Human-Enzyme Mediated, Systemic Depletion of Cystine for Cancer Treatment.

描述(申请人提供)：广泛的研究表明，与正常组织相比，前列腺癌(Pca)和其他高死亡率癌症，包括胶质母细胞瘤和小细胞肺癌，为增殖和生存严重依赖非必需氨基酸L-半胱氨酸(L-半胱氨酸)及其氧化形式L-半胱氨酸(CSSC)的摄取(由于对抗氧化剂的需求增加，以及在一些肿瘤中，它们显示内源性L-半胱氨酸合成缺陷)。我们假设，酶介导的血清L-半胱氨酸/CSSC池的全身性耗竭(使用一种经过改造的人酶，因此很可能具有良好的耐受性)构成了一种强大的、全新的癌症治疗方法，用于PCa作为一种单一治疗方法，并作为一种与现有化疗方法机械协同的药物。这一假说得到强有力的初步数据的支持，证实在PCa同种异体移植模型中(每隔4天)给药一次典型的工程化人类L-半胱氨酸/CSSC耗竭酶：(I)导致血清L-半胱氨酸/CSSC池的几乎完全耗尽；(Ii)介导持续和完全停止肿瘤的生长，使癌细胞经历细胞周期停止并伴随着氧化应激和自噬增加；(Iii)重要的是，治疗一个月以上耐受性非常好，没有体重减轻或严重毒副作用。为了建立在这些初步观察的基础上，我们提出了一项详细的研究计划，旨在提供临床相关的酶治疗候选方案，并通过体外和异体/异种移植的PCA模型提供验证，最终将有助于支持晚期临床前开发和IND应用。虽然L-半胱氨酸/CSSC耗竭可能适用于许多肿瘤的治疗，但我们选择关注PCa，首先是因为许多人PCa肿瘤已知存在L-半胱氨酸/CSSC动态平衡的缺陷，其次是因为需要有效的无毒治疗，因为PCa是美国男性癌症死亡的第二大原因。实现这些目标需要完成3个具体目标下的工作：SP。目标1将专注于设计一种可用于L半胱氨酸/CSSC耗竭的候选人酶，该酶具有最佳的酶动力学、底物选择性、稳定性 通过生物处理使其失活和聚集，并简化生产。将详细评估优化后的酶的PK和PD以及重复给药的任何毒性效应。在Sp.目的2我们将试图了解L半胱氨酸/CSSC缺失对PCa细胞的整体机制/代谢后果，哪些生物标志物可以预测敏感性/耐药性，并确定哪些与标准护理化疗药物联合可能提供协同/相加效应。最后，在Sp.目的：我们将在临床相关的不同表型(即P53基因状态、雄激素依赖/非依赖性)的原位PCA动物模型和小鼠原位PCA移植模型中评价优化的酶，以便在接近实际疾病的模型中研究该药物的疗效。

DESCRIPTION (provided by applicant): Extensive studies have revealed that in contrast to normal tissue, prostate carcinomas (PCa) and other high mortality cancers including glioblastomas, and small cell lung carcinomas are critically dependent on the uptake of the non-essential amino acid L-cysteine (L-Cys) and its oxidized form L-cystine (CSSC) for proliferation and survival (due to increased requirements for anti-oxidants and in some tumors because they display defects in endogenous L-Cys synthesis). We hypothesize that enzyme-mediated systemic depletion of the serum L-Cys/CSSC pool (using an engineered human enzyme, and thus likely well tolerated) constitutes a powerful and completely novel cancer therapeutic approach for PCa as a monotherapy and as an agent that is mechanistically synergistic with existing chemotherapeutic modalities. This hypothesis is supported by strong preliminary data demonstrating that the administration (every 4 days) of a prototypical engineered human L- Cys/CSSC depleting enzyme in an allograft model of PCa: (i) results in near complete depletion of the serum L-Cys/CSSC pool; (ii) mediates sustained and complete cessation of tumor growth with cancer cells experiencing cell cycle arrest accompanied by increased oxidative stress and autophagy and (iii) importantly treatment for over a month was very well tolerated with no weight loss or gross toxicities. To build on these preliminary observations we present a detailed research plan that is aimed at delivering a clinically relevant enzyme therapeutic candidate and providing the validation by in vitro and in allograft/xenograft PCa models that will eventually hel support late stage preclinical development and an IND application. While L-Cys/CSSC depletion may be applicable to the treatment of many tumors we elected to focus on PCa first because many human PCA tumors are known to experience defects in L-Cys/CSSC homeostasis and second because effective non-toxic treatments are needed as PCa is the second leading cause of cancer death in American men. Reaching these goals will require the completion of work under 3 Specific Aims: Sp. Aim 1 will focus on the engineering of a candidate human enzyme for L-Cys/CSSC depletion that displays optimal enzyme kinetics, substrate selectivity, stability to deactivation and aggregation and facile production by bioprocessing. The PK and PD of the optimized enzymes as well as any toxic effects upon repeated administration will be evaluated in detail. In Sp. Aim 2 we will seek to understand the global mechanistic/metabolic consequences of L-Cys/CSSC depletion on PCa cells, what biomarkers may predict susceptibility/resistance and to determine which combinations with standard of care chemotherapeutics may provide synergistic/additive effects. Finally, in Sp. Aim 3 we will evaluate the optimized enzyme in clinically relevant orthotopic PCa animal models that represent diverse phenotypes (i.e. p53 status, androgen dependence/independence) and mouse orthotopic PCa allografts such that the efficacy of the drug can be studied in models closely mimicking the actual disease.