Engineering bispecific antibodies for non-hormonal contraception

摘要 精子在上升到上尿路并使卵子受精之前必须游过粘液。此外，精子 必须过度激活才能穿透卵母细胞的透明带。多聚体抗体(Ab)，如IgM 这种结合精子表面抗原的物质可以将精子凝集成大到无法穿透毛孔的簇。 粘液。此外，还可以通过改造免疫球蛋白使单个精子与粘蛋白发生交联，并抑制 过度激活，从而阻止精子到达卵子并使其受精。话题被动 基于经阴道注射抗精子抗体(ASA)的免疫在动物模型中得到验证 20世纪80-90年代，直接克服了避孕药强度可变和可逆性不确定的问题 疫苗。然而，由于mAb生产的高成本和挑战，这一战略是不可行的。 使用免疫球蛋白M配方。鉴于生物处理的显著进步大大降低了成本 单抗制造的可能性，以及通过阴道内持续递送抗体的可能性 环，我们认为现在是开发新的、超有效的非激素避孕的ASA的时机已经成熟 基于阴道对人类精子的局部被动免疫。在试点研究中，我们已经表明 我们可以通过工程将ASA的精子凝集力提高15-50倍 由6到10个FAB结构域组成的基于Ig G的多价构建体(即链接到 亲本免疫球蛋白分子)。这些新构建物在加速加热条件下具有与免疫球蛋白类似的稳定性。 稳定性研究，以及与免疫球蛋白相似的生产和纯化产率。其中一个构造表示 波士顿大学避孕研究中心正在开发铅分子，分两个阶段进行 临床试验定于2020年开始。在这个项目中，我们寻求通过雇佣来进一步提高潜力 尖端双特异性抗体工程技术及酵母和哺乳动物细胞展示亲和力成熟 设计一个超强的双特异多价AsA构建库。我们的目标是CD52g，一口井 仅存在于人类精子上的特化和验证的抗原靶标，以及Eppin 通过抑制精子过度激活起作用的避孕靶。在目标1中，我们将利用酵母展示来 提高Fab对CD52g和Eppin的亲和力，构建多价双功能构建库 结合了这两个目标，并严格表征了这些分子。在目标2中，我们将表演一组 评估其潜在避孕效果的研究，包括精子凝集、捕获个体精子 在粘液中，并抑制精子过度激活。确定哪种构造将最有效地减少 在人类阴道中的进行性精子运动，我们将在目标3中进行体内剂量发现研究 在适应绵羊阴道的性交后生育测试中，来自目标1和目标2的最有效的构建。成功 这些研究的完成将可能使我们能够推进一个新的候选人，具有更大的 避孕效力比目前正在开发的ASA分子用于非激素避孕。

Summary Sperm must swim through mucus before ascending to the upper tract and fertilizing the egg. In addition, sperm must be hyperactivated to penetrate the zona pellucida of the oocyte. Polymeric antibodies (Ab) such as IgM that bind sperm surface antigens can agglutinate sperm into clusters too large to penetrate through the pores of mucus. In addition, IgG can also be engineered to crosslink individual sperm to mucins, as well as inhibit hyperactivation, consequently preventing sperm from reaching and fertilizing the egg. Topical passive immunization based on vaginal delivery of anti-sperm Ab (ASA) was validated in animal models in the 1980s-1990s, and directly overcomes the variable intensity and uncertain reversibility of contraceptive vaccines. However, this strategy was not practical due to the high costs of mAb production and challenges with IgM formulation. Given the remarkable advances in bioprocessing that have greatly reduced the costs of mAb manufacturing, and the possibility of sustained delivery of antibodies in the vagina via intravaginal rings, we believe the time is now ripe to develop novel, ultra-potent ASA for non-hormonal contraception based on topical passive immunization of the vagina against human sperm. In pilot studies, we have shown that we can greatly increase the sperm-agglutination potency of ASA by >15-50-fold by engineering multivalent, IgG-based construct comprised of six to ten Fab domains (i.e. 4 to 8 additional Fabs linked to the parent IgG molecule). These novel constructs possess comparable stability to IgG in accelerated thermal stability studies, and also similar production and purification yield as IgG. One of the constructs represent the lead molecule under development in the Boston University Contraceptive Research Center, with two Phase I clinical trials slated to begin in 2020. In this project, we seek to further improve the potencies by employing cutting edge bispecific Ab engineering technology and affinity maturation via yeast and mamallian cell display to engineer a library of ultra-potent bispecific multivalent ASA constructs. We will target both CD52g, a well characterized and validated antigen target present on human sperm only, and EPPIN, a well established contraceptive target that functions by inhibiting sperm hyperactivation. In Aim 1, we will utilize yeast display to improve the affinity of Fab against CD52g and EPPIN, engineer a library of multivalent bispecific constructs that bind both targets, and rigorously characterize these molecules. In Aim 2, we will perform a panel of studies to assess their potential contraceptive efficacy, including sperm agglutination, trapping individual sperm in mucus, and inhibition of sperm hyperactivation. To determine which construct will most effectively reduce progressive sperm motility in human vagina, we will perform in vivo dose finding studies in Aim 3 with the two most potent constructs from Aims 1 and 2 in a post-coital fertility test adapted to the sheep vagina. Successful completion of these studies will likely allow us to advance a novel ASA candidate with substantially greater contraceptive potency than the current ASA molecules under development for non-hormonal contraception.