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类感染新生隐球菌属中等有效。然而，由于几个原因，细胞因子的系统性传递可能会出现问题。细胞因子以局部方式起作用。此外，它们会迅速降解，副作用往往会限制它们的使用。为了克服这些局限性，我们建议构建和测试几种针对新生弧菌感染的抗体-细胞因子融合蛋白，目的是开发新的预防和治疗疫苗策略。对于这些融合蛋白，我们将使用针对新生弧菌外膜的抗体，将细胞因子直接靶向感染部位(S)，同时提高细胞因子的稳定性，从而在增加有效剂量的同时减少全身毒性。目前还没有针对新生葡萄球菌的有效疫苗。通过将细胞因子与新生葡萄球菌抗原联系起来，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-细胞因子融合蛋白预防和治疗新生弧菌感染的有效性，我们提出：1.构建、制备和鉴定针对新生弧菌胶囊的抗体-细胞因子融合蛋白。抗体-细胞因子融合蛋白由铰链柔性的人抗隐球菌壳多糖与白细胞介素2(IL-2)、粒细胞巨噬细胞集落刺激因子(GM-CSF)或IL-12基因融合而成。融合蛋白的特征是与新生葡萄球菌结合，以及它们作为细胞因子和抗体的功能。2.检测抗体-细胞因子融合蛋白对新生葡萄球菌的保护性免疫作用。用上述抗体-细胞因子融合蛋白调理热灭活的新生葡萄球菌或囊膜多糖，并给小鼠注射。或者，树突状细胞将在体外装载这些免疫复合体并输送给小鼠。将分析针对新生弧菌的宿主抗体和细胞免疫反应，并在肺部和播散性感染的小鼠模型中确定疫苗的保护效果。3.研究新型隐球菌胶囊特异性抗体融合细胞因子对隐球菌感染的疗效。抗体-细胞因子融合蛋白将被给予新感染或已确诊感染的小鼠，以确定它们是否可以直接预防播散性隐球菌感染。将定义宿主免疫反应并将其与疗效相关联。隐球菌性脑膜炎的死亡率为10%-20%，这表明需要更好的治疗方法。由于艾滋病毒感染者存活率的提高、新的艾滋病毒感染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而导致的免疫功能受损的人数持续增加，隐球菌病的风险人群继续增长。拟议的研究不仅与隐球菌感染有关，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.