DEVELOPING MULTI-ANTIGEN VACCINE FOR THE TREATMENT OF CHRONIC HBV

项目总结/摘要 该SBIR项目的最终目标是开发一种免疫疗法，基于我们的专利病毒样- 囊泡（VLV）平台，用于治疗慢性B型肝炎病毒感染的患者。在第一阶段，使用 慢性HBV感染的小鼠模型，我们已经建立了一个概念验证，用VLV治疗， 表达多种HBV抗原（聚合酶、HBcAg和MHBs）的载体-称为VLV-3xT 2A-导联 乙型肝炎B表面抗原（HBsAg）（HBV清除的替代标志物）的显著丧失[参见 进度报告]。在实现I期里程碑的过程中，我们证明了（i）诱导多特异性HBV T细胞 细胞，（ii）保护免受急性攻击感染，和（iii）HBsAg在小鼠模型中的显著降低。 在第二阶段，我们将使用相同的小鼠慢性模型来测试优化的初免， 加强方案以最大化清除病毒所需的免疫应答。同时，我们将确定 一种临床实用的最佳给药途径（肌内或皮下）。此外，本发明还提供了一种方法， 我们将进行药代动力学、毒性和体内生物分布研究， FDA对我们关于VLV-3xT 2A免疫疗法开发的Pre-IND会议要求的回应。的 该项目的目标是开发一种免疫疗法，以实现功能性治愈，其特征在于 使用新的基于VLV的HBV，持续丧失HBsAg（伴或不伴HBsAg抗体血清转化） 治疗第二阶段的具体目标如下：目标1：定义、优化和验证prime和boost 治疗方案。这一目标将包括四项任务：制定启动-加强策略， 关于评价产生的应答：1）T细胞应答幅度、特异性和功能性 表型（T细胞细胞因子应答、细胞溶解活性等），和2）抗体应答（即，抗体滴度 和抗体特异性）。我们还将确定VLV-3xT 2A预充-升压的顺序和时间 这意味着，免疫系统可以产生最佳的免疫应答。确定T细胞对HBV抗原的应答 在人MHC I类和MHC II类分子的背景下，来自VLV-3xT 2A。比较肌内注射与 通过测定未处理小鼠中的CD 4+和CD 8 + T细胞应答，通过皮下给药途径给药。评价 使用AAV介导的慢性HBV模型的VLV-3xT 2A初免-加强方案的功效。我们将 确定来自幼稚小鼠中最强的引发-加强的免疫应答是否可以清除持续的 通过AAV递送HBV在小鼠肝脏中启动HBV复制。目的2：生物分布、药代动力学， 安全性、毒理学和放大研究。这一目标涉及两项任务：进行生物分布， 使用目的1中确定的最佳给药途径进行的VLV-3xT 2A药代动力学研究，以及 在小鼠中进行VLV安全性和毒理学研究。这些结果将构成设计和 根据FDA对IND提交的要求，由GLP合同实验室进行未来毒理学研究， 在人类中进行VLV-3xT 2A的临床研究。确定扩大VLV生产规模的条件， 符合现行药品生产质量管理规范（cGMP）标准的工艺步骤，并表征 最佳预充-加强VLV治疗剂的质量和稳定性特征。

PROJECT SUMMARY/ABSTRACT The ultimate objective of this SBIR project is to develop an immunotherapy, based on our patented virus-like- vesicle (VLV) platform, for the treatment of patients infected with chronic hepatitis B virus. In Phase I, using a murine model of chronic HBV infection, we have established a Proof-of-Concept that treatment with a VLV vector expressing multiple HBV antigens (Polymerase, HBcAg and MHBs) - referred to as VLV-3xT2A - leads to a significant loss of hepatitis B surface antigen (HBsAg), a surrogate marker for HBV clearance [see Progress Report]. In fulfilling the Phase I milestones, we demonstrated (i) induction of multi-specific HBV T cells, (ii) protection from acute challenge infection, and (iii) significant reduction in HBsAg in a mouse model of persistent HBV replication.In Phase II, we will use the same murine chronic model to test an optimized prime- boost regimen to maximize the immune responses needed for clearance of the virus. In parallel, we will identify an optimal route of administration (intra-muscular or subcutaneous) that is practical for clinical use. In addition, we will perform pharmacokinetics, toxicity, and in vivo biodistribution studies in line with the feedback from the FDA’s response to our Pre-IND meeting request regarding development of VLV-3xT2A immunotherapy. The goal of this project is to develop an immunotherapy in order to achieve a functional cure, characterized by sustained loss of HBsAg (with or without HBsAg antibody seroconversion) using new VLV-based HBV therapies. The specific aims for Phase II are as follows: Aim 1: Define, optimize and validate prime and boost treatment regimens. This aim will include four tasks: Develop a prime-boost strategy and measure immune responses generated with respect to evaluating: 1) T cell response magnitude, specificity, and functional phenotype (T cell cytokine responses, cytolytic activity, etc.), and 2) antibody responses (i.e., antibody titers and antibody specificity). We will also determine the order and timing of VLV-3xT2A prime-boost administrations that results in the optimum immune response. Determine T cell responses to HBV antigens from VLV-3xT2A in the context of human MHC class I and MHC class II molecules. Compare intramuscular vs. subcutaneous routes of administration by assaying CD4+ and CD8+ T cell responses in naïve mice. Evaluate the efficacy of VLV-3xT2A prime-boost regimen using the AAV-mediated model of chronic HBV. We will determine whether the immune response from the strongest prime-boost in naïve mice can clear persistent HBV replication initiated in the mouse liver by AAV delivery of HBV. Aim 2: Biodistribution, pharmacokinetics, safety, toxicology, and scale-up studies. This aim involves two tasks: Conduct biodistribution and pharmacokinetic studies of VLV-3xT2A using the optimal route of administration determined in Aim 1 and conduct VLV safety and toxicology studies in mice. These results will form the basis for designing and conducting future toxicology studies by a GLP contract lab, per FDA requirements for IND submission to conduct clinical studies on VLV-3xT2A in humans. Identify conditions for scaling up VLV production and process steps amenable to current good manufacturing practice (cGMP) standards and characterize the quality and stability profiles of the optimal prime-boost VLV therapeutics.