Glycoengineered anti-CD39 promotes anticancer responses by depleting suppressive cells and inhibiting angiogenesis in tumor models
Haohai Zhang, Lili Feng, Paola de Andrade Mello, Changchuin Mao, Richard Near, Eva Csizmadia, Leo Li-Ying Chan, Keiichi Enjyoji, Wenda Gao, Haitao Zhao, Simon C. Robson
Haohai Zhang, Lili Feng, Paola de Andrade Mello, Changchuin Mao, Richard Near, Eva Csizmadia, Leo Li-Ying Chan, Keiichi Enjyoji, Wenda Gao, Haitao Zhao, Simon C. Robson
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Research Article Angiogenesis Immunology

Glycoengineered anti-CD39 promotes anticancer responses by depleting suppressive cells and inhibiting angiogenesis in tumor models

Abstract

Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5′-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcγ receptor–directed (FcγR-directed), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site–specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti–PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system–modulating strategy for cancer therapy.

Authors

Haohai Zhang, Lili Feng, Paola de Andrade Mello, Changchuin Mao, Richard Near, Eva Csizmadia, Leo Li-Ying Chan, Keiichi Enjyoji, Wenda Gao, Haitao Zhao, Simon C. Robson

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Figure 2

Afucosylated anti-mCD39 Ab (Afuc 5F2-m2c, αCD39) boosts ADCC function, inhibiting tumor growth in vivo.

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Afucosylated anti-mCD39 Ab (Afuc 5F2-m2c, αCD39) boosts ADCC function, i...
(A) CD39-overexpressing CHO cells were treated with antibodies for 30 minutes. Effector cells (Jurkat cells expressing mFcγRIV and NFAT-RE driving luciferase expression and luminescence signal) were added to the culture and incubated (6 hours). Afuc 5F2-m2c Ab exhibited the highest ADCC function in vitro. (B) MC38 tumor cells were engrafted in WT Foxp3EGFP–knockin reporter mice on day 0. Antibodies were administered (200 μg/mouse) intraperitoneally on days 9, 12, and 15. Saline (n = 7), WT 5F2-m1 (n = 4), Afuc 5F2-m1 (n = 4), WT 5F2-m2c (n = 5), Afuc 5F2-m2c (n = 4). (C and E) MC38 tumor cells were engrafted in WT mice (C and D) or Cd39–/– mice (E) (day 0). MC38 tumor–bearing mice received 5 mg/kg antibodies intraperitoneally (n = 7 per group) on days 8, 11, 14, and 17. Tumor growth (C and E) and/or time point when tumor size (2000 mm3) required euthanasia of mice (D) were measured. (F and G) Mice bearing subcutaneous B16F10 tumors received 10 mg/kg Ab treatment intraperitoneally on days 3, 6, and 9 after tumor inoculation. Tumor growth curve (F) and time to euthanasia (G) of mice in the control (CTRL) group (n = 12) and αCD39 mAb–treated group (n = 11) are presented. Data in B–E are shown as mean ± SEM. Two-way ANOVA (B, C, and E) and the log-rank test (D and G) were used for statistical analyses. (H) Mice bearing subcutaneous MC38 tumors received 5 mg/kg AF750-labeled αCD39 mAb (magenta) treatment intraperitoneally on day 11 after tumor inoculation (n = 4). Tissues were harvested on day 12. One hour before sacrifice, 15 mg/kg Hoechst 33342 (blue) was injected into tumor-bearing mice via tail vein. IHC images of representative fields of MC38 tumors and other organs. Scale bars: 100 μm. Data in A–H are representative of at least 2 independent experiments. *P < 0.05. NS, not significant.