With this Phase 2 submission, IsoPlexis proposes to deliver a single-cell proteomic solution for ultra-sensitive detection of unique correlative biomarkers in pre-clinical mouse models, in an automated system that can be used to accelerate the discovery of potential immune therapeutics. The recent revolution in new basic science discovery is failing to translate into improved patient treatment. The reduction of new drug and biologic applications submitted to the FDA every year is directly due to only 10% of promising preclinically approved treatments eventually advancing to market. As the pharmaceutical/biotech industry embarks on capitalizing on new immunotherapy discoveries, the complex human immune system presents challenges to the development of new treatments. While improved humanized mouse models (such as the Humanized Immune Systems, HIS) are a step forward, a need exists for an enabling biomedical technology to improve immune biomarker detection that de-risks therapy selection translating to success in human treatment. A platform which (1) measures secreted protein polyfunctionality in a single-cell platform for mice, and (2) gathers this data from limited mouse samples is necessary. (3) The technology should be able to measure multiplexed, emerging, protein biomarkers that have proven critical to such immune therapies in humans, drastically improving clinical success. With the successes from the Phase I part of the project, IsoPlexis has developed such a high-resolution immune response biomarker detection system (SCBC), delivering a comprehensive evaluation of the full polyfunctional protein secretion response of each immune cell to treatment, an analysis that has proven valuable in other immunotherapy arenas. This approach will maximize the potential data obtained from HIS mouse preclinical studies to advance the very best treatments. The two-year project will accomplish the following: Aim 1 Develop an integrated ?flow cell? consumable cartridge, with a >28-plex single-cell protein panel to evaluate human T cell response in HIS (humanized) mice and other genetically engineered mouse models (GEMMs). Aim 2: Develop an automated system to enable the handling of limited murine sample size and increase assay throughput, for larger-scale mouse screening studies in pre-clinical laboratories. Aims 3: Demonstrate proof-of-concept utility of this technology across four trials, for profiling polyfunctional response in HIS mice and other GEMMs, advancing biomarkers that lead to success in human treatments.