Abstract P2-09-04: Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer
Authors: Reimann H , Nguyen A , Hübner H , Erber R , Bausenwein J , Van der Meijden ED , Lux MP , Jud S , Griffioen M , Rauh C , Sanborn JZ , Benz SC , Rabizadeh S , Beckmann MW , Mackensen A , Rübner M , Fasching PA , Kremer AN 
Published: May 2019

Abstract
Background: Recent studies have demonstrated that the number of tumor infiltrating lymphocytes (TILs) positively correlates with outcome and response to chemotherapy in patients with HER2+ and Triple-Negative Breast Cancer (TNBC). Furthermore, first studies of immune-checkpoint inhibitors showed promising results in those patients. However, the targets of those TILs remain unknown. Neoantigens, which arise in the process of tumorigenesis, appear as potential targets. They can elicit high avidity, tumor-specific T-cell responses. Thus, it is the aim of our study to ascertainif these TILs are directed against tumor-specific mutations.
Methods: TILs from breast cancer biopsies taken at the time point of diagnosis were expanded by unspecific stimulation. Additionally, we used the Gentle Macs Dissociator in combination with flow cytometry to investigate the number of TILs in the tumor tissue. Furthermore, we performed whole-genome sequencing of tumor tissue and as reference autologous blood cells to determine tumor-specific mutations. Mutations leading to a non-synonymous amino acid change were analyzed for RNA expression of the encoding gene as well as to determine potential neoantigens. Neoantigens were evaluated for their potential binding to the patient's specific HLA molecules. Peptides for potential neoantigens were synthesized, loaded onto autologous antigen presenting cells (APCs) and cocultured with TILs. All IFNγ producing T-cells were clonally expanded and retested for peptide specificity to identify neoantigen specific T-cell clones.
Results: Our flow cytometric analysis of the tumor biopsy for more than 300 patients showed higher frequencies of TILs in TNBC as compared to other types of breast cancer or patients without malignancy. Screening for neoantigen specific T-cells in one patient led to identification of three peptide-specific CD4+ T-cell clones isolated from HER2+ breast cancer tissue taken at the time point of diagnosis. All T-cell clones specifically recognized the same tumor-specific mutation and not the wildtype counterpart. Furthermore, we demonstrated that these T-cell clones also recognized the endogenously expressed mutated antigen. This verified the ability of processing and presentation of the respective protein. Interestingly, we could also isolate a T-cell clone recognizing the same neoantigen in the resected tumor tissue after neoadjuvant therapy. Based on CDR3 sequencing we could prove that the four T-cell clones represented individual clones. This confirms the polyclonal nature of the immune response. Moreover, we showed that the same neoepitope was presented in two different HLA restriction molecules of the patient with three of the clones recognizing it in HLA-DPB1*0401 and one in HLA-DPB1*0201. These results further underline the immunogenicity of this neoantigen.
Conclusion: In conclusion, our data demonstrate tumor-specificity of TILs in a patient with HER2+ breast cancer. Furthermore, we show the feasibility to identify individual cancer specific T-cell targets in breast cancer patients. These results may contribute to the development of targeted patient-specific immunotherapies in the future.
Citation Format: Reimann H, Nguyen A, Hübner H, Erber R, Bausenwein J, Van der Meijden ED, Lux MP, Jud S, Griffioen M, Rauh C, Sanborn JZ, Benz SC, Rabizadeh S, Beckmann MW, Mackensen A, Rübner M, Fasching PA, Kremer AN. Identification of a neoantigen targeted by tumor-infiltrating lymphocytes in a patient with Her2+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-04.