Abstract 5456: Development of human antibodies targeting a novel cancer-associated fibroblast protein identified from spatial transcriptomics

Background Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and play a critical role in tumor progression and therapy resistance. Recent advances in spatial transcriptomics (ST) enable precise analysis of gene expression within the spatial context of tissues, offering new avenues for target discovery. Using spatial and single-cell transcriptomic data, we identified a novel target protein, POR-CAF-001, predominantly expressed in CAFs within solid tumor tissues and minimally in normal tissues, making it a promising candidate for therapeutic intervention. Method Spatial and single-cell transcriptomics data from human tumor samples were analyzed using our proprietary target discovery program to identify CAF-specific targets. Two human IgG antibodies (POR-CAF-001-01 and POR-CAF-001-02) against the POR-CAF-001 were generated through phage display, ELISA screening and recombinant production in mammalian cells. Surface plasmon resonance (SPR) was employed to validate a binding affinity of antibodies against POR-CAF-001 protein. Also, flow cytometry and immunocytochemistry (ICC) were performed to evaluate the binding specificity of the antibodies. Antibody internalization was assessed via an imaging-based internalization assay using pH-sensitive fluorophore-labeled antibodies, and their internalization was monitored over time. POR-CAF-001-positive, -negative and knockout cell lines were used to confirm binding specificity and internalization. Results Differential expression and spatial specificity analyses identified POR-CAF-001 with high expression in various types of tumor tissues with minimal expression in normal tissues. SPR analysis demonstrated binding affinities (KD) of 1.15 nM and 4.1 nM for POR-CAF-001-01 and POR-CAF-001-02, respectively. Flow cytometry and ICC analyses confirmed robust binding of both antibodies to POR-CAF-001-positive cells, with negligible binding in POR-CAF-001-negative and the target knockout cells. Both antibodies showed substantial internalization in POR-CAF-001-positive cells during the internalization assay, while minimal internalization was observed in the negative cell line. Conclusion Through our target discovery program with ST data, we identified POR-CAF-001 as a novel CAF-specific target protein and developed two human IgG antibodies against it. These antibodies demonstrated high specificity and successful internalization in target-positive cells. These findings provide a solid foundation for further preclinical development, including antibody-drug conjugates (ADCs), radiopharmaceutical therapy (RPT) or other therapeutic modalities targeting POR-CAF-001. Our study underscores the potential of ST in identifying novel targets and advancing cancer therapy. Citation Format: Jin Yeong Choi, Shengjun Li, MinKyu Kim, Yeonjae Jung, Jae Eun Lee, Daeseung Lee, Hongyoon Choi, Hyung-Jun Im. Development of human antibodies targeting a novel cancer-associated fibroblast protein identified from spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5456.