Antibody cytokine fusion proteins against Cryptococcus neoformans

描述（由申请方提供）：细胞因子治疗对小鼠和人新型隐球菌感染中度有效。然而，由于几个原因，细胞因子的全身递送可能是有问题的。细胞因子以局部方式起作用。此外，它们迅速降解，副作用往往限制了它们的使用。为了克服这些局限性，我们提出了构建和测试几个抗体-细胞因子融合蛋白抗C。新型人感染，目的是开发新的预防性和治疗性疫苗接种策略。对于这些融合蛋白，我们将使用对C.新形式，直接将细胞因子靶向感染部位，同时改善细胞因子稳定性;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C。新人类通过将细胞因子与C.新生儿抗原、抗体-细胞因子融合蛋白可有效产生保护性免疫。因此，为了检测抗体-细胞因子融合蛋白在预防和治疗C。我们提出：1.目的构建、制备和鉴定抗C.新囊将重组产生抗体-细胞因子融合蛋白，其由抗隐球菌荚膜多糖的铰链柔性人IgG 3组成，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遗传融合。将表征融合蛋白与C.新型人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C.新人类热死C。将用上述抗体-细胞因子融合蛋白调理新生儿或荚膜多糖并给予小鼠。或者，树突状细胞将离体负载这些免疫复合物并递送至小鼠。宿主抗体和细胞免疫反应特异性C。将分析新生儿，并在肺部和播散性感染的小鼠模型中确定疫苗接种的保护效力。3.确定与C.隐球菌感染中的neoformans荚膜。将抗体-细胞因子融合蛋白给予具有新的或已建立的感染的小鼠，以确定它们是否可以直接保护免受播散性隐球菌感染。将定义宿主免疫应答并将其与疗效相关联。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HIV感染者的存活率增加、新的HIV感染以及由于器官移植和风湿性疾病的免疫抑制疗法以及癌症的化疗而导致的免疫功能低下的人数持续增加，处于隐球菌病风险中的人群继续增长。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用于其他类型感染的抗体-细胞因子融合蛋白。

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.