RESUMO
The molecular rules driving TCR cross-reactivity are poorly understood and, consequently, it is unclear the extent to which TCRs targeting the same Ag recognize the same off-target peptides. We determined TCR-peptide-HLA crystal structures and, using a single-chain peptide-HLA phage library, we generated peptide specificity profiles for three newly identified human TCRs specific for the cancer testis Ag NY-ESO-1157-165-HLA-A2. Two TCRs engaged the same central peptide feature, although were more permissive at peripheral peptide positions and, accordingly, possessed partially overlapping peptide specificity profiles. The third TCR engaged a flipped peptide conformation, leading to the recognition of off-target peptides sharing little similarity with the cognate peptide. These data show that TCRs specific for a cognate peptide recognize discrete peptide repertoires and reconciles how an individual's limited TCR repertoire following negative selection in the thymus is able to recognize a vastly larger antigenic pool.
Assuntos
Antígeno HLA-A2/imunologia , Antígenos de Histocompatibilidade/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linhagem Celular , Humanos , Biblioteca de PeptídeosRESUMO
The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1ß, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 µM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects.IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatócitos/virologia , Antígeno B7-H1/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CCL4/genética , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Antígeno HLA-A2/imunologia , Hepacivirus/genética , Hepatócitos/imunologia , Humanos , Interferon gama/genética , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/genética , Mutagênese Sítio-Dirigida , Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T/genética , Transdução Genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T-cells is a major barrier to eradication. Latency-reversing agents that induce direct or immune-mediated cell death upon reactivation of HIV are a possible solution. However, clearance of reactivated cells may require immunotherapeutic agents that are fine-tuned to detect viral antigens when expressed at low levels. We tested the antiviral efficacy of immune-mobilizing monoclonal T-cell receptors against viruses (ImmTAVs), bispecific molecules that redirect CD8+ T-cells to kill HIV-infected CD4+ T-cells. T-cell receptors specific for an immunodominant Gag epitope, SL9, and its escape variants were engineered to achieve supraphysiological affinity and fused to a humanised CD3-specific single chain antibody fragment. Ex vivo polyclonal CD8+ T-cells were efficiently redirected by immune-mobilising monoclonal T-cell receptors against viruses to eliminate CD4+ T-cells from human histocompatibility leukocyte antigen (HLA)-A*0201-positive antiretroviral therapy-treated patients after reactivation of inducible HIV in vitro. The efficiency of infected cell elimination correlated with HIV Gag expression. Immune-mobilising monoclonal T-cell receptors against viruses have potential as a therapy to facilitate clearance of reactivated HIV reservoir cells.
Assuntos
Anticorpos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Anticorpos Monoclonais/farmacologia , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Latência ViralRESUMO
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/imunologiaRESUMO
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/imunologiaRESUMO
NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Superfície/biossíntese , Complexo CD3/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Distribuição Aleatória , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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/genéticaRESUMO
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/imunologiaRESUMO
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/genéticaRESUMO
Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics.
Assuntos
Anticorpos Biespecíficos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas/farmacologia , Anticorpos de Cadeia Única/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Técnicas In Vitro , Fluxo de TrabalhoRESUMO
Human CD8 is a T cell coreceptor, which binds to pHLA I and plays a pivotal role in the activation of cytotoxic T lymphocytes. Soluble recombinant CD8 alphaalpha has been shown to antagonize T cell activation, both in vitro and in vivo. However, because of a very low affinity for pHLA I, high concentrations of soluble CD8 alphaalpha are required for efficient inhibition. Based upon our knowledge of the wild-type CD8/pHLA I structure, we have designed and produced a mutated form of soluble CD8 alphaalpha that binds to pHLA I with approximately fourfold higher affinity. We have characterized the binding of the high affinity CD8 mutant using surface plasmon resonance and determined its structure at 2.1 A resolution using X-ray crystallography. The analysis of this structure suggests that the higher affinity is achieved by providing a larger side chain that allows for an optimal contact to be made between the HLA alpha3 loop and the mutated CDR-like loops of CD8.
Assuntos
Antígenos CD8/química , Antígenos CD8/genética , Substituição de Aminoácidos , Biologia Computacional , Simulação por Computador , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Engenharia de Proteínas/métodos , Dobramento de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
Naturally selected T-cell receptors (TCRs) are characterised by low binding affinities, typically in the range 1-100 microM. Crystal structures of syngeneic TCRs bound to peptide major histocompatibility complex (pMHC) antigens exhibit a conserved mode of binding characterised by a distinct diagonal binding geometry, with poor shape complementarity (SC) between receptor and ligand. Here, we report the structures of three in vitro affinity enhanced TCRs that recognise the pMHC tumour epitope NY-ESO(157-165) (SLLMWITQC). These crystal structures reveal that the docking mode for the high affinity TCRs is identical to that reported for the parental wild-type TCR, with only subtle changes in the mutated complementarity determining regions (CDRs) that form contacts with pMHC; both CDR2 and CDR3 mutations act synergistically to improve the overall affinity. Comparison of free and bound TCR structures for both wild-type and a CDR3 mutant reveal an induced fit mechanism arising from restructuring of CDR3 loops which allows better peptide binding. Overall, an increased interface area, improved SC and additional H-bonding interactions are observed, accounting for the increase in affinity. Most notably, there is a marked increase in the SC for the central methionine and tryptophan peptide motif over the native TCR.
Assuntos
Cristalografia por Raios X , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Escherichia coli/genética , Humanos , Ligação de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR-pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P4(1)/P4(3), with unit-cell parameters a = b = 120.4, c = 81.6 A. A complete data set was collected to 3.1 A and the structure is currently being analysed.
Assuntos
Antígenos de Neoplasias/química , Complexo Principal de Histocompatibilidade , Proteínas de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/isolamento & purificação , Cristalização , DNA Complementar , Humanos , Antígeno MART-1 , Melanócitos/fisiologia , Melanoma/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/isolamento & purificação , Plasmídeos , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Neoplasias Cutâneas/química , Difração de Raios XRESUMO
Natural T-cell responses generally lack the potency to eradicate cancer. Enhanced affinity T-cell receptors (TCRs) provide an ideal approach to target cancer cells, with emerging clinical data showing significant promise. Nevertheless, the risk of off target reactivity remains a key concern, as exemplified in a recent clinical report describing fatal cardiac toxicity, following administration of MAGE-A3 specific TCR-engineered T-cells, mediated through cross-reactivity with an unrelated epitope from the Titin protein presented on cardiac tissue. Here, we investigated the structural mechanism enabling TCR cross-recognition of MAGE-A3 and Titin, and applied the resulting data to rationally design mutants with improved antigen discrimination, providing a proof-of-concept strategy for altering the fine specificity of a TCR towards an intended target antigen. This study represents the first example of direct molecular mimicry leading to clinically relevant fatal toxicity, mediated by a modified enhanced affinity TCR designed for cancer immunotherapy. Furthermore, these data demonstrate that self-antigens that are expressed at high levels on healthy tissue should be treated with extreme caution when designing immuno-therapeutics.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Reações Cruzadas/imunologia , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Mimetismo Molecular , Receptores de Antígenos de Linfócitos T/metabolismo , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cardiotoxicidade , Linhagem Celular , Conectina/química , Conectina/imunologia , Conectina/metabolismo , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Engenharia Genética , Humanos , Modelos Moleculares , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Conectina/química , Reações Cruzadas/imunologia , Antígeno HLA-A1/imunologia , Proteínas de Neoplasias/imunologia , Peptídeos/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Conectina/imunologia , Reações Cruzadas/efeitos dos fármacos , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/química , Peptídeos/química , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
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/imunologiaRESUMO
Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.
Assuntos
Antígenos CD/genética , Antígenos CD/farmacologia , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Ativação Linfocitária/imunologia , Receptores Imunológicos/genética , Sequência de Aminoácidos , Antígenos CD/química , Autoimunidade , Bioensaio , Linhagem Celular , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Relação Dose-Resposta Imunológica , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Fatores Imunológicos/química , Cinética , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ativação Linfocitária/genética , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Dados de Sequência Molecular , Terapia de Alvo Molecular , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Polietilenoglicóis , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
HIV's considerable capacity to vary its HLA-I-restricted peptide antigens allows it to escape from host cytotoxic T lymphocytes (CTLs). Nevertheless, therapeutics able to target HLA-I-associated antigens, with specificity for the spectrum of preferred CTL escape mutants, could prove effective. Here we use phage display to isolate and enhance a T-cell antigen receptor (TCR) originating from a CTL line derived from an infected person and specific for the immunodominant HLA-A(*)02-restricted, HIVgag-specific peptide SLYNTVATL (SL9). High-affinity (K(D) < 400 pM) TCRs were produced that bound with a half-life in excess of 2.5 h, retained specificity, targeted HIV-infected cells and recognized all common escape variants of this epitope. CD8 T cells transduced with this supraphysiologic TCR produced a greater range of soluble factors and more interleukin-2 than those transduced with natural SL9-specific TCR, and they effectively controlled wild-type and mutant strains of HIV at effector-to-target ratios that could be achieved by T-cell therapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Sequência de Aminoácidos , Células Cultivadas , Produtos do Gene gag/química , Produtos do Gene gag/imunologia , Humanos , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Ligação Proteica , SolubilidadeRESUMO
Superantigens are known to activate a large number of T cells. The SAg is presented by MHC class II on the APC and its classical feature is that it recognizes the variable region of the beta-chain of the TCR. In this article, we report, by direct binding studies, that staphylococcal enterotoxin (SE) H (SEH), a bacterial SAg secreted by Staphylococcus aureus, instead recognizes the variable alpha-chain (TRAV27) of TCR. Furthermore, we show that different SAgs (e.g., SEH and SEA) can simultaneously bind to one TCR by binding the alpha-chain and the beta-chain, respectively. Theoretical three-dimensional models of the penta complexes are presented. Hence, these findings open up a new dimension of the biology of the staphylococcal enterotoxins.
Assuntos
Enterotoxinas/química , Antígenos de Histocompatibilidade Classe II/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Superantígenos/química , Animais , Humanos , Conformação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ressonância de Plasmônio de SuperfícieRESUMO
The use of recombinant T cell receptors (TCRs) to target therapeutic interventions has been hindered by the naturally low affinity of TCR interactions with peptide major histocompatibility complex ligands. Here, we use multimeric forms of soluble heterodimeric alphabeta TCRs for specific detection of target cells pulsed with cognate peptide, discrimination of quantitative changes in antigen display at the cell surface, identification of virus-infected cells, inhibition of antigen-specific cytotoxic T lymphocyte activation, and identification of cross-reactive peptides. Notably, the A6 TCR specific for the immunodominant HLA A2-restricted human T cell leukemia virus type 1 Tax(11-19) epitope bound to HLA A2-HuD(87-95) (K(D) 120 microm by surface plasmon resonance), an epitope implicated as a causal antigen in the paraneoplastic neurological degenerative disorder anti-Hu syndrome. A mutant A6 TCR that exhibited dramatically increased affinity for cognate antigen (K(D) 2.5 nm) without enhanced cross-reactivity was generated; this TCR demonstrated potent biological activity even as a monomeric molecule. These data provide insights into TCR repertoire selection and delineate a framework for the selective modification of TCRs in vitro that could enable specific therapeutic intervention in vivo.