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Background Colorectal cancer (CRC) remains the second leading cause of cancer-related deaths in the U.S., with rising incidence among young adults. While CAR-T cell therapy has been successful in treating some hematologic malignancies, its application to solid tumors is hindered, in part, by toxicity arising from on-target/off-tumor effects that stem from a lack of tumor antigen specificity. CD87, the receptor for the urokinase plasminogen activator (uPA), contributes to tumor progression and is highly upregulated in CRC. However, its expression is minimal in most normal tissues, suggesting that it might be an attractive target antigen for CAR-T cell therapy.Methods To assess the potential safety of CD87 as a therapeutic target, we analyzed publicly available single-cell RNA sequencing (scRNAseq) datasets of healthy human tissues predicted to have the highest CD87 expression based on data from the Human Protein Atlas. Next, we engineered CD87-directed CAR-T cells (CART87). Antigen-specific cytotoxicity in vitro was assessed in CRC cell lines (LS174T, SW480, T84), as well as a CD87-negative control cell line (HEK293). To further optimize CAR design, we employed a Jurkat NFAT-Lucia reporter system, which provides a quantifiable readout of CAR activation upon stimulation with recombinant uPAR, thus allowing unbiased, high-throughput screening of various scFv and structural domains.Results At the tissue and organ level, CD87 mRNA expression was highest in the bone marrow, followed by urinary bladder, appendix, gallbladder, and lung. scRNAseq analysis of healthy human bone marrow, whole blood leukocytes, lung and urinary bladder revealed that CD87 mRNA expression is mainly restricted to mature monocyte-lineage cells. These results suggest limited CD87 off-tumor expression. In preliminary cytotoxicity assays, CART87 demonstrated strong, specific killing of CD87-positive colorectal cancer cell lines while sparing CD87-negative HEK293 cells. CAR activation profiling using the Jurkat NFAT reporter system allowed for the rational selection of a lead CAR construct with enhanced CD87-specificity and activation potential in response to uPAR stimulation.Conclusions This work describes the rational design of a CAR87 construct using a high-throughput strategy that could be employed for CAR design for other target antigens. Furthermore, our work proposes CD87 as a potentially safe and effective target antigen for the development of CAR-T therapy in colorectal cancer, laying the groundwork for future preclinical in vivo studies and clinical development of CART87.