Protocol Amendment 1 was generated to ensure compliance with several deficiency requests received from the Paul-Ehrlich-Institute upon their review of the Initial Clinical Trial Application. One exclusion criteria had to be modified to exclude patients with any kind of brain metastases (symptomatic or asymptomatic). The patient observation after administration of each vaccination had to be described and it had to be specified that all treated patients, also those whose treatment was prematurely stopped, would be followed until Week 52. The stopping rules for the entire trial were added to the protocol and defined to be: ≥ 2 DLTs out of 2 to 6 patients in the first dose level (DL); unacceptable toxicity; new available data which may have led to a negative benefit risk assessment; and unsatisfactory enrollment. Also, the role of the Data Safety Monitoring Board (DSMB) was further defined and included decision-making authority for stopping the trial. It was defined that Phase I safety data had to be submitted to the German authority to decide on start of the Phase IIa part of the study. In addition to the changes resulting from the deficiency request, the recall skin test was removed due to the limited sensitivity of this assay, the urine pregnancy test was replaced by a serum pregnancy test, and an additional safety laboratory was added to the Week 26 visit. Amendment 1 was only submitted in Germany.

Protocol Amendment 2 was generated following discussion with the German Authority and included changes to the Inclusion and Exclusion Criteria and further clarification of the safety and efficacy assessments. The DSMB was renamed to CRC (Cohort Review Committee). The CRC as the new name was better aligned with the role of the committee in this study, and was a more common and accepted name for this type of oncology Phase I/IIa study. The exclusion criteria that were removed included the presence of mild allergy requiring seasonal (non-steroidal) medication, treatment of controlled pleural effusion by puncture, and specific radiation doses for tumor treatment. Patients with known brain metastasis and those who were > 75 years of age were to be excluded. If a patient was hospitalized for disease progression, this was no longer to be considered an AE or SAE. Amendment 2 was not submitted. Amendment 1 and 2 were combined to Amendment 3 and submitted in Germany and Switzerland.

Protocol Amendment 3 included the list of changes noted previously for Protocol Amendment 1 (required by the German Regulatory Authority, only implemented in Germany), and Protocol Amendment 2. Amendment 3 was submitted to the Swiss and German Regulatory Authority. The exclusion criteria that were removed included the presence of mild allergy requiring seasonal (non-steroidal) medication, treatment of controlled pleural effusion by puncture, and specific radiation doses for tumor treatment. Patients with brain metastases and those who were > 75 years of age were to be excluded. If a patient was hospitalized for disease progression, this was no longer to be considered an AE or SAE. The DSMB was renamed to the CRC as the new name was better aligned with the role of the committee in this study, and was a more common and accepted name for this type of oncology Phase I/IIa study.

Protocol Amendment 4 was implemented prior to starting Phase IIa. The duration of treatment with CV9201 was shortened to 7 weeks (Weeks 1, 2, 3, 5, and 7) leading to more frequent vaccinations in order to induce an immune response as quickly as possible since a considerable amount of time appears to be necessary to establish a persistent immune response. The CV9201 vaccine, which had been administered at the same location at each vaccination visit during Phase I, was now to be administered using an alternating pattern. CV9201 was to be applied intradermally into the thigh and upper arm of either side (4 sites in total) with each drug product component administered in 2 injections per treatment day, one into the thigh and one into the upper arm of the same side. The injection site for each component was to be alternating between the body halves for different treatment days. The rationale for this was that choosing 2 different injection sites per antigen should increase the number of lymph nodes exposed to the vaccine. Since recent preclinical results suggested that the immune response positively correlated with the number of lymph nodes being exposed to the vaccine, it was expected that this would contribute to efficacy. Prior to this preclinical data, this hypothesis was only supported in the published medical literature. The Phase IIa Week 5 procedures were revised to include autoimmunity assessments and blood sampling for immune response monitoring. Post vaccination monitoring was reduced to at least 2 hours at the study site, due to the good tolerability shown in Phase I. Patients who had progressive disease, and therefore needed other anticancer treatment (e.g., chemo- or radiotherapy), were no longer to be discontinued from the study. Patients were still prohibited from receiving treatment with other biological anti-cancer agents and/or cancer vaccines.

Protocol Amendment 5 was implemented to prolong the Survival Follow-Up period to 2 years and to include additional plasma testing on previously collected blood samples, that would allow judging the immune response to CV9201 more explicitly. Testing was for antibodies against other tumor specific antigens, vaccine specific antibody responses, and the induction of epitope spreading.