Irani Yazad D, Scotney Pierre D, Klebe Sonja, Mortimer Lauren A, Nash Andrew D, Williams Keryn A
Department of Ophthalmology, Flinders University, Adelaide, South Australia, Australia.
CSL Limited, Parkville, Melbourne, Victoria, Australia.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3404-3413. doi: 10.1167/iovs.16-21343.
We tested the ability of an antibody fragment with specificity for vascular endothelial growth factor-B (VEGF-B) to regress nascent and established corneal blood vessels in the rat.
A single chain variable antibody fragment (scFv) with specificity for VEGF-B was engineered from the 2H10 hybridoma. Binding to rat, mouse, and human VEGF-B was confirmed by surface plasmon resonance. Activity of the anti-VEGF-B scFv on developing and established corneal blood vessels was assessed following unilateral superficial cautery in male and female outbred Sprague Dawley rats. Groups (untreated, control scFv-treated, or anti-VEGF-B scFv-treated) comprised 6 to 22 rats. Treatment consisted of 5 μL scFv, 1 mg/mL, applied topically five times per day for 14 days, or two subconjunctival injections, 50 μg scFv each, applied 7 days apart, or combined topical and subconjunctival treatment. Corneal vessel area was quantified on hematoxylin-stained corneal flat-mounts, and groups were compared using the Mann-Whitney U test, with post hoc Bonferroni correction. Immunohistochemistry for cleaved caspase-3 was performed.
Topical anti-VEGF-B scFv therapy alone did not regress corneal blood vessels significantly (P > 0.05). Subconjunctival injection and combined treatment regressed 14-day established corneal blood vessels (25% reduction in vessel area [P = 0.04] and 37% reduction in vessel area [P < 0.001], respectively, compared to results in untreated controls). Cleaved caspase-3 was identified in vascular endothelial cells of anti-VEGF-B scFv-treated corneas. In scFv-treated rats, corneal endothelial cell function was maintained to 12 weeks after treatment and a normal blink reflex was present.
The anti-VEGF-B scFv significantly regressed established but not developing corneal blood vessels in rats.
我们测试了一种对血管内皮生长因子-B（VEGF-B）具有特异性的抗体片段使大鼠新生和已形成的角膜血管消退的能力。
从2H10杂交瘤构建出一种对VEGF-B具有特异性的单链可变抗体片段（scFv）。通过表面等离子体共振确认其与大鼠、小鼠和人类VEGF-B的结合。在雄性和雌性远交系Sprague Dawley大鼠单侧浅层烧灼后，评估抗VEGF-B scFv对发育中和已形成的角膜血管的活性。每组（未治疗、对照scFv治疗或抗VEGF-B scFv治疗）包含6至22只大鼠。治疗方法包括每天局部应用5 μL浓度为1 mg/mL的scFv，共5次，持续14天；或两次结膜下注射，每次50 μg scFv，间隔7天；或局部与结膜下联合治疗。在苏木精染色的角膜平铺标本上对角膜血管面积进行定量，并使用Mann-Whitney U检验进行组间比较，采用事后Bonferroni校正。进行了裂解型半胱天冬酶-3的免疫组织化学检测。
单独局部应用抗VEGF-B scFv治疗并未使角膜血管显著消退（P > 0.05）。结膜下注射和联合治疗使14天形成的角膜血管消退（与未治疗对照组相比，血管面积分别减少25% [P = 0.04]和37% [P < 0.001]）。在抗VEGF-B scFv治疗的角膜的血管内皮细胞中鉴定出裂解型半胱天冬酶-3。在scFv治疗的大鼠中，角膜内皮细胞功能在治疗后维持至12周，且存在正常的眨眼反射。
抗VEGF-B scFv可使大鼠已形成但非发育中的角膜血管显著消退。
