Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I dose escalation study

Purpose The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. Patients and methods Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. Results 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. Conclusions The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500 µg PRAME immunotherapeutic dose. Trial registration number NCT01149343, Results.

monoclonal antibody or a cancer vaccine containing a tumor antigen other than PRAME (such as the MAGE-A3 immunotherapeutic received as part of study www.clinicaltrials.gov NCT00796445) was allowed if the last dose was given eight weeks before the first dose of the PRAME immunotherapeutic. Isolated limb perfusion was allowed if performed at least four weeks before enrolment.
Patients requiring concomitant treatment with systemic corticosteroids or any other immunosuppressive agents were excluded (a maximum to 10 mg/day prednisone, or equivalent was permitted). Patients with previous or concomitant malignancies at other sites were excluded, as were patients with confirmed adrenal dysfunction, an autoimmune disease, patients who were known to be human immunodeficiency virus positive, patients with a family history of congenital or hereditary immunodeficiency or those with an uncontrolled bleeding disorder. Pregnant or lactating patients were not permitted to participate.
A maximum of 24 doses of PRAME immunotherapeutic could be administered according to the following schedule: the first 6 doses given at 2-week intervals, then 6 doses at 3-week intervals, then 4 doses at 6-week intervals, then every 3 months during year 2 and every 6 months for the next 2 years. Patients will be followed up for safety and clinical outcomes every 3 months for one year, and for survival for 5 years after the first treatment administration (to be published elsewhere).
Enrolment was staggered such that the first 3 patients of each dose-level received their first immunization on different days, allowing earlier identification of safety signals. Escalation to the next dose level occurred when 15 patients had commenced treatment with the previous dose level and when 3 patients had received at least 4 immunizations. Dose escalation only occurred if no case of dose limiting toxicity (DLT) had occurred in the first 3 patients, and if no more than 2 cases of DLT had occurred in all 15 patients enrolled at that dose-level. In the event of one case of DLT in the first 3 patients, the decision to proceed to the next dose-level was postponed until 6 patients at that dose-level had received at least 4 treatment administrations.

Safety monitoring
A Data and Safety Monitoring Committee (DSMC) composed of an independent group of experts reviewed the safety data, the clinical relevance of each DLT event and its relationship to the study treatment. The DSMC made recommendations to the sponsor concerning the continuation, modification or termination of the trial.
At each visit, blood and urine samples were collected for evaluation of hematologic, biochemical and coagulation parameters including serum cortisol, renal function tests, and urinalysis. Anti-nuclear antibodies were assessed at screening, at the time of doses 6, 12 and 16 and every six months thereafter.

Measurement of humoral immune responses
IgG antibody concentrations found in patient sera were measured in duplicate using a standard ELISA. Briefly, recombinant PRAME produced in Pichia pastoris was coated overnight at 2-8°C at 1.5µg/ml in appropriate ELISA plates. After washing and blocking the plates with assay diluent (1% BSA, 0.1% Tween 20, 0.2% Proclin 300 [Supelco]) during 1 hour at room temperature, clinical samples, control and standard samples were diluted in assay diluent complemented with 1mg/ml of Pichia lysate. Pre-immunization samples were pre-diluted 1:1000 and post-immunization serum specimens were pre-diluted 1:1000 and 1:10,000. Diluted samples were incubated for 60 minutes (10 minutes) at room temperature on antigen-coated plates. After washing, a secondary antibody (goat anti-human-IgG antibody conjugated to peroxidase -KPL) was incubated 60 minutes at room temperature.
After washing, the chromogen substrate solution (TMB, Biorad) was added and incubated 30 minutes at room temperature. The reaction was stopped with H 2 SO 4 (Merck) and absorbance optical density was measured at 450 / 620 nm within 60 minutes. The raw data were analyzed with SoftMaxPro software (Molecular Device).
IgG anti-PRAME antibody concentrations were expressed as ELISA Units per milliliter E.U/ml. The experimental strategy also allowed calculation of approximate frequencies of antigen specific T-cell precursors (assuming a clonal response).

Measurement of cell-mediated immune responses
For each pair of wells the ratio between the percentage of double positive cells in the specific and irrelevant stimulation was calculated. The geometric mean of the 24 ratios (GMR, considered as an immunogenicity score) was calculated to integrate the average responses observed in the 24 independent wells. For wells with fewer than 50 positive antigen-specific events the well ratio was arbitrarily set at 1.
GMR cut-offs were calculated for both CD4+ and CD8+ T cells from 23 healthy donors using the same analysis templates, and values were determined as 2.68 (for CD4+ T cell analysis) and 1.15 (for CD8+ T cell analysis). A patient was considered as an immune responder if the GMR 2 weeks post-dose 4 was both above the cut-off and at least 4 times higher than the GMR at baseline.

Statistical analysis
All statistical analyses were performed using SAS software version 9.2. The study was descriptive and no comparative tests were performed. The total treated cohort included all patients enrolled into the study who had received at least one treatment dose and the according-to-protocol cohort for immunogenicity included all patients who met eligibility criteria, who complied with protocol-defined procedures, and who had received at least the first 4 PRAME immunotherapeutic doses and had completed the visit 2 weeks post-dose 4. Geometric mean antibody concentrations were calculated for anti-PRAME IgG antibodies.

Results
The study commenced on 13 July 2010 and the data lock point for dose selection was 20 December 2011.

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