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: 8050650
• 负责人: KAM-MENG TCHOU-WONG
• 金额: $ 21.43万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050650
• 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; 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）：蓖麻毒素是蓖麻（Ricinus communis）的天然产物，属于B类毒素，因其热稳定性、全球可用性和易于生产而成为重要的生物武器。它可以以气溶胶的形式传播，这是恐怖分子可能使用的途径。蓖麻毒素是一种由二硫桥连接的两条多肽链组成的凝集素，需要进入细胞才能产生毒性。蓖麻毒素b链（RCB）促进毒素进入细胞，蓖麻毒素a链（RCA）具有RNA n -糖苷酶活性，攻击28S rRNA上的特定位点，阻止多肽链延伸，从而使核糖体失活（核糖体毒性）并导致细胞死亡。由于蓖麻毒素在细胞内化前仅在循环中游离一小段时间（数小时至数天），因此能够阻断细胞内部位RCA酶活性的治疗方法将获得最大的临床益处。我们最近有证据表明，在蓖麻毒素攻击后44小时和54小时，高剂量直接给药的新型抗蓖麻毒素a链抗体（43RCA IgG）可分别提供83%和75%的存活率。将来自猕猴的43RCA scFv的V结构域与人类IgG1的恒定区域融合，生成了43RCA IgG。43RCA scFv序列与人IgG种系基因的序列非常相似，在VH和VL区域具有90%的序列一致性，从而增加了这种类人抗体作为人类蓖麻毒素解毒剂的潜力。在人类接触的情况下，无论是偶然的还是故意的，预计会出现接触后治疗的延误。因此，必须开发治疗窗口超过2天的解毒剂，以便有足够的时间对暴露者进行治疗。因此，我们假设，通过细胞内递送中和性抗rca抗体来阻断细胞内蓖麻毒素活性，可以改善和延长暴露后治疗的治疗指数和/或窗口。为了实现我们的目标，我们将通过将抗体偶联到转运肽或细胞穿透肽（CPPs）来开发细胞渗透性单克隆抗体（或transmab），并将根据以下具体目标评估其治疗效果。特异性目的1：在细胞培养模型中确定细胞渗透性抗体对蓖麻毒素细胞毒性的保护作用。特异性目的2：利用肺吸入模型确定细胞渗透性抗体对暴露后蓖麻毒素诱导的肺损伤的治疗效果和致死率。特异性目的3：验证细胞渗透性抗体对雾化蓖麻毒素小鼠模型暴露后治疗的有效性。