SUPPLEMENTAL DIGITAL CONTENT
Safety and Immunogenicity of MAGE-A3 Cancer Immunotherapeutic with or without
Adjuvant Chemotherapy in Patients with Resected Stage IB to III MAGE-A3 -Positive
NSCLC
Jean-Louis Pujol MD, Johan F. Vansteenkiste MD, PhD, Tommaso Martino De Pas MD,
Djordje Atanackovic MD, Martin Reck MD PhD, Michiel Thomeer MD PhD, Jean-Yves
Douillard MD PhD, Gianpiero Fasola MD, Vanessa Potter MD PhD, Paul Taylor MD, Lionel
Bosquée MD, Robert Scheubel MD, Silvija Jarnjak MD, Muriel Debois MSc, Pedro de Sousa
Alves PhD, Jamila Louahed PhD, Vincent G. Brichard MD PhD, Frédéric F. Lehmann MD
PhD.
CONTENTS
1.
The detection of MAGE-A3 RNA expression by reverse transcription PCR .............. 2
2.
Study design details ..................................................................................................... 2
3.
Dose modification rules for chemotherapy .................................................................. 3
4.
Preparation of MAGE-A3 immunotherapeutic for injection ....................................... 4
5.
Safety assessment: serious adverse event definition ................................................... 5
1

SUPPLEMENTAL METHODS
1.
The detection of MAGE-A3 RNA expression by reverse transcription PCR
Routine protocols and reagents were used for the extraction of total RNA (with DNAse digestion step) from manually dissected formalin-fixed paraffin-embedded tumor sections
(containing at least 80% tumor cells) and from the human melanoma cell line MZ-2-3.0
(positive control; provided by Ludwig Institute of Cancer Research, Brussels, Belgium); and routine protocols and reagents were used for the preparation of cDNA from this RNA.
MAGE-A3 and ACTB (control) cDNA fragments were amplified by PCR using TaqMan reagents (Thermo Fisher Scientific, Gent, Belgium), the ABI 7900 system (Thermo Fisher
Scientific, Gent, Belgium), and the following primers; MAGE-A3 probe FAM-CCT-GTG-
ATC-TTC-AGC-AAA-MGB, MAGE-A3 forward TGT-CGT-CGG-AAA-TTG-GCA-GTA-T and MAGE-A3 reverse CAA-AGA-CCA-GCT-GCA-AGG-AAC-T, ACTB probe TGC-TCG-
CTC-CAA-CC, ACTB forward CTG-GAA-CGG-TGA-AGG-TGA-CA, and ACTB reverse
CGG-CCA-CAT-TGT-GAA-CTT-TG. The multi-well plates used in the PCR contained cDNA from tumor specimens and the positive control. A tumor specimen was defined as
MAGE-A3 positive if the relative expression of MAGE-A3 to ACTB in the specimen was ≥1% of the relative expression of MAGE-A3 to ACTB in the positive control.
2.
Study design details
The criteria for adequate bone-marrow reserve were; white blood-cell count > 3 x 10
9
/l, neutrophils > 1.5  10
9
/l, platelets > 50  10
9
/l, lymphocytes > 0.5  10
9
/l, and hemoglobin
> 10.0 g/dl. The criterion for adequate renal function was creatinine < 3-fold the upper limit of normal and the criteria for adequate hepatic function were serum bilirubin within normal range, and aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase all
<2-fold the upper limit of normal.
2

The surgical resection included radical mediastinal lymphadenectomy and lymph node sampling procedures, and was in line with the principles discussed in Vansteenkiste et al. J
Clin Oncol 2013;31:2396-2403. Hence lymph node sampling was good if the tumor was situated on the right, and limited if the tumor was situated on the left.
Patients with completely resected stage IB, II or III tumors and who were due chemotherapy, were proposed entry into Cohort 1 (if it included less than 18 patients) in preference to
Cohort 2. As soon as the target number of patients in a cohort had been reached, then enrolment was stopped for this cohort.
A patient was excluded if the patient had concomitant or previous malignancies at other sites
(unless the malignancy had been effectively treated); was pregnant or lactating; had a history of anaphylaxis or severe allergic reaction; had concurrent severe medical problems; had psychiatric or addictive disorders; required concomitant treatment with systemic corticosteroids, or any other immunosuppressive agents (except corticosteroids for antiemetic treatment; inhaled corticosteroids for chronic obstructive pulmonary disease; or prednisone, or equivalent, [<40 mg/day]); required home oxygenation; received any other investigational or non-registered drug or vaccine from 30 days before up to the end of the study period; or had a history of chronic alcohol consumption and/or drug abuse.
3.
Dose modification rules for chemotherapy
In Cohort 2, chemotherapy dose modification was decided according to standard practice at the investigation site. In Cohort 1, the doses of cisplatin (CDDP) and/or vinorelbine (VNR) for Day 1 of subsequent cycles were modified on the basis of the worst toxicity observed during the preceding cycle or of toxicities observed on the day of re-treatment.
The dose modification rules based on worst toxicity in preceding cycle were as follows.
Febrile neutropenia or grade 4 thrombocytopenia or any grade thrombocytopenia with
3

bleeding: 75% full dose. Even with optimal anti emetic prophylaxis and therapy, nausea or vomiting grade 3: 75% full dose or no change; or grade 4: 50% full dose. Any grade 3 toxicity, except hair loss: 75% full dose; or any grade 4 toxicity: 50% full dose or discontinue).
The dose modification rules, based on toxicity on Day 1 were as follows. Peripheral neurotoxicity grade 2: 50% of CDDP and no change in VNR; or grade 3 or 4: delay CDDP, if no recovery, stop CDDP and no change for VNR. Ototoxicity, e.g. tinnitus or significant hearing loss: 50% of CDDP and no change in VNR. Creatinine clearance, 35–49 ml/min:
50% of CDDP and no change in VNR; or <35 ml/min: delay CDDP, if no recovery stop
CDDP and no change in VNR.
The dose modification rules on Day 8 were as follows. Absolute neutrophil count 0.5–0.99 
10 9 /l or platelets 50–99  10 9 /l: VNR 75% full dose. Absolute neutrophil count < 0.5  10 9 /l or platelets <50  10
9
/l: omit VNR. Grade 3 non-hematological toxicity, except nausea/vomiting: 50% of VNR; or grade 4 non-hematological toxicity: omit VNR.
4.
Preparation of MAGE-A3 immunotherapeutic for injection
MAGE-A3 immunotherapeutic formulated with AS15 (which contains 50 µg MPL[3O desacyl-4’- monophosphoryl lipid A], 50 µg QS-21 Stimulon
®
[ Quillaja saponaria Molina , fraction 21] [licensed by GSK from Antigenics Inc, a wholly owned subsidiary of Agenus
Inc., a Delaware, USA corporation], 420 µg CpG7909 and liposome) was distributed as two components: vial (i) a lyophilized preparation (including recombinant MAGE-A3, 300 µg), and vial (ii) a liquid preparation. Prior to administration, the contents of vial (ii) were injected aseptically into vial (i) and mixed. This preparation was withdrawn with the same syringe and injected after the needle had been changed, immediately or within 4 hours after reconstitution
4

on condition that the final preparation had been kept at room temperature (20–30°C) or at
4°C.
5.
Safety assessment: serious adverse event definition
A serious adverse event (SAE) was any untoward medical occurrence that resulted in death, was life-threatening, required hospitalization or prolongation of existing hospitalization, resulted in disability/incapacity, was a congenital anomaly/birth defect in the offspring of a study patient, or was a Grade 4 AE.
5