Developing novel strategies for altering the cytokine microenvironment of tumors

 描述(由申请人提供)：使用具有强大和多效性作用的细胞因子增强抗肿瘤免疫反应在改进细胞免疫治疗方面具有巨大的潜力。不幸的是，由于这些强大的功能特性，全身注射细胞因子往往会产生严重的副作用，极大地限制了它们的使用和疗效。实验中实现的细胞因子的局部表达已显示出显着的效果，但治疗已经扩散和不易获得的肿瘤是极其困难的。我们正在开发一种新的融合蛋白(FP)策略，将这些方法结合在一起，从而系统地传递一种细胞因子，使其可能到达播散性肿瘤，但使其在肿瘤中优先发挥作用，从而将不良副作用降至最低。在这项提案中，我们正在开发一种新的方法，它使用一种FP，其中细胞因子连接到特定的结合部分(如单链可变片段(ScFv))，由蛋白酶位点分隔。在切割之前，细胞因子在很大程度上是不活跃的，因为它与抑制性单链抗体结合，但在被肿瘤部位优先表达的蛋白酶切割后，细胞因子变得在生物学上可与免疫细胞上亲和力极高的受体相互作用。我们正在开发的新的蛋白酶激活的细胞因子方法与以前在肿瘤部位增加细胞因子的方法有根本的不同。从机制上讲，这种策略使用单链抗体不是针对肿瘤的细胞因子，而是抑制细胞因子的活性，直到它被肿瘤微环境中过度表达的蛋白酶激活。我们正在确定含有IL-2、IL-12或趋化因子CXCL10的可激活FP的特征，这些FP旨在克服成功免疫治疗的两个重要障碍：许多肿瘤的免疫抑制微环境和T细胞经常低效地归巢到肿瘤部位。我们将基质金属蛋白酶(MMPs)结合在FPS中作为概念验证，因为MMPs在肿瘤微环境中经常在功能上过度表达，并与肿瘤进展密切相关。这种方法是通用的，因为它可以通过替换FP中的不同蛋白酶位点来定制几乎任何在肿瘤部位优先表达的蛋白酶。此外，考虑到使用噬菌体展示技术可以分离出的单链抗体的巨大多样性，我们假设这种方法基本上可以用于任何细胞因子或趋化因子。在体外验证了它们的活性后，我们将使用B16黑色素瘤模型(CD8细胞的浸润性相对较差)和Colon 38肿瘤模型(具有大量CD8细胞，但功能不佳)，采用一种综合的方法来检测这些融合蛋白的交付如何影响肿瘤微环境和抗肿瘤免疫反应。通过结合流式细胞仪分析、功能分析、定量逆转录聚合酶链式反应以及整体组织学和其他成像技术，我们将全面了解融合蛋白在体内的作用及其作用机制。如果成功，这种方法有可能为肿瘤治疗创造一种全新的生物制品类别。

 DESCRIPTION (provided by applicant): Enhancing anti-tumor immune responses using cytokines that have potent and pleiotropic effects has great potential to improve cellular immunotherapy. Unfortunately, as a result of these potent functional properties, systemic delivery of cytokines often has severe side-effects that have greatly limited their use and efficacy. Local expression of cytokines achieved experimentally has shown remarkable effects but it is extremely difficult to treat tumors that have already spread and are not easily accessible. We are developing a novel fusion protein (FP) strategy that combines these approaches whereby we deliver a cytokine systemically so that it might reach disseminated tumors, but have it function preferentially at the tumor thus minimizing unwanted side effects. In this proposal, we are developing a novel approach that employs a FP in which a cytokine is joined to a specific binding moiety (such as a single-chain Variable Fragment (scFv)), separated by a protease site. Before cleavage, the cytokine is largely inactive since it is bound to the inhibitory scFv, but after cleavage by proteases preferentially expressed at the tumor site, the cytokine becomes biologically available to interact with dramatically higher affinity receptors on immune cells. The novel protease activated cytokine approach we are developing is fundamentally different than previous approaches to increase cytokine at tumor sites. Mechanistically, this strategy employs a scFv not to target the cytokine to the tumor but to inhibi cytokine activity until it becomes activated by proteases that are over expressed in the tumor microenvironment. We are characterizing activatable FPs that contain either IL-2, IL-12, or the chemokine CXCL10, that are designed to overcome two important barriers to successful immunotherapy: the immunosuppressive microenvironment of many tumors and the often inefficient homing of T cells to tumor sites. We have incorporated a matrix metalloproteinase (MMP) site in the FPs as proof of concept since MMPs are often functionally over expressed in the tumor microenvironment and intimately involved in tumor progression. This approach is versatile since it can be customized to virtually any protease that is preferentially expressed at tumor sites by substituting different protease sites in the FP. Further, given the immense diversity of scFv that can be isolated using phage display, we hypothesize this approach could be used for essentially any cytokine or chemokine. After validating their activity in vitro, we wil employ an integrated approach to examine how delivery of these fusion proteins affect the tumor microenvironment and the anti-tumor immune response using the B16 melanoma model (which are relatively poorly infiltrated with CD8 cells) and the Colon 38 tumor model (which have numerous CD8 cells but which function suboptimally). By using a combination of flow cytometric analyses, functional analyses, qRT- PCR, as well as whole mount histology and other imaging technologies, we will gain a comprehensive insight of the effects of fusion proteins in vivo and their mechanism of action. If successful, this approach has the potential to create an entirely new class of biologics for tumor therapy.