Antibody cytokine fusion proteins against Cryptococcus neoformans

描述（由申请人提供）：细胞因子治疗对小鼠和人感染新型隐球菌有中等效果。然而，由于几个原因，细胞因子的全身递送可能会出现问题。细胞因子以局部方式起作用。此外，它们会迅速降解，副作用往往限制了它们的使用。为了克服这些限制，我们提出构建和测试几种针对新型C. neoformans感染的抗体-细胞因子融合蛋白，以开发新的预防和治疗疫苗策略。对于这些融合蛋白，我们将使用一种针对新生芽孢杆菌外囊的抗体，将细胞因子直接靶向感染部位，同时提高细胞因子的稳定性；从而增加有效剂量，同时降低全身毒性。目前还没有有效的疫苗用于预防新生弓形虫。通过将细胞因子与新生C.抗原连接，抗体-细胞因子融合蛋白可能有效地产生保护性免疫。因此，为了验证抗体-细胞因子融合蛋白在预防和治疗新生梭状菌感染中的作用，我们提出：目的：构建、制备和表征新生荚膜特异性抗体-细胞因子融合蛋白。抗体-细胞因子融合蛋白由针对隐球菌荚膜多糖的铰链柔性人IgG3组成，可与白细胞介素-2 （IL-2）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）或IL-12基因融合。融合蛋白的特点是与新生C.结合，以及它们作为细胞因子和抗体的功能。2. 确定抗体-细胞因子融合蛋白对新生梭状菌产生保护性免疫的潜力。用上述抗体-细胞因子融合蛋白对热杀死的新生C.或荚膜多糖进行调理，并给予小鼠。或者，树突状细胞将在体外装载这些免疫复合物并传递给小鼠。在小鼠肺和播散性感染模型中，将分析针对新型C.的宿主抗体和细胞免疫反应，并确定疫苗接种的保护效果。3. 确定细胞因子基因融合抗体特异性新生隐球菌胶囊治疗隐球菌感染的疗效。将抗体-细胞因子融合蛋白给予新感染或已感染的小鼠，以确定它们是否能直接保护小鼠免受播散性隐球菌感染。宿主免疫反应将被定义并与疗效相关。隐球菌脑膜炎的死亡率为10-20%，表明需要更好的治疗方法。由于艾滋病毒感染者的存活率增加、新的艾滋病毒感染病例增加，以及由于器官移植和风湿病的免疫抑制疗法以及癌症化疗导致免疫功能低下的人数继续增加，隐球菌病的风险人口继续增长。拟议的研究具有超越隐球菌感染的相关性，因为一个重要的长期目标是设计抗体-细胞因子融合蛋白用于其他类型的感染。

DESCRIPTION (provided by applicant): Treatment with cytokines is moderately effective against mouse and human infection with Cryptococcus neoformans. However, systemic delivery of cytokines can be problematic for several reasons. Cytokines act in a local manner. Furthermore, they are rapidly degraded and side effects often limit their use. To overcome these limitations, we propose to construct and test several antibody-cytokine fusion proteins against C. neoformans infection with the goal of developing novel prophylactic and therapeutic vaccination strategies. For these fusion proteins, we will use an antibody specific for the outer capsule of C. neoformans, targeting cytokines directly to the site(s) of infection, while improving cytokine stability; thereby increasing the effective dose while decreasing systemic toxicities. There are currently no effective vaccines in use against C. neoformans. By linking cytokines to C. neoformans antigens, antibody-cytokine fusion proteins may be effective in generating protective immunity. Therefore, to test the efficacy of antibody-cytokine fusion proteins in preventing and treating C. neoformans infection, we propose: 1. To construct, produce and characterize antibody-cytokine fusion proteins specific for C. neoformans capsule. Antibody-cytokine fusion proteins consisting of the hinge-flexible human IgG3 against cryptococcal capsular polysaccharide genetically fused to either interleukin-2 (IL-2), granulocyte- macrophage colony stimulating factor (GM-CSF) or IL-12 will be produced recombinantly. Fusion proteins will be characterized for binding to C. neoformans as well as their functionality as cytokines and antibodies. 2. To determine the potential of antibody-cytokine fusion proteins to generate protective immunity to C. neoformans. Heat-killed C. neoformans or capsular polysaccharide will be opsonized with antibody- cytokine fusion proteins described above and administered to mice. Alternatively, dendritic cells will be loaded ex vivo with these immune complexes and delivered to mice. Host antibody and cellular immune responses specific for C. neoformans will be analyzed and protective efficacy of vaccination determined in mouse models of pulmonary and disseminated infection. 3. To determine the efficacy of cytokines genetically fused to antibody specific for C. neoformans capsule in cryptococcal infection. Antibody-cytokine fusion proteins will be given to mice with new or established infection to determine whether they can directly protect against disseminated cryptococcal infection. Host immune responses will be defined and correlated with efficacy. Mortality from cryptococcal meningitis is 10-20% indicating the need for better therapies. The population at risk for cryptococcal disease continues to grow due to increased survival of persons infected with HIV, new HIV infections, and the continued expansion of the number of persons with immunocompromise due to immuno- suppressive therapies for organ transplantation and rheumatic diseases as well as chemotherapy for cancer. The proposed studies have relevance beyond cryptococcal infection, since a significant long-term objective is to design antibody-cytokine fusion proteins for use in other types of infections.