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. 2022 Aug 16:13:949140.
doi: 10.3389/fimmu.2022.949140. eCollection 2022.

Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab

Niklas Baumann  1 , Christian Arndt  1 , Judith Petersen  1 , Marta Lustig  1 , Thies Rösner  1 , Katja Klausz  2 , Christian Kellner  3 , Miriam Bultmann  4 , Lorenz Bastian  4 , Fotini Vogiatzi  5 , Jeanette H W Leusen  6 , Renate Burger  1 , Denis M Schewe  7 , Matthias Peipp  2 , Thomas Valerius  1
Affiliations

Myeloid checkpoint blockade improves killing of T-acute lymphoblastic leukemia cells by an IgA2 variant of daratumumab

Niklas Baumann et al. Front Immunol. .

Abstract

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 VSports手机版. 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. .

Keywords: CD38; CD47; IgA; SIRPα; T-cell acute lymphoblastic leukemia (T-ALL); daratumumab; immunotherapy. V体育安卓版.

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"VSports注册入口" Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
DARA-IgA2 mediates T-ALL cell killing by myeloid cells. (A) Expression of CD38 and CD47 on T-ALL cell lines HSB-2, MOLT-13 and P12-ICHIKAWA (P12) was quantified by indirect flow cytometry to determine the specific antigen-binding sites per cell (SABC). Mouse monoclonal antibodies against CD38 (clone HB-7) and CD47 (clone B6H12) were used at saturating concentrations (5 µg/ml) and detected with FITC-conjugated anti-mouse IgG F(ab’)2 fragments. The results from 3 independent experiments with means ± SEM are shown. The values of the control samples (ctrl. mAb) were subtracted from the values obtained with the antibodies. (B) Macrophage-mediated ADCP (E:T = 1:1) was analyzed by real-time fluorescence imaging over 5 h (300 min). Target cells were labelled with a pH-dependent red fluorescent dye (pHrodo), and phagocytosis was measured as red object counts per image (ROI) every 60 min. Antibodies were used at 10 µg/ml. Results are presented as means ± SEM of at least 3 experiments with effector cells from different donors. * indicates significant differences between DARA-IgG1 and IgG1 isotype control (ctrl. IgG1) or DARA-IgA2 and IgA2 isotype control (ctrl. IgA2) (p < 0.05 by two-way ANOVA), and & marks significant differences between DARA-IgG1 and DARA-IgA2 (p < 0.05 by two-way ANOVA). n. s., not significant. (C) PMN mediated ADCC (E:T = 80:1) was analyzed in 3 h 51Cr release assays with the indicated antibody concentrations (0-10 µg/ml) using GM-CSF (50 U/ml) stimulated PMN. Results are presented as means ± SEM of at least 3 experiments with effector cells from different donors. * indicates significant differences between DARA and isotype control (ctrl.) antibodies at the highest concentration (10 µg/ml) (p < 0.05 by one-way ANOVA), & marks significant differences between IgG1 and IgA2 (p < 0.05 by two-way ANOVA). n. s., not significant.
Figure 2
Figure 2
Genetic knock-out of CD47 or QPCTL improved myeloid cell-mediated T-ALL cell killing by IgG1 and IgA2 variants of daratumumab. (A) MOLT-13 QPCTL and CD47 knock-out (KO) cells were tested for the expression of CD38 and CD47 using antibody clones HB-7 and B6H12, respectively. The CD47 antibody B6H12 is pyro-glutamate independent and detects overall expression of the antigen. Control cells (ctrl.) were treated with Cas9 without QPCTL gRNA. Antibodies were used at saturating concentrations (5 µg/ml) and detected with FITC-conjugated goat anti-mouse Fcγ-specific F(ab)2 fragments (left panel, n=3). Knock-out of QPCTL was confirmed by immunoblotting (right panel), β-tubulin was used for loading control. (B) Binding of the pyro-glutamate dependent CD47 antibody CC2C6 (left panel) and soluble SIRPα-Fc fusion protein (right panel) on MOLT-13 control and knock-out cells was measured by indirect immunofluorescence and staining with FITC-conjugated goat anti-mouse or goat anti-human Fcγ specific F(ab)2 fragments. MFI values ± SEM of 3 independent experiments are shown. Background fluorescence of unlabeled cells was subtracted from each MFI value. (C) QPCTL KO and CD47 KO as well as control MOLT-13 cells were used as targets in macrophage-mediated ADCP (left panel) and in PMN-mediated ADCC (right panel) with DARA-IgA2 and DARA-IgG1 at 10 µg/ml. For ADCP, an E:T cell ratio of 1:1 was applied. Mean values ± SEM of the red object counts per image (ROI) after 4 h are shown (n=3 with different donors). PMN-mediated ADCC was determined by 51Cr release assays with GM-CSF (50 U/ml) stimulated PMN at an E:T ratio of 80:1. Percentage of mean lysis ± SEM of 4 independent experiments with effector cells from different donors are shown. * indicates significant differences between DARA and isotype control (p < 0.05 by two-way ANOVA). Significant differences between the KO variants and the control cells are indicated by § for DARA-IgA2 and by % for DARA-IgG1 (p < 0.05 by two-way ANOVA). n.s., not significant. KO, knock-out; QPCTL, glutaminyl-peptide cyclotransferase like; MFI, mean fluorescence intensity; ROI, red object count per image.
Figure 3
Figure 3
Blocking CD47/SIRPα interactions leads to efficient myeloid cell-mediated killing of T-ALL cell lines and patient samples by DARA-IgA2. (A) HSB-2, MOLT-13 and P12 cell lines were used as targets in ADCP (upper panel) and PMN-mediated ADCC (lower panel) assays in the absence (white circles) or presence (black circles) of the CD47 blocking antibody 5F9-IgG2σ (20 µg/ml). DARA and isotype controls were used at 10 µg/ml. For ADCP, an E:T ratio of 1:1 was applied while in ADCC assays the E:T ratio was 80:1. M0 macrophages were generated using 50 ng/ml M-CSF, PMN were activated with GM-CSF (50 U/ml). Values of at least three replicates using different donor effector cells are presented. * indicates significant differences between DARA and the respective isotype control (p < 0.05 by two-way ANOVA), # depicts significant differences between with and without CD47 blockade (p < 0.05 by two-way ANOVA). (B) T-ALL patient samples (n=3) were tested for expression of CD38 and CD47. Specific antigen-binding sites per cell (SABC) were quantified by CD38 antibody HB-7 and CD47 antibody B6H12 at saturating concentrations (5 µg/ml). FITC-conjugated anti-mouse IgG F(ab’)2 fragments were used for detection. Mean values ± SEM of three replicates are indicated in the table. ADCP assays (lower left panel) were performed in the absence or presence of the CD47 blocking antibody 5F9-IgG2σ (20 µg/ml). Patients were illustrated by different colors (color code included in table). DARA-IgA2 and isotype control were used at 10 µg/ml. An E:T cell ratio of 1:1 was applied. Shown are the red object counts per image (ROI) at maximal phagocytosis with macrophages from three different donors. Microscopic images (magnification 10x) show phagocytosed T-ALL patient cells (red dots) at the time of the highest phagocytosis rate. * indicates significant differences between DARA-IgA2 and isotype control (p < 0.05 by Wilcoxon matched-pairs signed rank test), # depicts significant differences between with and without CD47 blockade (p < 0.05 by Wilcoxon matched-pairs signed rank test. n. s., not significant.
Figure 4
Figure 4
Treatment of HSB-2 cells with all-trans retinoic acid (ATRA) enhances CD38 expression and DARA-IgA2 mediated tumor cell killing by myeloid cells in combination with CD47 blockade. (A) Concentration and time dependent increase of CD38 expression on T-ALL cell line HSB-2 after treatment with ATRA at indicated concentrations for 24 h, 48 h and 72 h (left panel). CD38, CD47 expression and determination of the specific antigen-binding sites per cell (SABC, right panel) after treatment with 1 µM ATRA for 72 h were quantified using QIFIKIT. For both CD38 stainings, antibody HB-7 at saturating concentration (5 µg/ml) was used, while for CD47 staining, both B6H12 and CC2C6 antibodies were used. FITC-conjugated goat anti-mouse IgG F(ab’)2 fragments as secondary reagent was used. Values of three independent experiments are shown. $ depicts significant differences between ATRA and DMSO control (p < 0.05 by two-way ANOVA in both panels). (B) Microscopic images of HSB-2 cells stained for CD38 with antibody HB-7 and FITC-conjugated goat anti-mouse IgG F(ab’)2 fragments (40x magnification). (C) HSB-2 cells were treated with 1 µM ATRA or DMSO for 72 h and were then used in ADCP (upper panel) or PMN-mediated ADCC (lower panel) experiments. Daratumumab variants and ctrl. antibodies were applied at 10 µg/ml while CD47 blockade antibody 5F9-IgG2σ was used at 20 µg/ml. Values of three (ADCP and ADCC) different donors are depicted. * indicates significant differences between DARA and the respective isotype control (p < 0.05 by two-way ANOVA), # indicates significant differences between ATRA + CD47 blockade versus ATRA alone or CD47 blockade alone (p < 0.05 by two-way ANOVA). n. s., not significant.
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