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1.
Protein Eng Des Sel ; 352022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35871543

RESUMO

TCR-like antibodies represent a unique type of engineered antibodies with specificity toward pHLA, a ligand normally restricted to the sensitive recognition by T cells. Here, we report a phage display-based sequential development path of such antibodies. The strategy goes from initial lead identification through in silico informed CDR engineering in combination with framework engineering for affinity and thermostability optimization, respectively. The strategy allowed the identification of HLA-DQ2.5 gluten peptide-specific TCR-like antibodies with low picomolar affinity. Our method outlines an efficient and general method for development of this promising class of antibodies, which should facilitate their utility including translation to human therapy.


Assuntos
Anticorpos , Bacteriófagos , Humanos , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T
2.
Sci Immunol ; 6(62)2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417258

RESUMO

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Celíaca/imunologia , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Epitopos de Linfócito T/imunologia , Glutens/química , Antígenos HLA-DQ/química , Humanos , Ativação Linfocitária/imunologia , Camundongos , Modelos Moleculares , Peptídeos/química , Receptores de Antígenos de Linfócitos T/química
3.
Structure ; 21(2): 257-65, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23313664

RESUMO

The DNA repair enzyme endonuclease V (EndoV) recognizes and cleaves DNA at deaminated adenine lesions (hypoxanthine). In addition, EndoV cleaves DNA containing various helical distortions such as loops, hairpins, and flaps. To understand the molecular basis of EndoV's ability to recognize and incise DNA structures with helical distortions, we solved the crystal structure of Thermotoga maritima EndoV in complex with DNA containing a one-nucleotide loop. The structure shows that a strand-separating wedge is crucial for DNA loop recognition, with DNA strands separated precisely at the helical distortion. The additional nucleotide forming the loop rests on the surface of the wedge, while the normal adenine opposite the loop is flipped into a base recognition pocket. Our data show a different principle for DNA loop recognition and cleavage by EndoV, in which a coordinated action of a DNA-intercalating wedge and a base pocket accommodating a flipped normal base facilitate strand incision.


Assuntos
Proteínas de Bactérias/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Thermotoga maritima/enzimologia , Adenina/química , Domínio Catalítico , Cristalografia por Raios X , Clivagem do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Ligação de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato
4.
Carcinogenesis ; 30(7): 1147-54, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19443904

RESUMO

The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform delta-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development.


Assuntos
Neoplasias dos Ductos Biliares/enzimologia , Colangiocarcinoma/enzimologia , Colangite Esclerosante/enzimologia , DNA Glicosilases/metabolismo , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/genética , Colangite Esclerosante/genética , Dano ao DNA , DNA Glicosilases/química , DNA Glicosilases/genética , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Íntrons , Masculino , Modelos Moleculares , Mutação , Conformação Proteica , Risco
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