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和抗MBG mAb与PE患者血浆中不同内源性类固醇（包括MBG）相互作用以及恢复这些患者红细胞中Na/K-ATP酶活性的能力。使用基于DigiFab、Digibind和抗MBG mAb的免疫测定法，我们研究了PE血浆高效液相色谱（HPLC）分级分离后内源性强心类固醇的洗脱曲线。 轻度子痫前期7例（28 +/- 2岁;胎龄39 +/- 0.5周;血压156 +/- 5 / 94 +/- 2 mm Hg）和6名血压正常的妊娠受试者（28 ± 1岁;胎龄39 ± 0.4周;血压111 ± 2 / 73 ± 2 mm Hg）。与正常血压妊娠对照组相比，PE与红细胞Na/K-ATP酶的实质性抑制（1.47 +/- 0.17 vs. 2.65 +/- 0.16 umol Pi/mL/hr，P<0.001）和血浆MBG水平的增加（0.67 ± 0.05 nmol/L vs. 1.83+/-0.35 nmol/L，P=0.03）相关。离体，在10 ug/mL浓度下，这与先前在PE中施用的Digibind的临床剂量一致，DigiFab和Digibind以及抗MBG mAb（0.5 ug/mL）恢复了PE患者中的红细胞Na/K-ATP酶活性。在合并PE和对照血浆的HPLC分级分离后，通过Digibind（176 pmole vs. 75 pmole）、DigiFab（221 pmole vs. 70 pmole）和抗MBG mAb（1056 pmole vs. 421 pmole）检测到PE相关的血浆甾体物质增加。因此，由于DigiFab与PE血浆中的内源性强心类固醇（包括MBG）相互作用并逆转PE诱导的NKA抑制，因此它可以替代Digibind用于PE患者中MBG的免疫中和。值得注意的是，我们的抗MBG mAb在恢复PE患者的红细胞Na/K-ATP酶活性方面更有活性，并且在PE血浆中检测到的甾体物质是抗地高辛抗体Digibind和DigiFab的4-5倍。抗MBG mAb具有很强的治疗应用潜力。我们的下一步是将这种抗MBG mAb人源化用于临床应用。 在第二项研究中，我们免疫中和了老年Dahl-S大鼠中升高的MBG水平，并研究了用抗MBG mAb抗体（OA; n=6）处理的老年Dahl-S中与媒介物处理的老年对照（OC; n=6）和年轻对照（YC; 3个月大; n=6）Dahl-S相比的促纤维化基因表达。断奶后，所有动物均保持低盐摄入量（0.1% NaCl）。在10天内对老Dahl-S施用抗体3次。治疗10天后，评估收缩压（SBP）、24小时MBG排泄、左心室（LV）中的mRNA表达（qPCR）以及主动脉中膜中的胶原蛋白和弹性蛋白丰度（组织化学）。 在OC与YC中，MBG水平增加3.6倍（p<0.01），SBP升高（175 +/- 3 vs. 117 +/- 6 mm Hg; p<0.001），主动脉弹性蛋白/胶原比例降低3.3倍，LV中涉及TGF-β信号传导的基因表达上调（TGF-β-1-3倍; TGF-β-2-8倍; CTGF - 7.5倍; SMAD 4、SMAD 5、MAPK 3和胶原蛋白-1-2倍），并且在MBG的免疫中和后下调。与YC相比，OC中胶原-1合成的负调节因子Fli-1下调2倍，并且在OA中恢复到YC中Fli-1的水平。OA组与OC组相比，SBP降低（156 +/- 5 mm Hg; p<0.05），主动脉弹性蛋白/胶原比例正常化。 MBG的免疫中和作用产生抗高血压和抗重塑作用，这与MBG在老年Dahl-S中启动的TGF-β和Fli 1促纤维化途径中涉及的基因表达正常化相关。弹性蛋白/胶原蛋白比率的恢复表明血管功能在衰老中通过抗MBG抗体而正常化。

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.