RESUMEN
Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naïve repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.
RESUMEN
Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.
Asunto(s)
Antineoplásicos , Ligando de CD40 , Humanos , Linfocitos T CD4-Positivos , Linfocitos T Colaboradores-Inductores , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Escamosas/terapia , Neoplasias Hematológicas/terapia , Inmunoterapia Adoptiva/métodos , Melanoma/terapia , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Sarcoma Sinovial/terapia , Factor de Crecimiento Transformador beta/metabolismo , Antígenos de Neoplasias/inmunología , Carcinoma de Células Escamosas/inmunología , Línea Celular Tumoral , Ingeniería Genética , Antígeno HLA-A2/metabolismo , Neoplasias Hematológicas/inmunología , Humanos , Tolerancia Inmunológica , Melanoma/inmunología , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Sarcoma Sinovial/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Microambiente TumoralRESUMEN
CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IEAsunto(s)
Presentación de Antígeno/inmunología
, Linfocitos T CD8-positivos/inmunología
, Infecciones por Virus de Epstein-Barr/inmunología
, Evasión Inmune/inmunología
, Western Blotting
, Desoxirribonucleasas/inmunología
, Técnicas de Silenciamiento del Gen
, Herpesvirus Humano 4/inmunología
, Humanos
, Reacción en Cadena en Tiempo Real de la Polimerasa
, Receptores Acoplados a Proteínas G/inmunología
, Proteínas de la Matriz Viral/inmunología
, Proteínas Virales/inmunología
RESUMEN
EBV elicits primary CD8(+) T cell responses that, by T cell cloning from infectious mononucleosis (IM) patients, appear skewed toward immediate early (IE) and some early (E) lytic cycle proteins, with late (L) proteins rarely targeted. However, L Ag-specific responses have been detected regularly in polyclonal T cell cultures from long-term virus carriers. To resolve this apparent difference between responses to primary and persistent infection, 13 long-term carriers were screened in ex vivo IFN-γ ELISPOT assays using peptides spanning the two IE, six representative E, and seven representative L proteins. This revealed memory CD8 responses to 44 new lytic cycle epitopes that straddle all three protein classes but, in terms of both frequency and size, maintain the IE > E > L hierarchy of immunodominance. Having identified the HLA restriction of 10 (including 7 L) new epitopes using memory CD8(+) T cell clones, we looked in HLA-matched IM patients and found such reactivities but typically at low levels, explaining why they had gone undetected in the original IM clonal screens. Wherever tested, all CD8(+) T cell clones against these novel lytic cycle epitopes recognized lytically infected cells naturally expressing their target Ag. Surprisingly, however, clones against the most frequently recognized L Ag, the BNRF1 tegument protein, also recognized latently infected, growth-transformed cells. We infer that BNRF1 is also a latent Ag that could be targeted in T cell therapy of EBV-driven B-lymphoproliferative disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Herpesvirus Humano 4/inmunología , Mononucleosis Infecciosa/inmunología , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/virología , Células Cultivadas , Ensayo de Immunospot Ligado a Enzimas , Antígenos HLA/metabolismo , Humanos , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Latencia del Virus/inmunologíaRESUMEN
Virus-specific CD4(+) T cells are key orchestrators of host responses to viral infection yet, compared with their CD8(+) T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II-epitope peptide tetramers to visualize human CD4(+) T cell responses to Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8(+) T cell responses. We find that, while not approaching virus-specific CD8(+) T cell expansions in magnitude, activated CD4(+) T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4(+) T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4(+) T cell priming.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Multimerización de Proteína , Enfermedad Aguda , Formación de Anticuerpos/inmunología , Antígenos Virales/inmunología , Proliferación Celular , Convalecencia , Epítopos/inmunología , Humanos , Inmunoglobulina G/inmunología , Memoria Inmunológica , Mononucleosis Infecciosa/inmunología , Mononucleosis Infecciosa/patología , Cinética , Fenotipo , Especificidad de la EspecieRESUMEN
The electron paramagnetic resonance technique of double electron-electron resonance (DEER) was used to measure nanometre-scale distances between nitroxide spin labels attached to the complement regulatory protein CD55 (also known as decay accelerating factor) and the von Willebrand factor A (vWF-A) domain of factor B. Following a thorough assessment of the quality of the data, distances obtained from good-quality measurements are compared to predicted distances from a previously hypothesised model for the complex and are found to be incompatible. The success of using these distances as restraints in multi-body docking routines is presented critically.
RESUMEN
Patients with the primary immunodeficiency X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2D1A, are highly susceptible to Epstein-Barr virus (EBV) infection. Nonetheless, some XLP patients demonstrate less severe clinical manifestations after primary infection. SH2D1A encodes the adaptor molecule SLAM-associated protein (SAP), which is expressed in T and natural killer cells and is required for cytotoxicity against B cells, the reservoir for EBV. It is not known why the clinical presentation of XLP is so variable. In this study, we report for the first time the occurrence of somatic reversion in XLP. Reverted SAP-expressing cells resided exclusively within the CD8(+) T cell subset, displayed a CD45RA(-)CCR7(-) effector memory phenotype, and were maintained at a stable level over time. Importantly, revertant CD8(+) SAP(+) T cells, but not SAP(-) cells, proliferated in response to EBV and killed EBV-infected B cells. As somatic reversion correlated with EBV infection, we propose that the virus exerts a selective pressure on the reverted cells, resulting in their expansion in vivo and host protection against ongoing infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/inmunología , Herpesvirus Humano 4 , Memoria Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/virología , Secuencia de Aminoácidos , Secuencia de Bases , Linfocitos T CD8-positivos/metabolismo , Cartilla de ADN/genética , Exones/genética , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Mutación/genética , Análisis de Secuencia de ADN , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Carga ViralRESUMEN
In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8(+) T-cell response. However, we noticed that in HLA-A 0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Ralpha; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Ralpha seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.
Asunto(s)
Antígenos CD/biosíntesis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Receptores de Interleucina-7/biosíntesis , Regulación hacia Arriba/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Epítopos/química , Humanos , Mononucleosis Infecciosa/inmunología , Receptor de Muerte Celular Programada 1 , Factores de TiempoRESUMEN
The early lytic cycle protein of Epstein-Barr virus (EBV), BNLF2a, has recently been shown to play a critical role in immune evasion by inhibiting the peptide transporter associated with antigen processing (TAP), thereby blocking antigen-specific CD8(+) T-cell recognition of many lytic cycle antigens. Surprisingly, we now show that a peptide ((50)VLFGLLCLL(58)) from the hydrophobic C-terminal region of this small (60-amino-acid) EBV protein is efficiently presented by the common class I allele HLA-A2 for recognition by cytotoxic T lymphocytes. The mechanism for this unexpected finding was revealed by experiments showing that this epitope is processed and presented independently of TAP.
Asunto(s)
Epítopos de Linfocito T/inmunología , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral/inmunología , Presentación de Antígeno , Antígeno HLA-A2/inmunología , Estructura Terciaria de Proteína , Linfocitos T Citotóxicos/inmunologíaRESUMEN
CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.
Asunto(s)
Antígenos CD/fisiología , Antígenos CD55/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Antígenos CD/genética , Linfocitos B/inmunología , Antígenos CD55/genética , Células Clonales , Cristalización , Citocinas/análisis , Citometría de Flujo , Variación Genética , Humanos , Leucocitos Mononucleares/inmunología , Glicoproteínas de Membrana/genética , Receptores Acoplados a Proteínas GRESUMEN
The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.
Asunto(s)
Antígenos CD55/fisiología , Magnesio/metabolismo , Factor de von Willebrand/química , Técnicas Biosensibles , Antígenos CD55/metabolismo , Factor D del Complemento/química , Proteínas del Sistema Complemento , Ácido Edético/química , Venenos Elapídicos/química , Humanos , Cinética , Magnesio/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Crystals of three epidermal growth-factor-like (EGF) domains of EMR2 (143 residues) have been grown. EMR2 is a member of the EGF-TM7 family of proteins. Different splice variants exist with between three and five consecutive EGF modules linked to a seven-span transmembrane G-protein-coupled receptor. Although its precise function is unknown, EMR2 is highly expressed in immune tissues and has been shown to weakly bind CD55, a complement-system regulator. Here, crystallization of EMR2 in the presence of Ca(2+), Ba(2+) and Sr(2+) ions is reported. A complete data set has been collected from all three crystal types, all of which belong to space group P2(1). An anomalous Patterson map from the Ba(2+) crystal data reveals three Ba(2+) ions bound within the asymmetric unit.