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1.
Nat Commun ; 11(1): 2135, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358509

RESUMO

A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/microbiologia , Fatores de Transcrição/metabolismo , Animais , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutação , Fosforilação , Fator de Transcrição STAT1/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcriptoma/genética
2.
Adv Exp Med Biol ; 1194: 41-58, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468522

RESUMO

Antibody V domain clustering is of paramount importance to a repertoire of immunology-related areas. Although several approaches have been proposed for antibody clustering, still no consensus has been reached. Numerous attempts use information from genes, protein sequences, 3D structures, and 3D surfaces in an effort to elucidate unknown action mechanisms directly related to their function and to either link them directly to diseases or drive the discovery of new medicines, such as antibody drug conjugates (ADC). Herein, we describe a new V domain antibody clustering method based on the comparison of the interaction sites between each antibody and its antigen. A more specific clustering analysis of the antibody's V domain was provided using deep learning and data mining techniques. The multidimensional information was extracted from the structural resolved antibodies when they were captured to interact with other proteins. The available 3D structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how antibody V domains recognize antigens as well as about which amino acids are involved in the recognition. As such, the antibody surface holds information about antigens' folding that reside with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, we propose a new simple philosophy to transform the conserved framework (fragment regions, complementarity-determining regions) of antibody V domain in a binary form using structural features of antibody-antigen interactions, toward identifying new antibody signatures in V domain binding activity. Finally, an advanced three-level hybrid classification scheme has been set for clustering antibodies in subgroups, which can combine the information from the protein sequences, the three-dimensional structures, and specific "key patterns" of recognized interactions. The clusters provide multilevel information about antibodies and antibody-antigen complexes.


Assuntos
Complexo Antígeno-Anticorpo , Análise por Conglomerados , Aprendizado de Máquina , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Regiões Determinantes de Complementaridade/química , Conformação Molecular
3.
Nucleic Acids Res ; 48(W1): W125-W131, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32432715

RESUMO

While antibodies are becoming an increasingly important therapeutic class, especially in personalized medicine, their development and optimization has been largely through experimental exploration. While there have been many efforts to develop computational tools to guide rational antibody engineering, most approaches are of limited accuracy when applied to antibody design, and have largely been limited to analysing a single point mutation at a time. To overcome this gap, we have curated a dataset of 242 experimentally determined changes in binding affinity upon multiple point mutations in antibody-target complexes (89 increasing and 153 decreasing binding affinity). Here, we have shown that by using our graph-based signatures and atomic interaction information, we can accurately analyse the consequence of multi-point mutations on antigen binding affinity. Our approach outperformed other available tools across cross-validation and two independent blind tests, achieving Pearson's correlations of up to 0.95. We have implemented our new approach, mmCSM-AB, as a web-server that can help guide the process of affinity maturation in antibody design. mmCSM-AB is freely available at http://biosig.unimelb.edu.au/mmcsm_ab/.


Assuntos
Anticorpos/genética , Afinidade de Anticorpos/genética , Mutação Puntual , Software , Anticorpos/química , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Engenharia de Proteínas
4.
Transfusion ; 60(4): 815-821, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32072650

RESUMO

BACKGROUND: Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown. STUDY DESIGN AND METHODS: Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay. RESULTS: Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind. CONCLUSION: Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype.


Assuntos
Isoantígenos/imunologia , Receptores de IgG/imunologia , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/genética , Sítios de Ligação de Anticorpos/genética , DNA Complementar , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Variação Genética , Células HEK293 , Humanos , Isoanticorpos/metabolismo , Isoantígenos/química , Receptores de IgG/genética
5.
J Allergy Clin Immunol ; 145(1): 301-311.e4, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31437490

RESUMO

BACKGROUND: Type I hypersensitivity is mediated by allergen-specific IgE, which sensitizes the high-affinity IgE receptor FcεRI on mast cells and basophils and drives allergic inflammation upon secondary allergen contact. CD23/FcεRII, the low-affinity receptor for IgE, is constitutively expressed on B cells and has been shown to regulate immune responses. Simultaneous binding of IgE to FcεRI and CD23 is blocked by reciprocal allosteric inhibition, suggesting that the 2 receptors exert distinct roles in IgE handling. OBJECTIVE: We aimed to study how free IgE versus precomplexed IgE-allergen immune complexes (IgE-ICs) target the 2 IgE receptors FcεRI and CD23, and we investigated the functional implications of the 2 pathways. METHODS: We performed binding and activation assays with human cells in vitro and IgE pharmacokinetics and anaphylaxis experiments in vivo. RESULTS: We demonstrate that FcεRI preferentially binds free IgE and CD23 preferentially binds IgE-ICs. We further show that those different binding properties directly translate to distinct biological functions: free IgE initiated allergic inflammation through FcεRI on allergic effector cells, while IgE-ICs were noninflammatory because of reduced FcεRI binding and enhanced CD23-dependent serum clearance. CONCLUSION: We propose that IgE-ICs are noninflammatory through reduced engagement by FcεRI but increased targeting of the CD23 pathway.


Assuntos
Alérgenos/imunologia , Anafilaxia/imunologia , Complexo Antígeno-Anticorpo/imunologia , Imunoglobulina E/imunologia , Lectinas Tipo C/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Alérgenos/genética , Anafilaxia/genética , Anafilaxia/patologia , Animais , Complexo Antígeno-Anticorpo/genética , Humanos , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Receptores de IgE/genética , Transdução de Sinais/genética
6.
Probl Radiac Med Radiobiol ; 24: 426-438, 2019 Dec.
Artigo em Inglês, Ucraniano | MEDLINE | ID: mdl-31841484

RESUMO

OBJECTIVE: Experimental study of the effect profile of bortezomib in the plasma cell myeloma (PCM) patients depend- ing on a specific phenotype carrier state and a pharmacochemical characteristics of ABO system glycoproteins. MATERIALS AND METHODS: The research was conducted on the 104 PCM patients, including the Chornobyl NPP acci- dent survivors (n = 49) and 65 study subjects in the comparison group. Immunogenetic criteria for positive response to the applied treatment protocols were issued according to the duration of remission, absence of infectious com- plications, and evidence of chronic renal failure as a disease complication. RESULTS: Possibility of glycoproteins A and B participation in the formation of human biological individuality at a level of protein-protein interaction with antineoplastic drug bortezomib, which is widely used in cancer management prac- tice, in particular in the PCM treatment is considered. The glycoprotein B was shown being a selective target for borte- zomib, slowing down the recognition and interaction of antigen B with monoclonal anti-B antibody, while the agglu- tination period lengthens at that by 66 %. Assumption that the formation of bortezomib complex with glycoprotein B provides a background for interaction with the key reaction of proteasome 26S inhibition, which to some extent con- tributes to the drug effect retardation was confirmed through the quantum-chemical calculations. Equilibrium is shift- ed toward the main reaction leading to a higher drug efficacy in patients with blood groups O (I) and A (II). CONCLUSIONS: Since the complexation occurs predominantly in alkaline medium the administration of drugs with alkaline reaction should be restricted for at least round the clock after administration of bortezomib according to its half-life in plasma in patients with B (III) blood group and chronic renal failure.


Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Acidente Nuclear de Chernobyl , Glicoproteínas/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Sistema ABO de Grupos Sanguíneos/genética , Sistema ABO de Grupos Sanguíneos/imunologia , Alelos , Complexo Antígeno-Anticorpo/genética , Antineoplásicos/química , Antineoplásicos/farmacocinética , Bortezomib/química , Bortezomib/farmacocinética , Estudos de Casos e Controles , Eritrócitos/imunologia , Eritrócitos/metabolismo , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Modelos Moleculares , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/imunologia , Plasmócitos/patologia , Ligação Proteica , Teoria Quântica , Exposição à Radiação/efeitos adversos , Sobreviventes , Termodinâmica , Resultado do Tratamento
7.
Nat Commun ; 10(1): 893, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792391

RESUMO

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.


Assuntos
Antígeno HLA-A11/química , Antígeno HLA-A11/metabolismo , Isoanticorpos/química , Isoanticorpos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Antígeno HLA-A11/genética , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Isoanticorpos/genética , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica
8.
Structure ; 27(3): 519-527.e5, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30595454

RESUMO

To investigate favorable single amino acid substitutions that improve antigen-antibody interactions, alanine (Ala) mutagenesis scanning of the interfacial residues of a cancer-targeted antibody, B5209B, was performed based on X-ray crystallography analysis. Two substitutions were shown to significantly enhance the binding affinity for the antigen, by up to 30-fold. One substitution improved the affinity by a gain of binding enthalpy, whereas the other substitution improved the affinity by a gain of binding entropy. Molecular dynamics simulations showed that the enthalpic improvement could be attributed to the stabilization of distant salt bridges located at the periphery of the antigen-antibody interface. The entropic improvement was due to the release of water molecules that were stably trapped in the antigen-antibody interface of the wild-type antibody. Importantly, these effects of the Ala substitutions were caused by subtle adjustments of the binding interface. These results will be helpful to design high-affinity antibodies with avoiding entropy-enthalpy compensation.


Assuntos
Alanina/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Neoplasias/imunologia , Substituição de Aminoácidos , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Neoplasias/terapia , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas
9.
Vaccine ; 37(1): 137-144, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30459071

RESUMO

Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Proteínas do Capsídeo/imunologia , Imunogenicidade da Vacina , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Complexo Antígeno-Anticorpo/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Feminino , Vetores Genéticos , Vírus da Hepatite B/genética , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes/imunologia , Tabaco/genética , Vacinas de Partículas Semelhantes a Vírus/genética
10.
Anal Chem ; 90(24): 14500-14506, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30427170

RESUMO

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Glucose Desidrogenase/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Complexo Antígeno-Anticorpo/genética , Aspergillus/enzimologia , Técnicas Biossensoriais , Receptores ErbB/análise , Receptores ErbB/imunologia , Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucose Desidrogenase/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Ressonância de Plasmônio de Superfície
11.
MAbs ; 10(7): 979-991, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30102105

RESUMO

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and ß-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or ß form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the ß-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three ß-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with ß-WTA, fulfill two recognition principles: binding to the ß-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.


Assuntos
Antibacterianos/química , Anticorpos Antibacterianos/química , Complexo Antígeno-Anticorpo/metabolismo , Parede Celular/química , Regiões Determinantes de Complementaridade/química , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/imunologia , Ácidos Teicoicos/química , Antibacterianos/sangue , Anticorpos Antibacterianos/sangue , Complexo Antígeno-Anticorpo/genética , Parede Celular/metabolismo , Cristalografia por Raios X/métodos , Humanos , Imunidade Humoral , Ligação Proteica , Conformação Proteica , Infecções Estafilocócicas/terapia , Relação Estrutura-Atividade , Ácidos Teicoicos/metabolismo , Transgenes/genética
12.
J Immunol ; 200(12): 3913-3925, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29712771

RESUMO

Prevalence of circulating immunocomplexes (ICs) strongly correlates with rheumatoid arthritis (RA) in humans. Deposits of IgG-ICs are abundant in affected joints of patients, yet molecular mechanisms for the pathogenic roles of such ICs are not fully understood. In this study, we present evidence that IgG-ICs precipitated from RA sera sensitized human monocytes for a long-lasting inflammatory functional state, characterized by a strong TNF-α response to cellular proteins representing damage-associated molecular patterns and microbe-derived pathogen-associated molecular patterns. Importantly, plate-coated human IgG (a mimic of deposited IC without Ag restriction) exhibited a similarly robust ability of monocyte sensitization in vitro. The plate-coated human IgG-induced functional programming is accompanied by transcriptomic and epigenetic modification of various inflammatory cytokines and negative regulator genes. Moreover, macrophages freshly isolated from synovia of patients with RA, but not sera-negative arthropathy, displayed a signature gene expression profile highly similar to that of IC-sensitized human monocytes, indicative of historical priming events by IgG-ICs in vivo. Thus, the ability of IgG-ICs to drive sustainable functional sensitization/reprogramming of monocytes and macrophages toward inflammation may render them key players in the development of RA.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Artrite Reumatoide/imunologia , Epigênese Genética/imunologia , Imunoglobulina G/imunologia , Inflamação/imunologia , Monócitos/imunologia , Transcriptoma/imunologia , Adulto , Idoso , Complexo Antígeno-Anticorpo/genética , Artrite Reumatoide/genética , Citocinas/imunologia , Epigênese Genética/genética , Feminino , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Inflamação/genética , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Transcriptoma/genética , Fator de Necrose Tumoral alfa/imunologia
13.
PLoS Comput Biol ; 14(4): e1006112, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29702641

RESUMO

A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native amino acid types for residues that contact the antigen in the native structures was 72% in simulations performed in the presence of the antigen and 48% in simulations performed without the antigen, for an ARR of 1.5. For the non-contacting residues, the ARR was 1.08. This shows that the sequence profiles are able to maintain the amino acid types of these conserved, buried sites, while recovery of the exposed, contacting residues requires the presence of the antigen-antibody interface. We tested RAbD experimentally on both a lambda and kappa antibody-antigen complex, successfully improving their affinities 10 to 50 fold by replacing individual CDRs of the native antibody with new CDR lengths and clusters.


Assuntos
Anticorpos/química , Software , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Regiões Determinantes de Complementaridade , Biologia Computacional , Simulação por Computador , Evolução Molecular Direcionada , Desenho de Fármacos , Humanos , Modelos Moleculares , Método de Monte Carlo , Conformação Proteica , Engenharia de Proteínas/métodos , Engenharia de Proteínas/estatística & dados numéricos
14.
PLoS One ; 13(1): e0191162, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29324815

RESUMO

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691-836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.


Assuntos
Camelídeos Americanos/imunologia , Proteínas do Tecido Nervoso/imunologia , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Reações Antígeno-Anticorpo , Fenômenos Biofísicos , Camelídeos Americanos/genética , Mapeamento de Epitopos , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas , Espalhamento a Baixo Ângulo , Anticorpos de Cadeia Única/genética , Ressonância de Plasmônio de Superfície , Difração de Raios X
16.
J Biol Chem ; 292(45): 18618-18627, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28931605

RESUMO

Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8+ cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/ß chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ∼40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50) expressing the TRAV13-1/TRBV27 gene combination bound to GIL-HLA-A2 to 1.7 Å resolution. Comparison of the F50-GIL-HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL-HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide-MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50-GIL-HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide-MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.


Assuntos
Antígenos Virais/metabolismo , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/metabolismo , Epitopos Imunodominantes/metabolismo , Modelos Moleculares , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas da Matriz Viral/metabolismo , Substituição de Aminoácidos , Afinidade de Anticorpos , Diversidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Antígenos Virais/química , Antígenos Virais/genética , Deleção Clonal , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Humanos , Ligação de Hidrogênio , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Vírus da Influenza A/imunologia , Vírus da Influenza A/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética
17.
Proc Natl Acad Sci U S A ; 114(24): E4812-E4821, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28559317

RESUMO

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.


Assuntos
Dependovirus/genética , Dependovirus/imunologia , Evolução Molecular Direcionada/métodos , Evasão da Resposta Imune/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Variação Antigênica/genética , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Dependovirus/classificação , Feminino , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Células HEK293 , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Sorotipagem
18.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 5): 294-299, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28471362

RESUMO

CD27 is a T-cell and B-cell co-stimulatory glycoprotein of the tumor necrosis factor (TNF) receptor superfamily that is dependent on the availability of the TNF-like ligand CD70. Therapeutic approaches to treating autoimmune diseases and cancers with antagonistic and agonistic anti-CD27 monoclonal antibodies (mAbs), respectively, have recently been developed. Mouse anti-human CD27 mAb 2177 shows potency in neutralizing CD70-induced signaling; however, it does not block the binding of soluble CD70. To provide insight into the mechanism of action of the mAb, the crystal structure of the CD27 extracellular domain in complex with the Fab fragment of mAb 2177 was determined at 1.8 Šresolution. CD27 exhibits the assembly of cysteine-rich domains characteristic of the TNF receptor superfamily. The structure reveals a unique binding site of mAb 2177 at the edge of the receptor molecule, which allows the mAb to sterically block the cell-bound form of CD70 from reaching CD27 while leaving the ligand epitope clear. This mode of action suggests a potential dual use of mAb 2177 either as an antagonist or as an agonist.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Complexo Antígeno-Anticorpo/química , Ligante CD27/química , Fragmentos Fab das Imunoglobulinas/química , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/química , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Complexo Antígeno-Anticorpo/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Ligante CD27/genética , Ligante CD27/imunologia , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Ligantes , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Células Sf9 , Spodoptera , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia
19.
Nucleic Acids Res ; 44(W1): W469-73, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27216816

RESUMO

Computational methods have traditionally struggled to predict the effect of mutations in antibody-antigen complexes on binding affinity. This has limited their usefulness during antibody engineering and development, and their ability to predict biologically relevant escape mutations. Here we present mCSM-AB, a user-friendly web server for accurately predicting antibody-antigen affinity changes upon mutation which relies on graph-based signatures. We show that mCSM-AB performs better than comparable methods that have been previously used for antibody engineering. mCSM-AB web server is available at http://structure.bioc.cam.ac.uk/mcsm_ab.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos/genética , Antígenos/imunologia , Internet , Mutação , Software , Anticorpos/química , Afinidade de Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/imunologia , Antígenos/química , Antígenos/genética , Benchmarking , Conjuntos de Dados como Assunto , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Engenharia de Proteínas , Vacinas/química , Vacinas/genética , Vacinas/imunologia
20.
Proc Natl Acad Sci U S A ; 113(19): E2636-45, 2016 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-27114511

RESUMO

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.


Assuntos
Anticorpos/química , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Alinhamento de Sequência/métodos , Sequência de Aminoácidos , Anticorpos/genética , Complexo Antígeno-Anticorpo/genética , Sequência de Bases , Simulação por Computador , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Químicos , Modelos Genéticos , Modelos Imunológicos , Homologia de Sequência de Aminoácidos
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