Abstract 1261: T cell responses against mutations in oncoproteins/tumor suppressor proteins and their induction by vaccination with long peptides.
Authors: Quandt Jasmin , Schlude Christoph , Bartoschek Michael , Cid-Arregui Angel , Beckhove Philipp , Momburg Frank 
Published: August 2014

Vaccination with peptides comprising tumor-associated antigens (TAAs) is a promising approach in cancer immunotherapy. TAAs derived from mutated genes are exclusively expressed in the tumor and are not shared with normal tissue. This reduces the risk of autoimmunity compared to non-mutated proteins when employed for cancer peptide vaccination. Moreover, the effectiveness of the vaccine can be enhanced by combining epitopes to induce cytolytic CD8+ as well as T helper (CD4+) cell responses. We therefore use long peptides (28-35 amino acids) to facilitate a presentation of MHC I and II epitopes. More precisely, we designed a panel of long peptides, with sequences derived from the most frequent mutated variants of the tumor suppressor Trp53 and the oncoproteins Kras and Braf described for colorectal (CRC) and pancreatic carcinomas. To investigate the relevance of immune responses against the chosen mutations we screened blood and bone marrow from 26 CRC patients for T cell responses against the long peptides using IFNγ ELISpot analysis. We found a tendency towards stronger responses against the mutated peptides compared to those against corresponding wild-type (wt) peptides. Furthermore, we correlated the ELISpot results with the abundance of Trp53 and Kras mutations identified in the patients’ primary tumors and metastases. This revealed that patients carrying mutations were more likely to be responsive against wt and mutated peptides than patients with no detectable mutation. In order to analyze the potency of our long peptides for active vaccination, we utilized C57BL/6J mice as well as a MHC-Class I/II humanized mouse strain (β2m-deficient, HLA-A2/Db/hβ2m [HHD chimera] and HLA-DR1 double transgenic) in a multi-peptide vaccination setting. Immune responses were monitored with flow cytometry by measuring cytokine secretion after in vitro restimulation of T cells from immunized mice. Thereby we observed responses for the majority of the long peptides tested. Interestingly, some of the mutated peptides showed a significantly higher induction of cytokine levels than corresponding wt sequences suggesting mutation-specific responses. In addition we were able to monitor both CD4+ and CD8+ T cell responses, in which the presence of CD4+ T cells enhanced CD8+ T cell responsiveness. This observation was made for several peptides in different combinations suggesting the induction of a multi-epitope response. As a final goal we plan to investigate the tumor-protective capacity of our vaccination approach. Therefore, the MHC-Class I/II transgenic mice were treated with the carcinogen 3-methylcholanthrene to generate a syngeneic tumor cell line. We were able to establish a fibrosarcoma cell line growing in vivo, which is engineered to express the most-immunogenic mutations from our vaccination panel to be tested in tumor challenge experiments.

Citation Format: Jasmin Quandt, Christoph Schlude, Michael Bartoschek, Angel Cid-Arregui, Philipp Beckhove, Frank Momburg. T cell responses against mutations in oncoproteins/tumor suppressor proteins and their induction by vaccination with long peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1261. doi:10.1158/1538-7445.AM2013-1261