However, befor e this, it is important to assess whether there is any interference with the immune response when TG01 is used concomitantly with gemcitabine . 3.2.2 Rationale for dose The traditional approach to dose selection for cytotoxic anticancer agents is to escalate the dose until dose limiting toxicity. This concept of maximum tolerated dose is not applicable for peptides that generally lack any inherent toxicity and that are for local administration in the skin. TG01 is for induction of cellular immune responses, and consequently the immune response rate is the most important parameter to consider for determining an appropriate dose. In a clinical study (CTN RAS 98010) a total of 38 pancreatic cancer patients, 13 after tumour resection and 25 with advanced disease, have been treated with TG01 with minimal toxicity and with good immunological outcome. In the resected group, 11 of 11 (100%) evaluable patients demonstrated an immune response (DTH response), and in the advanced disease group 14 of 25 (56%) patients demonstrated a positive DTH response. In this study the immunotherapy dose was 0.7 mg (0.1 mg of each peptide) and 30 µg GM-CSF was used an immunomodulator (Weden et al., 2011). The rationale for selecting the dose of 0.1 mg of each peptide per administration stems from the observations from a dose exploring study in colorectal cancer patients (CTN RAS 97005). In this t rial the patients were first given four weekly administrations of a single RAS peptide at the dose of 0.1 mg per administration and subsequently, after three weeks rest, four weekly administrations of 0.3 mg peptide. An additional dose of 0.1 mg peptide w as administered without GM -CSF at a separate site to elicit DTH. Indeed, more immune responders were seen after the second cycle, 13 versus 7 after the first cycle. Other studies with constant dose (0.1 mg per peptide) have demonstrated a similar increas e in number of recorded immune responders after administration of boosters after the initial 4 week cycle. In a study with a cocktail containing four RAS peptides (dose 0.10 mg per peptide) in patients with advanced pancreatic cancer (CTN RAS 97004) the number of DTH responders was increased from 11 after the initial 4 week cycle, to 16 responders after only two additional administrations (week 6 and 10). This suggested that the 0.10 mg and 0.30 mg per peptide were immunologically equivalent in the CTN RAS 98005 study and that increasing the TG01 dose beyond the current 0.7 mg dose (0.1 mg per peptide) is unlikely to heighten further the levels of specific immune responses. Alternatively, reducing the TG01 dose to levels lower than 0.1 mg per peptide may result in sub- optimal T cell responses. Given the acceptable safety profile and levels of T cell responses observed to date with the current dose, the clinical development will continue using a total TG01 dose of 0.7 mg (0.1mg each peptide) together with GM -CSF as immunomodulator. CT TG01 -01 Version v 10.0 27 June 2018 Confidential 45 / 87 3.2.3 Rationale for GM- CSF (Molgramostim) adjuvant Peptides are poorly immunogenic by themselves. To induce measurable levels of T-helper type 1 (Th1) or type 2 (Th2) immune responses against peptides, adjuvants are often required ( Gupta et al., 1996). Several adjuvants have been evaluated in humans for adjuvant activity when combined with carbohydrate and protein/peptide -based immunotherapy (Gupta et al., 1995; Singh & O'Hagan 1999). However, the only adjuvant licensed for use in human vaccines, aluminium hydroxide (Gupta et al., 1995) is a poor enhancer of the type of immune responses (Th1) that may be required for the efficacy of peptide -based ASCI, and, therefore, could not be selected for TG01 development. Studies in rats show that GM -CSF administered intradermally as a single dose with antigens, compared favourably with complete Freund’s adjuvant and aluminium hydroxide in eliciting cellular immunity ( Disis et al., 1996). GM-CSF has a number of biological properties making it a strong candidate to function as an immunomodulator for ASCI. It has been shown that GM -CSF can drive both humoral and cellular immune responses (Disis et al., 1996). Moreover, GM -CSF has been shown to induce anti -tumour immunity that has been associated with the induction of a broader T cell cytokine response (both Th1 and Th2), which has been implicated as important in mediating tumour rejection ( Hung et al., 1998; Mach et al., 2000; Soiffer et al., 1998). GM-CSF was selected as the immunomodulator fo r the development of TG01 based on its reported potent adjuvant activity when given in combination with protein/peptide antigens ( Dranoff et al., 1993; Gjertsen et al., 1995; Hunger et al., 2001). Studies in mice (Dranoff et al., 1993) have shown that T -cell responses could be generated in mice bearing human pancreatic tumour xenografts. Potent anti -tumour activity was shown in patients with metastatic melanoma following injection of autologous melanoma immunotherapy combined with GM- CSF ( Leong et al., 1999). GM-CSF has during recent years been used extensively as an immunomodulator for ASCI in clinical trials. GM -CSF has either been used alone ( Gjertsen et al., 2001; Bernhardt et al., 2006, Dutoit et al., 2002; Lomas et al., 2004; Schaed et al., 2002; Disis et al., 1999; Hunger et al., 2001; Brunsvig et al., 2006) or in combination with other adjuvants ( Chianese- Bullock et al., 2005), normally in the range of 30- 250 µg/injection. The dose to be used in this study is 30 µg which is consistent with a stimulatory effect of low doses of GM- CSF ( Parmiani G et al., 2007). 3.2.4 Rationale for dose regimen ASCI requires a more aggressive and continued treatment than traditional vaccination (prophylaxis) against infectious diseases. In vaccination the antigens used are foreign to the human immune system, the vaccinated subjects are healthy with normal immune functions and the induced immune response is allowed to down- regulate to a memory response ready to be reactivated upon a challenge. In contrast to this, in ASCI t he tumour associated antigens (TAA) that are used are “self”, or in the case of RAS peptides, slightly altered “self” protein/peptides to the treated subject and consequently immunological tolerance is much more difficult to break in this setting. The tre ated subjects are cancer patients that normally develop immune suppression and often have a rapid deteriorating immune system and, finally, a prevailing active anticancer immune CT TG01 -01 Version v 10.0 27 June 2018 Confidential 46 / 87 response is necessary in order to kill the cancer cells which on their side are continuously counteracting the anti- cancer immune response by inducing immune suppression. The scenario described above is highly relevant for patients with pancreatic cancer who have a short life expectancy and a rapidly deteriorating immune system. I n order to meet the challenges of inducing immune response in a limited time window available, the rapid disease progression and deterioration of the immune system, it is important to use an aggressive treatment regimen in order to induce efficient immune response as fast as possible and to keep the induced anti -cancer immune response in an active state. Whilst, complete resection and cure is the aim of pancreatic surgery, in reality this is rarely the case and the need for a rapid immune response to “mop” up any residual cancer cells add an urgency even in this setting. In vaccination either subcutaneous or intramuscular administration are normally used, and the vaccine is usually formulated with an adjuvant that provides a depot of antigens as well causi ng irritation and inflammatory reactions at the site of injection. In order to obtain optimal immunisation with TG01 the intradermal administration route is used. The resident Langerhans cells in the dermis are highly potent antigen presenting cells with strong capacity to endocytose antigens and to elicit subsequent T cell responses. Intradermal injection is accordingly the preferred route of administration ASCI intended to result in protective T- cell immunity. Due to the architecture (thickness) of th e skin only small volumes may be injected at a single site and irritants causing ulceration must be avoided. TG01 is reconstituted in water for injection and administered without any substances that provide a depot effect. The only “pharmacologically active” dose of peptides to reach the lymph nodes is the amount of peptides taken up by Langerhans cells locally before the injected peptides are degraded by tissue proteases. After migrating to the draining lymph nodes the activated Langerhans cells have a very limited life span. Thus each administration is followed by only a short period of expansion of specific T-cells before the stimulus is lost. In the absence of a depot effect and thus a continued release of antigen and stimulation of T- cells, repeated immunizations over an extended time period is required to generate a robust immune response. This will ensure a continued activation and expansion of antigen specific T cells to kill resident tumour cells and to replace T- cells that are tolerised/anergi sed by the tumour. The dosing regimen was developed using ASCI as standalone treatment but the rationale behind also applies when combined with chemotherapy. However, it is known that many anti-cancer chemotherapies also act via immunological pathways and, therefore, there is a potential synergy between chemotherapy and active immunotherapy for cancer (Lesterhuis et al., 2011 ). For adjuvant treatment of pancreatic cancer TG01 will be used in combination with gemcitabine . It has been shown in mice that gemcitabine can modulate antigen- specific ant i-tumo ur immune responses by enhancing cross presentation of tumour antigens to MHC class I and class II -restricted T-cells ( Nowak et al., 2002). In other animal studies gemcitabine could counteract immune suppression by depressing myeloid suppressor cells (Suzuki et al., 2005.) and successfully be combined with immunotherapy to yield synergistic anti -tumour effects ( Nowak et al., 2003; Broomfield et al., 2005). When combined with gemcitabine , the TG01 treatment regimen is adapted in order to optimize the potential synergistic effects of the two treatments. CT TG01 -01 Version v 10.0 27 June 2018 Confidential 47 / 87 Adjuvant gemcitabine treatment of pancreatic cancer is given in cycles of three weeks treatment and one week rest for up to six cycles (ESPAC3), starting wit hin 8 weeks after the resection has been done (Van Laethem et al., 2010; Neoptolemos et al., 2010; Oettle et al., 2007). It is considered to be beneficial for the patients to obtain an anti -cancer immune response as soon as possible after surgery and TG01 treatment will therefore be started as soon as possible but certainly between 1 to 8 weeks after the tumour has been removed. G emcitabine treatment will be started together with TG01 treatment preferably at least 3 weeks after initiation of TG01 immunotherapy . From when gemcitabine treatment is initiated TG01 will either be administered as booster injection s every 2 weeks or will be put on hold until end of chemotherapy. I n patients who have not shown recurrence or who had a positive immune response during the study treatment period, further booster injections will be given a fter gemcitabine has been completed . The current study started 2 years ago and patients in the main group participat ing for the whole length of the study (up to 2 years), could receive 26- 28 vaccinations with GM - CSF/TG01 as a maximum and an additional 9 injections of TG01 (see tables 1 and 2) for the purpose of detecting an immune response (which is the goal of the TG01 treatment). Vaccination started either before or at the same time as chemotherapy treatment and continued during chemotherapy and post -chemotherapy where possible. In total 19 patients were recruited under this treatment regimen and so far all except one patient have shown a positive immune response during the course of the study. It was also observed that 4 patients receiving this treatment regimen experienced allergic reactions related to TG01 or GM -CSF/TG01 after they received several injections. This type of allergic reactions are not uncommon when using these types of medications and, when they occur, can be usually prevented by using pre -medication with an antihistamine. It is believed that the number of vaccinations could be, in part, responsible for these events and as positive immune responses were clearly seen with the previous vaccination schedule, Targovax believe that the same immune response can be obtained with a reduced number of vaccinations, which will be tested in the proposed modified vaccination group. In addition, vaccinations during chemotherapy with gemcitabine will be reduced . Once an immune responses has been obtained by vaccinating up to 6 weeks and gemcitabine has been started , vaccination will be put on hold until chemotherapy completion . Vaccination will be restarted after chemotherapy has be en completed. 3.2.5 Use of Gemcitabine as adjuvant chemotherapy Gemcitabine has been shown to provide significantly better Disease Free Survival (DFS) versus observation alone with a mean DFS of 13.4 months compared with 6.9 months although there was no difference for overall survival (median OS 22.1 months ( Oettle et al., 2007). The use of 5- FU and folinic acid or gemcitabine –based chemoradiation has also been tested as adjuvant therapy but show no advantages over gemcitabine alone for either DFS or OS. The DF S for gemcitabine alone ranged from 10.9 months to 14. 3 months and median OS from 23 to 24.4 months.