Development of TGF-beta antagonists for cancer therapy

尽管TGF-β在肿瘤发生中具有肿瘤抑制和肿瘤促进的双重作用，但我们实验室和其他实验室的临床前数据先前表明，拮抗TGF-β的策略可以选择性地减少这种生长因子的不良肿瘤促进作用，同时保留对肿瘤抑制和正常体内平衡的理想作用。基于这些有希望的临床前结果，几种不同的TGF-β通路拮抗剂正处于治疗晚期癌症的早期临床试验中。然而，鉴于TGF-β的复杂生物学特性，成功开发用于癌症治疗的TGF-β拮抗剂将取决于对这些药物如何工作的清楚理解，以及如何选择将从这种治疗中受益的患者的相关问题。我们今年的工作主要分为三个方面：1.抗TGF-β治疗的优化和预测性生物标志物的开发。2013财年的一个主要重点是与一组转移性乳腺癌的可移植同基因小鼠模型合作，开发对抗TGF-β抗体治疗反应的预测性生物标志物，并探索与常规化疗药物的联合治疗，以提高整体疗效。转移负荷是我们的主要治疗终点。使用这种肿瘤面板，我们发现虽然TGF-β拮抗作用在某些模型中抑制转移，但在其他模型中对转移没有影响，甚至可以刺激转移。我们已经排除了一些对TGF-β拮抗作用有反应的合理候选预测生物标志物，并且正在进行基于发现的方法的工作，包括测试我们在相关项目ZIA BC 005785中鉴定的TGF-β介导的肿瘤抑制的基因特征。我们也正在通过研究在某些模型中观察到的TGF-β拮抗作用的不良作用的机制来解决这个问题。与主要涉及有效抗肿瘤免疫应答的再活化的TGF-β抗体的治疗作用不同，我们已经表明，不期望的刺激作用不依赖于免疫系统，并且似乎涉及中和TGF-β对肿瘤实质的残余肿瘤抑制作用。在联合治疗领域，我们已经表明，TGF-β抗体的治疗功效可以通过与亚治疗剂量的免疫调节化疗剂组合来增强，从而导致明显的生存益处。与其他治疗组合的工作正在进行中。作为该项目的一部分，我们还发现了B细胞标志物CD 79 a在未成熟髓系细胞的转移促进作用中的新功能作用。2.识别和定量非典型TGF-β信号传导的工具的开发。来自其他实验室的最新数据表明，通过非经典机制的TGF-β信号传导与TGF-β的肿瘤促进作用相关，而经典信号传导对于肿瘤抑制作用至关重要。因此，非典型TGF-β信号传导的激活可能有助于TGF-β从肿瘤抑制因子到肿瘤促进因子的转换。非经典信号传导机制包括涉及TGF-β Smads（Smad 2/3）和BMP Smads（Smad 1/5/8）的“混合Smad”信号传导复合物的形成。原位邻位连接（Duolink）是一种新技术，其使用成对抗体来检测和定量冷冻或固定组织切片中的蛋白质/蛋白质相互作用。我们已经产生并验证了用于检测混合Smad复合物的抗体，并成功开发了该技术的第一个明场应用，用于福尔马林固定的石蜡包埋组织，从而用于存档的临床标本。我们已经证明，混合的Smad复合物在胚胎中高度表达，但在正常成人组织中不表达，并且在疾病状态下重新激活。我们正在应用这种方法来观察临床前和临床乳腺癌材料中癌症进展不同阶段的非经典信号传导的开始。我们也在筛选我们的转移性乳腺癌小组，以确定非经典TGF-β信号传导是否可以作为TGF-β拮抗剂治疗的有用的预测生物标志物。3.肿瘤发生中TGF-β同种型效应有三种TGF-β亚型在体外具有非常相似的活性。相关的临床数据和文献证据表明，虽然TGF-β 1主要与不良结局相关，但TGF-β 3实际上可能与TGF-β 1相反，并与良好结局相关。为了解决这个问题，我们正在与XOMA公司合作，以评估具有不同亚型选择性的治疗性抗TGF-β抗体在我们的转移模型中的相对功效。同时，我们正在对不同模型的TGF-β亚型表达谱进行详细表征。通过这种方法，我们希望产生改进的TGF-β靶向疗法。

Despite the dual role for TGF-beta as both tumor suppressor and tumor promoter in carcinogenesis, preclinical data from our lab and others has previously suggested that strategies to antagonize TGF-beta may selectively reduce the undesirable tumor promoting effects of this growth factor while sparing the desirable effects on tumor suppression and normal homeostasis. Based on these promising preclinical results, several different TGF-beta pathway antagonists are in early phase clinical trials for the treatment of advanced cancer. However, given the complex biology of TGF-beta, the successful development of TGF-beta antagonists for cancer therapy will depend on a clear understanding of how these agents work, and the related question of how to select patients who will benefit from this type of treatment. Our work this year has fallen into three main areas: 1. OPTIMIZATION OF ANTI-TGF-BETA THERAPY AND DEVELOPMENT OF PREDICTIVE BIOMARKERS. One major focus in FY13 has been to work with a panel of transplantable syngeneic mouse models of metastatic breast cancer to develop predictive biomarkers of response to anti-TGF-beta antibody therapy, and to explore combination therapy with conventional chemotherapeutics in order to improve overall efficacy. Metastatic burden is our primary therapeutic endpoint. Using this tumor panel, we have found that while TGF-beta antagonism inhibits metastasis in some models, it has no effect on or can even stimulate metastasis in other models. We have excluded a number of plausible candidate predictive biomarkers of response to TGF-beta antagonism and work with discovery-based approaches is ongoing, including testing gene signatures of TGF-beta-mediated tumor suppression that we identified in the related project ZIA BC 005785. We are also approaching this problem by investigating the mechanisms that underlie the undesirable effects of TGF-beta antagonism that are seen in some models. Unlike the therapeutic effects of TGF-beta antibodies which largely involve reactivation of effective anti-tumor immune responses, we have shown that the undesirable stimulatory effects are independent of the immune system, and seem to involve neutralization of residual tumor suppressive effects of TGF-beta on the tumor parenchyma. In the area of combination therapy, we have shown that the therapeutic efficacy of TGF-beta antibodies can be enhanced by combination with subtherapeutic doses of immunomodulatory chemotherapeutics, leading to a clear survival benefit. Work with other therapeutic combinations is ongoing. As part of this project, we also uncovered a novel functional role for a B-cell marker, CD79a, in the metastasis-promoting effects of immature myeloid cells. 2. DEVELOPMENT OF TOOLS TO IDENTIFY AND QUANTITATE NON-CANONICAL TGF-BETA SIGNALING. Recent data from other labs has suggested that TGF-beta signaling via non-canonical mechanisms is associated with tumor promoting effects of TGF-beta, while canonical signaling is critical for tumor suppressor effects. Thus activation of non-canonical TGF-beta signaling may contribute to the switching of TGF-beta from tumor suppressor to tumor promoter. Non-canonical signaling mechanisms include the formation of 'mixed Smad' signaling complexes involving both TGF-beta Smads (Smad2/3) and BMP Smads (Smad1/5/8). In situ proximity ligation (Duolink) is a new technique that uses paired antibodies to detect and quantitate protein/protein interactions in frozen or fixed tissue sections. We have generated and validated antibodies for the detection of the mixed Smad complexes, and have successfully developed the first bright-field application of this technique for application to formalin-fixed, paraffin-embedded tissue and thus to archived clinical specimens. We have show that mixed Smad complexes are highly expressed in the embryo but not normal adult tissues, and are reactivated in disease states. We are applying this approach to look at the onset of non-canonical signaling at different stages in cancer progression in pre-clinical and clinical breast cancer material. We are also screening our metastatic breast cancer panel to determine whether non-canonical TGF-beta signaling might serve as a useful predictive biomarker for therapy with TGF-beta antagonists. 3. TGF-BETA ISOFORM EFFECTS IN TUMORIGENESIS. There are three isoforms of TGF-beta with very similar activities in vitro. Correlative clinical data and literature evidence suggests that whereas TGF-beta1 is primarily associated with poor outcome, TGF-beta3 may actually oppose TGF-beta1 and be associated with good outcome. To address this question, we are collaborating with XOMA Corp to assess the relative efficacy in our metastasis models of therapeutic anti-TGF-beta antibodies with different isoform selectivity. In parallel, we are performing detailed characterization of the TGF-beta isoform expression profiles of the different models. Through this approach, we hope to generate improved TGF-beta-targeted therapeutics.