannabinoid receptor types 1 and 2 (CB1 and CB2). Cannabinoid-protein complexes reveal the complex design of cannabinoid binding sites that are usually presented by conventional ligand-binding pockets on respective proteins. However, subtle differences in cannabinoid interaction with amino acids within the binding pocket often result in diverse consequences for protein function. The rapid increase in available structural data on cannabinoid-protein interactions will ultimately direct drug design efforts toward rendering highly potent cannabinoid-related pharmacotherapies that are devoid of side effects.


Assuntos
Canabinoides/química , Endocanabinoides/química , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , PPAR gama/química , Mapeamento de Interação de Proteínas , Receptores de Canabinoides/química , Proteínas Celulares de Ligação ao Retinol/química
6.
PLoS One ; 14(6): e0218613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31251757

RESUMO

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.


Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Olho , Imunoconjugados/química , Polietilenoglicóis/química , Proteínas/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Olho/efeitos dos fármacos , Feminino , Haplorrinos , Humanos , Imunoconjugados/isolamento & purificação , Imunoconjugados/farmacologia , Fragmentos Fab das Imunoglobulinas/química , Engenharia de Proteínas , Proteínas/isolamento & purificação , Proteínas/farmacologia
7.
MAbs ; 11(6): 1077-1088, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148507

RESUMO

We analyzed pairs of protein-binding, peptide-binding and hapten-binding antibodies crystallized as complex and in the absence of the antigen with and without conformational differences upon binding in the complementarity-determining region (CDR)-H3 loop. Here, we introduce a molecular dynamics-based approach to capture a diverse conformational ensemble of the CDR-H3 loop in solution. The results clearly indicate that the inherently flexible CDR-H3 loop indeed needs to be characterized as a conformational ensemble. The conformational changes of the CDR-H3 loop in all antibodies investigated follow the paradigm of conformation selection, because we observe the experimentally determined binding competent conformation without the presence of the antigen within the ensemble of pre-existing conformational states in solution before binding. We also demonstrate for several examples that the conformation observed in the antibody crystal structure without antigen present is actually selected to bind the carboxyterminal tail region of the antigen-binding fragment (Fab). Thus, special care must be taken when characterizing antibody CDR-H3 loops by Fab X-ray structures, and the possibility that pre-existing conformations are present should always be considered.


Assuntos
Anticorpos/química , Antígenos/química , Regiões Determinantes de Complementaridade/química , Fragmentos Fab das Imunoglobulinas/química , Simulação de Dinâmica Molecular , Cristalografia por Raios X , Humanos
8.
Nature ; 571(7764): 279-283, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168089

RESUMO

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Oxisteróis/química , Receptor Smoothened/química , Receptor Smoothened/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Ligantes , Modelos Moleculares , Oxisteróis/metabolismo , Receptor Patched-1/metabolismo , Conformação Proteica , Transdução de Sinais , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
9.
Immunol Invest ; 48(8): 771-780, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31044633

RESUMO

Immunoglobulin (Ig) molecules are composed of Fab and Fc portions tethered by a hinge region that enables them to rotate and flex, relative to each other. Variable (V) and constant (C) domains of the Fab are connected by a flexible elbow region that is responsible for the movements of the V and C heterodimers. Significant movements of Fc domains have also been documented. The Ig portion's rotational freedom greatly enhances its ability to react with antigens and cell receptors, often simultaneously. The antigen-combining site also displays a dynamic structure. The ability of its various parts to change position greatly facilitates their complexation with various antigenic compounds.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Domínios Proteicos , Multimerização Proteica , Antígenos/química , Antígenos/imunologia , Antígenos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Ligação Proteica , Rotação
10.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013630

RESUMO

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death with minimal damage to normal cells; however, some cancer cells are resistant to TRAIL. TRAIL resistance may be overcome by agonistic antibodies to TRAIL receptors. In this study, we report the toxic effects of a novel recombinant agonistic human anti-TRAIL receptor 1 (DR4) monoclonal antibody Fab fragment, DR4-4, on various TRAIL-resistant and -sensitive cancer cell lines. The mechanisms of DR4-4 Fab-induced cell death in a human T cell leukemia cell line (Jurkat) were investigated using cell viability testing, immunoblotting, immunoassays, flow cytometry, and morphological observation. DR4-4 Fab-induced caspase-independent necrosis was observed to occur in Jurkat cells in association with p38 mitogen-activated protein kinase activation, cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein degradation, decreased mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production. Increased cytotoxic effects of DR4-4 Fab were observed in combination with TRAIL or γ-irradiation. Our results indicate that the novel DR4-4 Fab might overcome TRAIL-resistance and induce death in leukemia cells via cellular mechanisms different from those activated by TRAIL. DR4-4 Fab may have application as a potential therapeutic antibody fragment in single or combination therapy for cancer.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Antineoplásicos Imunológicos/química , Apoptose/efeitos dos fármacos , Biomarcadores , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Ligação Proteica
11.
PLoS One ; 14(2): e0213215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30818380

RESUMO

BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity. METHODOLOGY: Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments. PRINCIPAL FINDINGS: IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion. CONCLUSIONS/SIGNIFICANCE: Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Anticorpos Anticitoplasma de Neutrófilos/química , Imunoglobulina G/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anticitoplasma de Neutrófilos/sangue , Especificidade de Anticorpos , Configuração de Carboidratos , Sequência de Carboidratos , Estudos de Coortes , Feminino , Glicosilação , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/sangue , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Mieloblastina/antagonistas & inibidores , Mieloblastina/imunologia , Peroxidase/antagonistas & inibidores , Peroxidase/imunologia , Polissacarídeos/química , Adulto Jovem
12.
PLoS One ; 14(2): e0210749, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30730999

RESUMO

Globally, human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in newborns, young children, and the elderly for which there is no vaccine. The RSV fusion (F) glycoprotein is a major target for vaccine development. Here, we describe a novel monoclonal antibody (designated as R4.C6) that recognizes both pre-fusion and post-fusion RSV F, and binds with nanomole affinity to a unique neutralizing site comprised of antigenic sites II and IV on the globular head. A 3.9 Å-resolution structure of RSV F-R4.C6 Fab complex was obtained by single particle cryo-electron microscopy and 3D reconstruction. The structure unraveled detailed interactions of R4.C6 with antigenic site II on one protomer and site IV on a neighboring protomer of post-fusion RSV F protein. These findings significantly further our understanding of the antigenic complexity of the F protein and provide new insights into RSV vaccine design.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Sítios de Ligação de Anticorpos , Fragmentos Fab das Imunoglobulinas/química , Vírus Sinciciais Respiratórios/química , Proteínas Virais de Fusão/química , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Spodoptera , Proteínas Virais de Fusão/imunologia
13.
J Virol ; 93(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30787153

RESUMO

Parvovirus B19, one of the most common human pathogens, is a small DNA virus that belongs to the Parvoviridae As a result of previous infections, antibodies to B19 are present in most adults. B19 has a strong tropism to erythroid progenitor cells and is able to cause a series of medical conditions, including fifth disease, arthritis, myocarditis, hydrops fetalis, and aplastic crisis. No approved vaccine is currently available for B19, and there is a lack of structural characterization of any B19 epitopes. Here we present the first cryo-electron microscopy (cryo-EM) structure of a B19 virus-like particle (VLP) complexed with the antigen-binding fragment (Fab) of a human neutralizing antibody, 860-55D. A model was built into the 3.2-Å-resolution map, and the antigenic residues on the surface of the B19 capsid were identified. Antibody 860-55D bridges the capsid of B19 by binding to a quaternary structure epitope formed by residues from three neighboring VP2 capsid proteins.IMPORTANCE Parvovirus B19 is a common human pathogen and a particular threat to children, pregnant women, and patients with sickle cell disease or AIDS. Currently, neutralizing antibody is the most efficient treatment for acute B19 infections. Research on the antigenic properties of B19 will guide the usage of these antibodies and facilitate vaccine development. We have determined and report here the high-resolution structure of B19 virus-like particles (VLPs) complexed with the Fab of a human neutralizing antibody. The structure shows a quaternary structure epitope formed by three VP2 proteins and provides details on host recognition of human B19 virus.


Assuntos
Anticorpos Antivirais/química , Capsídeo , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Parvovirus B19 Humano , Capsídeo/química , Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Humanos , Parvovirus B19 Humano/química , Parvovirus B19 Humano/ultraestrutura , Estrutura Secundária de Proteína
14.
Protein J ; 38(2): 134-141, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30805759

RESUMO

In medicine, gold nanoparticles are widely used because of its unique properties. They are usually attached to a monoclonal antibody in treatment and diagnosis. Computational and laboratory work has demonstrated that the structure of the protein can change after interaction with gold nanoparticle and the effect of nanoparticle on the protein is dependent on the type of bond between them. Thus, finding out how nanoparticles affect the protein structure can help us to design the optimal complex of gold nanoparticle-antibody. In the present study, docking and molecular dynamic simulation were performed to obtain an insight at the molecular level in the binding of immunoglobulin G to the Gold nanoparticles, the structure change in immunoglobulin G, and binding energies of Fab and Fc domains of Immunoglobulin G to the GNP. We found the Fab region was more stable than the Fc region when bound to the GNP surface and it also had less structural changes. In neutral pH, Van der Waals interactions contribute more to the Fab-GNP interaction compared to electrostatic interactions; However, in Fc-GNP interaction, the main contributor is the electrostatic energy.


Assuntos
Ouro , Imunoconjugados/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Nanopartículas Metálicas/química , Ouro/química , Ouro/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Eletricidade Estática
15.
Biotechnol J ; 14(5): e1800647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30810268

RESUMO

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 n m and a dissociation rate that is 55- to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.


Assuntos
Afinidade de Anticorpos/imunologia , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sítios de Ligação , Sítios de Ligação de Anticorpos , Biotina , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Humanos , Imobilização , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Peptídeo Hidrolases , Ligação Proteica , Especificidade por Substrato , Leveduras
16.
MAbs ; 11(3): 477-488, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30794086

RESUMO

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer's disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396/Ser404 has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer404 tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer422, which was shown to have a ß-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer396 epitope, which is in tandem with pSer404. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation.


Assuntos
Anticorpos Monoclonais Murinos/química , Fragmentos Fab das Imunoglobulinas/química , Fosfoproteínas/química , Proteínas tau/química , Animais , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Domínios Proteicos , Estrutura Secundária de Proteína
17.
PLoS Biol ; 17(2): e3000139, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716060

RESUMO

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.


Assuntos
Anticorpos Neutralizantes/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Fragmentos Fab das Imunoglobulinas/química , Vírus da Influenza A/química , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Especificidade de Anticorpos , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Ligações de Hidrogênio , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Sf9 , Spodoptera
18.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 2): 80-88, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30713158

RESUMO

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts produced by ultraviolet light are mutagenic and carcinogenic. The crystal structures of the anti-(6-4) photoproduct antibody 64M-5 Fab and of its complex with dT(6-4)T were determined at 2.5 and 2.0 Šresolution, respectively. A comparison between the dT(6-4)T-liganded and unliganded structures indicates that the side chain of His93L is greatly rotated and shifted on binding to dT(6-4)T, leading to the formation of an electrostatic interaction with the phosphate moiety of dT(6-4)T, which shows a remarkable induced fit. Based on a comparison of the dT(6-4)T-liganded structures of the 64M-5 and 64M-2 Fabs, the electrostatic interaction between the side chain of His93L in 64M-5 and the phosphate moiety of dT(6-4)T is lost for Leu93L in 64M-2, while Arg90L in 64M-5 instead of Gln90L in 64M-2 stabilizes the conformation of complementarity-determining region (CDR) L3. These differences contribute to the higher affinity of 64M-5 for dT(6-4)T compared with that of 64M-2.


Assuntos
DNA/química , Fragmentos Fab das Imunoglobulinas/química , Dímeros de Pirimidina/química , Sequência de Aminoácidos , Afinidade de Anticorpos , Cristalografia por Raios X , DNA/efeitos da radiação , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Dímeros de Pirimidina/metabolismo , Homologia de Sequência , Eletricidade Estática , Raios Ultravioleta
19.
MAbs ; 11(3): 463-476, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30636503

RESUMO

Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 á´¼C). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the Fab region. At higher temperatures (e.g., 65 á´¼C), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.


Assuntos
Dietil Pirocarbonato/química , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Rituximab/química , Espectrometria de Massas , Estrutura Quaternária de Proteína
20.
Bioconjug Chem ; 30(3): 800-807, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30649877

RESUMO

Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')2 production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')2 production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.


Assuntos
Anticorpos/química , Pepsina A/química , Animais , Afinidade de Anticorpos , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/química
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