{
  "abstract": "Background T cell engagers (TCEs) are powerful therapeutics which can kill cells expressing low levels of target antigen. To limit on-target off-tumor toxicity, it is critical to identify antigens with limited normal tissue expression or apply strategies for conditional engagement. While few antigens are completely tumor-specific, tumor cells may co-express pairs of antigens which are not co-expressed on normal cells. By selectively targeting such antigen pairs but not the individual antigens, AND-gated TCEs can impart anti-tumor efficacy with reduced toxicity. Here, we aimed to identify and validate a tumor-specific antigen pair for a potential AND-gated TCE.Methods Gene signatures were used to identify tumor cells in publicly available single cell RNA-sequencing (scRNA-seq) datasets for colorectal (CRC), 1 2 non-small cell lung (NSCLC),3–5 and breast cancer (BC).6 7 Next, we compiled genes predicted to encode cell-surface proteins.8 To find co-expressed genes, Weighted Gene Correlation Network Analysis (WGCNA) was applied.9 were then analyzed for normal tissue expression using published scRNA-seq data.10–12 For one tumor-specific pair, membrane co-expression was examined via multiplex immunohistochemistry (IHC) on normal and tumor tissue-derived microarrays (TMAs). In addition, five adenocarcinoma CRC tissue blocks with known mesothelin (MSLN) expression and five unbiasedly chosen mucinous CRC tissue blocks were also assessed.Results From 66 co-expressed tumor antigen pairs identified by WGCNA in more than one dataset, only three were tumor-specific. Target validation was pursued for mucin 13 (MUC13) and MSLN, due to expression of the pair in CRC and NSCLC and known toxicities of therapies targeting MSLN. 12–15 On a normal TMA, low levels of dual-positive staining for MUC13xMSLN were observed in 3 non-essential tissues. On tumor-derived TMAs, 37% (11/30) of CRC and 0% (0/30) of NSCLC samples were double-positive, consistent with the bioinformatics data where 18% (10/55) of CRC and 4% (2/50) of NSCLC patients showed co-expression. Double-positive tissue blocks for adenocarcinoma (4/5) and mucinous CRC (3/5) showed respective fractions of double-positive cells of 1-15% and 1-50%.Conclusions To our knowledge, this is the first report of MUC13xMSLN as a potential tumor-specific antigen-pair in CRC. An AND-gated MUC13xMSLN TCE may reduce toxicities seen with MSLN therapies, although the percentage of patients expressing the target pair and the frequency of dual-positive cancer cells in these patients is relatively low. Overall, this data suggests the described bioinformatics and protein validation approach is suitable for identifying targets for AND-gated therapies in other cancer subtypes.Acknowledgements Excelra conducted the scRNA-seq analysisReferences Pelka K, et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell. 2021;184(18):4734–4752.e20.Joanito I, et al. Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer. Nat Genet. 2022;54:963–975.Wu F, et al. Single-cell profiling of tumor heterogeneity and the microenvironment in advanced non-small cell lung cancer. Nat Commun. 2021;12:2540.Kim N, et al. Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma. Nat Commun. 2020;11:2285.Laughney et al. Regenerative lineages and immune-mediated pruning in lung cancer metastasis. Nat Med. 2020;26(2):259–269.Wu S, et al. A single-cell and spatially resolved atlas of human breast cancers. Nat Genet. 2021;53(9):1334–1347.Pal B, et al. A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast. EMBO J. 2021;40(11):e107333Dannenfelser R, et al. Discriminatory power of combinatorial antigen recognition in cancer T cell therapies. Cell Syst. 2020;11(3):215–228.e5.Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinform. 2008;9:559.The Tabula Sapiens Consortium. The tabula sapiens: a multiple-organ, single cell transcriptomic atlas of humans. Science. 2022;376:eabl4896.Lee N, et al. Establishing a bone marrow single cell reference atlas to study ageing and diseases. Front. Immunol. 2023;14:1127879.Lake B, et al. Integrative single-cell analysis of transcriptional and epigenetic states in the human adult brain. Nat Biotechnol. 2018;36(1):70–80.Hassan R, et al. Mesothelin-targeting T cell receptor fusion construct cell therapy in refractory solid tumors: phase 1/2 trial interim results. Nat Med. 2023;29(8):2099–2109.Haas A, et al. Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells. Mol Ther. 2023;31(8):2309–2325.Tanyi J, et al. Phase I study of autologous T cells bearing fully-humanized chimeric antigen receptors targeting mesothelin in mesothelin-expressing cancers. Gynecol Oncol. 2022;166:S164-S165.",
  "authors": [
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Catherine C Henry"
    },
    {
      "affiliations": [
        "Cullinan Oncology, Philadelphia, PA, USA"
      ],
      "name": "Kavya Rakhra"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Patrick A Baeuerle"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Jennifer S Michaelson"
    },
    {
      "affiliations": [
        "Cullinan Therapeutics, Cambridge, MA, USA"
      ],
      "name": "Karsten Sauer"
    }
  ],
  "title": "973 Identification of MUC13xMSLN as a novel tumor-specific antigen pair for AND-gated T cell engagers in colorectal cancer",
  "uid": "caf325cf-b7a2-50ab-945a-fd528221dfb4"
}