Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma

Abstract

Background and purpose: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development.

Experimental approach: In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys.

Key results: CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia.

Conclusions and implications: CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

Figure 1

CAT-354 is a potent and selective neutralizer of IL-13. (A) Baculovirus-derived human, human R130Q and cynomolgus IL-13-induced TF-1 cell proliferation (n= 6). (B) Recombinant mouse IL-13-induced HUVEC VCAM-1 upregulation (n= 3). (C) Endogenous IL-13-driven HDLM-2 cell proliferation (n= 2). (D) Recombinant human IL-13, IL-4 and IL-1β-induced HUVEC VCAM-1 upregulation (n= 5, 3, 6 respectively). Data are shown as mean average points ± SEM from n independent experiments.

Figure 2

CAT-354 prevents IL-13-induced activation of a variety of cell types implicated in human asthma. Inflammatory cell recruitment and activation: (A) eotaxin-1 release from normal human lung fibroblasts (NHLF) (n= 4); (B) shape change in human eosinophils (n= 4). Activation of bronchial smooth muscle: (C and D) calcium flux in human bronchial smooth muscle cells (HBSM) (n = 3). Modulation of the IgE axis: (E) CD23 upregulation in human monocytes(n = 6); (F) isotype switching in B cells (n= 6). Data are shown as mean average points ± SEM from n independent experiments. In C and D differences in cell responses were compared using a one-way anova with Dunnett's multiple comparisons post-test. *P < 0.05; ***P < 0.001 significantly different from values with recombinant human IL-13.

Figure 3

CAT-354 inhibits human IL-13-induced leukocytic inflammation in the mouse air pouch. (A) Total leukocytes and (B) eosinophils. Data are shown as mean number of infiltrating leukocytes ± SEM (n= 9–10 from two blocked experiments). **P < 0.01; ***P < 0.001, significantly different from recombinant human IL-13 (huIL-13) tested by one-way anova followed by Dunnett's multiple comparisons test.

Figure 4

Effects of CAT-354 on the allergic phenotype in humanized BALB/c mice. (A) Model schematic. Daily mAb dosing. (B) Airway hyperresponsiveness (n= 4–8). 72h mAb dosing. (C) BAL eosinophilia with inset % eosinophils (n= 20–25); (D) Airway hyperresponsiveness (n= 14–18 except dex n= 8); (E) Forced expiratory volume (n= 6–8). Data are shown as mean average ± SEM from 1 (B) or 2 (C–E) blocked experiments. *P < 0.05; **P < 0.01; ***P < 0.001 versus s/c group using one-way (two-way for AHR) anova followed by Dunnett's (Bonferroni's for AHR) multiple comparisons test using s/c as the control group.

Figure 5

Differential effects of modulating the IL-13 axis on the allergic phenotype in WT BALB/c (A–D) and huIL-13 knock-in BALB/c (E-H) mice. (A and E) BAL eosinophilia with inset % eosinophils (n= 14–16); (B and F) FEV (n= 8); (C and G) AHR (n= 4–8); (D) BAL muIL-13 and (H) BAL huIL-13 (n= 15–16). Data are shown as mean average ± SEM from two blocked experiments. *P < 0.05; **P < 0.01; ***P < 0.001, significantly different from values in the sensitization and challenge (s/c) group; one-way (two-way for AHR) anova followed by Dunnett's (Bonferroni's for AHR) multiple comparisons test.

Figure 6

CAT-354 decreases AHR and eosinophilia in a cynomolgus model of antigen challenge. (A) Model schematic. (B) BAL inflammation (n= 21). (C) Histamine AHR in phase I (n= 18) and (D) Phase II (n= 18). (E) Histamine PC₃₀ (n= 18). (F) Histamine AHR by R_L AUC (n= 14). (G) Antigen priming (n= 20). (H) BAL IL-13 (n= 22). (I) BAL eotaxin (n= 22). Data displayed as the arithmetic mean ± SEM of individual animal changes at day 11 compared with day 1 for the phase I and II changes, respectively and phase II compared with phase I in the endpoint column. Statistical testing was performed only on the endpoint and was a two-way Student's t-test with the null hypothesis that the endpoint contained the number zero. *P < 0.05; **P < 0.01; ***P < 0.001.