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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to the field of cancer therapy and immunology.
• the present invention relates to cancer therapy by active immunotherapy, namely therapy of non-small cell lung cancer by active immunization with a peptide derived from the catalytic subunit of human telomerase.
• Non-small cell lung cancer (NSCLC) accounts for about 80% of cases, and most subjects present with inoperable stage III or stage IV disease.
• Metastatic disease (stage IV) carries a dismal prognosis, with a five year survival of 1%.
• stage III carries a dismal prognosis, with a five year survival of 1%.
• the successful treatment of stage III patients depends on the control of both local disease and occult metastases. If the disease can be encompassed within an appropriate radiation volume, i.e. stage I-IIIA, curatively intended radiotherapy (>60 Gy) is the treatment of choice.
• the 5-year survival for stage III patients treated with radiotherapy alone is less than 5%. About one third of patients will relapse locally, one third will develop distant metastases and one third will develop both.
• Several approaches to multimodality treatment have been investigated. These include induction chemotherapy and concurrent chemo-radiotherapy as well as consolidation chemotherapy. However, progress has been limited. Most patients die from relapsed disease,
• telomere The enzyme telomerase is expressed in most human cancers, including NSCLC, and is considered as an attractive target for a universal cancer vaccine. Telomeric DNA confers stability to chromosomes, and normal somatic cells can undergo a limited number of cell divisions because the telomeres are shortened at each mitosis. Tumor cells bypass this biological clock by expressing the enzyme telomerase that synthesises new telomere units.
• Peptide GV1001 (SEQ ID NO: 1) consists of 16 amino acids derived from the active site of hTERT. Previously two GV1001 trials have been reported. The GV1001 peptide is recognized on multiple HLA class II molecules encoded by both DP, DQ and DR subloci. This promiscuous HLA-binding profile suggests that the GVlOOl vaccine may be applicable to the general patient population and may elicit a broad T- helper response within each individual. Further, GVlOOl includes nested HLA class I epitopes, facilitating recruitment of CD8+ cytotoxic T cells.
• Fig. 1 GVlOOl-specific T-cell responses.
• PBMCs were obtained prior to start of therapy, at weeks 6, 10, and at every vaccination thereafter.
• the PBMCs were stimulated once in vitro and tested for proliferation against irradiated PBMCs ⁇ peptide GVlOOl.
• Columns represent mean SI (response with GV100 divided by response without GVlOOl). If the recorded SI exceeds the upper limit of the respective chart, the exact SI is annotated at the top end of columns.
• the diagram shows pre- and postvaccination T-cell responses from all evaluable patients. For each subject, the time point with the highest SI is displayed. Responses with SI>2 were considered GVlOOl specific.
• Fig. 2 GVlOOl-specific T-cell responses.
• PBMCs were obtained prior to start of therapy, at weeks 6, 10, and at every vaccination thereafter.
• the PBMCs were stimulated once in vitro and tested for proliferation against irradiated PBMCs ⁇ peptide GVlOOl.
• Columns represent mean cpm or mean SI (response with GV100 divided by response without GVlOOl). If the recorded cpm or SI exceeds the upper limit of the respective chart, the exact cpm or SI is annotated at the top end of columns.
• B and C show long-term T-cell memory. The diagrams show development of T-cell responses as recorded from follow-up samples.
• D-F show that samples stored overnight prior to PBMC isolation(*) mostly tested negative, even in subjects where freshly isolated samples tested positive.
• G depicts parallel testing of PBMC samples isolated upon arrival or stored overnight.
• Fig. 3 Survival of patients.
• PFS was assessed by Kaplan-Meier analysis, immune responders were compared with non- responders. PFS was defined as clinical endpoint in the protocol described herein because standard treatment after progression was likely to influence OS (stage III patients). The plot shows PFS for study patients with or without a GV1001 T-cell response. Observed PFS is extended for the immune responders.
• cancer vaccines work through different mechanisms and may therefore be effective against cancer cells resistant to chemo- and radiotherapy. Moreover, the chemo-radiation may possibly enhance rather than preclude the immune response.
• tissue damage may induce "danger signals" that provide a pro-inflammatory
• Docetaxel which is applied in the present treatment regime (cf. below), may potentially enhance the vaccine response through other mechanisms.
• the present invention is based on data form a phase II vaccine trial with the telomerase peptide GV1001 (EARPALLTSRLRFIPK, SEQ ID NO: 1) in stage III NSCLC patients. The study evaluated GV1001 vaccination shortly after chemo-radiotherapy.
• results are presented from a phase II trial that investigated vaccination of NSCLC patients with the telomerase peptide GV1001.
• the clinical study included 23 patients, and no treatment related serious adverse effects were observed.
• the study demonstrated an 80% immune response rate per protocol. This response rate is considerable compared to most cancer vaccine trials, including those investigating telomerase-based approaches.
• peptide vaccines represent short HLA-class I matched epitopes
• long HLA II- matched peptides like GV1001 may according to the findings herein be particularly suited for combined protocols.
• Long peptides recruit CD4+ T-helper cells that are known to interact extensively with other immune cells.
• GVlOOl-specific T-helper cells may engage APCs presenting antigens from apoptotic tumor cells and induce epitope spreading.
• the present inventors address this issue in ongoing studies on long term survivors from GV1001 trials and have indeed identified responses against hTERT epitopes outside GV1001.
• a new phase I/II trial in NSCLC patients we plan to combine chemo- or radiotherapy and vaccination with some of these novel hTERT peptides.
• Thl Thl effector cytokines
• Th2 cytokines may arise in response to powerful immunoactivation.
• the wide range of Thl/Th2 cytokines may also point to a polyfunctional response.
• Several studies, in particular of infectious diseases, have suggested that polyfunctional cytokine profiles are associated with protective immunity.
• the frequency of immune responders was similar as assessed by DTH recordings or T-cell assays.
• most subjects in the presently presented trial, where vaccination followed shortly after chemoradiotherapy were DTH negative. This observation points to a possible immune modulating effect of
• the present invention relates to a method for treatment of cancer in a human patient, said method comprising administering an effective amount of the peptide EARPALLTSRLRFIPK (SEQ ID NO: 1) and an immunological adjuvant to said patient, wherein said patient has received radiotherapy and optionally chemotherapy, and wherein said patient has received the radiotherapy 4-28 days prior to said treatment.
• SEQ ID NO: 1 has the potential of inducing a beneficial anti-tumour immunity in patients, who suffer from a number of other cancer types and who are being treated with radiotherapy and/or chemotherapy.
• SEQ ID NO: 1 will be useful in combination treatment involving chemotherapy and/or radiotherapy in most if not all cancer types where chemotherapy and/or radiotherapy is/are used to combat the disease. It is preferred that the patients in question have been or are undergoing chemotherapy and that they have received radiotherapy within a period fo 4-28 days prior to initiation of treatment with SEQ ID NO: 1.
• the cancer trated is non-small cell lung cancer (NSCLC).
• NSCLC non-small cell lung cancer
• SEQ ID NO: 1 is administered intradermal ⁇ , but other convenient administration routes may also be applied.
• SEQ ID NO: 1 may be administered subcutaneously, or by other parenteral routes commonly used in the art, such as via the intraperitoneal or intramuscular routes.
• a preferred administration location is in the lower abdomen, but the location of
• administration may be varied as convenient or practical. This means that the administration may be in the limbs or dorsally.
• the immunological adjuvant is administered at the same site as SEQ ID NO: 1.
• the "same site” is herein meant both the same location on the human body and the same type of administration. So, if SEQ ID NO: 1 is administered intradermal ⁇ in the lower abdomen, then the adjuvant is also administered intradermal ⁇ in the lower abdomen.
• said immunological adjuvant is typically administered shortly prior to administration of SEQ ID NO: 1, such as between 1-30 or 2-29 or 3-28 or 4-27 or 5- 26 or 6-25 or 7-26 or 8-25 or 9-24 minutes prior to SEQ ID NO: 1.
• the adjuvant is administered 10-15 minutes prior to SEQ ID NO: 1.
• the amount of SEQ ID NO: 1 administered must be effective, which means that the amount is at leat 10 nmol per administration, such as at least 50 nmol, or at least 200 or at least 250 nmol.
• the amount administered is often at most 2000 nmol, such as at most 1500 nmol, at most 1000 nmol, or at most 800 nmol, such as at most 500 nmol.
• the an amount is about 300 nmol.
• the adjuvant is any convenient immunological adjuvant, but especially immunostimulating adjuvants are relevant. Examples of suitable immunological adjuvants are discussed extensively in WO 98/20027.
• a particularly interesting immunologically adjuvant is granulocyte macrophage-colony stimulating factor (GM-CSF) - this adjuvant is in certain embodiments of the invention administered in an amount of 75 pg per administration, but may be administered in both higher or lower dosages as determined by testing the potency of GM-CSF when administered via a particular route and in a particular location on the body. As discussed above, the patients treated according to the present invention have been previously treated with chemotherapy and radiotherapy.
• GM-CSF granulocyte macrophage-colony stimulating factor
• any chemotherapeutical regime useful in treatment of NSCLC may have been used, but in the practice of the current invention, the beneficial effects have been observed when immunizing with SEQ ID NO: 1 after treatment with docetaxel, in particular after weekly administrations of docetaxel 20 mg/m 2 (where the m 2 indication is skin surface area of the treated individual).
• the radiotherapeutic treatment regime may be any regime convenient in the treatment of NSCLC, but in the practice of the present invention, the results reported below was obtained after radiotherapy that consisted of 3D radiotherapy 2 Gy x 30.
• the method of the invention typically entails repeated immunizations after immunization 1 in order to induce an effective immune response.
• the immunization scheme followed in the examples below entails that patients are subsequently administered the same doses of SEQ ID NO: 1 and immunological adjuvant via the same route as in the first immunization and according to the following scheme: twice within the week following the first administration, once a week in weeks 2, 3, 4, 6, 8, 10, 14, 18, 22, once at month 6, and once at month 9.
• immunization schemes may be optimized, and as indicated in the examples, there is a possibility that the number of follow-up or booster immunizations can be reduced - a skilled immunologist will be capable of determining from further experiments an optimized immunization scheme.
• the immunological status of the individual patients may be monitored and based on result from this monitoring the immunization protocol for each patient may be adjusted individually.
• the invention also relates to GV1001 (SEQ ID NO : 1) for use in the method disclosed herein and as set forth in the claims. Further the invention also relates to use of SEQ ID NO: 1 for the preparation of a pharmaceutical composition for the treatment of NSCLC according to the method of the invention disclosed above and as defined in the claims.
• GV1001 denotes the peptide EARPALLTSRLRFIPK (SEQ ID NO: 2), which is derived from the amino acid sequence of human telomerase protein (hTERT).
• Radiotherapy generally denotes tumour treatment by use of ionizing radiation, which can be applied locally or systemically.
• the exact type of radiation therapy useful differs from cancer type to cancer type and is generally well known to the skilled person.
• “Chemotherapy” is used generally for treatment of cancer patients with a variety of cytotoxic or cytostatic drugs.
• chemotherapy for instance denotes anticancer therapy using the following groups of drugs:
• Anti-metabolites mimic purines (e.g. azathioprine and mercaptopurine) or pyrimidines, which are the building-blocks of DNA.
• the anti-metabolites prevent these substances from becoming incorporated into DNA during the "S" phase of the cell cycle, stopping normal development and division. They also affect RNA synthesis.
• Plant alkaloids and terpenoids (L01C). These alkaloids prevent microtubule function.
• the main examples are vinca alkaloids and taxanes.
• Vinca alkaloids (L01CA) are derived from the Madagascar periwinkle, Catharanthus roseus (formerly known as Vinca rosea) and include vincristine, vinblastine, vinorelbine, and vindesine.
• Podophyllotoxin is a plant-derived compound that is said to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide. They prevent the cell from entering the Gl phase (the start of DNA replication) and the replication of DNA (the S phase).
• Taxanes are derived from the natural product paclitaxel, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semisynthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase of the cell cyclus.
• Topoisomerase inhibitors (L01CB and L01XX). These include type I topoisomerase inhibitors such as the camptothecins irinotecan and topotecan, and the type II inhibitors such as amsacrine, etoposide, etoposide phosphate, and teniposide.
• Cytotoxic antibiotics include actinomycin (L01DA01), anthracyclines such as doxorubicin (L01DB01), daunorubicin (L01DB02), valrubicin, idarubicin, epirubicin (L01DB03), and other cytotoxic antibiotics such as bleomycin (LOIDCOI), plicamycin (L01DC02), and mitomycin (L01DC03).
• actinomycin L01DA01
• anthracyclines such as doxorubicin (L01DB01), daunorubicin (L01DB02), valrubicin, idarubicin, epirubicin (L01DB03), and other cytotoxic antibiotics such as bleomycin (LOIDCOI), plicamycin (L01DC02), and mitomycin (L01DC03).
• an "immunological adjuvant” has its usual meaning in the art: A substance or composition which, when administered to an individual assists in the induction/elicitation of a specific immune response towards an antigen.
• a preferred adjuvant in the present invention is GM- CSF.
• the primary objective of the phase II trial was immunological response. Toxicity and time to progression were secondary objectives. Twenty-three subjects with inoperable stage IIIA/B NSCLC were enrolled between November 2006 and July 2008 from 3 different centres in Norway. Twelve patients were enrolled at The Norwegian Radium Hospital, four at St Olav's Hospital and seven at The Southern Hospital of Norway, Kristiansand. The trial was approved by the Norwegian Medicines Agency, the Regional Committee for Medical Research Ethics and the Hospital Review Board. It was performed in compliance with the World Medical
• the study population had been treated with weekly docetaxel 20mg/m2 and 3 D radiotherapy 2 Gy x 30 within the last 4 weeks.
• Subjects with metastatic disease were excluded based on a pre-study CT scan of the thorax and upper abdomen and an MRI scan of the brain.
• the eligibility criteria also included Eastern Oncology Group (ECOG) performance status 0-2, age > 18 years, WBC > 1.5 x 10 9 /L; platelets > 100 x 10 9 /L, Hb > 9g/dL (> 5.6 mmol/L);
• the strategy behind the study design was to pave the way for a phase III trial in stage III NSCLC patients, evaluating the vaccine within a multimodal treatment regime.
• the dosage of GV1001 was based on data from our previous dose-escalation trials in NSCLC and pancreatic cancer.
• the chemoradiotherapy represented institutional standard treatment in 2006 for inoperable stage III NSCLC.
• Our decision to include 20 evaluable patients was based on the main study objectives: to show that combined treatment with chemoradiotherapy and GVlOOl is feasible and may yield immunization, to provide safety data and to obtain an estimate for PFS and immune response rate. In a given sample size, the number of subjects with immune response and serious adverse events (SAE) will follow a binomial distribution.
• GVlOOl 300 nmol peptide in 0.20 ml saline
• GM-CSF 75 pg Leukine; Bayer, Oslo, Norway
• the vaccine peptide GVlOOl corresponds to the 16 amino acid residue 611-626
• PBMCs Peripheral blood mononuclear calls
• the PBMCs were isolated and frozen as previously described. Thawed PBMCs were stimulated once in vitro with the vaccine peptide prior to T cell assays, as described earlier . At this initial stimulation, the PBMCs were cultured with GVlOOl (25 pmol/l) for 7 ot 10 days, with addition of IL-2 (10 U/ml) from day 3.
• T-cell proliferation assays 3 H Thymidine were performed essentially as previously described. Pre- and post-vaccination samples were analyzed in parallel for response to peptide stimulation. Irradiated autologous PBMCs were used as antigen presenting cells (APCs).
• Stimulation with Staphylococcal enterotoxin C was used as positive control and as a measure of immunocompetence. All patients responded to SEC. T cell cultures were tested in triplicates. SEM was usually below 10%. Proliferation counts after stimulation with the irrelevant peptide (K-RAS 508) were generally not significantly different from controls without peptide. T cell responses were considered antigen-specific when the stimulatory index (SI; response with antigen divided by response without antigen) was above 2. Bioplex cytokine analyses were done on supernatants harvested 48 hours after T-cell stimulation, according to the manufacturer's protocol (Bio-Rad Laboratories). Supernatants were analysed in duplicates/triplicates, each parallel kept separate through T-cell stimulation and Bioplex assays.
• Delayed-Type Hypersensitivity Delayed-type hypersensitivity (DTH) skin test was performed at baseline, at weeks 2, 3, 4, 6, 10 and at the time of later vaccinations.
• DTH testing 60 nmol GV1001 in 0.10 ml saline was injected i.d. at a site separate from the site of vaccination, without GM-CSF.
• the patients registered the DTH skin reaction 48 hours after administration.
• a positive DTH test was defined as an erythema/induration with average diameter > 5 mm.
• PFS was calculated from start of vaccination. Kaplan-Meyer/log-rank analysis was applied from comparing immune responders versus nonimmune responders with regard to PFS. To assess whether the immune response represented an independent prognostic factor, Cox regression with enter analysis was conducted. Disease stage represented the most important identifiable prognostic factor, apart from immune response. The subjects were staged as stage IIIA or IIIB.
• a GV1001-specific T cell response was demonstrated in 16 patients after vaccination, compared to no patients in pre-vaccination samples (Fig. 1). A positive DTH response was observed in one patient only. Three subjects were not evaluable according to the protocol (see above). The immunological response rate was 70% by intention to treat (ITT) analysis, and 80% per protocol.
• PFS progression-free survival

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