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Interleukin-10 (IL-10) is a highly pleiotropic cytokine that regulates immunological homeostasis through anti-inflammatory and/or immunostimulatory functions. Moreover, IL-10 is well known to exert diverse roles in tumor immunology and immunotherapy. The present study investigated the presence of circulating tumor antigen-specific IL-10-producing T cells in patients with head and neck squamous cell carcinoma (HNSCC), and determined factors that may influence the immunodynamics of IL-10-producing T cells. In vitro, peripheral blood mononuclear cells (PBMCs) stimulated with the tumor antigens p53 and MAGE-A4 were evaluated for interferon (IFN)-γ/IL-10 production using the IFN-γ/IL-10 double-color enzyme-linked immunosorbent spot assay. The proportion of T cells expressing immune checkpoint molecules in PBMCs was analyzed using flow cytometry. Of the 18 patients with HNSCC, 2 (11.1%) and 9 (50.0%) exhibited p53-specific IFN-γ and IL-10 production, respectively. Meanwhile, MAGE-A4-specific IFN-γ and IL-10 production was detected in 4 (28.6%) and 7 (50.0%) of 14 patients. In the p53-specific responses, IL-10-producing T cells were observed in significantly more patients than IFN-γ producing T cells (P=0.0275). In both CD4+ and CD8+ T cells, the proportion of T cells expressing lymphocyte activation gene-3 (Lag-3) was significantly lower in patients with p53-specific IL-10 production than in those without. In certain patients, Lag-3 blockade enhanced tumor antigen-specific IL-10. Taken together, the present study successfully demonstrated that tumor antigen-specific IL-10-producing T cells exist in the peripheral blood of patients with HNSCC and that Lag-3+ T cells may serve an important role in modulating IL-10-producing T cells. These findings provide novel insights into the roles of IL-10 and Lag-3 in mediating antitumor immune responses.
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T cell receptor (TCR) T cell therapies target tumor antigens in a human leukocyte antigen (HLA)-restricted manner. Biomarker-defined therapies require validation of assays suitable for determination of patient eligibility. For clinical trials evaluating TCR T cell therapies targeting melanoma-associated antigen A4 (MAGE-A4), screening in studies NCT02636855 and NCT04044768 assesses patient eligibility based on: (1) high-resolution HLA typing and (2) tumor MAGE-A4 testing via an immunohistochemical assay in HLA-eligible patients. The HLA/MAGE-A4 assays validation, biomarker data, and their relationship to covariates (demographics, cancer type, histopathology, tissue location) are reported here. HLA-A∗02 eligibility was 44.8% (2,959/6,606) in patients from 43 sites across North America and Europe. While HLA-A∗02:01 was the most frequent HLA-A∗02 allele, others (A∗02:02, A∗02:03, A∗02:06) considerably increased HLA eligibility in Hispanic, Black, and Asian populations. Overall, MAGE-A4 prevalence based on clinical trial enrollment was 26% (447/1,750) across 10 solid tumor types, and was highest in synovial sarcoma (70%) and lowest in gastric cancer (9%). The covariates were generally not associated with MAGE-A4 expression, except for patient age in ovarian cancer and histology in non-small cell lung cancer. This report shows the eligibility rate from biomarker screening for TCR T cell therapies and provides epidemiological data for future clinical development of MAGE-A4-targeted therapies.
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Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell-surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/major histocompatibility complex (MHCs) in a T cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific single-chain variable fragment (scFv) antibody against the MAGE-A4p230-239/human leukocyte antigen (HLA)-A∗02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4+ tumor cells in an HLA-A∗02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4+ HLA-A∗02:01+ tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors.
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Neoplasias , Receptores Quiméricos de Antígenos , Anticuerpos de Cadena Única , Ratones , Animales , Humanos , Anticuerpos de Cadena Única/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T , Antígenos HLA-A , Inmunoterapia AdoptivaRESUMEN
TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Neoplasias/metabolismo , Inmunoterapia Adoptiva , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS. DESIGN: A retrospective cohort study. SETTING: Infertility Clinic and Institute for Regenerative Medicine. PATIENT(S): This study consisted of 79 adult subjects with KS and 12 adult control subjects. INTERVENTION(S): The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4-stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software. MAIN OUTCOME MEASURE(S): The primary outcome was the presence of MAGE-A4-positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4- negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules. RESULT(S): The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16-48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens. CONCLUSION(S): We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results.
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Síndrome de Klinefelter , Espermatogonias , Adulto , Humanos , Masculino , Adolescente , Adulto Joven , Persona de Mediana Edad , Espermatogonias/patología , Síndrome de Klinefelter/complicaciones , Estudios de Cohortes , Espermatogénesis , Estudios Retrospectivos , Hematoxilina , Eosina Amarillenta-(YS) , Parafina , Semen , Testículo/patología , Hormona Folículo Estimulante , Testosterona , Hormona LuteinizanteRESUMEN
We aimed to investigate the clinical significance of the expression of NY-ESO-1 and MAGE-A4 in soft tissue sarcoma (STS). Immunostaining for NY-ESO-1, MAGE-A4, and Ki67 was performed using pathological specimens harvested from 10 undifferentiated pleomorphic sarcoma (UPS), nine myxofibrosarcoma (MFS), and three malignant peripheral nerve sheath tumor (MPNST) patients treated at our hospital. We examined the correlation of NY-ESO-1 and MAGE-A4 expression levels with tumor size, histological grade, and SUVmax values. Positive cell rates of various markers were also compared between patients in remission and those who were not in remission. The rates of cases positive for NY-ESO, MAGE-A4, and Ki67 were 50%, 63.6%, and 90.9%, respectively. The average rates of cells positive for NY-ESO, MAGE-A4, and Ki67 in all STS types were 18.2%, 39.4%, and 16.8%, respectively. A positive correlation was observed between rates of cells positive for NY-ESO-1 and MAGE-A4 and between NY-ESO-1 and MAGE-A4 expression levels and clinical features. There was no significant difference in the positive cell rate of NY-ESO-1 or MAGE-A4 between remission and non-remission cases. Our results suggest that NY-ESO-1 and MAGE-A4 expression may be useful for the diagnosis and prognostication of UPS, MFS, and MPNST.
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BACKGROUND: Cancer testis (CT) antigens are promising targets for cancer immunotherapies such as cancer vaccines and genetically modified adoptive T cell therapy. In this study, we evaluated the expression of three CT antigens, melanoma-associated antigen A4 (MAGE-A4), New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) and sarcoma antigen gene (SAGE). METHODS: MAGE-A4, NY-ESO-1 and/or SAGE antigen expression in tumour samples was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Informed consent was obtained from individuals prior to study enrolment. RESULTS: In total, 585 samples in 21 tumour types were evaluated between June 2009 and March 2018. The positive expression rates of these CT antigens were as follows: MAGE-A4, 34.6% (range, 30.7-38.7); NY-ESO-1, 21.0% (range, 17.2-25.1); and SAGE, 21.8% (range, 18.5-25.4). The MAGE-A4 antigen was expressed in 54.9% of oesophageal cancers, 37.5% of head and neck cancers, 35.0% of gastric cancers and 34.2% of ovarian cancers; the NY-ESO-1 antigen was expressed in 28.6% of lung cancers, 25.3% of oesophageal cancers and 22.6% of ovarian cancers; and the SAGE antigen was expressed in 35.3% of prostate cancers, 32.9% of oesophageal cancers and 26.3% of ovarian cancers. The most common tumour type in this study was oesophageal cancer. MAGE-A4, NY-ESO-1 and SAGE antigen expression were assessed in 214 oesophageal cancer samples, among which 24 (11.2%) were triple-positive, 58 (27.1%) were positive for any two, 59 (27.6%) were positive for any one, and 73 (34.1%) were triple negative. CONCLUSIONS: Oesophageal cancer exhibited a relatively high rate of CT antigen mRNA expression positivity.
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Antígenos de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/patología , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies.
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Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Antígenos de Neoplasias/química , Cristalografía por Rayos X , Antígeno HLA-A2/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
A substantial obstacle to the success of adoptive T cell-based cancer immunotherapy is the sub-optimal affinity of T-cell receptors (TCRs) for most tumor antigens. Genetically engineered TCRs that have enhanced affinity for specific tumor peptide-MHC complexes may overcome this barrier. However, this enhancement risks increasing weak TCR cross-reactivity to other antigens expressed by normal tissues, potentially leading to clinical toxicities. To reduce the risk of such adverse clinical outcomes, we have developed an extensive preclinical testing strategy, involving potency testing using 2D and 3D human cell cultures and primary tumor material, and safety testing using human primary cell and cell-line cross-reactivity screening and molecular analysis to predict peptides recognized by the affinity-enhanced TCR. Here, we describe this strategy using a developmental T-cell therapy, ADP-A2M4, which recognizes the HLA-A2-restricted MAGE-A4 peptide GVYDGREHTV. ADP-A2M4 demonstrated potent anti-tumor activity in the absence of major off-target cross-reactivity against a range of human primary cells and cell lines. Identification and characterization of peptides recognized by the affinity-enhanced TCR also revealed no cross-reactivity. These studies demonstrated that this TCR is highly potent and without major safety concerns, and as a result, this TCR is now being investigated in two clinical trials (NCT03132922, NCT04044768).
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Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Antígenos de Neoplasias , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
Testicular biopsy may be a component of the work-up of male infertility. However, no reliable diagnostic tools are available for objective quantitative assessment of spermatogenic cells. It is well known that MAGE-A4 is selectively expressed in spermatogonia and our group has previously demonstrated that DOG1 differentially stains germ cells. Therefore, we performed DOG1 and a double stain cocktail (DOG1 and 57b murine monoclonal anti-MAGE-A4) immunohistochemical stains on 40 testicular infertility biopsies (10 each with active spermatogenesis, Sertoli cell-only, hypospermatogenesis, and maturation arrest), 25 benign seminiferous tubules from radical orchiectomies, and 5 spermatocytic tumors (ST). In biopsies/resections with active spermatogenesis, DOG1 stained spermatocytes and spermatids and was absent in spermatogonia, while MAGE-A4 stained spermatogonia and primary spermatocytes (weak). In hypospermatogenesis, DOG1 highlighted decreased spermatocytes/spermatids and MAGE-A4 highlighted decreased spermatogonia. DOG1 staining confirmed decreased to absent spermatocytes in maturation arrest and MAGE-A4 staining established the presence of preserved spermatogonia in all cases. All STs were negative for DOG1 and positive for MAGE-A4, while all Sertoli cell-only cases were negative for DOG1 and the double stain cocktail. In conclusion, we confirmed that DOG1 is expressed in spermatocytes and spermatids and MAGE-A4 highlights primarily spermatogonia. Usage of these stains facilitates confirmation of maturation arrest, assessment of the percentage of testis involvement in hypospermatogenesis and identification of mixed patterns. Finally, this study supports that the differentiation of STs is more closely related to spermatogonia than the more mature spermatocytes.
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Anoctamina-1/metabolismo , Biomarcadores de Tumor/metabolismo , Infertilidad Masculina/veterinaria , Proteínas de Neoplasias/metabolismo , Biopsia , Humanos , Inmunohistoquímica , Infertilidad Masculina/diagnóstico , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patología , Células de Sertoli/metabolismo , Células de Sertoli/patología , Espermatogénesis , Espermatogonias/metabolismo , Espermatogonias/patología , Coloración y Etiquetado , Testículo/metabolismo , Testículo/patologíaRESUMEN
MAGE-A4 antigen is a cancer-testis antigen that is frequently expressed in tumor tissues. Cholesteryl pullulan (CHP) is a novel antigen delivery system for cancer vaccines. This study evaluated the safety, immune responses and clinical outcomes of patients who received a CHP-MAGE-A4 vaccine. Twenty-two patients with advanced or metastatic cancer were enrolled, and were subcutaneously vaccinated with either 100 µg or 300 µg of CHP-MAGE-A4. Seven and 15 patients, respectively, were repeatedly vaccinated with 100 µg or 300 µg of CHP-MAGE-A4; patients in both groups received a median of 7 doses. No serious adverse events related to the vaccine were observed. Of 7 patients receiving the 100 µg dose, 2 (29%) showed immune responses, compared with 3 of the 14 (21%) patients who received the 300 µg dose. In total, MAGE-A4-specific antibody responses were induced in 5 of 21 (24%) patients. No differences in survival were seen between patients receiving the 100 µg and 300 µg doses, or between immune responders and non-responders. Eleven (50%) patients had pre-existing antibodies to NY-ESO-1. In 16 patients with esophageal or head/neck squamous cell carcinoma, the survival time was significantly shorter in those who had NY-ESO-1-co-expressing tumors. Patients with high pre-existing antibody responses to NY-ESO-1 displayed worse prognosis than those with no pre-existing response. Therefore, in planning clinical trials of MAGE-A4 vaccine, enrolling NY-ESO-1-expressing tumor or not would be a critical issue to be discussed. Combination vaccines of MAGE-A4 and NY-ESO-1 antigens would be one of the strategies to overcome the poor prognosis.
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BACKGROUND: Aim was to analyze the expression of different cancer testis antigens (CTA) and to assess its prognostic value in salivary gland carcinomas. METHODS: Patients with salivary gland carcinomas diagnosed 1994 to 2010 were included. Baseline characteristics, pathohistological, clinical, and outcome data were assessed. Tissue microarrays were constructed and immunohistochemistry for different CTA (NY-ESO1, NY-BR1, MAGE A1, MAGE A3, MAGE A4, MAGE C1/CT7, and MAGE C2/CT10) was performed. CTA expression was assessed and statistically correlated with pathological and outcome data. RESULTS: Expression rates of CTA in salivary gland tumors ranged from 0% to 40%. MAGE A4 expression was associated with a lower tumor grade tumor grading (P = .017), and a favorable recurrence-free (P = .003), disease-specific (P = .046) and overall survival (P = .028). CONCLUSIONS: MAGE A4 is a highly significant prognostic marker in salivary gland carcinoma; its expression is associated with low-grade histology, a low rate of distant metastasis and a favorable survival. LEVEL OF EVIDENCE: 4.
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BACKGROUND/AIM: The aim of this study was to explore whether the treatment effect or immune response to a cancer vaccine can be predicted by the percentage of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) after vaccination. PATIENTS AND METHODS: Sixteen patients (9 men, 7 women; median age 61.5 years) enrolled in the CHP-MAGE-A4 cancer vaccine clinical trial who had a fixed dose (300 µg of CHP-MAGE-A4 cancer vaccine and 0.5 Klinische Einheit (KE) of OK432 and received at least four vaccinations were investigated. Safety, immune response, and clinical effects were assessed before and after the cancer vaccination. RESULTS: Treg ratios that remained low both before and after vaccination were associated with a good prognosis, and a low Treg/CD4 lymphocyte ratio 7-weeks after the initial vaccination was correlated with a better prognosis. CONCLUSION: The Treg ratio following vaccination appears to have some utility for predicting patient prognosis.
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Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Vacunación , Adulto , Anciano , Vacunas contra el Cáncer/administración & dosificación , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Estimación de Kaplan-Meier , Leucocitos Mononucleares/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Neoplasias/terapia , Evaluación de Resultado en la Atención de Salud/métodos , Pronóstico , Linfocitos T Reguladores/metabolismoRESUMEN
INTRODUCTION: Lung cancer is the leading cause of cancer mortality worldwide; therefore, understanding the biological or clinical role of tumor-associated antigens and autoantibodies is of eminent interest for designing antitumor immunotherapeutic strategies. METHODS: Here we prospectively analyzed the serum frequencies of New York esophageal squamous cell carcinoma 1 (NY-ESO-1), human epidermal growth factor 2/neu, and melanoma-associated antigen A4 (MAGE-A4) antibodies and expression of the corresponding antigens in tumors of 121 patients with NSCLC undergoing an operation without prior neoadjuvant chemotherapy and compared them with those in 57 control age-matched patients with no history of a malignant disease. RESULTS: We found that only antibodies specific for NY-ESO-1 (19.8% [n = 24 of 121]) were significantly increased in the group of patients with NSCLC compared with in the controls. NY-ESO-1 seropositivity was significantly positively associated with an active smoking history in patients with NSCLC but not in smokers from the control group. In tumors, the frequency of NY-ESO-1 mRNA expression was 6.3% (in four of 64 patients), the frequency of human epidermal growth factor 2/neu (HER 2/neu) expression was 11.9% (five of 42), and the frequency of MAGE-A4 expression was 35.1% (20 of 57). MAGE-A4 expression in tumors correlated with smoking status and male sex in patients with NSCLC. Patients with squamous cell carcinoma displayed higher expression of NY-ESO-1 and MAGE-A4 in tumors than did patients with adenocarcinoma. On the other hand, 94.7% of nonsmoking patients in our study had adenocarcinoma (of whom 73.7% were women). CONCLUSION: These results confirm the reported high immunogenicity of NY-ESO-1 and suggest that a smoking-induced chronic inflammatory state might potentiate the development of NY-ESO-1-specific immune responses. Moreover, smoking might contribute to the expression of other cancer/testis antigens such as MAGE-A4 at early stages of NSCLC development.
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Antígenos de Neoplasias/sangre , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/sangre , Neoplasias Pulmonares/sangre , Proteínas de la Membrana/sangre , Proteínas de Neoplasias/sangre , Receptor ErbB-2/sangre , Fumar/efectos adversos , Adenocarcinoma/sangre , Adenocarcinoma/etiología , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/inmunología , Carcinoma de Pulmón de Células no Pequeñas/etiología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/patología , Masculino , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios ProspectivosRESUMEN
AIM: To investigate the antigen spreading pattern in the CHP-MAGE-A4-vaccinated patients and analyze the clinical relevance of antigen spreading pattern as a surrogate marker of patient survival. MATERIALS & METHODS: 12 patients who had been injected with 300 µg of CHP-MAGE-A4 and 0.5 Klinische Einheit of OK-432 in more than five vaccinations were analyzed. RESULTS: Increases in the anti-MAGE-A4-specific antibody response were observed in eight patients (66.7%), compared with six patients (50%) for anti-NY-ESO-1 and five patients (41.7%) for anti-MAGE-A3 after five vaccinations. We identified frequent antigen spreading following MAGE-A4 vaccinations without associations with the clinical response or patient prognosis. CONCLUSION: Antigen spreading pattern might reflect tumor shrinkage as a response to treatment and treatment history (clinical trial registration number: UMIN000001999).
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Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Neoplasias del Colon/terapia , Proteínas de la Membrana/inmunología , Proteínas de Neoplasias/inmunología , Anciano , Anticuerpos/sangre , Biomarcadores Farmacológicos/metabolismo , Neoplasias del Colon/inmunología , Neoplasias del Colon/mortalidad , Epítopos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Supervivencia , Carga TumoralRESUMEN
The 'selfish spermatogonial selection'- model was proposed to explain the paternal age effect (PAE) of some congenital disorders associated with point mutations in male germ cells. According to this, spermatogonia carrying pathogenic mutations gain a selection advantage over non-mutated spermatogonia which leads to an increased number of mutated spermatogonia and consequently spermatozoa over time. Recently, an immunohistochemical approach using the premeiotic marker melanoma antigen family A4 (MAGE A4) was undertaken by the Wilkie group to confirm the presence of microclones of putatively mutated spermatogonia in testes of elderly men. The objective of our study was the age-dependent assessment of testes from men with normal spermatogenesis using MAGE A4 immunohistochemistry to identify and corroborate cellular clusters indicative for 'selfish spermatogonial selection' in our cohort. We analyzed testicular tissues obtained from men with normal spermatogenesis assigned to three age groups [(1) 28.8 ± 2.7 years; (2) 48.1 ± 1 years; (3) 71.9 ± 6.8 years, n/group = 8]. We could detect very similar distribution patterns of MAGE A4-positive cells and the presence of several types of microclusters as reported previously. However, these cellular clusters, indicative for clonal expansion, were not only present in testes from elderly men but also in those from age group 1 and 2. Using graphical three-dimensional modelling, we identified that cross-section directions e.g. longitudinal sections might provoke misleading interpretation of spermatogonial clusters, in particular when the tissue processing is limited. Thus, appropriate fixation and embedding is needed for reliable analysis of testicular sections. We therefore propose a more careful interpretation of such spermatogonial clusters and recommend a 3-D analysis to unequivocally determine 'selfish spermatogonial selection'-manifestations.
Asunto(s)
Envejecimiento/patología , Imagenología Tridimensional , Mutación , Espermatogénesis/genética , Espermatogonias/patología , Testículo/patología , Adulto , Anciano , Envejecimiento/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biopsia , Células Clonales , Estudios de Cohortes , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
A phase I+II clinical trial of vaccination with MAGE-A4 protein complexed with cholesteryl pullulan melanoma antigen gene-A4 nanogel (CHP-MAGE-A4) is currently underway in patients with MAGE-A4-expressing cancer. In the present study, the primary phase I endpoint was to test the safety of the administration of 300 µg CHP-MAGE-A4 with and without OK-432. Another aim of the study was to clarify the details of the specific humoral immune response to vaccination. The 9 patients enrolled for phase I were vaccinated 6 times, once every 2 weeks: 3 patients with 100 µg and 3 patients with 300 µg CHP-MAGE-A4, and 3 patients with 300 µg CHP-MAGE-A4 plus 0.5 clinical units of OK-432. Toxicities were assessed using Common Terminology Criteria for Adverse Events v3.0. Clinical response was evaluated by modified Response Evaluation Criteria in Solid Tumours. Immunological monitoring of anti-MAGE-A4-specific antibodies was performed by ELISA of pre- and post-vaccination patient sera. The 6 vaccinations produced no severe adverse events. Stable disease was assessed in 4/9 patients. Anti-MAGE-A4 total immunoglobulin (Ig)G titers increased in 7/9 patients. Efficacious anti-MAGE-A4 IgG1, 2 and 3 antibody responses were observed in 7/9 patients. Among them, positive conversions to T helper 2 (Th2)-type antibody responses (IgG4 and IgE) were observed after frequent vaccination in 4/7 patients. The Th2 conversion was possibly associated with undesirable clinical observations, including progressive disease and the appearance of a new relapse lesion. The present study suggested that frequent vaccinations activated a Th2-dominant status in the cancer patients. The identification of a time-dependent IgG subclass and IgE antibody production during vaccination protocols may be a useful surrogate marker indicating a potentially undesirable change of the immunological environment for an effective antitumor immune response in cancer patients.
RESUMEN
Trans-lesion synthesis (TLS) is a DNA damage-tolerant and error-prone mode of DNA replication. Recent work shows that many cancer cells coopt an aberrantly expressed germ cell protein, melanoma antigen-A4 (MAGE-A4), to activate TLS. MAGE-A4-induced "pathological TLS" provides a potential mechanism through which neoplastic cells can tolerate intrinsic and therapeutic genotoxicity while acquiring mutability.
RESUMEN
PURPOSE: We conducted a cancer vaccine clinical trial with MAGE-A4 protein. Safety, clinical response, and antigen-specific immune responses were analyzed and the prognostic factors by vaccination were investigated. EXPERIMENTAL DESIGN: Twenty patients with advanced esophageal, stomach or lung cancer were administered MAGE-A4 vaccine containing 300µg protein subcutaneously once every 2 weeks in six doses. Primary endpoints of this study were safety and MAGE-A4 immune responses. RESULTS: The vaccine was well tolerated. Fifteen of 20 patients completed one cycle of vaccination and two patients showed SD. A MAGE-A4-specific humoral immune response was observed in four patients who had high expression of MAGE-A4 and MHC class I on tumor cells. These four patients showed significantly longer overall survival than patients without an antibody response after vaccination (p=0.009). Patients with tumor cells expressing high MAGE-A4 or MHC class I antigen showed significantly longer overall survival than those with low expression. Induction of CD4 and CD8T cell responses was observed in three and six patients, respectively, and patients with induction of MAGE-A4-specific IFNγ-producing CD8T cells, but not CD4T cells, lived longer than those without induction. CONCLUSIONS: The CHP-MAGE-A4 vaccine was safe. Expression of MAGE-A4 and MHC class I in tumor tissue and the induction of a MAGE-A4-specific immune response after vaccination would be feasible prognostic markers for patients vaccinated with MAGE-A4.