RESUMEN
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.
Asunto(s)
Epítopos Inmunodominantes/metabolismo , Inmunoterapia Adoptiva/métodos , Antígeno MART-1/metabolismo , Melanoma/inmunología , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Aminoácidos , Presentación de Antígeno , Sitios de Unión , Células Cultivadas , Células Clonales , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Activación de Linfocitos , Antígeno MART-1/química , Melanoma/terapia , Péptidos/química , Unión Proteica , Conformación Proteica , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/trasplanteRESUMEN
Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed ß-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl ß-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl ß-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general.
Asunto(s)
Aldehídos/metabolismo , Ésteres/metabolismo , Oxidorreductasas/metabolismo , Compuestos de Sulfhidrilo/química , Aldehídos/química , Sitios de Unión , Biocatálisis , Dominio Catalítico , Cefalosporinas/biosíntesis , Cefalosporinas/química , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Ésteres/química , Hierro/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación Molecular , Mutagénesis , Oxidación-Reducción , Oxidorreductasas/genética , Oxígeno/química , Oxígeno/metabolismo , Penicilinas/biosíntesis , Penicilinas/química , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Regiones Determinantes de Complementariedad/metabolismo , Antígenos HLA/metabolismo , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Cristalografía por Rayos X , Antígenos HLA/química , Antígenos HLA/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Humanos , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T , Alanina , Biotinilación , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Complejo Mayor de Histocompatibilidad , Conformación Molecular , Mutación , Biblioteca de Péptidos , Péptidos/metabolismo , Unión Proteica , Solventes , Resonancia por Plasmón de Superficie , Termodinámica , AguaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Proteínas de la Membrana/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/biosíntesis , Antígenos de Superficie/biosíntesis , Complejo CD3/inmunología , Línea Celular Tumoral , Epítopos/inmunología , Femenino , Antígeno HLA-A2/inmunología , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Melanoma/inmunología , Melanoma/metabolismo , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Distribución Aleatoria , Proteínas Recombinantes de Fusión/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.
Asunto(s)
Antígenos de Neoplasias/química , Vacunas contra el Cáncer/farmacología , Epítopos de Linfocito T/química , Proteínas de la Membrana/química , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/farmacología , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/química , Línea Celular Tumoral , Quimiocina CCL4 , Cristalografía por Rayos X , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/química , Antígeno HLA-A2/inmunología , Inmunización , Interferón gamma/análisis , Proteínas Inflamatorias de Macrófagos/análisis , Complejo Mayor de Histocompatibilidad/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Transfección , Vacunas Sintéticas/químicaRESUMEN
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.
Asunto(s)
Antígenos HLA/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Especificidad de Anticuerpos , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular , Línea Celular Tumoral , Cristalografía por Rayos X , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Indicadores y Reactivos , Modelos Moleculares , Simulación de Dinámica Molecular , Imitación Molecular/genética , Imitación Molecular/inmunología , Péptidos/química , Péptidos/genética , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunologíaRESUMEN
Activation of cytotoxic T cells is initiated by engagement of the T-cell receptor (TCR) with peptide-major histocompatibility class I complexes (pMHCI). The CD8 co-receptor also binds to pMHCI, but at a distinct site, and allows the potential for tripartite TCR/pMHCI/CD8 interactions, which can increase T cell antigen sensitivity. There has been a substantial interest in the effect of the pMHCI/CD8 interaction upon TCR/pMHCI engagement, and several conflicting studies have examined this event, using the soluble extracellular domains of CD8 and the TCR, by surface plasmon resonance. However, the evidence to date suggests that the TCR engages cognate pMHCI before CD8 recruitment, so the question of whether TCR engagement alters CD8 binding is likely to be more relevant to the biological order of T cell antigen encounter. Here, we have examined the binding of CD8 to several variants of the HLA A2-restricted telomerase(540-548) antigen (ILAKFLHWL) and the HLA A2-restricted NY-ESO-1(157-165) antigen (SLLMWITQC) that bind to their cognate TCRs with distinct affinities and kinetics. These interactions represent a range of agonists that exhibit different CD8 dependency for activation of their respective T cells. By using engineered affinity enhanced TCRs to these ligands, which have extended off-rates of approximately 1h compared to seconds for the wildtype TCRs, we have examined pMHCI/CD8 binding before and during TCR-engagement. Here we show that the binding of the extracellular domain of the TCR to pMHCI does not transmit structural changes to the pMHCI-CD8 binding site that would alter the subsequent pMHCI/CD8 interaction.
Asunto(s)
Antígenos CD8/metabolismo , Antígeno HLA-A2/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T Citotóxicos/inmunología , Sitios de Unión , Antígenos CD8/inmunología , Antígeno HLA-A2/química , Antígeno HLA-A2/inmunología , Humanos , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
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.
Asunto(s)
Antígenos CD8/química , Antígenos CD8/genética , Sustitución de Aminoácidos , Biología Computacional , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Cristalografía por Rayos X , Complejo Mayor de Histocompatibilidad/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Escherichia coli/genética , Humanos , Enlace de Hidrógeno , Cinética , Ligandos , Modelos Moleculares , Mutación , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/genética , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/química , Complejo Mayor de Histocompatibilidad , Proteínas de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/aislamiento & purificación , Cristalización , ADN Complementario , Humanos , Antígeno MART-1 , Melanocitos/fisiología , Melanoma/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/aislamiento & purificación , Plásmidos , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Neoplasias Cutáneas/química , Difracción de Rayos XRESUMEN
The mammalian alpha/beta T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.
Asunto(s)
Regiones Determinantes de Complementariedad/química , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/química , Antígenos/metabolismo , Línea Celular Transformada , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Humanos , Cinética , Ligandos , Complejo Mayor de Histocompatibilidad , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso , Biblioteca de Péptidos , Péptidos/inmunología , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta , Sensibilidad y Especificidad , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Reacciones Cruzadas/inmunología , Inmunoterapia/efectos adversos , Inmunoterapia/métodos , Imitación Molecular , Receptores de Antígenos de Linfocitos T/metabolismo , Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cardiotoxicidad , Línea Celular , Conectina/química , Conectina/inmunología , Conectina/metabolismo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Ingeniería Genética , Humanos , Modelos Moleculares , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión Proteica/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR-pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6(4)22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 A. VM calculations indicated one molecule per asymmetric unit. A 2 A data set was collected and the structure is currently being determined using molecular replacement.
Asunto(s)
Complejo Mayor de Histocompatibilidad , Receptores de Antígenos de Linfocitos T/química , Linfocitos T CD8-positivos/inmunología , Clonación Molecular , Cristalización , Humanos , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Receptores de Antígenos de Linfocitos T/aislamiento & purificación , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A(∗)0201) complexed with human T cell lymphotropic virus type 111-19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3ß loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3ß loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies.
RESUMEN
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.
Asunto(s)
Citotoxicidad Inmunológica , Neoplasias Experimentales/terapia , Receptores de Antígenos de Linfocitos T/fisiología , Animales , Linfocitos T CD8-positivos/inmunología , Humanos , Memoria Inmunológica , Inmunoterapia , Interferón gamma/biosíntesis , Activación de Linfocitos , Ratones , Ratones SCID , Neoplasias Experimentales/inmunologíaRESUMEN
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.
Asunto(s)
Antígenos CD/genética , Antígenos CD/farmacología , Factores Inmunológicos/genética , Factores Inmunológicos/farmacología , Activación de Linfocitos/inmunología , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Antígenos CD/química , Autoinmunidad , Bioensayo , Línea Celular , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Relación Dosis-Respuesta Inmunológica , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Factores Inmunológicos/química , Cinética , Receptor Leucocitario Tipo Inmunoglobulina B1 , Activación de Linfocitos/genética , Complejo Mayor de Histocompatibilidad/genética , Complejo Mayor de Histocompatibilidad/inmunología , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Polietilenglicoles , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
T cell recognition is initiated by the binding of TCRs to peptide-MHCs (pMHCs), the interaction being characterized by weak affinity and fast kinetics. Previously, only 16 natural TCR/pMHC interactions have been measured by surface plasmon resonance (SPR). Of these, 5 are murine class I, 5 are murine class II, and 6 are human class I-restricted responses. Therefore, a significant gap exists in our understanding of human TCR/pMHC binding due to the limited SPR data currently available for human class I responses and the absence of SPR data for human class II-restricted responses. We have produced a panel of soluble TCR molecules originating from human T cells that respond to naturally occurring disease epitopes and their cognate pMHCs. In this study, we compare the binding affinity and kinetics of eight class-I-specific TCRs (TCR-Is) to pMHC-I with six class-II-specific TCRs (TCR-IIs) to pMHC-II using SPR. Overall, there is a substantial difference in the TCR-binding equilibrium constants for pMHC-I and pMHC-II, which arises from significantly faster on-rates for TCRs binding to pMHC-I. In contrast, the off-rates for all human TCR/pMHC interactions fall within a narrow window regardless of class restriction, thereby providing experimental support for the notion that binding half-life is the principal kinetic feature controlling T cell activation.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase I/química , Receptores de Antígenos de Linfocitos T/química , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Cinética , Receptores de Antígenos de Linfocitos T/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
HLA-A*2402 is the most commonly expressed HLA allele in oriental populations. It is also widely expressed in the Caucasian population, making it one of, if not the most abundant HLA I types. In order to study its structure in terms of overall fold and peptide presentation, a soluble form of this HLA I (alpha1, alpha2, alpha3 and beta(2)m domains) has been expressed, refolded and crystallized in complex with a cancer-related telomerase peptide (VYGFVRACL), and its structure has been solved to 2.8 A resolution. The overall structure of HLA-A*2402 is virtually identical to other reported peptide-HLA I structures. However, there are distinct features observable from this structure at the HLA I peptide binding pockets. The size and depth of pocket B makes it highly suitable for binding to large aromatic side chains, which explains the high prevalence of tyrosine at peptide position 2. Also, for HLA binding at peptide position 5, there is an additional anchor point, which allows the proximal amino acids to protrude out, providing a prominent feature for TCR interaction. Finally, pocket F allows the anchor residue at position 9 to be bound unusually deeply within the HLA structure.
Asunto(s)
Antígenos HLA-A/química , Telomerasa/química , Secuencia de Aminoácidos , Cristalización , Epítopos de Linfocito T/genética , Humanos , Péptidos/química , Estructura Cuaternaria de ProteínaRESUMEN
Antibody and T-cell receptors (TCRs) are the primary recognition molecules of the adaptive immune system. Antibodies have been extensively characterized and are being developed for a large number of therapeutic applications. This has been possible because of the ability to manufacture stable, soluble, monoclonal antibodies which retain the antigen specificity of B cells. Unlike antibodies, TCRs are not expressed in a soluble form, but are anchored to the T-cell surface by an insoluble trans-membrane domain. Characterization and development of TCRs has been hampered by the lack of suitable methods for producing them as soluble and stable proteins. Here we report the engineering of soluble human TCRs suitable for crystallization studies and potentially for in vivo therapeutic use.