Upregulation of surface expressed sialoglycans on tumor cells is one of the mechanisms which promote tumor growth and progression. Specifically, the interactions of sialic acids with sialic acid-binding immunoglobulin-like lectins (Siglecs) on lymphoid or myeloid cells transmit inhibitory signals and lead to suppression of anti-tumor responses. Here, we show that neutrophils express among others Siglec-9, and that EGFR and HER2 positive breast tumor cells express ligands for Siglec-9. Treatment of tumor cells with neuraminidases or a sialyl transferase inhibitor significantly reduced binding of a soluble recombinant Siglec-9-Fc fusion protein, while EGFR and HER2 expression remained unchanged. Importantly, the cytotoxic activity of neutrophils driven by therapeutic EGFR or HER2 antibodies in vitro was increased by blocking the sialic acid/Siglec interaction, either by reducing tumor cell sialylation or by a Siglec-9 blocking antibody containing an effector silenced Fc domain. In vivo a short-term xenograft mouse model confirmed the improved therapeutic efficacy of EGFR antibodies against sialic acid depleted, by a sialyltransferase inhibitor, tumor cells compared to untreated cells. Our studies demonstrate that sialic acid/Siglec interactions between tumor cells and myeloid cells can impair antibody dependent tumor cell killing, and that Siglec-9 on polymorphonuclear cells (PMN) is critically involved. Considering that PMN are often a highly abundant cell population in the tumor microenvironment, Siglec-9 constitutes a promising target for myeloid checkpoint blockade to improve antibody-based tumor immunotherapy.

Immunotherapy with therapeutic antibodies has shown a lack of durable responses in some patients due to resistance mechanisms. Checkpoint molecules expressed by tumor cells have a deleterious impact on clinical responses to therapeutic antibodies. Myeloid checkpoints, which negatively regulate macrophage and neutrophil anti-tumor responses, are a novel type of checkpoint molecule. Myeloid checkpoint inhibition is currently being studied in combination with IgG-based immunotherapy. In contrast, the combination with IgA-based treatment has received minimal attention. IgA antibodies have been demonstrated to more effectively attract and activate neutrophils than their IgG counterparts. Therefore, myeloid checkpoint inhibition could be an interesting addition to IgA treatment and has the potential to significantly enhance IgA therapy.

Despite the curative potential of checkpoint blockade immunotherapy, the majority of patients remain unresponsive to existing treatments. Glyco-immune checkpoints — interactions of cell-surface glycans with lectin, or glycan binding, immunoreceptors — have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a tumor-targeting arm as well as a lectin ″decoy receptor″ domain that directly bind tumor glycans and block their ability to engage lectin receptors on immune cells. AbLecs elicited tumor killing in vitro via macrophage phagocytosis and NK cell and granulocyte cytotoxicity, matching or outperforming combinations of monospecific antibodies with lectin-blocking or glycan-disrupting therapies. Furthermore, AbLecs synergized with blockade of the ″don′t eat me″ signal CD47 for enhanced tumor killing. AbLecs can be readily designed to target numerous tumor-associated antigens and glyco-immune checkpoint ligands, and therefore represent a new modality for cancer immune therapy.

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.

The addition of monoclonal antibodies daratumumab, elotuzumab and isatuximab to the treatment of patients with multiple myeloma significantly improved the outcome and prolonged survival. Unfortunately, although many patients benefit, depth and duration of response are a problem. In order to improve efficacy of antibody-based immunotherapy, we aimed to combine CD38-directed antibodies daratumumab and isatuximab as well as SLAMF7-targeting elotuzumab with a CD47 blocking antibody to enhance phagocytosis of myeloma cells. Antibody-dependent cellular phagocytosis (ADCP) of malignant plasma cells is described to be one important mode of action of daratumumab, isatuximab and elotuzumab, respectively. Of note, CD47 is highly expressed on myeloma cells and allows evading immune recognition by myeloid cells, i.e. monocytes, macrophages and neutrophils. Binding of CD47 to SIRPα expressed on myeloid cells provides a strong 'don't eat me' signal and diminishes phagocytosis of tumor cells. Blocking the CD47-SIRPα axis, by a monoclonal antibody against CD47 or a SIRPα-Fc fusion protein can restore recognition of tumor cells by macrophages and enhance phagocytosis. In patients with Non-Hodgkin's lymphoma the combination of CD20 antibody rituximab with CD47 antibody magrolimab was clinically successful (Advani et al., NEJM 379:1711, 2018). To test the applicability of blocking the CD47-SIRPα axis and improve ADCP of myeloma cells by CD38-targeting or SLAMF7-directed myeloma antibodies, we generated a CD47 IgG2σ antibody carrying an engineered Fc domain not binding to Fcγ receptors (FcγR). This CD47 antibody was subsequently used in phagocytosis experiments in combination with antibodies daratumumab, isatuximab as well as elotuzumab and various myeloma cell lines. The cell lines AMO-1, JK-6L, L363, RPMI-8226, and U266 express different levels of CD47, CD38 and SLAMF7 as determined by quantitative flow cytometry. M0 macrophages expressing FcγRs were generated from healthy donor PBMC monocytes by cultivation with M-CSF for 10-14 days prior use in 6 hour real-time live cell imaging phagocytosis experiments with pHrodo-labeled myeloma cells - turning red only when engulfed by macrophages. Macrophages and myeloma cells were used at an effector-to-target cell ratio of 1:1. Importantly, ADCP of myeloma cells induced by all three monoclonal antibodies, daratumumab, isatuximab or elotuzumab, can be enhanced by the addition of the CD47 blocking antibody. However, improvement in phagocytosis strongly differs between myeloma cell lines although all have high CD47 level on their cell surface. In responsive myeloma cell lines, ADCP mediated by CD38 antibodies daratumumab or isatuximab was found more efficient than that by SLAMF7 antibody elotuzumab. This may be related to the significantly higher CD38 than SLAMF7 expression at the myeloma cell surface. Our findings demonstrate that ADCP of approved IgG antibodies targeting CD38 or SLAMF7 can be enhanced by blocking the CD47-SIRPα axis and this may depend on the particular malignant plasma cell phenotype. The inhibition of this myeloid 'don't eat me' signal with a CD47 blocking antibody may open a new avenue for powerful myeloma immunotherapy. Since combination treatments with proteasome inhibitors and IMiDs are commonly used, these interactions also require attention. Initial data indicate that pre-treatment of myeloma cells with proteasome inhibitor carfilzomib did not negatively impact improvement of ADCP by blocking the CD47-SIRPα axis in responsive cell lines. Taken together, particularly CD38-targeting antibodies may have a significant potential to further improve immunotherapy in multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.