Novel monocyte effector function in CLL immune therapy

描述（由申请人提供）：慢性淋巴细胞白血病（CLL）是成人白血病最常见的形式，目前可用的疗法无法治愈。一种有前途的治疗形式是抗体疗法，其中 CLL 细胞被抗 CD20 抗体（如利妥昔单抗和奥法木单抗）靶向。这导致了生存率的显着改善，特别是与其他疗法相结合，但完全缓解仍然相对罕见。单核细胞和巨噬细胞是抗体治疗的关键介质，这取决于 Fc?受体（Fc？R）活性。我们发现这些 Fc?R 的激活会导致 sFlt-1 的产生，sFlt-1 是 VEGF 受体的可溶性抑制形式。 sFlt-1 可以抑制 VEGF 信号传导，该信号传导在 CLL 细胞存活中发挥重要作用。这些结果使我们推测FcγR介导的sFlt-1产生可以抑制CLL细胞中的抗凋亡信号，并且这解释了抗体介导的抗肿瘤作用的重要部分。因此，在抗体治疗的背景下加强单核细胞/巨噬细胞 sFlt-1 的产生可能是增强其有效性的有力手段。为了检验这一假设的预测，我们提出： 目标 1：分析 sF​​lt-1 的产生和响应抗 CD20 的功能。在这里，我们将 a) 鉴定结合抗 CD20 抗体后主要负责 sFlt-1 产生的细胞，检查 sFlt-1 对 CLL 细胞存活的影响，并研究抑制 VEGF 介导的肿瘤细胞存活的机制。我们还将b)检查单核细胞产生的sFlt-1是否使CLL细胞更容易响应临床前和临床上用于治疗CLL的药物而直接凋亡。目标 2：分析体内抗体治疗中 sFlt-1 的产生和功能。我们将使用已建立的 CLL 小鼠模型来 a) 使用小鼠 CD20 抗体测试在抗体介导的 B 细胞耗竭期间是否产生 sFlt-1，b) 鉴定负责 sFlt-1 产生的相关 Fc?R 效应细胞，c) 测试中和 sFlt-1 是否会减少 抗 CD20 抗体在小鼠 CLL 模型中的功效，以及 d) 接受奥法木单抗单一疗法的 CLL 患者中 sFlt-1 水平是否与反应和无进展生存期相关。目标 3：通过 Fc?R 聚类阐明 sFlt-1 诱导机制。在这里，我们将确定 a) 哪种激活性 Fc?R 负责 sFlt-1 诱导，以及这种诱导是否受到 Fc?RIIb 和 SHIP 的负向调节，b) Fc?R 诱导的 sFlt-1 产生是直接还是间接作用，以及 c) 参与 sFlt-1 诱导的信号通路。摘要：这项研究完成后，我们将充分探索抗 CD20 介导的单核细胞和巨噬细胞杀伤肿瘤细胞的全新机制。这些机制研究将提供信息，以进一步增强两种抗 CD20 抗体（例如奥法木单抗）在 CLL 中的功效，但也可能在使用类似抗体的治疗的多种其他肿瘤中发挥功效。

DESCRIPTION (provided by applicant): Chronic Lymphocytic Leukemia (CLL) is the most common form of adult leukemia and is incurable with currently available therapies. One promising form of treatment has been antibody therapy, where CLL cells are targeted by anti-CD20 antibodies such as rituximab and ofatumumab. This has led to significant improvements in survival, especially in combination with other therapies, but complete remissions are still relatively rare. Monocytes and macrophages are critical mediators of antibody therapy, and this depends upon Fc? receptor (Fc?R) activity. We have found that activation of these Fc?R leads to production of sFlt-1, a soluble, inhibitory form of the VEGF receptor. sFlt-1 can inhibit VEGF signaling, which has been shown to play a major role in CLL cell survival. These results led us to hypothesize that Fc?R-mediated sFlt-1 production can dampen anti- apoptotic signals in CLL cells, and that this accounts for a significant portion of antibody-mediated antitumor effects. Hence, strengthening monocyte / macrophage sFlt-1 production within the context of antibody therapy may be a powerful means of enhancing its effectiveness. To test the predictions of this hypothesis we propose: Aim 1: Analysis of sFlt-1 production and function in response to anti-CD20. Here, we will a) identify the cells primarily responsible for sFlt-1 production upon binding anti-CD20 antibodies, examine the effect of sFlt-1 on CLL cell survival, and study the mechanism(s) by which VEGF-mediated survival in tumor cells is inhibited. We will also b) examine whether sFlt-1 production by monocytes makes CLL cells more susceptible to direct apoptosis in response to agents in use preclinically and clinically for the treatment of CLL. Aim 2: Analysis of sFlt-1 production and function in antibody treatment in vivo. We will use established murine models of CLL to a) test whether sFlt-1 is produced during antibody-mediated B cell depletion using a murine CD20 antibody, b) identify relevant Fc?R-bearing effector cell(s) responsible for sFlt-1 production, c) test whether neutralizing sFlt-1 reduces the efficacy of anti-CD20 antibody in a murine CLL model, and d) Whether sFlt-1 levels in CLL patients receiving monotherapy with ofatumumab correlate with response and progression free survival. Aim 3: Elucidation of mechanism of sFlt-1 induction by Fc?R clustering. Here, we will determine a) which activating Fc?R is responsible for sFlt-1 induction and whether this induction is negatively regulated by Fc?RIIb and SHIP, b) whether Fc?R-induced sFlt-1 production is a direct or indirect effect, and c) the signaling pathway(s) involved in sFlt-1 induction. Summary: At completion of this study we will have fully explored an entire new mechanism of anti-CD20 mediated killing of tumor cells by monocytes and macrophages. These mechanistic studies will provide information to further enhance the efficacy of both anti-CD20 antibodies such as ofatumumab in CLL but potentially a wide variety of other tumors where similar antibody based treatments are utilized.