The main goal of this open-label, first-in-human, phase 1 umbrella trial (ClinicalTrials.gov: NCT02316457) was to evaluate the feasibility, safety, and tolerability of two types of mRNA–LPX-based vaccines: a standard warehouse vaccine made from non-mutated tumor-associated antigens (TAAs) and a custom-made, individualized neoantigen vaccine. The study particularly reports on the outcomes of the individualized neoantigen vaccine arm. Conducted in Germany and Sweden, the trial adhered to the Declaration of Helsinki and Good Clinical Practice Guidelines, with approval from independent ethics committees and regulatory authorities in both countries. Each patient provided written informed consent.
Eligibility criteria included: histologically confirmed invasive adenocarcinoma triple-negative breast cancer (TNBC), prior standard care treatment (neoadjuvant chemotherapy followed by surgery, or surgery plus adjuvant chemotherapy), at least 18 years old, and tumors expressing a minimum of five neoantigens. Patients could be enrolled within a year after completing their treatment, and key exclusion criteria involved any breast cancer recurrence before treatment commencement and significant autoimmune diseases or active viral infections.
The individualized neoantigen vaccine was made up of two single-stranded RNA molecules, with each encoding up to ten neoantigen targets based on analysis of the patient’s tumor mutations. Tumor samples were obtained from diagnostic biopsies or surgery, depending on quality. Electronic data capture was managed using the agCapture 3.4.2.6 system (ArisGlobal). The clinical trial report on primary and secondary endpoints was submitted to health authorities in spring 2021, covering findings up to end of treatment and a follow-up period of 56 days. After this, three previously treated patients were followed passively, while 11 others actively participated in long-term follow-up until 2023, encapsulated in an addendum submitted in spring 2024.
For next-generation sequencing (NGS), tumor DNA was extracted from FFPE samples using a modified QIAamp DNA kit. RNA extraction also occurred in duplicates, and various libraries were created for targeted RNA sequencing and whole-exome sequencing. DNA and RNA data analysis coordinated by a proprietary bioinformatic pipeline yielded significant sequencing results.
Mutation detection involved aligning DNA reads to a reference genome and the use of various computational tools for analyzing somatic variants. Identified mutations underwent further assessment for their potential as neoantigen vaccine targets, leading to the development of a customized vaccine. The expression of each neoantigen was also estimated through RNA sequencing, revealing insights into mutation-specific responses.
The assessment of immune responses involved frequent blood sampling during the study. Synthetic overlapping peptides and IVS cultures were used for various immunogenicity tests, including ELISpot assays. Specific T cell populations were tracked using advanced staining and molecular techniques, while gene expression analysis leveraged robust software packages.
Innovatively, the study emphasized personalized approaches throughout the trial—from vaccine manufacturing methods to TCR profiling—ultimately demonstrating the individualized nature of immunotherapeutic strategies. The entire process—from blood sampling for immune response assessment to sequential data analysis—reflected a dynamic interplay of personalized medicine and cutting-edge technology.
