Depletion of major pathogenic cells in asthma by targeting CRTh2
Tao Huang, Meredith Hazen, Yonglei Shang, Meijuan Zhou, Xiumin Wu, Donghong Yan, Zhonghua Lin, Margaret Solon, Elizabeth Luis, Hai Ngu, Yongchang Shi, Arna Katewa, David F. Choy, Nandhini Ramamoorthi, Erick R. Castellanos, Mercedesz Balazs, Min Xu, Wyne P. Lee, Marissa L. Matsumoto, Jian Payandeh, Joseph R. Arron, Jo-Anne Hongo, Jianyong Wang, Isidro Hötzel, Cary D. Austin, Karin Reif
Tao Huang, Meredith Hazen, Yonglei Shang, Meijuan Zhou, Xiumin Wu, Donghong Yan, Zhonghua Lin, Margaret Solon, Elizabeth Luis, Hai Ngu, Yongchang Shi, Arna Katewa, David F. Choy, Nandhini Ramamoorthi, Erick R. Castellanos, Mercedesz Balazs, Min Xu, Wyne P. Lee, Marissa L. Matsumoto, Jian Payandeh, Joseph R. Arron, Jo-Anne Hongo, Jianyong Wang, Isidro Hötzel, Cary D. Austin, Karin Reif
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Research Article Immunology Therapeutics

Depletion of major pathogenic cells in asthma by targeting CRTh2

Abstract

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2–specific antibodies may be a novel and more efficacious treatment for asthma.

Authors

Tao Huang, Meredith Hazen, Yonglei Shang, Meijuan Zhou, Xiumin Wu, Donghong Yan, Zhonghua Lin, Margaret Solon, Elizabeth Luis, Hai Ngu, Yongchang Shi, Arna Katewa, David F. Choy, Nandhini Ramamoorthi, Erick R. Castellanos, Mercedesz Balazs, Min Xu, Wyne P. Lee, Marissa L. Matsumoto, Jian Payandeh, Joseph R. Arron, Jo-Anne Hongo, Jianyong Wang, Isidro Hötzel, Cary D. Austin, Karin Reif

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Figure 8

Characterization of h19A2, a humanized hCRTh2-specific Ab.

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Characterization of h19A2, a humanized hCRTh2-specific Ab. (A) Flow cyto...
(A) Flow cytometry analyses of humanized anti-hCRTh2 Ab h19A2 bound to hCRTh2 expressed on 293 cells but not mock-transfected WT 293 cells. Anti-CRTh2 Ab concentrations were 1 μg/ml (black line) and 0.1 μg/ml (gray line); isotype control Ab was 1 μg /ml (tinted histogram). (B) Reactivity by flow cytometry of humanized anti-hCRTh2 Ab h19A2 with primary human basophils or eosinophils from peripheral blood leukocytes. Anti-CRTh2 Ab concentrations were 5 μg/ml (solid line), 0.6 μg/ml (dotted line), and 0.06 μg/ml (dashed line); isotype control Ab was 5 μg/ml (tinted histogram). (C) Scatchard analysis of the Fab fragment of humanized hCRTh2-specific Ab h19A2 binding to hCRTh2−-transfected 293 cells. The mean binding affinity from 2 separate experiments is 11 nM. (D) Humanized h19A2 Ab does not block PGD2-mediated reduction of forskolin-induced cAMP levels in hCRTh2+ cAMP Hunter cells. The graph shows the measurement of intracellular cAMP levels in the presence of increasing concentrations of hCRTh2-specific antibodies h19A2 or h8B1 after cAMP levels were controlled with the concomitant administration of forskolin and PGD2. Results are mean of triplicates ± SD. (E) Humanized h19A2 or h8B1 antibodies show no agonistic activity on forskolin-induced cAMP levels in hCRTh2+ cAMP Hunter cells. The graph shows the measurement of intracellular cAMP levels after induction of cAMP with forskolin in the presence of increasing concentrations of hCRTh2-specific antibodies h19A2 or h8B1, or with increasing concentrations of PGD2. (F) Afucosylation increases the potency of h19A2-mediated ADCC. Activity curves for hCRTh2-specific WT h19A2 (circles), afucosylated h19A2.af (triangles), and control Rituximab (squares) human IgG1 antibodies in an autologous flow cytometry–based ADCC assay using human primary basophils (left panel) and eosinophils (right panel) as target cells. A representative experiment is shown. The mean ADCC activity from 5 separate experiments is shown in Table 2. PGD2, prostaglandin D2; ADCC, antibody-dependent cell-mediated cytotoxicity.