Novel chimerized IgA CD20 antibodies : Improving neutrophil activation against CD20-positive malignancies

ABSTRACT Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different in vivo mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing in vitro, both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.Peer reviewe

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

Treatment strategies and clinical outcomes in consecutive patients with locally advanced pancreatic cancer:A multicenter prospective cohort

Introduction: Since current studies on locally advanced pancreatic cancer (LAPC) mainly report from single, high-volume centers, it is unclear if outcomes can be translated to daily clinical practice. This study provides treatment strategies and clinical outcomes within a multicenter cohort of unselected patients with LAPC. Materials and methods: Consecutive patients with LAPC according to Dutch Pancreatic Cancer Group criteria, were prospectively included in 14 centers from April 2015 until December 2017. A centralized expert panel reviewed response according to RECIST v1.1 and potential surgical resectability. Primary outcome was median overall survival (mOS), stratified for primary treatment strategy. Results: Overall, 422 patients were included, of whom 77% (n = 326) received chemotherapy. The majority started with FOLFIRINOX (77%, 252/326) with a median of six cycles (IQR 4-10). Gemcitabine monotherapy was given to 13% (41/326) of patients and nab-paclitaxel/gemcitabine to 10% (33/326), with a median of two (IQR 3-5) and three (IQR 3-5) cycles respectively. The mOS of the entire cohort was 10 months (95%CI 9-11). In patients treated with FOLFIRINOX, gemcitabine monotherapy, or nab-paclitaxel/gemcitabine, mOS was 14 (95%CI 13-15), 9 (95%CI 8-10), and 9 months (95%CI 8-10), respectively. A resection was performed in 13% (32/252) of patients after FOLFIRINOX, resulting in a mOS of 23 months (95%CI 12-34). Conclusion: This multicenter unselected cohort of patients with LAPC resulted in a 14 month mOS and a 13% resection rate after FOLFIRINOX. These data put previous results in perspective, enable us to inform patients with more accurate survival numbers and will support decision-making in clinical practice. (C) 2020 The Authors. Published by Elsevier Ltd

Causes and consequences of delayed diagnosis in breast cancer screening with a focus on mammographic features and tumour characteristics

PURPOSE: To study the prevalence, causes and consequences of delayed breast cancer diagnosis in the screening population. METHODS: This retrospective study was performed in women who underwent biennial screening mammography between January 1, 2009 and June 30, 2019. Patients were divided into 3 groups; screen-detectedbreast cancer (SDC) without a diagnostic delay, a primary diagnostic delay(i.e. missed cancer at previous screening round)and a delay in diagnostic work-up after recall. Women with a true interval cancer (IC; i.e. not visible on prior examinations) were excluded. Outcome parameters included mammographic and tumour characteristics, lymph node status and surgical treatment. RESULTS: In our sample of 4491 women with breast cancer (4292 SDC and 199 'missed' IC), respectively, a total of 1112 women experienced a diagnostic delay of = 4 months. Compared to women without a diagnostic delay (n = 2720), the 176 women with a delay in diagnostic work-up showed overall similar mammographic abnormalities (P = 0.052). These groups show similar distributions in invasive tumours, tumour stage and lymph node status (P = 0.25, P = 0.95 and P = 0.93, respectively). Women with a primary diagnostic delay (n = 936) showed less calcifications (P < 0.001), and more masses with calcifications and architectural distortions on mammography (P = 0.01 and P = 0.04, respectively). Moreover, this group comprised larger tumours (P < 0.001) and lymph node metastases (P < 0.001), and more often underwent mastectomy (P < 0.001). CONCLUSIONS: A primary diagnostic delay in breast cancer diagnosis results in less favourable tumour characteristics and relatively more mastectomies compared to no delay in breast cancer diagnosis and a delay in diagnostic work-up after recall

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

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action

Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG

Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab')(2)-mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (Fc alpha RI). In addition to monocytes, macrophages and eosinophils as Fc alpha RI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. Fc alpha RI expression on neutrophils is similar to 2 times and similar to 20 times lower than that of Fc gamma receptors Fc gamma RIIa and Fc gamma RIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA- mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the Fc alpha R/FcR gamma-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for Fc gamma RIIa, combined with a possible decoy role of the highly expressed Fc gamma RIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment

Fc gamma receptor IIIA genotype is associated with rituximab response in antimyelin-associated glycoprotein neuropathy

Background Treatment with anti-B cell antibody rituximab may ameliorate the disease course in a subgroup of patients with polyneuropathy associated with IgM monoclonal gammopathy. Polymorphisms of leukocyte IgG receptors (Fc gamma R) that influence efficiency of antibody-dependent cell-mediated cytotoxicity determine rituximab efficacy in patients with lymphoma and autoimmune disease. Objective To investigate the association of Fc gamma RIIA and Fc gamma RIIIA polymorphisms with the response to rituximab treatment in a cohort of patients with polyneuropathy associated with IgM monoclonal gammopathy (PNP-IgM) with and without antimyelin-associated glycoprotein antibodies. Methods We determined Fc gamma RIIA-R/H131 and Fc gamma RIIIA-V/F158 genotypes in 27 patients with PNP-IgM using allele-specific PCR and Sanger sequencing. Results The Fc gamma RIIIA-V/V158 genotype was associated with functional improvement (p=0.02) after 1 year. Conclusions Fc gamma RIIIA polymorphisms are potential biomarkers for response to rituximab treatment in polyneuropathy associated with IgM monoclonal gammopath