Development of a therapeutic anti-marinobufagenin antibody

在我们的第一项临床研究中，我们比较了DigiFab、Digibind和anti-MBG mAb与PE患者血浆中不同内源性类固醇（包括MBG）相互作用的能力，以及恢复这些患者红细胞中Na/ k - atp酶活性的能力。采用基于DigiFab、Digibind和anti-MBG mAb的免疫分析，我们研究了PE血浆高效液相色谱（HPLC）分离后内源性强心剂类固醇的洗脱谱。

In our first clinical study we compared DigiFab, Digibind, and anti-MBG mAb with respect to their ability to interact with different endogenous steroids, including MBG, in plasma from PE patients, and to restore Na/K-ATPase activity in erythrocytes from these patients. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of endogenous cardiotonic steroids following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Seven patients with mild preeclampsia (28 +/- 2 years; gestational age, 39 +/- 0.5 weeks; blood pressure 156 +/- 5 / 94 +/- 2 mm Hg) and 6 normotensive pregnant participants (28 +/- 1 years; gestational age, 39 +/- 0.4 weeks; blood pressure 111 +/- 2 / 73 +/- 2 mm Hg) were enrolled. PE was associated with a substantial inhibition of erythrocyte Na/K-ATPase (1.47 +/- 0.17 vs. 2.65 +/- 0.16 umol Pi/mL/hr in control group, P<.001) and increase in plasma MBG levels (0.670.05 nmol/L vs. 1.83+/-0.35 nmol/L, P=0.03) compared to normotensive pregnant control. Ex vivo, at 10 ug/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 ug/mL) restored erythrocyte Na/K-ATPase activity in PE patients. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in plasma steroidal material was detected by Digibind (176 vs. 75 pmoles), DigiFab (221 vs. 70 pmoles), and anti-MBG mAb (1056 vs. 421 pmoles). Therefore, because DigiFab interacts with endogenous cardiotonic steroids, including MBG, from PE plasma and reverses PE-induced NKA inhibition, it can substitute for Digibind for immunoneutralization of MBG in patients with PE. Notably, our anti-MBG mAb was more active in restoration of erythrocyte Na/K-ATPase activity in PE patients, and detected 4-5-folds more steroidal material in PE plasma than both anti-digoxin antibodies, Digibind and DigiFab. Anti-MBG mAb has strong potential for therapeutic application. Our next step is humanization of this anti-MBG mAb for clinical application. In the second study we immunoneutralized heightened MBG levels in old Dahl-S rats, and studied pro-fibrotic gene expression in old Dahl-S treated with anti-MBG mAb antibody (OA; n=6) in comparison to vehicle-treated old control (OC; n=6) and to young control (YC; 3 months old; n=6) Dahl-S. All animals were kept on a low salt intake (0.1% NaCl) after weaning. Antibody was administered 3 times during 10 days to old Dahl-S. Following 10 days of treatment, systolic blood pressure (SBP), 24-hr MBG excretion, mRNA expression (qPCR) in left ventricles (LV), and collagen and elastin abundance (histochemistry) in aortic media were assessed. In OC vs. YC, MBG level increased 3.6-fold (p<0.01), SBP elevated (175 +/- 3 vs. 117 +/- 6 mm Hg; p<0.001), aortic elastin/collagen ratio decreased 3.3-fold, and expression of genes, implicated in TGF-beta-signaling in LV were upregulated (TGF-beta-1 - 3-fold; TGF-beta-2 - 8-fold; CTGF - 7.5-fold; SMAD4, SMAD5, MAPK3, and Collagen-1 - 2-fold), and were down-regulated following immunoneutralization of MBG. The negative regulator of collagen-1 synthesis Fli-1 was 2-fold down-regulated in OC vs. YC, and restored in OA to the level of Fli-1 in YC. In OA vs. OC, SBP decreased (156 +/- 5 mm Hg; p<0.05), and aortic elastin/collagen ratio was normalized. Immunoneutralization of MBG produces anti-hypertensive and anti-remodeling effects associated with normalization of gene expression implicated in TGF-beta- and Fli1-pro-fibrotic pathways initiated by MBG in aged Dahl-S. Restoration of elastin/collagen ratio indicates that vascular function is normalized by anti-MBG antibody in aging.