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Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

IntroductionSeveral CD19 targeted antibody-based therapeutics are currently available for patients with diffuse large B-cell lymphoma (DLBCL), including the Fc-modified antibody immunotherapy tafasitamab. This therapeutic landscape warrants the evaluation of potential sequencing approaches. Prior to a subsequent CD19-targeted therapy, CD19 expression on tafasitamab-treated patient biopsy samples may be assessed. However, no standardized methods for its detection are currently available. In this context, selecting a tafasitamab-competing CD19 detection antibody for immunohistochemistry (IHC) or flow cytometry (FC) may lead to misinterpreting epitope masking by tafasitamab as antigen loss or downregulation.MethodsWe analyzed a comprehensive panel of commercially available CD19 detection antibody clones for IHC and FC using competition assays on tafasitamab pre-treated cell lines. To remove bound tafasitamab from the cell surface, an acidic dissociation protocol was used. Antibody affinities for CD19 were measured using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI).ResultsWhile CD19 was successfully detected on tafasitamab pre-treated samples using all 7 tested IHC antibody clones, all 8 tested FC antibody clones were confirmed to compete with tafasitamab. An acidic dissociation was demonstrated essential to circumvent CD19 masking by tafasitamab and avoid false negative FC results.DiscussionThe current study highlights the importance of selecting appropriate CD19 detection tools and techniques for correct interpretation of CD19 expression. The findings presented herein can serve as a guideline to investigators and may help navigate treatment strategies in the clinical setting.

BackgroundDespite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects.MethodsAntibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).ResultsThree different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model.ConclusionThis study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo.

Background: Antibody Fc-driven engagement of macrophages is critical for evoking cellular activation and effector functions and influencing tumour-associated macrophage (TAM) recruitment. We previously reported that IgE class antibodies promote restriction of cancer growth in rodent models associated with significant TAM infiltration. However, the human macrophage-associated IgE-Fc Receptor (FcεR) axis remains unexplored. We investigated the effects of anti-tumour IgE stimulation on human macrophage activation. Methods: Human blood monocyte-differentiated quiescent (M0), classically-(M1) and alternatively-(M2) activated macrophages were crosslinked with IgE and polyclonal antibodies to mimic immune complex formation. We examined surface marker expression, cytokine secretion, protein kinase phosphorylation and gene expression in IgE-stimulated macrophages and IgE antibody-dependent macrophage-mediated cytotoxicity (ADCC) against tumour cells. Findings: A proportion (40%) of M2 and (