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1.
J Biol Chem ; 295(33): 11486-11494, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32532817

RESUMO

T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies.


Assuntos
Antígenos de Neoplasias/imunologia , Antígeno HLA-A2/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Antígenos de Neoplasias/química , Cristalografia por Raios X , Antígeno HLA-A2/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química
2.
J Biol Chem ; 289(43): 29912-26, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25160627

RESUMO

The membrane-proximal external region (MPER) of the human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein subunit gp41 is targeted by potent broadly neutralizing antibodies 2F5, 4E10, and 10E8. These antibodies recognize linear epitopes and have been suggested to target the fusion intermediate conformation of gp41 that bridges viral and cellular membranes. Anti-MPER antibodies exert different degrees of membrane interaction, which is considered to be the limiting factor for the generation of such antibodies by immunization. Here we characterize a fusion intermediate conformation of gp41 (gp41(int)-Cys) and show that it folds into an elongated ∼ 12-nm-long extended structure based on small angle x-ray scattering data. Gp41(int)-Cys was covalently linked to liposomes via its C-terminal cysteine and used as immunogen. The gp41(int)-Cys proteoliposomes were administered alone or in prime-boost regimen with trimeric envelope gp140(CA018) in guinea pigs and elicited high anti-gp41 IgG titers. The sera interacted with a peptide spanning the MPER region, demonstrated competition with broadly neutralizing antibodies 2F5 and 4E10, and exerted modest lipid binding, indicating the presence of MPER-specific antibodies. Although the neutralization potency generated solely by gp140(CA018) was higher than that induced by gp41(int)-Cys, the majority of animals immunized with gp41(int)-Cys proteoliposomes induced modest breadth and potency in neutralizing tier 1 pseudoviruses and replication-competent simian/human immunodeficiency viruses in the TZM-bl assay as well as responses against tier 2 HIV-1 in the A3R5 neutralization assay. Our data thus demonstrate that liposomal gp41 MPER formulation can induce neutralization activity, and the strategy serves to improve breadth and potency of such antibodies by improved vaccination protocols.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos/imunologia , Feminino , Cobaias , Proteína gp41 do Envelope de HIV/química , Humanos , Soros Imunes/imunologia , Imunização , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Difração de Nêutrons , Estrutura Terciária de Proteína , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Espalhamento a Baixo Ângulo
3.
PLoS Pathog ; 9(3): e1003202, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23505368

RESUMO

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.


Assuntos
Anticorpos Neutralizantes/imunologia , Camelídeos Americanos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunização , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Neutralização , Proteolipídeos/administração & dosagem , Proteolipídeos/imunologia , Anticorpos de Domínio Único , Ressonância de Plasmônio de Superfície
4.
J Exp Med ; 220(9)2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37382893

RESUMO

Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRß-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.


Assuntos
Células T Invariantes Associadas à Mucosa , Humanos , Membrana Celular , Comunicação Celular , Reações Cruzadas , Reparo do DNA , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Menor
5.
Nat Commun ; 13(1): 5333, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088370

RESUMO

Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.


Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Antígenos de Linfócitos T/genética
6.
J Clin Invest ; 130(5): 2673-2688, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32310221

RESUMO

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.


Assuntos
Antígenos HLA/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Especificidade de Anticorpos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Cristalografia por Raios X , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Indicadores e Reagentes , Modelos Moleculares , Simulação de Dinâmica Molecular , Mimetismo Molecular/genética , Mimetismo Molecular/imunologia , Peptídeos/química , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia
7.
Antibodies (Basel) ; 8(2)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31544844

RESUMO

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

8.
Virology ; 397(1): 64-72, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19945724

RESUMO

The Borna disease virus (BDV) nucleoprotein (N) monomer resembles the nucleoprotein structures from rabies virus (RABV) and vesicular stomatitis virus (VSV). We show that BDV N assembles into ring- and string-like structures in the presence of 5' genomic BDV RNA. RNA induced polymerization is partly RNA-specific since polymerization is inefficient in the presence of 3' genomic BDV RNA or E. coli RNA. Mutagenesis of basic residues located in the cleft made up by the N- and C-terminal domains of N abrogate RNA-induced polymerization indicating that BDV N binds RNA similarly as observed in case of RABV and VSV N-RNA complexes. Bound RNA is not protected and sensitive to degradation. N-RNA polymers form complexes with the phosphoprotein P as required for functional transcription or replication units. Our data indicate that BDV N utilizes similar structural principles for N-RNA and N-P-RNA complex formation as observed for related negative strand RNA viruses.


Assuntos
Vírus da Doença de Borna/fisiologia , Nucleoproteínas/metabolismo , Multimerização Proteica , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Sítios de Ligação , Mutagênese Sítio-Dirigida , Fosfoproteínas/metabolismo , Proteínas Estruturais Virais/metabolismo
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