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. 2021 Aug;112(8):3029-3040.
doi: 10.1111/cas.14999. Epub 2021 Jun 18.

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

Niklas Baumann  1 , Thies Rösner  1 , J H Marco Jansen  2 , Chilam Chan  2 , Klara Marie Eichholz  1 , Katja Klausz  1 , Dorothee Winterberg  3 , Kristina Müller  3 , Andreas Humpe  4 , Renate Burger  1 , Matthias Peipp  1 , Denis M Schewe  3 , Christian Kellner  4 , Jeanette H W Leusen  2 , Thomas Valerius  1
Affiliations

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

Niklas Baumann et al. Cancer Sci. 2021 Aug.

Abstract

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.

Keywords: CD47; EGFR antibody; glutaminyl cyclase; immunotherapy; myeloid cell.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Glutaminyl cyclase inhibition by SEN177 reduces signal regulatory protein alpha (SIRPα) binding to CD47‐positive tumor cells. A, Spatial interactions between the N‐terminal pyro‐glutamate modification at CD47 (brown) and its binding site for SIRPα (purple) (adapted from NCBI Structure PDB ID 2JJT). B, Treatment of MDA‐MB‐468, Kyse‐30, and A431 cancer cells with SEN177 (0.08‐50 µM) for 3 d does not affect cell growth as measured by MTS assays. Mean values are shown as percentage of DMSO control ± SEM of three independent experiments. C, Treatment of MDA‐MB‐468 cells with SEN177 (0.08‐50 µM, 72 h) results in dose‐dependent reduction of SIRPα‐Fc (5 µg/mL) binding compared to DMSO control, as analyzed by indirect immunofluorescence analyses. D, Time‐dependent (0‐96 h) inhibition of SIRPα‐Fc (5 µg/mL) binding to SEN177 (10 µM) treated MDA‐MB‐468 cells. E, Reconstitution of SIRPα‐Fc (5 µg/mL) binding after SEN177 (10 µM, 72 h) treatment of MDA‐MB‐468 cells when cell culture medium was refreshed and SIRPα‐Fc binding to target cells was analyzed at the indicated time points. F, Dose‐dependent binding of soluble SIRPα‐Fc (0.04‐25 µg/mL) on SEN177 (10 µM, 72 h) treated A431 cells is significantly reduced compared to DMSO treated cells. G, Reduction of SIRPα‐Fc (5 µg/mL) binding on different SEN177 (10 µM, 72 h) treated tumor cell lines. Mean values as a percentage of the DMSO control ± SEM of at least three independent experiments are displayed. #P ≤ .05 (two‐way ANOVA). n.s., nonsignificant difference
FIGURE 2
FIGURE 2
Glutaminyl cyclase inhibition does not affect epidermal growth factor receptor (EGFR) Ab‐triggered growth inhibition, complement‐dependent cytotoxicity (CDC), or Ab dependent cell‐mediated cytotoxicity (ADCC). A, Colon cancer cell line DiFi was treated with 10 µM SEN177 or DMSO as a control in combination with increasing concentrations of cetuximab (CTX) for 3 d. Cell growth was measured in a colorimetric, MTS‐based assay. Mean values are shown as percentage of control without Ab ± SEM of three independent experiments. B, SEN177 (10 µM, 72 h) or DMSO pretreated A431 cells served as target cells in CDC experiments. Normal human serum (25% v/v) was used as a source of complement. The EGFR Abs CTX and matuzumab (MTZ) or their combination were applied. Rituximab was used as a human isotype control Ab (ctrl. IgG1). C, In ADCC assays, A431, PBMC, or purified natural killer (NK) cells were used as effector cells in classical chromium release experiments (effector : target cell ratios of 40:1 and 10:1, respectively). In B and C, all Abs were used at a final concentration of 10 µg/mL. Mean values ± SEM of at least three independent experiments are shown. Data were analyzed by two‐way ANOVA. *P ≤ .05, control vs specific Abs. n.s., nonsignificant difference between DMSO control and SEN177 treated cells
FIGURE 3
FIGURE 3
Glutaminyl cyclase inhibition enhances tumor cell phagocytosis by macrophages using epidermal growth factor receptor (EGFR) Abs of different isotypes. A, Fc receptor profiling of macrophage colony‐stimulating factor (M‐CSF) differentiated macrophages was undertaken by indirect flow cytometry using Abs (10 µg/mL) against the indicated Fc receptors. Results shown are histograms from one representative experiment with an isotype control Ab (ctrl. mAb, white area) and the specific Ab (spec. mAb, black area). FI, fluorescence intensity. A summary of the mean fluorescence intensities (MFI) ± SEM obtained from experiments with five different donors is provided. B, Tumor cell lines MDA‐MB‐468 and Kyse‐30 were treated with SEN177 (10 µM) or DMSO for 3 d before serving as target cells in Ab‐dependent cellular phagocytosis experiments with M‐CSF differentiated macrophages at an effector : target cell ratio of 1:2. EGFR Abs of human IgG1 (cetuximab [CTX]), IgG2 (panitumumab [PANI]), and IgA2 (CTX‐IgA2) as well as a nonbinding control Ab (ctrl. IgG1) were used at a concentration of 50 µg/mL. Mean values ± SEM of at least three independent experiments are shown. Data were analyzed by two‐way ANOVA. *P ≤ .05, control vs specific Abs; #P ≤ .05, DMSO vs SEN177 treated cells
FIGURE 4
FIGURE 4
Glutaminyl cyclase inhibition improves polymorphonuclear neutrophilic granulocyte (PMN)‐mediated Ab‐dependent cell‐mediated cytotoxicity (ADCC) by epidermal growth factor receptor Abs depending on their isotype. A, Expression of Fc receptors on PMN was analyzed by flow cytometry. Results are presented as histograms from one representative experiment showing the fluorescence intensity (FI) and as a summary of the mean fluorescence intensities (MFI) ± SEM obtained from five experiments each with a different donor. B, Kyse‐30 cells were treated with SEN177 (10 µM) or an equivalent amount of DMSO as a control for 3 d and subsequently served as target cells in PMN‐mediated ADCC experiments using varying effector : target (E:T) cell ratios (fixed E:T ratio of 40:1 for isotype control). C, Tumor cell lines A431 and MDA‐MB‐468 were treated with SEN177 (10 µM) or DMSO for 3 d and subsequently used as target cells in PMN‐mediated ADCC experiments. E:T cell ratios of 40:1 (A431) and 80:1 (MDA‐MB‐468) were applied. In ADCC experiments, all Abs and isotype controls (ctrl.) were used at a concentration of 1 µg/mL. Bars represent mean values ± SEM of at least three independent experiments. Data were analyzed by two‐way ANOVA. *P ≤ .05, control vs specific Abs; #P ≤ .05, DMSO vs SEN177 treated cells; §P ≤ .05 between isotypes (SEN177 treated cells). CTX, cetuximab; PANI, panitumumab
FIGURE 5
FIGURE 5
Glutaminyl cyclase inhibition in tumor cells improves epidermal growth factor receptor (EGFR) Ab therapeutic efficacy in a xenogeneic tumor model. A, MBA‐MD‐468 tumor cells were incubated with 10 µM SEN177 or DMSO for 3 d prior to intraperitoneal (IP) injection into FcαRI transgenic (CD89 Tg) or nontransgenic (non‐Tg) SCID mice. Untreated EGFR‐negative murine Ba/F3 cells served as a recovery control. Immediately after cell inoculation, mice were treated with 100 µg EGFR Abs or PBS as a control and killed 16 h later. B, C, Numbers of tumor cells in mice were evaluated by flow cytometry of cells from the peritoneal fluid 16 h after treatment with Abs or PBS as indicated. Results are presented as the median ratio of MBA‐MD‐468 to Ba/F3 cells (n = 5 per group) with an initial ratio of 5:1. D, Tumor cell recovery from mice treated with EGFR Abs irrespective of their isotype was significantly (P ≤ .05) reduced in mice who received SEN177 pretreated vs DMSO treated tumor cells (right panel), while a nonsignificant difference was observed in mice treated with PBS (left panel). Data shown are the individual and mean values of the experiments shown in (B) and (C) with 15 mice per group (Ab treatment panel) and five mice (DMSO) and 10 mice (SEN177) in the PBS treated panel. Data were analyzed by two‐tailed, unpaired t test. *P ≤ .05, PBS control vs specific Abs; #P ≤ .05, DMSO vs SEN177 treatment. n.s., not significant. CTX, cetuximab; PANI, panitumumab
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