Targeting of ED-A+ Fibronectin as novel disease-modifying concept in preclinical Pulmonary Hypertension: head-to-head comparison of three antibody-based strategies

Background: Among the five clinical groups of Pulmonary Hypertension (PH), groups 2 and 3 account for over 90% of cases. While therapeutic management of these patients is restricted to addressing the underlying disease, specific treatments are exclusively available for group 1 and group 4. Except for group 4, pulmonary vascular and right ventricular remodeling shares various similarities among the different clinical groups and is accompanied by an extensive re-occurrence of extra-domain A containing fibronectin (ED-A⁺ Fn), a molecule being virtually absent in healthy adults but critically involved in PH-associated remodeling.

Aim of the current study was to analyze treatment effects of different antibody-based strategies addressing ED-A⁺ Fn focusing on both, functional blocking of ED-A⁺ Fn and targeted pharmaco-delivery directly to the site of disease by using ED-A⁺ Fn as structural target.

Methods: Induction of PH in mice was realized using the Monocrotaline approach. Sham-treated controls (ctrl., n=10) were compared to untreated PH (PH, n=12) and mice treated with Macitentan as comparator (MAC, n=12) and with either the recombinant @ED-A⁺ Fn antibody F8 in IgG format (F8IgG, n=8), a fusion protein of F8 (scFv format) and Interleukin-9 (F8IL9F8, n=11) or F8 (IgG format) fused to Interleukin-9 (F8-IL9, n=6). The study focused on head-to-head comparison of the three F8-based treatment strategies with each other and with MAC as comparison group. Treatment outcomes were assessed, among others, by transthoracic echocardiography, hemodynamic measurements, histological analysis as well as immunohistochemical evaluation of immune cell infiltration (leukocytes, Leu; macrophages, MC; and regulatory T cells, Tregs).

Results: PH induction led to a significant increase in right ventricular systolic pressure (RVPsys) and a deterioration in echocardiographic parameters of right ventricular form and function, including right ventricular basal diameter (RVbasal) or tricuspid annular plane systolic excursion (TAPSE) compared to controls (p<0.05). All of the three F8 based treatment strategies significantly reduced RVPsys (p<0.05) compared to the PH group with F8IgG achieving the most pronounced reduction (p<0.002). Also, echocardiographic parameters, e.g., RVbasal or TAPSE, significantly improved consistently (p<0.05). Histologically, lung tissue damage was significantly attenuated by F8IgG and F8IL9 (p<0.05) while all three F8 formats significantly reduced cardiac tissue damage (p<0.05). Immunohistochemical analysis revealed a notable decrease in Leu recruitment in the F8IgG group (p=0.021), while there were no effects of all three F8-based strategies regarding MC infiltration (p=n.s.). For Tregs, in all of the three F8 based treatment groups, a significant increase in tissue infiltration could be shown (p<0.05).

Conclusion: Different antibody-based treatment strategies targeting ED-A⁺ Fn in PH associated tissue remodeling could be shown to have significant beneficial disease-modifying effects. There was no superiority of antibody based targeted delivery of Interleukin-9 compared to functional blocking of ED-A⁺ Fn. Thus, the observed effects are most probably caused by the attenuation of ED-A⁺ Fn dependent activation of vascular smooth muscle cells (pulmonary vasculature) and fibro-/myofibroblasts (right ventricle) as previously demonstrated by our group. Our novel concept holds the potential to address a broad spectrum of PH phenotypes.