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1.
MAbs ; 14(1): 2143009, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36394299

RESUMEN

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.


Asunto(s)
Inmunoglobulina G , Neoplasias , Humanos , Ratones , Animales , Interleucina-2 , Ratones Transgénicos , Leucocitos Mononucleares , Inmunoterapia
2.
Nat Commun ; 12(1): 708, 2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514724

RESUMEN

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.


Asunto(s)
Anticuerpos Biespecíficos/farmacología , Neovascularización Coroidal/tratamiento farmacológico , Desarrollo de Medicamentos/métodos , Fragmentos Fab de Inmunoglobulinas/farmacología , Ingeniería de Proteínas , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/ultraestructura , Becaplermina/antagonistas & inhibidores , Sitios de Unión de Anticuerpos/genética , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Concentración 50 Inhibidora , Inyecciones Intravítreas , Masculino , Modelos Moleculares , Prueba de Estudio Conceptual , Conformación Proteica , Ratas , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores
3.
MAbs ; 11(8): 1402-1414, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31526159

RESUMEN

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales/química , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química
4.
PLoS One ; 8(4): e61953, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23613981

RESUMEN

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.


Asunto(s)
Angiopoyetina 2/inmunología , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Terciaria de Proteína , Electricidad Estática , Relación Estructura-Actividad , Temperatura
5.
Nucleic Acids Res ; 40(21): 11036-46, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22977180

RESUMEN

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8-Arp4-actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3-H4 tetramers over H2A-H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3-H4)(2) over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.


Asunto(s)
Proteínas de Microfilamentos/química , Nucleosomas/metabolismo , Actinas/química , Actinas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Humanos , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia
6.
Bioarchitecture ; 1(4): 192-195, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22069513

RESUMEN

The function of nuclear actin is poorly understood. It is known to be a discrete component of several chromatin-modifying complexes. Nevertheless, filamentous forms of actin are important for various nuclear processes as well. Nuclear actin is often associated with nuclear actin-related protein Arp4 and other actin-related proteins like Arp8 in the INO80 chromatin remodeler. We recently determined the crystal structure of S. cerevisiae Arp4 that explains why Arp4 is unable to form actin like filaments and shows that it is constitutively bound to an ATP nucleotide. More interestingly, in vitro activities of Arp4 and Arp8 seem to be directed towards stabilizing monomeric actin and to integrate it stoichiometrically into the INO80 complex. Based on this activity, we discuss possible roles of nuclear Arps in chromatin modifying complexes and in regulating more general aspects of nuclear actin dynamics.

7.
EMBO J ; 30(11): 2153-66, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21499228

RESUMEN

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.


Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Actinas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño
8.
J Biol Chem ; 283(42): 28757-66, 2008 Oct 17.
Artículo en Inglés | MEDLINE | ID: mdl-18701464

RESUMEN

Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions.


Asunto(s)
Regulación de la Expresión Génica , Proteínas de Unión al ARN/química , Secuencia de Aminoácidos , ADN/química , Dimerización , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido
9.
RNA ; 13(7): 1043-51, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17526645

RESUMEN

Poly(C)-binding proteins (PCBPs) are KH (hnRNP K homology) domain-containing proteins that recognize poly(C) DNA and RNA sequences in mammalian cells. Binding poly(C) sequences via the KH domains is critical for PCBP functions. To reveal the mechanisms of KH domain-D/RNA recognition and its functional importance, we have determined the crystal structures of PCBP2 KH1 domain in complex with a 12-nucleotide DNA corresponding to two repeats of the human C-rich strand telomeric DNA and its RNA equivalent. The crystal structures reveal molecular details for not only KH1-DNA/RNA interaction but also protein-protein interaction between two KH1 domains. NMR studies on a protein construct containing two KH domains (KH1 + KH2) of PCBP2 indicate that KH1 interacts with KH2 in a way similar to the KH1-KH1 interaction. The crystal structures and NMR data suggest possible ways by which binding certain nucleic acid targets containing tandem poly(C) motifs may induce structural rearrangement of the KH domains in PCBPs; such structural rearrangement may be crucial for some PCBP functions.


Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Poli C/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Poli C/química , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN/química , ARN/metabolismo , Homología de Secuencia de Aminoácido
11.
Nucleic Acids Res ; 35(8): 2651-60, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17426136

RESUMEN

KH (hnRNP K homology) domains, consisting of approximately 70 amino acid residues, are present in a variety of nucleic-acid-binding proteins. Among these are poly(C)-binding proteins (PCBPs), which are important regulators of mRNA stability and posttranscriptional regulation in general. All PCBPs contain three different KH domains and recognize poly(C)-sequences with high affinity and specificity. To reveal the molecular basis of poly(C)-sequence recognition, we have determined the crystal structure, at 1.6 A resolution, of PCBP2 KH3 domain in complex with a 7-nt DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the C-rich strand of human telomeric DNA. The domain assumes a type-I KH fold in a betaalphaalphabetabetaalpha configuration. The protein-DNA interface could be studied in unprecedented detail and is made up of a series of direct and water-mediated hydrogen bonds between the protein and the DNA, revealing an especially dense network involving several structural water molecules for the last 2 nt in the core recognition sequence. Unlike published KH domain structures, the protein crystallizes without protein-protein contacts, yielding new insights into the dimerization properties of different KH domains. A nucleotide platform, an interesting feature found in some RNA molecules, was identified, evidently for the first time in DNA.


Asunto(s)
ADN/química , Modelos Moleculares , Proteínas de Unión al ARN/química , Telómero/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia
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