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

In the published article, there was an error in Figure 5 as published. The mitochondrion was misplaced in this figure. The corrected Figure 5 and its caption appear below The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

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

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

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

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

Clitocine reversal of P-glycoprotein associated multi-drug resistance through down-regulation of transcription factor NF-κB in R-HepG2 cell line.

Multidrug resistance (MDR) is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB

Reversal effect of clitocine in drug resistant cancer cells.

<p>(A) P-gp was detected in HepG2, R-HepG2, MES-SA and MES-SA/Dx5 cells by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040720#s2" target="_blank">Materials and Methods</a>. (B) After treatment with different concentrations of clitocine in R-HepG2 cells for 48 h, cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 6. (C) R-HepG2 cells were treated with different concentrations of doxorubicin together with 0, 0.05, 0.1, 0.2 or 0.4 µM clitocine respectively for 48 h. Cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (D) After treatment with different concentrations of clitocine in MES-SA/Dx5 cells for 48 h, cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (E) MES-SA/Dx5 cells were treated with different concentrations of doxorubicin together with 0, 0.05, 0.1, 0.2 or 0.4 µM clitocine respectively for 48 h. Cell viability was determined by MTT assay. Data are mean ± SD, <i>N</i> = 3. (F) Cellular doxorubicin accumulation level in R-HepG2 cells was measured by flow cytometry. The cells were incubated with 2 µM doxorubicin (Dox) alone or together with 0.2 µM clitocine for 24 h. The amount of doxorubicin accumulated in treated cells was quantified by flow cytometric method. (G) MDR1 mRNA level in R-HepG2 cells after clitocine treatment. After treatment with 0.2 µM clitocine for 48 h, total RNA in R-HepG2 cells was extracted. The MDR1 mRNA level in R-HepG2 cells was then measured by qRT-PCR analysis. Data are mean ± SD, <i>N</i> = 6. (H) The relative luciferase activity of full length MDR1 promoter reporter as determined by Dual-Luciferase Reporter Assay System. Cells were transiently transfected with full length MDR1 promoter reporter followed by treatment with 0.2 µM clitocine for 24 h. After that, the cells were lysed and the luciferase activity was measure. Data are mean ± SD, <i>N</i> = 6. ^*<i>P</i><0.05 vs. control, ^**<i>P</i><0.01 vs. control.</p

Truncation analysis of the MDR1 promoter with clitocine treatment.

<p>(A) The relative luciferase activity of 5′-end truncated MDR1 promoters was detected by Dual-Luciferase Reporter Assay System. R-HepG2 cells were transiently transfected with the truncated MDR1 promoter reporters followed by treatment with 0.2 µM clitocine for 24 h. After that, the cells were lysed and the luciferase activity was measured. Data are mean ± SD, <i>N</i> = 8. (B) Computational analysis of the putative transcription factor binding sites in the full length MDR1 promoter was performed. A single consensus binding site for transcription factor NF-κB was identified in the region −450 to −193. (C) R-HepG2 cells were transiently transfected with wild type (WT) and mutated type (MT) full length (−988∼+525) MDR1 promoter reporter followed by treatment with 0.2 µM clitocine in R-HepG2 cells for 24 h. After that, the cells were lysed and the luciferase activity was measured. Data are mean ± SD, <i>N</i> = 4. ^*<i>P</i><0.05 vs. control. (D) Immunoprecipitaition (CHIP) assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040720#s2" target="_blank">Materials and Methods</a>. Chromatin from R-HepG2 cells was cross-linked, sheard and immunoprecipitated with anti-NF-κB p65 antibody. Normal rabbit IgG was included as the negative control (IgG) and the input DNA from fragmented chromatin before immunoprecipitation was used as internal control. The recovered chromatin was subjected to PCR analysis using primers covering the putative NF-κB binding motif of the MDR1 promoter and the PCR products were resolved in 1.5% agarose gel. A representative experiment is shown, and similar results were obtained from three independent experiments.</p

The structure of clitocine.

<p>The structure of clitocine.</p