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
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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
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(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
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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).
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