Mechanism-based Changes in Anti-METH Monoclonal Antibody Therapy During Pregnancy

说明(申请人提供)：(+)-甲基苯丙胺(冰毒)是孕妇中最容易上瘾和滥用最严重的药物之一，但目前还没有批准的药物来保护脆弱的胎儿或母亲免受其不利的健康影响。这些研究的长期目标是开发基于抗体的药物，这些药物可以在怀孕期间给予，以保护母亲和胎儿免受冰毒的毒性和成瘾影响。虽然人类怀孕期间的单抗(MAb)治疗未经测试，但我们实验室在怀孕大鼠身上进行的开创性研究揭示了mAb药代动力学(PKs)的新的和具有治疗意义的变化，这些变化损害了抗体的效力。具体地说，单抗的半衰期、分布体积和清除量都随着妊娠阶段的不同而发生显著变化。类似的妊娠期特异性PK变化以前还没有报道，驱动这些变化的机制目前尚不清楚。在测试单抗功能的相关研究中，某些抗冰毒单抗药物(如mAb6H4)在给药后24小时内体内冰毒结合功能显著降低。即使94%的mAb剂量仍然存在，这种情况也会发生。重要的是，尽管所有单抗的剂量相同并且表现出相似的PK特征，但对甲基(Kd)的结合亲和力与体内功能并不相关。这一建议的中心假设是，新生儿Fc受体(FcRN)挽救途径既是孕期mAb清除变化的关键调节因素，也有助于体内mAb甲基结合功能的降低。FcRN是一种具有良好特性的受体，它通过一种依赖于pH的挽救机制来从分解代谢消除(清除)中拯救免疫球蛋白并促进免疫球蛋白从母亲到胎儿的被动转移。然而，FcRN在妊娠期间调节mAbPKs的作用尚不清楚。此外，在回收途径中发现的低pH环境表明它可以促进在体内对单抗的修饰。为了验证我们的假设，我们将执行以下目标：1-在单抗清除变化最大的时候确定FcRN在怀孕期间的组织表达水平，2-确定FcRN挽救途径在调节妊娠期间IgG清除中的作用，以及3-定义FcRN挽救途径在降低mAb6H4甲基结合功能中的作用。这项拟议的研究将利用免疫组织化学和Western blotting技术，结合怀孕和未怀孕大鼠的体内PK实验。作为这些研究的工具，抗冰毒mAb6H4将被重新设计为单一氨基酸突变，消除FcRN结合并抑制通过挽救途径。除了为医学/博士候选人提供极好的培训机会外，这些创新研究还将有助于单抗药物的临床前开发，以保护母亲和胎儿免受药物滥用对健康的危险影响。 公共卫生相关性：这些研究将确定FcRN挽救途径在调节孕期单抗清除和功能中的作用，对影响抗体治疗效果的体内生理过程产生新的见解。这一知识有望推动安全有效的基于抗体的药物的临床前开发，用于治疗孕妇及其易受药物滥用的胎儿。此外，由于FcRN在调节内源性和外源性Ig G类抗体方面的普遍作用，这些研究也将有助于我们理解怀孕和非怀孕生理状态下体液免疫系统的调节。

DESCRIPTION (provided by applicant): (+)-Methamphetamine (METH) is one of the most addictive and heavily abused drugs among pregnant women, yet there are no approved medications for protecting the vulnerable fetus or mother from its adverse health effects. The long-term objective of these studies is to develop antibody-based medications that can be given during pregnancy to protect the mother and fetus from the toxic and addictive effects of METH. While monoclonal antibody (mAb) therapy during human pregnancy is untested, seminal studies from our laboratory in pregnant rats reveal novel and therapeutically important changes to mAb pharmacokinetics (PKs) that compromise antibody efficacy. Specifically, mAb half-life, volume of distribution, and clearance all dramatically change with each gestation stage. Similar gestation-stage specific PK changes have not been reported previously, and the mechanisms that drive these changes are currently unknown. In related studies testing mAb function, certain anti-METH mAb medications (e.g., mAb6H4) show significant in vivo reductions in METH-binding function during the first 24 hrs after administration. This occurred even though >94% of the mAb doses were still present. Importantly, binding affinity for METH (KD) did not correlate with in vivo function despite all mAbs being administered at equal doses and displaying similar PK characteristics. The central hypothesis of this proposal is that the neonatal Fc receptor (FcRn) salvage pathway is both a key regulator of mAb clearance changes during pregnancy and facilitates in vivo reductions in mAb METH- binding function. FcRn is a well-characterized receptor that acts via a pH-dependent salvage mechanism to rescue IgG from catabolic elimination (clearance) and facilitate the passive transfer of IgG from mother to fetus. However, the role of FcRn in regulating mAb PKs during pregnancy is unknown. Furthermore, the low pH environment found in the salvage pathway suggests it could facilitate in vivo modifications to mAbs. To test our hypothesis, we will perform the following aims: 1 - Determine FcRn tissue expression levels during rat pregnancy at times when mAb clearance changes are maximal, 2 - Define the role of the FcRn salvage pathway in regulating IgG clearance during pregnancy, and 3 - Define the role of the FcRn salvage pathway in reducing mAb6H4 METH- binding function. The proposed studies will utilize immunohistochemistry and Western blotting techniques in conjunction with in vivo PK experiments in both pregnant and non-pregnant rats. Instrumental to these studies, anti-METH mAb6H4 will be re-engineered with a single amino acid mutation that eliminates FcRn binding and inhibits passage through the salvage pathway. In addition to providing an excellent training opportunity for a MD/PhD candidate, these innovative studies will contribute to the pre-clinical development of mAb medications for the protection of both the mother and fetus from the dangerous health effects of drugs of abuse. PUBLIC HEALTH RELEVANCE: These studies will define the role of the FcRn salvage pathway in regulating monoclonal antibody (mAb) clearance and function during pregnancy, yielding new insights into the in vivo physiologic processes that affect antibody therapeutic efficacy. This knowledge is expected to advance the preclinical development of safe and effective antibody-based medicines for use in treating pregnant women and their vulnerable fetus(es) from drugs of abuse. Furthermore, due to the ubiquitous role of FcRn in regulating both endogenous and exogenously administered antibodies of the IgG class, these studies will also contribute to our understanding of the regulation of the humoral immune system during pregnant and non-pregnant physiologic states.