Using+Telomerase+for+Targeted+Gene+Therapy+in+Cancer+Patients

by Ashley Leslie and Charles Walker

Introduction
We began our investigation into the use of telomerase as a target for cancer therapies with a 2008 review article published in the British Journal of Cancer. While the article discussed multiple approaches to telomeric therapeutics, we chose to focus on immunotherapy, in particular, the use of peptide vaccines, as shown below.



=Background =

About Telomerase
Telomerase is an enzyme that maintains telomeres, the sequences of repeated DNA at the ends of chromosomes (geron). Telomerase consists of an RNA subunit (telomerase RNA, or TERC) and a protein subunit (telomerase reverse transcriptase, or TERT) (Allison). Through the RNA template (AAUCCC) provided by TERC, TERT uses reverse transcriptase to add on repeating bases (TTAGGG) to the ends of chromosomes in the 5'-3' direction (Allison).

Telomerase and Cancer
Telomeres are an essential part of a chromosome—they protect and stabilize chromosomes, but each time a cell’s DNA is replicated, the telomeres are shortened (Shay). With successive divisions the telomeres become so short that the cell either stops dividing (senesces) or dies (apoptosis) (Shay). Thus, telomerase works to elongate telomeres and stall this cell fate. Telomerase activity normally peaks during embryonic development when cells are dividing extremely rapidly to promote growth and then is downregulated in mature cells, with the exception of germ and stem cells (geron). However, telomerase is also found to be active in 85-90% of tumors from cancers of all types (medscape). It is the upregulation of telomerase that provides cancer cells with the potential for immortality—their telomeres are continually extended, permitting unlimited rounds of cell division (Allison). Given this disparity in telomerase activity between normal cells and cancerous cells, telomerase is an appealing target for cancer therapeutics and is the focus of much current research. 

Telomerase Immunotherapy
Immunotherapy involves the treatment of disease by either eliciting or suppressing an immune response. In the case of telomerase immunotherapy, a drug product would be designed that causes an immune response against the hTERT protein (the catalytic subunit of telomerase). Thus, any telomerase-positive cells expressing hTERT would be killed. Researchers have already demonstrated that hTERT peptides can be presented as epitopes by major histocompatibility complexes (MHCs), resulting in an immune response (medscape).

Telomerase immunotherapy holds promise as hTERT is such a widely expressed tumor-associated antigen, but, given manufacturing complexities coupled with complicated biological mechanisms, designing such a therapy is easier said than done. There is also a need for more innovative clinical trial designs to determine how exactly telomeric immunotherapies should be used as a treatment against cancer.

Telomerase Peptide Vaccination (GV1001)
<span style="font-family: 'Times New Roman',Times,serif;">One of the most advanced telomeric immunotherapy products is the telomerase peptide vaccine GV1001. GV1001 <span style="font-family: 'Times New Roman',Times,serif;">contains the 16 amino acid long peptide hTERT (611-626), which <span style="font-family: 'Times New Roman',Times,serif;">is a "promiscuous HLA (human leukocyte antigen) class II epitope", (cancer.gov, Bernhardt). This means that it is able to bind various HLA molecules and potentially raise an immune response against telomerase-positive cells (cancer.gov). In 2006, researchers published a paper on a phase I/II clinical trial involving the administration of the telomerase peptide GV1001 as a vaccine in patients with inoperable pancreatic cancer (Bernhardt). More recently, phase III trials have been conducted as well.

=<span style="font-family: 'Times New Roman',Times,serif;">The Research =

<span style="font-family: 'Times New Roman',Times,serif;">GV1001 Phase I/II Clinical Trial
<span style="font-family: 'Times New Roman',Times,serif;">The goals of this trial were to determine the safety and tolerability of GV1001, as well as the vaccine's optimal dosage and any potential survival benefits.

<span style="font-family: 'Times New Roman',Times,serif;">Methods
<span style="font-family: 'Times New Roman',Times,serif;">48 patients with inoperable pancreatic cancer were divided into three groups for the trial: low dose (60 nmol, n=11), medium dose (300 nmol, n=17), and high dose (1µmol, n=20). GV1001 vaccinations were therapeutically administered to patients through intradermal injections (injected between the layers of skin). Three vaccinations were given in the first week, followed by only one in weeks 2, 3, 4, 6 and 10. While data from all patients was used for the safety analysis, only those who received at least 6 injections over a four week period were considered for the evaluation of immunogenicity and survival. The number of evaluable patients in the low, medium and high dose groups were eight, 16, and 14, respectively.

<span style="font-family: 'Times New Roman',Times,serif;">As a measure of safety and toxicity, patients were monitored for adverse reactions during and after receiving each of their vaccine injections. Immune response was measured by delayed-type hypersensitivity (DTH) skin test—DTH is an injury of one's tissue caused by the body's mounting of a T cell mediated defense. These tests were performed on each patient at their first visit and all visits from week 2 onwards. T cell response was also monitored via proliferation assays of Acid Citrate Dextrose-blood samples.

<span style="font-family: 'Times New Roman',Times,serif;">Results
<span style="font-family: 'Times New Roman',Times,serif;">None of the 48 patients showed any signs of toxicity or a particularly severe reaction to the injection. However, every one of the patients were found to have signs of inflammation at the injection site. Observed side effects included fever in 2% of patients, chills in 10%, pain in 6%, fatigue in 2%, nausea in 12%, and vomiting in 2%. Interestingly, when separated by dose, the low dose group had the highest frequency of such adverse events at 45% of patients, followed by the intermediate group (41%), and then the high dose group with only 10%. Just looking at the number of adverse effects, the intermediate group had approximately four times more than either of the the other groups. This pattern could be partially explained by a sort of culmination effect—the intermediate group received more injections and was followed over a longer period of time.

<span style="font-family: 'Times New Roman',Times,serif;">Patients that tested positive for DTH, or were found to have GV1001-specific T cells in their assayed blood samples, were considered immune responders (Figure 3). In some cases, DTH tests were positive while T-cell responses were negative and vise-versa. Because researchers didn't know what to attribute this discrepancy to—different biological responses or the sensitivity in assays used to test T-cell response—patient cytokine profiles from two different patients with different immune reactions were taken. Researchers were able to conclude that GV1001 vaccinations produced identical cytokine profiles in both patients despite their different immune responses and further testing confirmed that different T-cells could recognize different GV1001 fragments. Overall, immune responses were detected in 63% of the evaluable patients. The intermediate <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">group showed the most promising results with the highest proportion of immune responders—75% of evaluable patients in that group. This group also developed immune responses faster, stronger, and more often than either of the other dose groups.

<span style="font-family: 'Times New Roman',Times,serif;">A treatment-related survival benefit from the GV1001 vaccine was found for the intermediate treatment where the median survival time was 8.6 months, compared to 4.0 months for the low dose and 5.1 months for the high dose, which were not considered statistically significant (Figure 4A). When the data is stratified by immune response, rather than level of dosage, immune responders were found to have a significantly higher median survival time of 7.2 months, compared to 2.9 months for all non-immune responders (Figure 4B).





<span style="font-family: 'Times New Roman',Times,serif;">Discussion
<span style="font-family: 'Times New Roman',Times,serif;">The GV1001 vaccine was found to be a safe therapeutic product without major physiological side effects and the finding of increased survival time points to its efficacy as a possible cancer vaccine. Overall, the result of this study were very encouraging and support continued research in the form of a phase III trial. Given that GV10001 is a promiscuous epitope, it could also be used in the creation of a broad spectrum cancer vaccine, but further research is needed.

<span style="font-family: 'Times New Roman',Times,serif;">GV1001 Phase III Clinical Trial
<span style="font-family: 'Times New Roman',Times,serif;">Given the positive results of the combined phase I/II trial, an international, randomized phase III trial was initiated to compare the efficacy of the current standard of treatment, Gemcitabine (a common chemotherapy drug), alone versus GV1001, followed by Gemcitabine, in patients with unresectable, metastatic pancreatic cancer. The trial was meant to compare the primary outcome of overall survival and secondarily, progression free survival.

<span style="font-family: 'Times New Roman',Times,serif;">Methods
<span style="font-family: 'Times New Roman',Times,serif;">520 patients were randomly assigned to one arm of the trial—either Gemcitabine alone or GV1001 plus Gemcitabine. Gemcitabine was only added to the experimental treatment if the patient's condition worsened. Gemcitabine was to be given in accordance with the Burris regime and the SmPC, followed by cycles of 1 week of rest and 3 weeks of chemotherapy. 0.56mg of GV1001 would be administered to patients in the experimental group on day 1, 3, 5, 8, 15, 22, week 6, and every 4 weeks after that. CT scans were taken every eight weeks to measure cancer progression.

<span style="font-family: 'Times New Roman',Times,serif;">Results
<span style="font-family: 'Times New Roman',Times,serif;">The study began in June 2006 and ran until May 2008 when a preliminary analysis found that there was no survival benefit for 178 patients given GV1001 and the trial was terminated (Buanes). 365 patients were enrolled in the study at the time of termination (Buanes). By August 2008, 238 of the patients had died. Median overall survival was 7.3 months for Gemcitabine and 5.9 months for GV1001 and median progression free survival was 3.7 versus 1.9 months (Buanes).

<span style="font-family: 'Times New Roman',Times,serif;">Discussion
<span style="font-family: 'Times New Roman',Times,serif;">In comparison with the current standard of treatment for pancreatic cancer, GV1001 failed to demonstrate efficacy. It is possible that the GV1001 treatment would prove to be beneficial if given in parallel with chemotherapy.

=<span style="font-family: 'Times New Roman',Times,serif;">Conclusions =

<span style="font-family: 'Times New Roman',Times,serif;">Final Thoughts on the GV1001 Peptide Vaccine
<span style="font-family: 'Times New Roman',Times,serif;">The GV1001 peptide vaccine originally appeared to be an effective treatment. In the phase I/II trial, the peptide vaccine was found to be safe and nontoxic—even at the highest dosage. However, the phase III trial debunked those previous suppositions by showing that GV1001 was not any better of a treatment for pancreatic cancer than current chemotherapy drugs.

<span style="font-family: 'Times New Roman',Times,serif;">Going forward, it would be interesting for studies to focus on administering the vaccine to patients with various stages of pancreatic cancer. The vaccine could even be tested prophylactically, rather than therapeutically, for individuals with a genetic predisposition. I <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">n addition, it was mentioned earlier that a GV1001 peptide vaccine could potentially be used on a wide variety of cancers—I would be curious to see whether it shows any better efficacy when used as a treatment for a different type of cancer.

<span style="font-family: 'Times New Roman',Times,serif;">More clinical trials have to be done to determine the vaccine's efficacy in other cancers and at different stages. The main problem with conducting more trials will be the investment of time and money given the recent more discouraging findings. Finally, future trials should determine if the vaccine has any synergistic effects when combined with other standard cancer treatments, for example chemotherapy or surgical debulking of tumors. Again, innovative clinical trials will be imperative to answering these questions

<span style="font-family: 'Times New Roman',Times,serif;">Although the phase III trial of the GV1001 peptide vaccine was a failure, the preliminary phase I/II trial success and sound conceptual base for this treatment make it worth pursuing along. The idea of a cancer vaccine is exciting and a very hot button research topic. Only time will tell if the GV1001 peptide vaccine will work as intended and become the next highly effective cancer therapy.

<span style="font-family: 'Times New Roman',Times,serif;">References
<span style="font-family: 'Times New Roman',Times,serif;"><range type="comment" id="512766460_1">Allison, Elizabeth Ann. Fundamental Molecular Biology, 2e. Hoboken, NJ: Wiley, 2012. Print. <span style="font-family: 'Times New Roman',Times,serif;">http://www.medscape.com/viewarticle/575367_2 <span style="font-family: 'Times New Roman',Times,serif;">http://www.geron.com/telomerase <span style="font-family: 'Times New Roman',Times,serif;">http://hmg.oxfordjournals.org/content/10/7/677.full ---> shay <span style="font-family: 'Times New Roman',Times,serif;">http://www.nature.com/bjc/journal/v98/n4/pdf/6604209a.pdf ---> shay and keith <span style="font-family: 'Times New Roman',Times,serif;">S L Bernhardt, M K Gjertsen, S Trachsel, M Møller, J A Eriksen, M Meo, T Buanes, et al. (2006). Telomerase peptide vaccination of patients with non-resectable pancreatic cancer: a dose escalating phase I&sol;II study. British Journal of Cancer. doi:10.1038/sj.bjc.6603437 http://clinicaltrials.gov/ct2/show/record/NCT00358566 http://meeting.ascopubs.org/cgi/content/abstract/27/15S/4601 ---><span style="background-color: #ffffff; color: #403838; font-family: Helvetica,'Lucida Sans Unicode',Verdana,Arial,'Lucida Grande',Tahoma,sans-serif; vertical-align: baseline;">Buanes</range id="512766460_1">

http://www.cancer.gov/drugdictionary?cdrid=502114