chun yang - US grants

? DESCRIPTION (provided by applicant): Sleep problems occur in most people with mental disorders (e.g. Schizophrenia, Alzheimer's and Parkinson's diseases) and are common in many other disorders (e.g. pain, cancer, brain injury, stroke). Drowsiness caused by neuropsychiatric conditions, other disorders or work/life-style has a negative impact on our health & cognitive performance and sometimes can even lead to death (e.g. falling asleep during driving). Many people self-medicate with stimulants such as caffeine to temporarily restore alertness, while clinically, patients are prescribed medications such as modafinil to treat excessive sleepiness. However, current treatment strategies produce many side effects (physical tolerance, dependence, etc.). Thus, there is a substantial need to identify new pharmacological mechanisms that can be utilized to prevent drowsiness, and improve cognitive performance. Previous work from our laboratory and others has demonstrated the significance of basal forebrain (BF) as a wakefulness-promoting center. Its cortically-projecting systems play an important role in regulating cortical activity, plasticity and cognition. Our novel preliminary dat suggest that a purinergic P2 receptor (P2R) agonist applied into BF increases wakefulness in vivo and excites BF putative cortically-projecting neurons in vitro. Therefore, investigation of BF P2Rs' role in sleep-wake regulation may contribute to the discovery of novel therapeutic targets, which can greatly benefit the general public in removing drowsiness, improving cognitive performance and preventing errors/accidents. We will test our overarching hypothesis that activation of BF P2Rs induces wakefulness by exciting cortically-projecting BF neurons. Both specific aims (SAs) will be tested in mouse models, with Swiss-Webster mice for SA1 and a state-of-the-art transgenic mouse model (GAD67-GFP knock-in mice) which allows the identification of GABA neurons prior to recording for SA2. SA1 will directly test the role of P2Rs by specifically stimulating the BF P2R system with local drug infusion using reverse microdialysis and measuring the changes of sleep-wake states and power spectra with electroencephalogram (EEG)/electromyogram (EMG) recording techniques. We will use multiple P2R agonists (with different selectivity to class P2XRs vs class P2YRs) and co-infusion of a P2XR antagonist into BF to determine whether P2XRs or P2YRs in BF are involved in promoting wakefulness. We will also investigate if acute and chronic partial sleep-deprivation-induced drowsiness can be reversed with BF P2R activation, an essential first step towards translational studies. SA2 will investigate the cellular mechanisms by focusing on BF cortically-projecting cholinergic and GABAergic neurons. We will use whole-cell patch clamp to record the individual cellular responses to the P2R agonists and determine if P2XRs or P2YRs are responsible for these responses. Our study proposed here will advance our understanding of the BF purinergic system in manipulating cortical activity and could contribute to the identification of novel therapeutic targets for removing drowsiness and improving cognition.

PROJECT SUMMARY This proposal aims to develop a novel caveolae targeting antibody for rapid clinical translation. Its long-term objective is to develop the caveolae pumping system, an active transendothelial transport pathway, to provide an effective solution to the delivery and toxicity problem of systemically administered chemotoxins. Work will be based on the current proprietary mouse monoclonal antibody that targets a truncated form of Annexin A1 (mAnnA1) that is concentrated in the endothelial cell caveolae of tumor vasculature. Previous work has demonstrated mAnnA1 to be the first antibody to penetrate solid tumors actively, rapidly, and specifically, and concentrate attached cargo directly into target tissue. Humanization of mAnnA1 is expected to yield a variant with similar binding affinity, stability, and purity that can then be used for further commercial development to generate novel immunoconjugates to treat many forms of primary and metastatic cancers. We hypothesize that enhancing precision delivery will confer significant survival advantages for patients through the antibody's ability to concentrate chemotoxins directly into solid tumors and metastatic lesions, thus sparing exposure to healthy tissues. The overall objective of this Phase I application is to advance translational studies of mAnnA1 through development and selection of a humanized antibody with optimized binding affinity and therapeutic potential. In Aim 1, antibody humanization will proceed using CDR grafting technology, producing 9-16 variants. Selection of hAnnA1 candidates with binding affinities similar to mAnnA1 will be performed using ELISA and surface plasmon resonance analysis. In Aim 2, hAnnA1 variants will be further screened by their ability to retain key biophysical properties (binding affinity, stability, purity) after chemical conjugation. The therapeutic potencies of the top 2 variants, in the form of therapeutically active immunoconjugates, will be validated in vivo using a metastatic model of breast cancer. The proposed study is highly significant because a first-in-class humanized AnnA1 antibody with minimized immunogenicity could deliver therapeutic cargo precisely across in vivo barriers and concentrate them inside tumors, and thus obviate the current reliance on passive transvascular delivery. If successful, it will create the first precision drug delivery platform based on the caveolae pumping system. The resulting lead hAnnA1 antibody will be advanced in an STTR Phase II project for further commercial and preclinical development, pharmacological and toxicological testing, and other studies needed for IND filing and clinical utility. Potential partnering or out-licensing points for pharmaceutical company facilitation of commercialization of hAnnA1 can occur after each completed phase of the overall project.