Developing novel strategies for altering the cytokine microenvironment of tumors

 描述（由申请人提供）：使用具有强效和多效性作用的细胞因子增强抗肿瘤免疫应答具有改善细胞免疫疗法的巨大潜力。不幸的是，由于这些有效的功能特性，细胞因子的全身递送通常具有严重的副作用，这极大地限制了它们的使用和功效。通过实验实现的细胞因子的局部表达已经显示出显著的效果，但是治疗已经扩散并且不容易接近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，该策略结合了这些方法，从而我们全身递送细胞因子，使其可能到达播散性肿瘤，但使其优先在肿瘤中发挥作用，从而最大限度地减少不必要的副作用。在这个提议中，我们正在开发一种新的方法，该方法采用FP，其中细胞因子与特异性结合部分（如单链可变片段（scFv））连接，由蛋白酶位点分开。在切割之前，细胞因子基本上是无活性的，因为它与抑制性scFv结合，但是在被优先在肿瘤部位表达的蛋白酶切割之后，细胞因子变得生物学上可用于与免疫细胞上显著更高亲和力的受体相互作用。我们正在开发的新型蛋白酶激活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不同。从机制上讲，这种策略采用scFv不将细胞因子靶向肿瘤，而是抑制细胞因子活性，直到它被肿瘤微环境中过表达的蛋白酶激活。我们正在表征含有IL-2，IL-12或趋化因子CXCL 10的可活化FP，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细胞通常无效的肿瘤部位归巢。我们在FP中加入了基质金属蛋白酶（MMP）位点作为概念证明，因为MMP通常在肿瘤微环境中功能性过度表达，并密切参与肿瘤进展。这种方法是通用的，因为它可以通过取代FP中的不同蛋白酶位点而定制为几乎任何优先在肿瘤位点表达的蛋白酶。此外，鉴于可以使用噬菌体展示分离的scFv的巨大多样性，我们假设这种方法可以用于基本上任何细胞因子或趋化因子。在体外验证它们的活性后，我们将采用一种综合方法来检查这些融合蛋白的递送如何影响肿瘤微环境和使用B16黑素瘤模型（其被CD 8细胞浸润相对较差）和Colon 38肿瘤模型（其具有大量CD 8细胞但功能次优）的抗肿瘤免疫应答。通过使用流式细胞术分析、功能分析、qRT-PCR以及整体组织学和其他成像技术的组合，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剂。

 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.