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Identifier 000416898
Title Establishment of the optimal administration schedule for cryptic telomerase peptides (hTERT) as cancer immunotherapy
Alternative Title Καθορισμός του βέλτιστου τρόπου χορήγησης τροποποιημένων κρυπτικών πεπτιδίων της Τελομεράσης (hTERT) σαν ανοσοθεραπεία σε ασθενείς με νεοπλασματική νόσο
Author Παπαδημητράκη, Ελισάβετ
Thesis advisor Μαυρουδής, Δημήτριος
Γεωργούλιας, Βασίλειος
Παπαματθαιάκης, Ιωσήφ
Reviewer Σαμώνης, Γεώργιος
Αγγελάκη, Σοφία
Θεοδωρόπουλος, Παναγιώτης
Κοτσάκης, Αθανάσιος
Abstract Establishment of the optimal administration schedule for cryptic telomerase peptides (hTERT) as cancer immunotherapy Introduction In most human cancers, activation of telomerase appears to be a hallmark, associated with unlimited cell proliferation of tumour cells (Blasco, 2005; Shay & Wright, 2000). By ensuring maintenance of telomeres’ length above a critically short point, telomerase prevents the induction of cellular senescence or apoptosis for the cancer cells, therefore allowing for tumour progression. Telomerase, and more specifically its catalytic subunit hTERT, is found to be overactive in 85–90% of cancers, marking it as a popular target for anticancer therapies. TERT572-based vaccine- Rationale Nearly all human tumour-associated antigens, including telomerase, derive from non-altered self-proteins, thereby are subjects of the immune tolerance. The HLA-I molecules can bind both dominant and cryptic peptides. The dominant peptides have a strong affinity for HLA-I alleles, are abundant on the cell surface, and are strongly immunogenic, whereas cryptic peptides are not as abundant on the cell surface, have weak HLA-I affinity, demonstrating weak immunogenicity or complete lack of immunogenicity. In contrast to dominant peptides, cryptic peptides are poorly expressed, thereby do not induce immune tolerance escaping massive clonal deletion. These characteristics of the cryptic peptides make them a favourable target, candidate for the development of a specific, peptide antitumor vaccine therapy. Moreover, the use of tumour nonspecific antigens may be a better choice for anticancer vaccines since they are not dependant on adjuvants or the efficacy of delivery (Mavroudis et al., 2006; Menez-Jamet & Kosmatopoulos, 2009; Ruden & Puri, 2013). In our studies with the peptide-based vaccine (hTERT- based), we tried to overcome the tolerance-related blunting of T cell responses, by using cryptic (low affinity for HLA) peptides for the induction of an antitumor immune response. However, binding of wild type cryptic peptide antigens to HLA is usually unstable, with weak immunogenicity, and therefore 6 challenging in regard to immune response possibly hampering T cell priming and activation. More recent research has focused on the development of optimized cryptic peptides with higher affinity binding to HLA. Based on this approach, our peptide-based anticancer vaccine, known as Vx-001 (Vaxon Biotech, Paris, France), consists of a low affinity cryptic peptide hTERT572 (RLFFYRKSV) and its optimized version, the hTERT572Y(1) (YLFFYRKSV), which has the first amino-acid residue replaced with a modified tyrosine (Y1) residue. This sequence aims to enhance the peptide’s affinity for HLA-I molecules and potentially can circumvent the self-tolerance issue. The TERT572Y peptide has been found to induce tumour immunity in HLA-A*0201 transgenic mice but luckily not autoimmunity (Gross et al., 2004). In addition, Vx-001 leads to enhanced immunogenicity of the cryptic peptide when presented by HLA-A*0201 molecules (the most frequently expressed allele, present in 40–45% of population) without altering antigen’s specificity (Mavroudis et al., 2006). In the current study, our primary goal was to establish the optimal vaccination protocol, for administration of the two TERT peptides (the native TERT572 and its optimized variant TERT572Y) regarding its ability to elicit the best immunologic response in respect to ex vivo reactivity of peptide-induced CTLs. Following establishment of the best vaccination schedule, the study aims to 1) assess the safety profile of the TERT vaccine, 2) correlate the immunologic outcome with the clinical outcome of the patients who received the TERT vaccine. Patients and Methods Patients In the first phase of the study for the establishment of the optimal vaccination schedule, 48 patients were enrolled, while overall 142 patients with various types of advanced solid tumours and previous exposure to standard treatment were enrolled in the telomerase peptide (hTERT) vaccination protocol. The inclusion criteria included HLA-A*0201 haplotype, histologically proven malignancy, advanced disease (Stage IV or locally advanced/unresectable), older than 18 years, performance status by WHO of 0-2, at least one chemotherapy regimen prior to vaccination, adequate bone marrow/liver/renal function. Peptides The 9-mer cryptic native TERT572 (RLFFYRKSV) peptide and its optimized variant TERT572Y (YLFFYRKSV), were synthesized initially by Epytop (Nimes, France) and later by Pepscan 7 (Lelystad, The Netherlands). Each peptide was prepared as a lyophilized powder (2 mg/vial) for reconstitution with 0.5 ml sterile water. Blood samples for Immunomonitoring Before each vaccination, 100ml peripheral blood in EDTA (ethylene diamine tetra acetic acid) was collected from each patient through a peripheral venous puncture. The time points of blood collection were set at baseline, prior to 3rd and 6th vaccination and before each boost administration of the peptide. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma, UK) density centrifugation and cryo-preserved in freezing medium at - 80oC until their future use the immune-assessment assays. Vaccination protocol (Schemes A, B) All HLA A*0201 patients (no= 48) received two subcutaneous (s.c) injections with 2mg of the optimized TERT572Y peptide followed by four s.c injections with 2mg of either the native TERT572 peptide (scheme A) or the optimized TERT572Y (scheme B), depending on the randomization schedule, every three weeks until disease progression as indicated in each result section. Patients who completed the 6-vaccination schedule and experienced disease stabilization or objective clinical response, received boost vaccinations (re-vaccinations) with 2mg native TERT572 peptide every three months until disease progression. Methods The evaluation of interferon-γ enzyme linked-TERT-specific T cell immunologic response was performed mainly by the Enzyme-linked immunosorbent (ELIspot) assay. To ensure high accuracy, 3 independent experiments were performed for each test. Results Our results revealed that vaccination with the optimized TERT572Y followed by the native TERT572 peptides can induce strong T cell responses, with higher avidity and frequencies of T cell responses, after the completion of 6-vaccinations. T cell responses after the sixth vaccination were detected more frequently (44% vs. 17%), and with higher number of peptide-specific reactive T cells (60 T cells/2 × 105 peripheral blood mononuclear cell vs. 10 T cells/ 2 × 105 peripheral blood mononuclear cell, p = 0.04), and higher avidity in the patients who received 4 more vaccinations with the TERT572 peptide compared with patients who received only TERT572Y vaccinations. These results demonstrate that the best vaccination schedule involves 8 first the administration of the optimized TERT572Y followed by the native TERT572 peptide in patients who are candidates for cancer immunotherapy. The association between immunologic response and clinical outcome (PFS and OS) was evaluated. Overall, there was no significant difference in either PFS or OS for patients who developed an immunologic response at any time during vaccination between the 2 schemes. However, in the subgroup analysis of patients who enrolled in scheme A vaccination, those who developed an immune response had a significantly longer PFS compared with those without an immune response (13.5 vs. 3.5mo; log-rank test p=0.01). In the next phase of the study, the best vaccination schedule was used in clinical trials with different tumour types and a cohort of NSCLC patients. Our studies confirmed a favourable toxicity profile of the TERT vaccine, without serious acute or late adverse events and without evidence of autoimmune reactions even after its administration for up to 2 years. Acute adverse events (AAE) were observed in 29 (52%) patients, and they were mild (grade 1). The most common AAE was grade 1 local skin reaction (n = 15; 27%). In our study, those patients who developed an immunologic response at any time during vaccination had a significantly higher PFS (5.2 months; range, 0.9–51.8) compared with those who failed to develop any response following vaccination (2.2 months, range, 1.4–6.5; p = 0.0001. Multivariate analysis demonstrated that the development of immunological response was an independent factor associated with better PFS (HR = 3.35, 95% CI 1.7–6.7; p = 0.001), while there was a trend for worse OS in patients who did not develop immunologic response during the vaccination (HR = 2.0, 95% CI 1.0–4.0; P = 0.057).
Language English
Subject Cancer vaccine
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Issue date 2018-07-18
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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