{
  "abstract": "Background Antibody-based therapeutics have emerged as highly effective strategies in the field of Oncology. These include monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), bispecifics, radioimmunotherapies, and chimeric antigen receptor (CAR)-T cell therapies. 1 However, a significant bottleneck in the advancement of antibody-based therapies lies in the difficulty of identifying antibodies with precise specificity to tumor-associated antigens. This challenge stems in part from the vast diversity of the antibody repertoire. To address the challenges in antibody discovery, we conducted proof-of-concept experiments to establish a platform for interrogating the antibody repertoire. This platform combines high-throughput B cell receptor (BCR) sequencing and characterization of antibody repertoires via mass spectrometry.Methods We aimed to integrate the Ig-seq platform, initially developed by Georgiou and others, 2 for investigating the circulating antibody repertoire elicited by vaccination. Blood samples were collected from healthy donors (Providence cohort) and cancer patients (SIIREN study, OSU cohort3) who had received the SARS-CoV-2 S-protein mRNA vaccine. Single-cell B cell receptor sequencing (scBCR-seq) was performed on antigen-specific B cells. Circulating S-protein-specific antibodies were isolated by affinity purification using an antigen-affinity column. We digested the recovered antibodies using trypsin for the mass spec analysis. The resulting mass spectra were analyzed using PEAKS with a donor-specific custom database that encompassed donor-specific BCR sequencing, in addition to the UniProt proteome database.Results scBCR-seq analyses of healthy donors revealed the evolution of S-protein-specific memory B cells at various time points with different durations of persistence. We then analyzed the repertoire of antibodies that bind to the S-protein. As expected, minimal S-protein-reactive antibodies were detected before vaccination, suggesting limited potential for cross-reactivity of pre-existing anti-common cold coronavirus antibodies against the SARS-CoV-2 S-protein. We observed de novo antibody production upon the primary series of vaccines. The booster vaccine elicited the reemergence of antibodies temporarily induced by the primary series and also induced new SARS-CoV-2 S-protein-reactive antibodies. The breadth of antibodies generated after the booster vaccine was much broader than that of those elicited by the primary series.Conclusions Our next step will be to optimize the assay further, 1) by improving the coverage of CDR3 by adding digesting antibodies, and 2) by bioinformatically recovering potential BCR sequences. We will then compare repertoire dynamics between healthy donors and cancer patients. Ultimately, we envision that this platform allows us to characterize the tumor-reactive antibody repertoire using patient-derived cancer cell lines and antibodies from intra-tumoral B cells.References Paul S, Konig MF, Pardoll DM, Bettegowda C, Papadopoulos N, Wright KM, Gabelli SB, Ho M, van Elsas A, Zhou S. Cancer therapy with antibodies. Nat Rev Cancer 2024;24:399–426. 10.1038/s41568-024-00690-x.Georgiou G, Ippolito GC, Beausang J, Busse CE, Wardemann H, Quake SR. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat Biotechnol 2014;32:158–168. 10.1038/nbt.2782.Azar JH, Evans JP, Sikorski MH, Chakravarthy KB, McKenney S, Carmody I, Zeng C, Teodorescu R, Song NJ, Hamon JL, et al. Selective suppression of de novo SARS-CoV-2 vaccine antibody responses in patients with cancer on B cell-targeted therapy. JCI Insight 2023;8. 10.1172/jci.insight.163434.",
  "authors": [
    {
      "affiliations": [
        "Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA"
      ],
      "name": "Gaurav Shrestha"
    },
    {
      "affiliations": [
        "Shimadzu Corporation, Kyoto, Japan"
      ],
      "name": "Noriko Iwamoto"
    },
    {
      "affiliations": [
        "Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "Lauren Hamilton"
    },
    {
      "affiliations": [
        "Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "Tanisha L Christie"
    },
    {
      "affiliations": [
        "Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "P Dominick Sanders"
    },
    {
      "affiliations": [
        "Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "Brian Piening"
    },
    {
      "affiliations": [
        "Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "Bernard A Fox"
    },
    {
      "affiliations": [
        "Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR, USA"
      ],
      "name": "William L Redmond"
    },
    {
      "affiliations": [
        "Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA",
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Jordan Krull"
    },
    {
      "affiliations": [
        "Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA",
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Qin Ma"
    },
    {
      "affiliations": [
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Mitch A Phelps"
    },
    {
      "affiliations": [
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Zhiwei Hu"
    },
    {
      "affiliations": [
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Zihai Li"
    },
    {
      "affiliations": [
        "Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA",
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Mark P Rubinstein"
    },
    {
      "affiliations": [
        "Shimadzu Corporation, Kyoto, Japan"
      ],
      "name": "Takashi Shimada"
    },
    {
      "affiliations": [
        "The Ohio State University, Columbus, OH, USA"
      ],
      "name": "Yoshinobu Koguchi"
    }
  ],
  "title": "192 Development of the proteogenomic antibody repertoire assay for anti-tumor antibody discovery",
  "uid": "12309413-a4f9-536b-8764-a285535c3f60"
}