Detailed protocol

were seen. No objective tumour responses were detected, though 3 patients have demonstrable stable disease. On an intention-to-treat basis, the median survival was 10 months. However, there were some cases of prolonged survival (of those who completed the trial, the median survival was 25 months). We believe that the anti-cancer immune response to the vaccine could be improved by the induction of HSPs within the tumour sample, and the addition of the immune adjuvant aIFN.

Immune Adjuvants: HSPs, GM-CSF and aIFN

3.1. HSPs

3.1.1        Theory

HSPs are natural chaperones of intracellular antigenic peptides.⁴ HSP-peptide complexes have been shown to be highly immunogenic.⁵ When exposed to HSPs, macrophages and dendritic cells are stimulated to secrete cytokines and to express antigen presenting and co-stimulatory molecules.⁶ These antigen presenting cells (APC) then take up the HSP-peptide complex, and the peptide is re-presented by the class I MHC of the APC.⁷ This has the potential to induce an antigen-specific cytotoxic (CD8+) T cell response, though CD4+ T lymphocytes and natural killer cells may also be involved in the generalised immune response.⁵

Every cancer expresses a unique group of antigens.⁵ Immunisation with cancer-derived HSP-peptide complexes can elicit a specific immune response that is directed only against the cancer from which the HSPs were obtained and not against antigenically distinct tumours or normal tissues.⁴ Ideally this specific immune response would lead to the recognition and destruction of tumour cells. Four classes of HSP preparation (gp96, HSP70, HSP90 and calreticulin) have been used successfully in anti-cancer vaccines.^4,8

Relative to normal tissues, some primary human tumour cell lysates are enriched in the HSPs gp96 and HSP70.⁹ HSP production in tumour cells can be further increased by exposure to a stressor, such as heat, cold and radiation.¹⁰ The metabolism of a cell slows significantly upon heating, with the production of HSPs increasing rapidly upon return to normal temperature (37°C). Therefore, exposure of a tumour to heat stress, followed by a subsequent rest period at 37°C prior to cell lysis, will further increase the total HSP content in the final cell lysate vaccine.¹¹

3.1.2        Pre-clinical studies of HSP tumour vaccines

Vaccination with autologous tumour-derived HSP-peptide complexes has been shown to result in both prophylactic and therapeutic antitumour activity in multiple animal models.^4,12 Preventive immunisation against tumours using HSP vaccines in mice was initially demonstrated by Srivastava et al.¹³ Tamura et al⁵ later showed that treatment of mice with pre-existing cancers with HSP preparations derived from autologous cancer resulted in delayed progression of the primary cancer, reduced metastatic load and prolonged life span. Tumour types included melanoma, fibrosarcoma, colon, lung and spindle cell carcinomas. The HSP vaccine could be derived from either the primary or a metastatic lesion. However, treatment with HSP preparations derived from cancers other than the autologous cancer did not provide significant protection.

Observations from our own animal experiments have indicated a 40% survival rate of mice vaccinated with tumour cell lysate enriched with HSPs (through heat-shocking of live cells prior to lysis) and subsequently challenged with viable tumour cells. Non-survivors also displayed slower tumour growth kinetics (unpublished data).

C57Bl/6J mice were vaccinated with placebo (saline) or autologous tumour lysates (AE17sOVA lysate), some of which were prepared from tumour cells that had been exposed to various stressors (UV, radiation, heat). Mice were inoculated with live tumour on day 14. Mice that received the heat shocked lysate displayed slower tumour growth (Fig A) and prolonged survival (Fig B).

3.1.3        Clinical trials of HSP tumour vaccines

Janetzki et al¹⁴ performed a pilot study evaluating a gp96-autologous tumour vaccine in patients with assorted malignancies. A CD8+ restricted immune response was seen in 6 out of 12 patients. In four of these patients the response intensified with successive vaccinations. Four of the patients had stabilisation of disease for 3-7 months. Amato et al¹⁵ studied a similar vaccine in patients with renal cell carcinoma. Of 16 patients, 1 had a complete response, 3 had partial remissions, and 3 had prolonged stabilisation of disease (>52 weeks). Similar results were found in a larger follow-up trial by the same authors¹⁶.

An autologous tumour-derived HSP-gp96 vaccine (Oncophage) is currently being studied in a number of phase I and II trials involving >500 patients worldwide. In melanoma trials, responses have been seen in 14 - 36% of patients.^17,18 In a group of melanoma patients who were disease-free post-surgery and vaccinated with Oncophage, >75% of the group were alive and disease-free 17 months later¹⁹.  In renal cell carcinoma, response rates of 10 % were seen, with disease stabilisation in an additional 25%²⁰. In 29 patients with metastatic colorectal carcinoma who underwent surgery followed by Oncophage, overall 2-year survival was 79%. A significant, tumour-specific T cell immune response was seen in 60% of patients and was associated with a lower rate of recurrence.^21,22 A similar immune response has been demonstrated in gastric carcinoma^23,24, pancreatic carcinoma²⁵ and melanoma^17,18.

Our vaccine will differ somewhat from the above studies. As mentioned above, 4 different classes of HSPs have been used successfully in vaccines. Rather than restrict the vaccine to a single class of HSP, we are interested in the effect of exposure to multiple HSPs. As such, we are not going to isolate and purify the individual HSPs within our vaccine.

Importantly, no autoimmune reactions or severe side effects have been observed in any of the human trials of HSP vaccines to date.

3.2             GM-CSF

3.2.1        Preclinical studies of adjuvant GM-CSF in tumour vaccines

GM-CSF activates APCs, which in turn take up, process and present tumour antigens in local draining lymph nodes. A classical experiment demonstrating the importance of GM-CSF as an adjuvant in tumour vaccination was published in 1993²⁶. In this murine melanoma model tumour cells were genetically engineered by viral transduction to secrete a variety of cytokines. GM-CSF secreting tumours were the most strongly immunogenic, resulting in both therapeutic and protective immunity. These results have been confirmed in other preclinical models.

Other tumour vaccine studies have used soluble GM-CSF. In a B16 mouse melanoma model, immunisation with tumour cells together with a slow release preparation of GM-CSF resulted in a level of immunity comparable to that of transduced tumour cells that secreted GM-CSF²⁷. Similar results were seen in a mouse lymphoma model, where mice were vaccinated with a lymphoma-derived immunoglobulin idiotype combined with 4 days of GM-CSF. This was associated with significantly enhanced protective tumour immunity and prolonged survival. Local (subcutaneous) administration of GM-CSF was more effective than systemic (intraperitoneal) administration²⁸.  Rhesus monkeys have been vaccinated with an anti-idiotype antibody mimicking Lewis Y antigen (which is expressed in adenocarcinoma cells) and given two schedules of GM-CSF at 10 mg/kg on day 1 only or the same dose for 4 days. Only the prolonged schedule resulted in protective antibodies²⁹.

3.2.2        Clinical studies of adjuvant GM-CSF in tumour vaccines

A number of clinical studies have used autologous tumour cells and GM-CSF as immunotherapy for early stage or advanced cancer. Simons et al³⁰ treated patients with prostate cancer with irradiated autologous tumour cells genetically engineered to secrete GM-CSF. This resulted in strong cell mediated and humoral immunity to prostate cancer. Leong et al³¹ treated patients with metastatic melanoma with autologous tumour cells, recombinant GM-CSF and BCG. This resulted in a 20% clinical response rate with an extra 10% having stable disease. Soiffer et al³² vaccinated melanoma patients with irradiated autologous tumour cells engineered to secrete GM-CSF. A potent antimelanoma immune response (both cytotoxic T cells and antibodies) was associated with destruction of both primary and metastatic lesions. In patients with early stage colorectal cancer, Samanci et al³³ trialled a vaccine containing rCEA (a colorectal tumour antigen) alone or with GM-CSF (80mg/d for 4 days). The GM-CSF group developed strong humoral and cell mediated immunity as opposed to the rCEA alone group. No autoimmune reactions were seen.  Mellstedt³⁴ et al treated colorectal patients with a vaccine containing GA-73.3 antigen plus soluble GM-CSF (75mg/d for 4 days). Strong cell mediated responses were seen. Side effects were minimal in all of these trials. More recently, vaccination with irradiated autologous tumour cells engineered to secrete GM-CSF was demonstrated to enhance antitumour immunity in patients with metastatic non-small cell lung cancer.³⁵ Positive DTH responses were seen in 18 of 22 patients. Infiltrates of T cells associated with tumour necrosis were demonstrated in metastatic lesions in 3 of 6 patients. 2 patients rendered disease-free by surgery remained disease-free at >40 months and 5 patients showed stable disease. Toxicity was limited to grade 1-2 local skin reactions.

Collectively, these pre-clinical and clinical data strongly support the use of GM-CSF as an adjuvant for cancer vaccines. We have chosen to use soluble GM-CSF as opposed to virally transduced cells engineered to secrete GM-CSF because: i) it is easier, ii) the amount of GM-CSF delivered is predictable, and iii) it has been used effectively in other trials with minimal side effects.

3.3             aIFN

3.3.1        Preclinical studies of adjuvant aIFN in tumour vaccines

aIFN is known to have immune modulating activity including the augmentation of T cell responses to cancer. It can also have a direct inhibitory effect on tumour growth. In murine tumour models, aIFN has been demonstrated to result in a significant delay in onset of clinical symptoms and death compared to placebo. Some tumour inhibitory effect has also been demonstrated.³⁶ In combination with cisplatinum it has resulted in tumour regression in human mesothelioma xenografts in nude mice.³⁷ An aIFN-producing tumour cell vaccine +/- lung radiation was studied in a murine model of metastatic renal cell carcinoma.³⁸ The growth rate of the aIFN-secreting tumour cells was significantly reduced in vitro and in vivo. The host anti-tumour response to subcutaneous vaccination with aIFN-secreting tumour cells was systemic, with a significant reduction in lung metastases. The effect was enhanced by lung irradiation. Santodonato et al³⁹ demonstrated that vaccination with irradiated, aIFN-producing tumour cells inhibited the growth of primary and metastatic tumours, whereas control (ie non-aIFN producing) cells were ineffective. Both CD4+ and CD8+ T lymphocytes were involved in the anti-tumour response.

3.3.2        Clinical studies of adjuvant aIFN in tumour vaccines

In patients with gastrointestinal and lung malignancies, Itoh et al⁴⁰ evaluated a vaccine containing carcinoembryonic antigen in combination with adjuvant aIFN (1 million units twice weekly). Positive DTH responses were seen in 2 out of 10 patients, who remained stable for 6 and 9 months. No toxicity was observed. Anton et al⁴¹ used a weekly autologous tumour cell vaccine combined with aIFN (1 million units) and IL2 in 208 patients undergoing surgery for renal cell carcinoma. Toxicity was mild. Compared with historical controls, there was an improvement in disease-free survival (21 months).

Like GM-CSF, pre-clinical and clinical data demonstrates that aIFN is an effective adjuvant for tumour vaccines. We have chosen a low dose (250,000 units) of aIFN to exploit its immunomodulatory effects as opposed to its cytotoxic effects. The low dose will mainly act locally and therefore have minimal systemic side effects.

3.4       Potential adverse events

3.4.1    Procurement of Tumour Tissue for Vaccine manufacture

-        Video assisted thoracoscopy: risks associated with a general anaesthetic, the surgery (pain, bleeding), the intercostal catheter in the postoperative period (pain, infection, bleeding); potential for tumour to track along the thoracoscopy site – this can be prevented by postoperative irradiation to the area.

-        Resection/ Biopsy of a Subcutaneous Tumour Deposit: reaction to the local anaesthetic, postoperative pain, and wound infection.

3.4.2   The Vaccine

·        local cutaneous skin reaction/ discomfort at the injection site

·        local infection (rare)

·        HSPs: no significant side effects, in particular no autoimmune reactions, have been reported in HSP vaccine trials to date.

·        systemic side effects of the cytokines: malaise, fever, fatigue, bone pain, myalgia and headache (uncommon at the low doses we plan to use)

·        GM-CSF: increase in white cell count or eosinophil count (uncommon). In our recently completed mesothelioma trial, GM-CSF (80mg/d for 4 days/ fortnight) was well tolerated with no significant side effects.

·        aIFN: adverse effects are dose related and unlikely at the very low dose to be used. Rare, but serious, side effects include: depression, liver, heart, bone marrow and lung toxicity. These are more common at high dose and in those with preexisting disease of these organs.

·        theoretical risk of reintroduction of live tumour cells: the methods we will use to inactivate tumour cells have been well validated in other published clinical trials, with no reports of tumour growth at injection sites. In our experience, 34 patients have been treated with no evidence of tumour growth at vaccination sites. Every possible precaution will be taken to ensure cells are non-viable prior to reintroduction.

3.4.3    Safety of the GM-CSF / aIFN Combination

The combination of GM-CSF and aIFN has not previously been used in malignant mesothelioma. However, this combination has been studied in human trials of malignant melanoma, renal cell carcinoma (RCC), chronic myelogenous leukaemia (CML) and chronic hepatitis B (HBV) and hepatitis C (HCV) viral infection. In these trials, much higher doses of a IFN were used (³1 million units (MU) vs 250,000 units in our trial). A high dose is more likely to have a systemic effect, and therefore side effects, than a low dose that would mainly act locally. In some trials the cytokines were given sequentially, rather than concurrently. Many of the melanoma and renal cell carcinoma trials included interleukin2 (IL2). IL2 is known to commonly cause side effects. When a patient experiences an adverse event whilst receiving a combination of IL2, GM-CSF and aIFN, it is more likely to be due to the IL2, though this can be hard to prove conclusively. We will not be using IL2 in this study. Lastly, some of the studies included chemotherapy and the toxicity reported may be due to this, rather than the aIFN/ GM-CSF combination.

Toxicity in Human Malignancy Trials using combined GM-CSF/aIFN

1. Malignant Melanoma:

·        de Gast et al⁴² trialled 5 days of oral temozolomide followed by 12 days of a combination of IL2 (4 MU/m²/d), aIFN (5 MU/d) and GM-CSF (2.5mg/kg/d) in 74 patients. All patients experienced flu-like symptoms, fatigue and anorexia. Two patients withdrew due to malaise. 58 patients had transient abnormalities of liver function tests.

·        Groenewegen et al⁴³ studied dacarbazine, GM-CSF (2.5 mg/kg/d for days 2-12), IL2 (1.8 MU/d for days 8-18) and aIFN (6 MU/d for days 15-20) in 32 patients. Therapy was well tolerated without significant toxicity. One patient had a skin rash attributed to GM-CSF. Mild flu-like symptoms occurred due to aIFN.

·        Vaughan et al⁴⁴ combined cisplatin, dacarbazine, tamoxifen and IL2 (9 MU/m²/d) with intermittent aIFN (5 MU/m² on days 6-10 and 17-21) and varying doses of GM-CSF in 19 patients. Constitutional symptoms and lymphopaenia were the main side effects.

These studies all included IL2 & used much higher doses of aIFN than we are planning to use.

·        Schachter et al⁴⁵ used aIFN (3 MU/d on days 1,3,5) followed by chemotherapy then GM-CSF in 40 patients with acceptable toxicity. The aIFN and GM-CSF were not concurrent in this trial.

2. Melanoma and RCC

·        De Gast et al⁴⁶ This study was designed to determine maximally tolerated dose (MTD) and dose-limiting toxicity (DLT) of combined GM-CSF, IL2 and aIFN. Therapy was given for 12 consecutive days every 3 weeks. MTD was 2.5 mg/kg/d of GM-CSF, 4 MU/m²/d of IL2 and 5 MU/d of aIFN. DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/ malaise and fluid retention. There was one episode of angioedema that was attributed to GM-CSF. One patient died from a cerebrovascular accident during the study. This was thought to be possibly treatment related, though in an ongoing phase 2 study (70 patients) by the same authors there have been no cerebrovascular accidents. The cytokine doses we are planning to use are well below the MTDs in this study. When not using IL2, the MTD of the other cytokines may actually be higher.

3. Renal Cell carcinoma

·        Ryan et al⁴⁷ trialled chemoimmunotherapy in a poor prognosis group of patients with metastatic RCC. Patients received daily cis-retinoic acid combined with GM-CSF (125 mg/d) for 2 weeks, followed by IL2 (11 MU/d for 4 days/wk) and aIFN (10 MU/d for 2 days/wk) for 4 weeks. Dose reductions of IL2 and aIFN were required for dehydration, respiratory difficulty, fatigue, fever, myalgia and mental status changes. Eosinophilia occurred in most patients and mild leucocytosis not requiring dose reduction in one patient. Other side effects included fever, fatigue and anorexia. Mucositis, chelitis and dermatitis were due to cis-retinoic acid. There was one possible therapy-related death from a cardiopulmonary arrest. This patient had had previous cardiovascular toxicity (tachycardia and hypotension) and a dose reduction had been planned. This study used very high doses of cytokines combined with chemotherapy in unwell patients.

·        Lummen et al⁴⁸ studied a IFN (10 MU) plus GM-CSF (15-300 mg) 3 times per week in 21 patients with advanced RCC. 24% of patients dropped out due to grade 3 toxicities associated with high dose GM-CSF and aIFN. Doses of 120-150mg GM-CSF 3 times per week were tolerated.

·        Westermann et al⁴⁹ studied varying combinations of GM-CSF (5 mg/kg 3x/wk), IL2 (4 MU/m² 5x/wk) and aIFN (5 MU/m² 3x/wk) in metastatic RCC. There was no severe organ toxicity, though there was one case of marked eosinophilia. Fatigue and flu-like symptoms were more common when the three cytokines were combined.

4. CML

·        Cortes et al⁵⁰ used GM-CSF (30-60 mg/m² /d) in patients with CML already taking aIFN (8 MU 2x/wk to 11 MU/d). The combination was well tolerated, with less myelosuppression than aIFN alone. These patients had already been stabilised on aIFN, so there may have been selection bias for patients who would tolerate the combination.

4.      Objectives

To assess the response of a vaccine containing heat shock enhanced autologous tumour lysate       with adjuvant GM-CSF and αIFN on the outcome of patients with malignant mesothelioma.

The primary outcomes will be:

1.      Stimulation of a tumour specific immune response

2.      Disease response

3.      Time taken to disease progression.

4.      Overall survival 

5.      Study Plan & Schedule of Assessments

5.1             Methods of collecting data

The stimulation of a tumour specific immune response will be measured by way of a delayed type hypersensitivity skin test (DTH) using non-viable autologous tumour tissue. The material is rendered non-viable by the same way as the autologous tumour lysate material (see laboratory protocol). The tests will be read as either positive or negative, a positive test being an erythematous, raised skin reaction measuring > 5mm 48 hours after its administration. Western blots will also be performed on patients’ serum to determine the presence of protein bands that will indicate antibody formation. Again the test will be either positive or negative.

Tumour response will be assessed by serial CT scan measurements. The now standard RECIST⁵¹ criteria will be used to measure tumour response (Appendix 2).

Time to progression will be measured as the time from the first vaccination to tumour progression as assessed by radiological means (see above) or the development of a new lesion.

5.1             Study Plan

5.2             Schedule of assessments

Notes:     FVC: forced vital capacity

            DTH: delayed-type hypersensitivity skin test

            WB: western blot

            FBP: full blood profile; LFTs: liver function tests; U&Es: urea and electrolytes

* At week 14 a decision will be made as to whether a patient continues with fortnightly vaccinations or has follow up at monthly intervals.

5.3             Clinical Protocol

Eligible patients will consent to a video assisted thoracoscopy (VAT) or subcutaneous tumour deposit resection or will be having a surgical pleurectomy as a therapeutic, non-experimental procedure. The surgeon will be asked to provide us with at least 5 cc of tumour tissue that will be placed in a sterile jar and retrieved by one of our staff and transported immediately to the laboratory. Therapy will begin within 2 weeks. The vaccines will be administered as a sub-cutaneous injection over the deltoid once a fortnight for 12 weeks. A dose of 0.25 x 10⁶ U aIFN and 80ug of GM-CSF will be administered as a daily subcutaneous injection at the vaccine site  for 5 days beginning on the day of the vaccine. CT scans of the thorax will be obtained at baseline (ie in the immediate post surgical period), and at the end of the 12 week vaccination period. In those patients with measurable disease tumour thickness will be measured by RECIST criteria. Lung function, as measured by forced vital capacity (FVC), will be tested at fortnightly intervals. DTH testing will be performed at baseline, followed by 3 further tests at monthly intervals. Patients will return to clinic 2 days after each DTH skin test to have the result read and recorded. FBP and U&Es will be performed at baseline and monthly for 3 months to monitor for any potential haematological and renal toxicity or electrolyte disturbance.

Those patients assessed as having responding or stable disease (by RECIST criteria) at the end of the vaccination period will be offered further vaccinations at fortnightly intervals until the vaccine supply runs out, or there is clear evidence of disease progression. CT scans will be performed at 2 monthly intervals, or monthly if lung function has deteriorated by more than 25%, during this prolonged vaccination phase.

Patients with progressive disease will be followed up monthly until time of death. A history, physical examination and details of subsequent therapies will be obtained. All patients will also be referred back to their original treating doctor for ongoing follow-up.