Plasmacytic markers as immunotherapeutic targets for primary CD20 ‐negative aggressive B‐cell lymphomas

Aggressive B-cell lymphomas (B-NHLs) that lose classical B-cell markers pose major therapeutic challenges. Among them, plasmablastic lymphoma (PBL), primary effusion lymphoma (PEL), KSHV/HHV8-positive diffuse large B-cell lymphoma (DLBCL) and anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma represent rare and often treatment-refractory subtypes.1-4 These entities are often associated with immune deficiency and characterized by downregulation of the transcription factors Paired Box Protein 5 (PAX5) and B-Cell Lymphoma 6 (BCL-6) and subsequent loss of CD19 and CD20 precluding the use of rituximab or other CD20- or CD19-directed therapies. Morphologically and phenotypically, they share features of plasmacytic/blastic differentiation and thus commonly express CD38, Signaling Lymphocytic Activation Molecule Family Member 7 (SLAMF7) (CD319) and B-cell maturation antigen (BCMA/CD269), caused by upregulation of Interferon Regulatory Factor 4 (IRF4) and B-lymphocyte-induced maturation protein 1 (BLIMP1).5 Targeting these plasmacytic features by bortezomib6, 7 or by the addition of CD38 antibodies has been studied in clinical case series and early clinical trials for PBL.8, 9 Furthermore, translational studies investigated SLAMF7 as target,10 but all these strategies do not represent a current established standard. The aim of the present study was to systematically examine the therapeutic potential of targeting plasmacytic antigens in primary CD20-negative aggressive B-NHL. We evaluated whether clinically approved immunotherapeutic antibodies for multiple myeloma—namely anti-CD38 (daratumumab, isatuximab),11, 12 anti-SLAMF7 (elotuzumab)13 or the bispecific BCMA×CD3 antibody (teclistamab)14—exert in vitro cytotoxic activity against CD20-negative aggressive B-cell lymphoma cell lines, with a focus on PEL, because the largest number of established cell lines is available for this subtype, but also limiting the transferability of the analyses to other primarily CD20-negative entities. Furthermore, we explored whether pharmacologic activation of immune effector cells by lenalidomide or vitamin D supplementation could enhance antibody-dependent cellular cytotoxicity (ADCC). Four well-characterized PEL cell lines (BC-3, BCBL-1, CRO-AP2, CRO-AP5) and control lines of CD20+ DLBCL and Burkitt lymphoma were analysed by flow cytometry for surface expression of CD19, CD20, CD79b, CD38, SLAMF7 and BCMA. The multiple myeloma cell line NCI-H929 served as a positive control for BCMA expression. In vitro ADCC assays were performed using natural killer (NK) cells isolated untouched from healthy donors as effector cells. Target cells were preincubated with daratumumab, isatuximab or elotuzumab at concentrations ranging from 0.0001 to 1 μg/mL. To assess the impact of effector cell modulation, peripheral blood mononuclear cells (PBMCs) were treated in vitro with lenalidomide (1 μM, 24 h) prior to NK isolation, and in selected donors, vitamin D3 supplementation in vivo was performed to reach serum 25-OH-vitamin D3 levels ≥65 ng/mL.15 For BCMA × CD3 bispecific antibody assays, untouched isolated T cells or CD16+ NK cells from healthy donors were used as effectors at an effector-to-target ratio of 10:1 and 5:1. Flow cytometry confirmed that all four PEL cell lines lacked CD20 expression, consistent with their plasmablastic origin. CD19 and CD79b were only very weakly expressed (Figure 1.1). In contrast, strong and uniform expression of CD38 and SLAMF7 and moderate but consistent BCMA expression were observed across all lines. Compared with the NCI-H929 myeloma control, PEL cells expressed lower BCMA (CD269) surface levels (Figure S1), yet the presence of CD38, SLAMF7 and BCMA provided potential alternative immunotherapeutic targets. The very weak expression of CD19 correlates with the absence of significant NK-cell activity under tafasitamab regardless of the dose and the PEL cell line (Figure 1.2). The same applies to CD79b and polatuzumab vedotin. A concentration of 1 μg/mL of polatuzumab vedotin significantly inhibited BCBL-1 proliferation by 10%, compared to untreated cells (Figure 1.3), but this relatively weak inhibition only applied to BCBL-1. Consistent with the weak expression of CD19 and CD79b, they are not promising targets for immunotherapy of PEL. Daratumumab and isatuximab induced dose-dependent NK-cell cytotoxicity proportional to CD38 surface density (Figure 1.4). Daratumumab was slightly more effective, reaching lysis rates of up to 15%–25% at 1 μg/mL, whereas slightly lower lysis rates were achieved with isatuximab at comparable concentrations (Figure 1.4, Figure S2.1–3). Elotuzumab-induced NK-cell activity varied. While less than 20% of BCL-1, BC-3 and CRO-AP2 cells was lysed even at 1 μg/mL, this antibody induced lysis rates of up to 50% against CRO-AP5 (Figure 1.5). This modest lysis in SLAMF7+ lines is consistent with its reliance on NK-cell activation rather than direct ADCC (Figure 1.6). Although not strongly expressed on PEL cells, BCMA/CD269 was a somewhat more reliable target. Thus, the BCMA/CD3-bispecific BiTE teclistamab induced a significant, T-cell-mediated ADCC of almost 25% against BCBL-1 at a dose of 0.0001 μg/mL (Figure 1.7). Against BC-3, the subjects' T cells lysed up to 17% of these PEL cells in a concentration-dependent manner starting at 0.001 μg/mL teclistamab (Figure 1.6). Interestingly, despite its Fc-silenced design, teclistamab induced comparable in vitro cytotoxicity when CD16+ NK cells were used as effectors, mirroring the T-cell response pattern (Figure S3.1 however, we tested not the combination with lenalidomide for this antibody. The enhancement of ADCC by lenalidomide and, to a lower extent, by vitamin D supports prior observations of immune effector modulation and suggests clinically achievable strategies to improve antibody efficacy in these lymphoma subtypes. However, the previously described effect of vitamin D substitution on ADCC remains controversial, as in indolent lymphoma, the ILyAD trial did not report a benefit, and the results of the OPTIMAL > 60 trial regarding vitamin D substitution are awaited. The consistent in vitro activity of the BCMA × CD3 bispecific antibody further expands the spectrum of potential targets. While BCMA expression was lower in PEL than in myeloma, T-cell–mediated lysis was still significant, implying that even moderate antigen density may suffice for therapeutic engagement. The observation that NK cells can also mediate BCMA-directed cytotoxicity despite Fc silencing of teclistamab suggests secondary mechanisms of activation or residual Fc interactions. A limitation of this study is that G-protein-coupled receptor family C group 5 member D (GPRC5D), the target of another bispecific antibody approved for multiple myeloma, was not investigated. From a translational perspective, our data indicate that PEL and hypothetically also other primary CD20-negative aggressive B-cell NHL entities might benefit from therapeutic strategies already approved for multiple myeloma. Our results show that anti-CD38, anti-SLAMF7 and BCMA × CD3 antibodies or BiTEs, respectively, alone or combined with lenalidomide or vitamin D exhibit strong in vitro cytotoxic activity against primary CD20-negative aggressive B-cell lymphomas. The combination of anti-CD38 antibodies with IMiDs and chemotherapy as CHO(E)P/EPOCH,9 or the use of BCMA-directed bispecific antibodies, could represent rational approaches for evaluation in early-phase clinical trials for patients with either therapy-naïve or relapsed/refractory disease, as conventional B-cell–directed immunotherapy fails. The findings establish a functional rationale for repurposing multiple myeloma immunotherapies in these rare, but high-risk lymphoma subgroups, addressing a major unmet clinical need. Moritz Bewarder: Writing – review and editing; data curation. Claudia Schormann: Methodology; investigation; formal analysis; data curation; writing – review and editing. Stefan Lohse: Data curation; methodology; writing – review and editing. Sigrun Smola: Writing – review and editing; methodology. Igor A. Kos: Data curation; writing – review and editing. Frank Neumann: Conceptualization; methodology; formal analysis; validation; investigation; visualization; writing – original draft; writing – review and editing; data curation. Joerg T. Bittenbring: Writing – review and editing. Philipp B. Staber: Writing – review and editing; resources; funding acquisition. Onur Cetin: Writing – review and editing; data curation. Eva C. Schwarz: Data curation; writing – review and editing. Markus Hoth: Data curation; writing – review and editing; funding acquisition; conceptualization. Stephanie Maurer: Data curation; writing – review and editing. Lorenz Thurner: Conceptualization; methodology; formal analysis; visualization; writing – review and editing; writing – original draft; resources; funding acquisition. Konstantinos Christofyllakis: Writing – review and editing; data curation. Open Access funding enabled and organized by Projekt DEAL. Funding was provided by the Schwiete Foundation. Data are available upon request. Figure S1. Figure S2. Figure S3. Table S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.