Development of cell-permeable antibodies for post-exposure treatment of ricin

开发用于蓖麻毒素暴露后处理的细胞渗透性抗体

• 批准号: 7896982
• 负责人: KAM-MENG TCHOU-WONG
• 金额: $ 20万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896982
• 关键词: Aerosols; Alexa594; Antibodies; Antidotes; Biological; Biological Assay; Biological Factors; Blood Circulation; Breathing; Categories; Cell Culture Techniques; Cell Death; Cells; Chad; Clinical; Confocal Microscopy; Data; Development; Disulfides; Dose; Edema; Genes; Goals; Heating; Hour; Human; IgG1; Immunoglobulin Constant Region; Immunoglobulin G; In Vitro; Individual; Label; Lead; Lectin; Link; Lung; Lung Inflammation; Macaca; Measures; Military Personnel; Modeling; Monoclonal Antibodies; Mus; Narration; Peptide Transport; Peptides; Plants; Population; Production; RNA glycosylase; Ribosomal RNA; Ribosomes; Ricin; Ricin A Chain; Ricin B Chain; Ricinus communis; Route; Site; Survival Rate; Tandem Repeat Sequences; Testing; Therapeutic; Therapeutic Effect; Therapeutic Index; Time; Toxic effect; Toxin; Universities; Validation; Weight; aerosolized; animal facility; antibody conjugate; base; caspase-3; cytotoxicity; exposed human population; extracellular; improved; in vivo; lung injury; mouse model; neutralizing antibody; novel; polypeptide; prevent; protective effect; public health relevance; uptake; weapons; wet lung

DESCRIPTION (provided by applicant): Ricin, a natural product of the castor bean (Ricinus communis) and a Category B toxin, is significant as a biological weapon because of its heat stability, worldwide availability, and ease in production. It can be disseminated as an aerosol, a likely route that terrorists may use. The ricin toxin is a lectin consisting of two polypeptide chains linked by a disulfide bridge and cellular entry is required for toxicity. The ricin B-chain (RCB) facilitates entry of the toxin into the cell and the ricin A-chain (RCA) possesses RNA N-glycosidase activity that attacks a specific site on the 28S rRNA, preventing polypeptide chain elongation, thereby inactivating ribosomes (ribotoxic) and leading to cell death. Since the ricin toxin is free in the circulation for only a brief period of time (hours to days) before cellular internalization, the greatest clinical benefits will be derived from therapeutics capable of blocking RCA enzymatic activity at intracellular sites. We have recent evidence that a high dose of a novel primatized anti-ricin A-chain antibody (43RCA IgG) administered directly to the lung can offer 83% and 75% survival when administered at 44 hr and 54 hr post-ricin challenge, respectively. The primatized 43RCA IgG was generated by fusing the V domains from macaque-derived 43RCA scFv to the constant regions of the human IgG1. The 43RCA scFv sequence is very similar to that for human IgG germline genes, with 90% sequence identity for the VH and VL regions, thereby increasing the potential for this human-like antibody to be used as ricin antidote in humans. In the case of human exposure, whether it is accidental or deliberate, it is anticipated that there will be delays in post-exposure treatment. It is, therefore, imperative to develop antidotes with a therapeutic window beyond 2 days to allow sufficient time for treatment of exposed individuals. We therefore hypothesize that the therapeutic index and/or window for post- exposure treatment can be improved and extended by cytosolic delivery of neutralizing anti-RCA antibodies to block intracellular ricin activity. To achieve our goal, we will develop cell-permeable monoclonal antibodies (or TransMabs) by conjugating antibodies to transport peptides or cell-penetrating peptides (CPPs) and the therapeutic effects will be evaluated as described in the following specific aims. Specific Aim 1: To Ascertain the Protective Effects of Cell-Permeable antibodies against Ricin Cytotoxicity in Cell Culture Models. Specific Aim 2: To Ascertain the Efficacy of Cell-Permeable Antibodies for Post-Exposure Treatment of Ricin-Induced Lung Injury and Lethality using the Lung Aspiration Model. Specific Aim 3: To Validate the Efficacy of Cell-Permeable Antibodies for Post-Exposure Treatment in an Aerosolized Ricin Mouse Model. PUBLIC HEALTH RELEVANCE: Since there is currently no treatment for the ricin toxin, development of a specific antidote for the treatment of ricin after exposure will contribute significantly to the protection of our civilian and military populations. Additionally, the availability of an effective antidote may also significantly reduce the threat of the use of ricin as a biological weapon.

描述（由申请人提供）：Ricin是蓖麻豆（Ricinus communis）和B类毒素的天然产物，由于其热稳定性，全球可用性和生产易于生产而成为生物武器。它可以作为气溶胶传播，这是恐怖分子可能使用的一条可能的路线。 ricin毒素是一种凝集素，由两个由二硫键连接的两个多肽链组成，毒性需要细胞进入。 Ricin B链（RCB）有助于将毒素进入细胞，而Ricin A链（RCA）具有RNA N-Glycosidase酶活性，可在28S RRNA上攻击特定位点，从而防止多肽链的延长，从而阻止核糖体（核糖毒素）和导致细胞死亡。由于在细胞内在化之前仅短时间（小时至几天），在循环中ricin毒素是免费的，因此最大的临床益处将来自能够阻止细胞内部位的RCA酶活性的治疗剂。我们最近有证据表明，分别在44小时和54小时的联合蛋白后挑战时给予高剂量的高剂量的新型抗癌A链抗体（43RCA IgG）可提供83％和75％的存活率。通过将V域从猕猴衍生的43RCA SCFV融合到人IgG1的恒定区域，从而产生了Primatized 43RCA IgG。 43RCA SCFV序列与人类IgG系基因的序列非常相似，VH和VL区域具有90％的序列同一性，从而增加了这种人类样抗体用作人类ricin抗体的潜力。对于人类暴露，无论是偶然的还是故意的，预计暴露后治疗将会延迟。因此，必须用超过2天的治疗窗口开发解毒剂，以便有足够的时间治疗暴露的个体。因此，我们假设可以通过胞质递送中和中和中和的抗RCA抗体来阻止细胞内Ricin活性来改善和/或窗口进行暴露后治疗。为了实现我们的目标，我们将通过结合运输肽或细胞穿透肽（CPP）的抗体来开发可渗透的单克隆抗体（或透射），并将如下特定目标所述评估治疗效应。具体目的1：确定细胞渗透性抗体对细胞培养模型中细胞毒素的保护作用。具体目的2：使用肺抽吸模型确定可渗透性抗体在暴露后治疗Ricin诱导的肺损伤和致死性的功效。具体目标3：验证可渗透性抗体在雾化的莱西蛋白小鼠模型中暴露后处理的疗效。 公共卫生相关性：由于目前尚无治疗Ricin毒素的治疗，因此在暴露后开发用于治疗Ricin的特定解毒剂将对我们的平民和军事人群的保护产生重大贡献。此外，有效的解毒剂的可用性也可能会大大减少使用利辛蛋白作为生物武器的威胁。