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1.
Eur J Immunol ; 49(7): 1052-1066, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31091334

RESUMO

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.


Assuntos
Epitopos Imunodominantes/metabolismo , Imunoterapia Adotiva/métodos , Antígeno MART-1/metabolismo , Melanoma/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Aminoácidos , Apresentação de Antígeno , Sítios de Ligação , Células Cultivadas , Células Clonais , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Melanoma/terapia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplante
2.
J Biol Chem ; 289(2): 628-38, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24196962

RESUMO

αß T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short "foreign" peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.


Assuntos
Regiões Determinantes de Complementaridade/metabolismo , Antígenos HLA/metabolismo , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Cristalografia por Raios X , Antígenos HLA/química , Antígenos HLA/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T
3.
J Biol Chem ; 288(26): 18766-75, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23698002

RESUMO

The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nM to 270 µM). We used phage-display to produce a melanoma-specific TCR (α24ß17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nM) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24ß17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Alanina , Biotinilação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Complexo Principal de Histocompatibilidade , Conformação Molecular , Mutação , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Solventes , Ressonância de Plasmônio de Superfície , Termodinâmica , Água
4.
Blood ; 119(15): 3420-30, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22318202

RESUMO

We investigated whether TCRs restricted to the more ubiquitously expressed MHC class I molecules could be used to redirect human regulatory T cells (Tregs). Using a series of HLA-A2-restricted TCRs that recognize the same peptide-MHC class I complex (pMHC) with affinities varying up to 3500 fold, we observed that TCR affinity had no effect on the ability of the introduced TCRs to confer potent Ag-specific suppressive activity. Surprisingly, we found a naturally occurring, low-affinity MHC class I-restricted TCR specific for an NY-ESO-1 epitope that was unable to redirect a functional CD4 T-effector cell response could confer potent antigen-specific suppressive activity when expressed in Tregs and severely impair the expansion of highly functional HIV-1(GAG)-specific CD8 T cells expressing a high-affinity TCR. This suppressive activity was only observed when both Ags were presented by the same cell, and no suppression was observed when the target Ags were put in distinct cells. These studies underscore the clinical utility of using MHC class I-restricted TCRs to endow Tregs with specificity to control autoimmune disease and highlight the conditions in which this approach would have most therapeutic benefit.


Assuntos
Especificidade do Receptor de Antígeno de Linfócitos T/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/fisiologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células K562 , Ativação Linfocitária/genética , Ativação Linfocitária/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Transfecção , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
5.
Eur J Immunol ; 42(12): 3174-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22949370

RESUMO

T-cell destiny during thymic selection depends on the affinity of the TCR for autologous peptide ligands presented in the context of MHC molecules. This is a delicately balanced process; robust binding leads to negative selection, yet some affinity for the antigen complex is required for positive selection. All TCRs of the resulting repertoire thus have some intrinsic affinity for an MHC type presenting an assortment of peptides. Generally, TCR affinities of peripheral T cells will be low toward self-derived peptides, as these would have been presented during thymic selection, whereas, by serendipity, binding to pathogen-derived peptides that are encountered de novo could be stronger. A crucial question in assessing immunotherapeutic strategies for cancer is whether natural TCR repertoires have the capacity for efficiently recognizing tumor-associated peptide antigens. Here, we report a comprehensive comparison of TCR affinities to a range of HLA-A2 presented antigens. TCRs that bind viral antigens fall within a strikingly higher affinity range than those that bind cancer-related antigens. This difference may be one of the key explanations for tumor immune escape and for the deficiencies of T-cell vaccines against cancer.


Assuntos
Apresentação de Antígeno , Antígeno HLA-A2/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Humanos , Neoplasias/terapia , Timo/imunologia
6.
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
7.
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
8.
Nat Biotechnol ; 23(3): 349-54, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15723046

RESUMO

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to approximately 1-100 muM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax(11-19) peptide-HLA-A(*)0201 complex and the NY-ESO-1(157-165) tumor-associated peptide antigen-HLA-A(*)0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.


Assuntos
Afinidade de Anticorpos , Formação de Anticorpos , Regiões Determinantes de Complementaridade/genética , Evolução Molecular Direcionada/métodos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/imunologia , Microquímica/métodos , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Ligação Proteica , Receptores de Antígenos de Linfócitos T/biossíntese , Recombinação Genética/genética
9.
Mol Cancer Ther ; 6(7): 2081-91, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17620437

RESUMO

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540-548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT(540-548) peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT(540-548) on tumor cells: (a) a CD8(+) CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT(540-548) complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT(540-548) peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications.


Assuntos
Antígenos HLA-A/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Telomerase/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Separação Celular , Células Clonais , Ensaio de Imunoadsorção Enzimática , Epitopos , Antígeno HLA-A2 , Humanos , Interferon gama/farmacologia , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Inibidores de Proteassoma , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Linfócitos T Citotóxicos/efeitos dos fármacos , Transfecção
10.
Nat Med ; 18(6): 980-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22561687

RESUMO

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.


Assuntos
Citotoxicidade Imunológica , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Memória Imunológica , Imunoterapia , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos SCID , Neoplasias Experimentais/imunologia
11.
Mol Biotechnol ; 45(2): 140-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20143183

RESUMO

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed alpha and beta chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Glutationa Redutase/genética , Receptores de Antígenos de Linfócitos T/biossíntese , Tiorredoxina Dissulfeto Redutase/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glutationa Redutase/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Ressonância de Plasmônio de Superfície , Tiorredoxina Dissulfeto Redutase/metabolismo
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