Vaccines and Therapeutics for Anthrax

炭疽疫苗和治疗方法

基本信息

批准号：
9354779
负责人：
Stephen Leppla
金额：
$ 75.43万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9354779
关键词：
Acute Myelocytic Leukemia Adenovirus Vector Adenylate Cyclase Affinity Alpaca Anthrax Vaccines Anthrax disease Antibodies Antigens Antineoplastic Agents Bacillus anthracis Bacillus anthracis spore Bacteria Bacterial Infections Binding Binding Sites Biochemical Biological Assay Blast Cell Blood capillaries Bone Marrow Budgets Cell Line Cell Proliferation Cell surface Cells Chimeric Proteins Cleaved cell Cyclic AMP Cyclophosphamide Cytosol Data Development Disease Edema Endothelial Cells Engineering Epitopes Genetic Glutamine Heating Human Immune response Implant In Vitro Inbred Strains Mice Individual Infection Infection prevention Intervention Isoleucine Knock-out Knockout Mice Libraries Link MEK inhibition MEKs Malignant Neoplasms Matrix Metalloproteinases Methods Mitogen-Activated Protein Kinase Kinases Modeling Monoclonal Antibodies Morphogenesis Mus Mutate Pathway interactions Pentostatin Peptide Hydrolases Peripheral Blood Mononuclear Cell Phage Display Property Protein C Inhibitor Proteins Rattus Recombinants Resistance Rodent Septic Toxemia Serine Protease Solid Neoplasm Specificity Symptoms Testing Therapeutic Therapeutic Index Toxic Actions Toxic effect Toxin Tumor-Derived Urokinase Vaccine Therapy Valine Variant Virulence Virulence Factors Work angiogenesis anthrax lethal factor anthrax toxin anthrax toxin receptors base cancer cell capillary cell type cytotoxicity drug candidate edema factor gene therapy immunogenicity improved in vivo killings macrophage mutant neoplastic cell pathogen preference prevent prophylactic receptor screening sensor success testisin therapeutic development therapeutic vaccine tool tumor tumor endothelial marker 8 vaccine development

项目摘要

Anthrax lethal toxin (LT) and edema toxin (ET) are made of three protein components: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the cell binding moiety which binds to cellular receptors Tumor Endothelial Marker-8 (TEM8) and Capillary Morphogenesis Protein-2 (CMG2). After binding these cellular receptors, PA is proteolytically cleaved by ubiquitously expressed cell surface proteases, and forms an active oligomer which allows binding of the two enzymatic moieties, LF and EF and their transport into the cell cytosol. LF leaves several mitogen activated protein kinase kinases (MEKs) and rodent inflammasome sensor Nlrp1. EF is an adenylate cyclase which converts ATP to cAMP. These two toxins are the major virulence determinants of anthrax, so vaccine and therapeutic development against this disease primarily targets these proteins. Re-engineering of PA to selectively be activated by tumor cells also allows an opportunity for development of anti-cancer agents. In the last budget period we continued our work in identification and characterization of variable domains of camelid heavy chain-only antibodies (VHHs) against anthrax toxins. These proteins are small single chain, heat- and pH- stable entities that can access epitopes that conventional antibodies cannot. Furthermore, they can be produced in recombinant form and purified rapidly once their sequence is known. Fifteen VHHs with subnanomolar affinity for LF and EF were obtained from immunized alpacas and screened for anthrax neutralizing activity. Multiple classes of neutralizing VHHs were identified, including VHHs cross reactive against both toxins and preventing toxin entry into cells. Individual VHHs as well as bispecific neutralizing agents made from two linked neutralizing VHHs were tested in lethal anthrax spore infection models as well as mouse footpad edema models with success. In a separate related study, gene therapy with an adenoviral vector expressing a bispecific VHH-based neutralizing agent consisting of two linked VHHs targeting different PA-neutralizing epitopes was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. This study demonstrated the potential for genetic delivery of antibodies as an effective method for prophylactic protection from anthrax. We also extended our studies with tumor-targeted anthrax lethal toxin to demonstrate that tumor cells which are normally resistant to anthrax toxins in vitro are highly sensitive to toxins when implanted in mice and that the host TEM8 and CMG2 receptors are required for toxin efficacy against these solid tumors. Using mice in which anthrax toxin receptors were selectively knocked out or exclusively expressed in various stromal compartments, we demonstrated that tumor sensitivity to toxin therapy requires CMG2 expression in host-derived tumor endothelial cells. Toxin inhibits proliferation of these cells through inhibition of the MEK pathways. Thus, the impact of the toxin therapeutics on tumor vessels and angiogenesis is the primary mechanism of action against solid tumors. In the same studies, we show that suppression of the host immune response with a regime of pentostatin and cyclophosphamide to overcome the immunogenicity of the toxin allows for an increase in the number of rounds of toxin therapy, and a significant improvement in therapeutic potential. In a related study on development of the anthrax toxins as anti-cancer agents, we used a phage display selection method to isolate PA variants with selective enhanced binding to CMG2 relative to TEM8. PA isoleucine residue 656 when mutated to valine or glutamine was found to have significantly reduced activity against TEM8-expressing cells while maintaining activity against CMG2-expressing cells. The mutant PA preference for CMG2 over TEM8 was also demonstrated in CMG2 and TEM8 knockout mice. We improved the specificity of previously constructed toxin anti-cancer agents in other studies. LF binding sites which require three subsites on two adjacent PA molecules were mutated in urokinase-activated and matrix metalloproteinase-activated PA variants in order to achieve higher specificity by requiring intermolecular complementation of these tumor-cell protease dependent PA molecules for inducing toxicity. Screening of a library of PA variants allowed selection of two optimal low activity mutants which when used in combination provided potent anti-tumor activity. We also collaborated on two studies related to anthrax toxin targeting of cancer cells. In the first, the toxicity of the urokinase-activated anthrax toxin against acute myeloid leukemia (AML) lines was correlated with urokinase plasminogen activator (uPAR) expression and MEK activity of each cell line. Bone marrow blasts and peripheral blood mononuclear cells were found to lack uPAR expression and were resistant to the toxin, demonstrating tumor selectivity in targeting of AML. In a separate study, a PA variant in which the sequence derived from protein C inhibitor replaced the native PA cleavage sequence was created. This variant was cleaved by the tumor associated serine protease testisin and showed increased killing of testisin-expressing cancer cells both in vitro and in vivo.

炭疽致死毒素（LT）和水肿毒素（ET）由三种蛋白质成分组成：保护性抗原（PA），致死因子（LF）和水肿因子（EF）。 PA是与细胞受体肿瘤内皮标记物8（TEM8）和毛细管形态发生蛋白2（CMG2）结合的细胞结合部分。结合了这些细胞受体后，PA被普遍表达的细胞表面蛋白酶蛋白水解裂解，并形成活性低聚物，该寡聚物允许两个酶促部分，LF和EF及其转运及其转运到细胞胞质醇。 LF留下了几种有丝分裂激活的蛋白激酶激酶（MEK）和啮齿动物炎症体传感器NLRP1。 EF是一种将ATP转换为CAMP的腺苷酸环化酶。这两种毒素是炭疽的主要毒力决定因素，因此针对该疾病的疫苗和治疗性发育主要针对这些蛋白质。重新设计PA以选择性地被肿瘤细胞选择性激活，还可以机会开发抗癌剂。在最后一个预算期间，我们继续在鉴定和表征骆驼型重链抗体（VHHS）对炭疽毒素的可变域（VHHS）方面的工作。这些蛋白质是小型单链，热和ph稳定的实体，可以访问常规抗体无法使用的表位。此外，它们可以以重组形式产生，并在序列已知后迅速纯化。从免疫羊驼获得了15个对LF和EF的亚摩尔亲和EF的VHH，并筛选了炭疽中和中和活性。鉴定了多种中和VHHS的类别，包括对毒素的VHHS交叉反应性和防止毒素进入细胞。在致命的炭疽孢子感染模型中测试了由两个连接的中和VHH制成的单独的VHHS以及双连接的中和VHH的双特异性中和剂，以及成功的小鼠FOODPAD水肿模型。在另一项相关研究中，用腺病毒载体表达了双特异性VHH的基因治疗，该基于双特异性VHH的中和剂由两种靶向不同的PA中和表位的连接的VHH组成，以两种近近交布小鼠菌株，BALB/CJ和C57BL/6J，发现以保护小鼠的抗sporce和Anthrax sporce和Anthrax cearbles anthrax cearble andthraxin spection和Anthrax sporce andtheraxin。这项研究表明，抗体遗传递送作为预防性保护免受炭疽的有效方法的潜力。我们还通过靶向肿瘤的炭疽致死毒素扩大了研究，以证明通常在植入小鼠中对毒素具有对毒素的抗性细胞，并且宿主TEM8和CMG2受体对毒素具有高度敏感性。使用在各种基质室中选择性地敲除或专门表达炭疽毒素受体的小鼠，我们证明肿瘤对毒素疗法的敏感性需要在宿主衍生的肿瘤内皮细胞中CMG2表达。毒素通过抑制MEK途径抑制这些细胞的增殖。因此，毒素疗法对肿瘤血管的影响和血管生成是对实体瘤的作用机理。在同一项研究中，我们表明，抑制pentostatin和环磷酰胺方面对宿主免疫反应的抑制，以克服毒素的免疫原性，从而可以增加毒素治疗的回合数量，并显着改善治疗势。在一项关于炭疽毒素作为抗癌剂的相关研究中，我们使用噬菌体显示选择方法来分离具有选择性增强的与CMG2相对于TEM8的PA变体。 PA异亮氨酸残基656在突变为缬氨酸或谷氨酰胺时，对表达TEM8的细胞的活性显着降低，同时维持对表达CMG2表达CMG2的细胞的活性。在CMG2和TEM8敲除小鼠中也证明了CMG2高于TEM8的突变体PA偏好。我们在其他研究中提高了先前构建的毒素抗癌剂的特异性。 LF结合位点需要在两个相邻的PA分子上进行三个亚矿石的LF结合位点在尿蛋白酶激活和基质金属蛋白酶激活的PA变体中突变，以通过需要对这些肿瘤细胞蛋白酶依赖蛋白酶的PA分子的分子互补来实现更高的特异性，以诱导诱导毒性。对PA变体库进行筛选允许选择两个最佳低活性突变体，这些突变体在组合使用时提供了有效的抗肿瘤活性。我们还合作进行了两项与炭疽毒素靶向癌细胞有关的研究。首先，尿激酶激活的炭疽毒素对急性髓细胞性白血病（AML）线的毒性与尿激酶纤溶酶原激活剂（UPAR）表达和MEK活性相关。发现骨髓爆炸和外周血单核细胞缺乏UPAR表达，并且对毒素具有抗性，表现出在靶向AML时的肿瘤选择性。在另一项研究中，产生了蛋白C抑制剂衍生的序列取代天然PA裂解序列的PA变体。这种变体被肿瘤相关的丝氨酸蛋白酶睾丸裂解，并显示出表达睾丸蛋白的癌细胞在体外和体内增加。