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Recent advances in cellular immunotherapy show that cancer cells can be efficiently eliminated by adoptive transfer of modified patient T cells. The main effector functions can be provided by genetically engineered lymphocytes expressing T cell receptors (TCR) isolated from tumor antigen-specific cytotoxic CD8+ lymphocytes, which recognize tumor-associated antigens presented on HLA class I molecules. Nevertheless, for efficient and sustained cytotoxic and memory CD8+ T cell responses, T cell help delivered by CD4+ T lymphocytes plays a critical role. Multifunctional CD4+ T cells are essential to activate, control and maintain immune responses. CD4+ T cells recognize antigen-derived peptides presented by HLA class II molecules, thereby HLA class II tumor antigen-positive cells may also be efficiently eliminated by direct CD4+ cytotoxic mechanisms. Recently it has been shown that adoptive transfer of tumor antigen-specific CD4+ T cells alone can lead to substantial regression of epithelial tumors (Science. 2014 May 9;344(6184):641-5). To appropriately employ CD4+ T cells in tumor defense, detailed molecular knowledge regarding the antigens and corresponding epitopes they recognize is essential. We recently expanded our TCR platform by introducing a fast and efficient method for direct identification of antigens and respective epitopes recognized by CD4+ T cells. During method development, elucidated viral antigens allowed rapid clinical application, and the methodology was also applied for rapid identification of epitopes of CD4+ T cell clones recognizing multiple cancer-testis antigens (CT). For this, PBL from a healthy donor were stimulated with a mixture of autologous APCs transfected with in vitro transcribed RNA encoding various CT antigens fused to targeting signals for directed HLA class II cross-presentation. Antigen-reactive CD4+ T cells were isolated and expanded. In order to identify the antigen and epitope specificity of each T cell clone, DNA sequences of each CT antigen were randomly digested and DNA fragments introduced in a vector mixture that allows expression of the fragments in all possible reading frames fused to a bacterial selection marker. These vectors were then expressed in XL1 blue bacteria and fed as pools to autologous APCs. Thereby the APCs phagocytosed and directly presented random CT epitopes on their HLA class II molecules. Subsequently, these pool-loaded APCs were tested with CT-antigen-specific CD4+ T cell clones. In a second step, the single bacterial colonies of a positive pool were re-tested individually and the antigen fragments recognized were identified by DNA sequencing, and confirmed by testing of synthetic peptides. HLA restriction was determined by testing responses against antigen-positive HLA allogenic cells. Using this approach, TCR sequences, epitope sequences and respective MHC-restriction elements were identified for multiple antigens in a single round procedure. This method has broad application for identification of: 1) antigens and epitopes recognized by CD4 tumor infiltrating lymphocytes, 2) immune relevant patient-specific mutations recognized by CD4+ T cells, 3) defined epitopes recognized by T helper cells for generation of tumor vaccines, 4) epitope-specificity of CD4+ T cells, against any selected antigen. This technology enables the generation and molecular characterization of TCRs originating from tumor-specific CD4+ helper T cells, complementing our already established approach for isolation of high affinity TCRs derived from cytotoxic CD8+ T cells. Citation Format: Milosevic Slavoljub, Ellinger Christian, Wehner Carina, Raffegerst Silke, Wilde Susanne, Weis Manon, Sailer Nadja, Schendel Dolores. Method for molecular characterization of antigens, epitopes and T cell receptors for adoptive CD4 immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A017.