Abstract As the development of cancer immunotherapy and urgent demand of precision medicine, cancer vaccines have been envisioned as a novel and effective tool to cure cancer disease or prevent cancer from recurrence or metastasis. In order to support the research and development of new modality in drug discovery, we have established an advanced and integrated in vitro and in vivo platform for various types of cancer vaccine, such as peptide vaccine, mRNA vaccine, DC vaccine and so on. Ovalbimin (OVA) is a key model antigen which has been widely used in vaccination experiments. Here we established an OVA overexpressed system based on different types of tumor cells, such as B16F10-OVA and MC38-OVA. Firstly, we proved that MHC-I:OVA complex were presented on the surface of stable cell lines successfully through IFN stimulation. The results of in vitro CD8+ T cells killing assay showed the B16F10-OVA cells can be largely killed which indicates OVA protein has been presented and can be utilized as a specific target for analysis. In vitro ELISPOT assay is well characterized for efficiency tool for immunogenicity determination. Using ELISPOT assay, we compared the immunogenicity of OVA specific peptide and mRNA administration via different injection manners. Furthermore, in vivo tumor model of B16F10-OVA and MC38-OVA have also been established to evaluate the anti-tumor efficacy of peptide and mRNA vaccine. The mechanism of cancer vaccine has also been investigated via T cell, B cell and other cell populations blocking. Additionally, we explored the target protein biodistribution and in vivo behavior of mRNA vaccine carrier with Lumina LT system. In summary, the integrated OVA-specific system can be used for new adjuvants and peptide/mRNA carrier exploration in cancer vaccine discovery. Citation Format: Jian Xiang, Jun Huang, Wenrong Zhou, Zhengang Peng, Qingyang Gu, Qunsheng Ji. Development of an integrated in vitro and in vivo OVA-specific system for cancer vaccine discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5563.