Engineering bispecific antibodies for non-hormonal contraception

用于非激素避孕的双特异性抗体工程

• 批准号: 10618849
• 负责人: Samuel Lai
• 金额: $ 49.38万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10618849
• 关键词: Acceleration; Affinity; Agglutination; Anatomy; Animal Model; Antibodies; Antibody Formation; Antigen Targeting; Antigens; Binding; Biological Assay; Bispecific Antibodies; Boston; Cells; Cervix Mucus; Clinical Trials; Computer Assisted; Contraceptive Agents; Contraceptive Vaccines; Contraceptive methods; Deposition; Development; Dose; Embryo; Engineering; Enzyme-Linked Immunosorbent Assay; Fab domain; Federal Government; Fertility; Formulation; Histopathology; Human; Immune; Immunoglobulin G; Immunoglobulin M; Immunologic Contraception; Individual; Infertility; Inflammation; Inflammatory; Kinetics; Lead; Libraries; Link; Macaca; Male Genital Organs; Measures; Methods; Modeling; Monoclonal Antibodies; Mucins; Mucous body substance; Oocytes; Oryctolagus cuniculus; Parents; Passive Immunization; Penetration; Phase I Clinical Trials; Pilot Projects; Polymers; Production; Protease Inhibitor; Research; Sampling; Seminal fluid; Sheep; Specificity; Sperm Agglutination; Sperm Motility; Spermatozoa antibody; State Government; Surface; Surface Antigens; Swimming; Technology; Testing; Tight Junctions; Time; Tissues; Universities; Vaccines; Vagina; Vaginal Ring; Vaginal delivery procedure; Woman; Yeasts; Zona Pellucida; antibody engineering; bioprocess; cell motility; cervicovaginal; contraceptive efficacy; contraceptive target; cost; crosslink; cytokine; gel electrophoresis; genital secretion; improved; in vivo; manufacture; next generation; novel; pathogen; pre-clinical assessment; prevent; reproductive tract; reversible contraceptive; safety assessment; sperm analysis; sperm cell; unintended pregnancy; vaccine response; zygote

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.

总结 精子在上升到上生殖道并与卵子结合之前，必须游过粘液。此外，精子 必须被超活化才能穿透卵母细胞的透明质膜。聚合抗体（Ab），如IgM 结合精子表面抗原的蛋白质可以使精子凝集成大到无法穿过小孔的团 粘液。此外，IgG还可以被工程化以将个体精子交联到粘蛋白，以及抑制精子的生长。 过度激活，从而阻止精子到达卵子并使卵子受精。话题被动语态 基于抗精子抗体（阿萨）阴道给药的免疫接种在动物模型中得到了验证， 1980 - 1990年代，并直接克服了避孕的可变强度和不确定的可逆性 疫苗。然而，由于mAb生产的高成本和挑战，这种策略并不实用。 IgM的配方。鉴于生物加工技术的显著进步大大降低了成本 单克隆抗体的生产，以及通过阴道内持续递送抗体的可能性 戒指，我们相信现在开发用于非激素避孕的新型、超强效阿萨的时机已经成熟 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， 我们可以通过工程改造将阿萨的精子凝集效力大大提高>15-50倍 多价的基于IgG的构建体包含6至10个Fab结构域（即4至8个连接至Fab结构域的另外的Fab）。 亲本IgG分子）。这些新的构建体在加速热老化中具有与IgG相当的稳定性。 稳定性研究，以及与IgG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一种领先的分子， 临床试验定于2020年开始。在这个项目中，我们试图通过使用 尖端的双特异性抗体工程技术和通过酵母和哺乳动物细胞展示的亲和力成熟 以工程改造超有效双特异性多价阿萨构建体的文库。我们将靶向CD 52 g， 已表征和验证的抗原靶点仅存在于人精子上，而EPPIN是一种成熟的 通过抑制精子过度活化发挥作用的避孕靶点。在目标1中，我们将利用酵母展示， 提高Fab对CD 52 g和EPPIN的亲和力，工程化多价双特异性构建体文库 结合两个靶点，并严格表征这些分子。在目标2中，我们将执行一组 评估其潜在避孕效果的研究，包括精子凝集、捕获单个精子 和抑制精子过度活化。为了确定哪种结构将最有效地减少 在人类阴道中的精子运动性，我们将在目标3中使用两种药物进行体内剂量探索研究 在适应绵羊阴道的性交后生育力试验中，来自目的1和2的最有效的构建体。成功 这些研究的完成将可能使我们能够推进一种新的阿萨候选药物， 避孕效力比目前正在开发的非激素避孕的阿萨分子。