Enhancing IgA-mediated neutrophil cytotoxicity against neuroblastoma by CD47 blockade.

BACKGROUND: Approximately half of the neuroblastoma patients develop high-risk neuroblastoma. Current treatment involves a multimodal strategy, including immunotherapy with dinutuximab (IgG ch14.18) targeting GD2. Despite achieving promising results, the recurrence rate remains high and poor survival persists. The therapeutic efficacy of dinutuximab is compromised by suboptimal activation of neutrophils and severe neuropathic pain, partially induced by complement activation. METHODS: To enhance neutrophil cytotoxicity, IgG ch14.18 was converted to the IgA isotype, resulting in potent neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), without complement activation. However, myeloid checkpoint molecules hamper neutrophil cytotoxicity, for example through CD47 that is overexpressed on neuroblastomas and orchestrates an immunosuppressive environment upon ligation to signal regulatory protein alpha (SIRPα) expressed on neutrophils. In this study, we combined IgA therapy with CD47 blockade. RESULTS: In vitro killing assays showed enhanced IgA-mediated ADCC by neutrophils targeting neuroblastoma cell lines and organoids in comparison to IgG. Notably, when combined with CD47 blockade, both IgG and IgA therapy were enhanced, though the combination with IgA resulted in the greatest improvement of ADCC. Furthermore, in a neuroblastoma xenograft model, we systemically blocked CD47 with a SIRPα fusion protein containing an ablated IgG1 Fc, and compared IgA therapy to IgG therapy. Only IgA therapy combined with CD47 blockade increased neutrophil influx to the tumor microenvironment. Moreover, the IgA combination strategy hampered tumor outgrowth most effectively and prolonged tumor-specific survival. CONCLUSION: These promising results highlight the potential to enhance immunotherapy efficacy against high-risk neuroblastoma through improved neutrophil cytotoxicity by combining IgA therapy with CD47 blockade.

IgA antibody immunotherapy targeting GD2 is effective in preclinical neuroblastoma models.

BACKGROUND: Immunotherapy targeting GD2 is very effective against high-risk neuroblastoma, though administration of anti-GD2 antibodies induces severe and dose-limiting neuropathic pain by binding GD2-expressing sensory neurons. Previously, the IgG1 ch14.18 (dinutuximab) antibody was reformatted into the IgA1 isotype, which abolishes neuropathic pain and induces efficient neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) via activation of the Fc alpha receptor (FcαRI/CD89). METHODS: To generate an antibody suitable for clinical application, we engineered an IgA molecule (named IgA3.0 ch14.18) with increased stability, mutated glycosylation sites and substituted free (reactive) cysteines. The following mutations were introduced: N45.2G and P124R (CH1 domain), C92S, N120T, I121L and T122S (CH2 domain) and a deletion of the tail piece P131-Y148 (CH3 domain). IgA3.0 ch14.18 was evaluated in binding assays and in ADCC and antibody-dependent cellular phagocytosis (ADCP) assays with human, neuroblastoma patient and non-human primate effector cells. We performed mass spectrometry analysis of N-glycans and evaluated the impact of altered glycosylation in IgA3.0 ch14.18 on antibody half-life by performing pharmacokinetic (PK) studies in mice injected intravenously with 5 mg/kg antibody solution. A dose escalation study was performed to determine in vivo efficacy of IgA3.0 ch14.18 in an intraperitoneal mouse model using 9464D-GD2 neuroblastoma cells as well as in a subcutaneous human xenograft model using IMR32 neuroblastoma cells. Binding assays and PK studies were compared with one-way analysis of variance (ANOVA), ADCC and ADCP assays and in vivo tumor outgrowth with two-way ANOVA followed by Tukey's post-hoc test. RESULTS: ADCC and ADCP assays showed that particularly neutrophils and macrophages from healthy donors, non-human primates and patients with neuroblastoma are able to kill neuroblastoma tumor cells efficiently with IgA3.0 ch14.18. IgA3.0 ch14.18 contains a more favorable glycosylation pattern, corresponding to an increased antibody half-life in mice compared with IgA1 and IgA2. Furthermore, IgA3.0 ch14.18 penetrates neuroblastoma tumors in vivo and halts tumor outgrowth in both 9464D-GD2 and IMR32 long-term tumor models. CONCLUSIONS: IgA3.0 ch14.18 is a promising new therapy for neuroblastoma, showing (1) increased half-life compared to natural IgA antibodies, (2) increased protein stability enabling effortless production and purification, (3) potent CD89-mediated tumor killing in vitro by healthy subjects and patients with neuroblastoma and (4) antitumor efficacy in long-term mouse neuroblastoma models.

Sialic Acids on Tumor Cells Modulate IgA Therapy by Neutrophils via Inhibitory Receptors Siglec-7 and Siglec-9.

Immunotherapy with targeted therapeutic antibodies is often ineffective in long-term responses in cancer patients due to resistance mechanisms such as overexpression of checkpoint molecules. Similar to T lymphocytes, myeloid immune cells express inhibitory checkpoint receptors that interact with ligands overexpressed on cancer cells, contributing to treatment resistance. While CD47/SIRPα-axis inhibitors in combination with IgA therapy have shown promise, complete tumor eradication remains a challenge, indicating the presence of other checkpoints. We investigated hypersialylation on the tumor cell surface as a potential myeloid checkpoint and found that hypersialylated cancer cells inhibit neutrophil-mediated tumor killing through interactions with sialic acid-binding immunoglobulin-like lectins (Siglecs). To enhance antibody-dependent cellular cytotoxicity (ADCC) using IgA as therapeutic, we explored strategies to disrupt the interaction between tumor cell sialoglycans and Siglecs expressed on neutrophils. We identified Siglec-9 as the primary inhibitory receptor, with Siglec-7 also playing a role to a lesser extent. Blocking Siglec-9 enhanced IgA-mediated ADCC by neutrophils. Concurrent expression of multiple checkpoint ligands necessitated a multi-checkpoint-blocking approach. In certain cancer cell lines, combining CD47 blockade with desialylation improved IgA-mediated ADCC, effectively overcoming resistance that remained when blocking only one checkpoint interaction. Our findings suggest that a combination of CD47 blockade and desialylation may be necessary to optimize cancer immunotherapy, considering the upregulation of checkpoint molecules by tumor cells to evade immune surveillance.

Disruption of the sialic acid/Siglec-9 axis improves antibody-mediated neutrophil cytotoxicity towards tumor cells.

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.

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody.

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

Corrigendum: Targeting myeloid checkpoint molecules in combination with antibody therapy: A novel anti-cancer strategy with IgA antibodies?

[This corrects the article DOI: 10.3389/fimmu.2022.932155.].

Targeting Myeloid Checkpoint Molecules in Combination With Antibody Therapy: A Novel Anti-Cancer Strategy With IgA Antibodies?

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.

Anti-GD2 IgA kills tumors by neutrophils without antibody-associated pain in the preclinical treatment of high-risk neuroblastoma.

BACKGROUND: The addition of monoclonal antibody therapy against GD2 to the treatment of high-risk neuroblastoma led to improved responses in patients. Nevertheless, administration of GD2 antibodies against neuroblastoma is associated with therapy-limiting neuropathic pain. This severe pain is evoked at least partially through complement activation on GD2-expressing sensory neurons. METHODS: To reduce pain while maintaining antitumor activity, we have reformatted the approved GD2 antibody ch14.18 into the IgA1 isotype. This novel reformatted IgA is unable to activate the complement system but efficiently activates leukocytes through the FcαRI (CD89). RESULTS: IgA GD2 did not activate the complement system in vitro nor induced pain in mice. Importantly, neutrophil-mediated killing of neuroblastoma cells is enhanced with IgA in comparison to IgG, resulting in efficient tumoricidal capacity of the antibody in vitro and in vivo. CONCLUSIONS: Our results indicate that employing IgA GD2 as a novel isotype has two major benefits: it halts antibody-induced excruciating pain and improves neutrophil-mediated lysis of neuroblastoma. Thus, we postulate that patients with high-risk neuroblastoma would strongly benefit from IgA GD2 therapy.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Fc Engineering Strategies to Advance IgA Antibodies as Therapeutic Agents.

In the past three decades, a great interest has arisen in the use of immunoglobulins as therapeutic agents. In particular, since the approval of the first monoclonal antibody Rituximab for B cell malignancies, the progress in the antibody-related therapeutic agents has been incremental. Therapeutic antibodies can be applied in a variety of diseases, ranging from cancer to autoimmunity and allergy. All current therapeutic monoclonal antibodies used in the clinic are of the IgG isotype. IgG antibodies can induce the killing of cancer cells by growth inhibition, apoptosis induction, complement activation (CDC) or antibody-dependent cellular cytotoxicity (ADCC) by NK cells, antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages, or trogoptosis by granulocytes. To enhance these effector mechanisms of IgG, protein and glyco-engineering has been successfully applied. As an alternative to IgG, antibodies of the IgA isotype have been shown to be very effective in tumor eradication. Using the IgA-specific receptor FcαRI expressed on myeloid cells, IgA antibodies show superior tumor-killing compared to IgG when granulocytes are employed. However, reasons why IgA has not been introduced in the clinic yet can be found in the intrinsic properties of IgA posing several technical limitations: (1) IgA is challenging to produce and purify, (2) IgA shows a very heterogeneous glycosylation profile, and (3) IgA has a relatively short serum half-life. Next to the technical challenges, pre-clinical evaluation of IgA efficacy in vivo is not straightforward as mice do not naturally express the FcαR. Here, we provide a concise overview of the latest insights in these engineering strategies overcoming technical limitations of IgA as a therapeutic antibody: developability, heterogeneity, and short half-life. In addition, alternative approaches using IgA/IgG hybrid and FcαR-engagers and the impact of engineering on the clinical application of IgA will be discussed.