Harnessing tumor-informed B cell receptors for discovery and antibody-based immunotherapy in sarcoma

Background: Soft tissue sarcomas (STS) are rare and heterogenous cancers, with limited effective treatments and poor prognosis. While some patients with STS respond to immune checkpoint blockade (ICB), the majority do not benefit. This highlights an urgent need to elucidate mechanisms of response and resistance to ICB and develop novel immunotherapeutic approaches for patients with STS. We and others have shown that tumor infiltrating B cells (TIL-Bs), particularly those in tertiary lymphoid structures (TLS) are associated with response to ICB and longer survival. However, the antigen specificity, mechanisms of action, and therapeutic potential of these TIL-Bs and their B cell receptors (BCRs) remain unknown. Methods: We recently completed a phase 2 clinical trial evaluating ICB administered in the neoadjuvant setting in patients with resectable undifferentiated pleomorphic sarcoma (UPS) of the extremity/trunk or dedifferentiated liposarcoma (DDLPS) of the retroperitoneum (Roland et al. Nature Cancer 2024). Leveraging a unique biospecimen resource from this trial, we reconstructed intratumoral BCR heavy and light chain (V H and V L ) repertoires using pre-treatment baseline tumor biopsies (n = 27), RNA-sequencing, and an in-house pipeline including TRUST4. We then performed single intratumoral B cell immunoglobulin sequencing using cell suspensions from 4 TLS-positive tumors to reconstruct and express 105 recombinant BCR antibodies (rBCR Abs) as IgG1. Flow cytometry (FC) was performed to characterize the binding features of a subset of 20 rBCR Abs with human DDLPS (LPS224, LPS246) and UPS cell lines (UPS186, RIS819.1). Binding was quantified as the ratio of the mean fluorescent intensity (MFI) of the antibody to the MFI of the isotype control for each cell line. Abs with an MFI ratio ≥ 1.2 were classified as binders and tumor surface binding was further assessed by live cell microscopy. Antibody-dependent cellular cytotoxicity (ADCC) assays were performed to assess the functional capacity of this cohort of rBCR Abs to mediate NK-cell dependent tumor killing. The anti-tumor efficacy of 1 lead rBCR Ab was assessed using an MCA205 immunocompetent murine STS model. Results: Of 105 rBCR Abs screened, 23 (22%) exhibited reproducible surface binding to at least one sarcoma cell line by FC. Among these, 13 (12%) bound LPS lines and 18 (17%) bound UPS lines. To date, 20 rBCR Abs have been confirmed to bind the surface of sarcoma cell lines by live cell staining of which 5 mediated tumor cell killing in vitro. One candidate rBCR Ab has been evaluated in vivo, localizing to tumor and synergized with PD-1/PD-L1 blockade to suppress tumor growth when administered intraperitoneally. Conclusion: These findings highlight the potential of rBCR Abs to serve as a novel therapeutic strategy and as tools to elucidate the roles of B cells and the BCR repertoire within the tumor immune microenvironment of patients with STS.