Development of TGF-beta antagonists for cancer therapy

尽管转化生长因子-β作为肿瘤抑制因子和肿瘤促进剂在肿瘤发生中的双重作用，我们实验室和其他人的临床前数据已经表明，拮抗转化生长因子-β的策略可能选择性地减少这种生长因子的不良促癌作用，同时避免对肿瘤抑制和正常体内平衡的预期作用。基于这些有希望的临床前结果，几种不同的转化生长因子-β途径拮抗剂正在进行治疗晚期癌症的早期临床试验。然而，考虑到转化生长因子-β的复杂生物学，成功开发用于癌症治疗的转化生长因子-β拮抗剂将取决于对这些药物如何发挥作用的清楚了解，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.抗转化生长因子-β治疗的优化和预测生物标记物的开发。2013财年的一个主要重点是与转移性乳腺癌的可移植同基因小鼠模型小组合作，开发对抗转化生长因子-β抗体治疗反应的预测生物标志物，并探索与传统化疗药物的联合治疗，以提高整体疗效。转移负担是我们的主要治疗终点。使用这个肿瘤小组，我们发现，尽管转化生长因子-β拮抗剂在某些模型中抑制转移，但在其他模型中它对转移没有影响，甚至可以刺激转移。我们已经排除了一些对转化生长因子-β拮抗反应有可能的候选预测生物标记物，基于发现的方法的工作正在进行中，包括测试我们在相关项目Zia BC 005785中确定的转化生长因子-β介导的肿瘤抑制的基因签名。我们也正在通过研究在某些模型中看到的转化生长因子-β拮抗的不良影响的机制来解决这个问题。与主要涉及重新激活有效的抗肿瘤免疫反应的转化生长因子-β抗体的治疗作用不同，我们已经证明，这种不良的刺激作用独立于免疫系统，并且似乎涉及中和转化生长因子-β对肿瘤实质的残留肿瘤抑制作用。在联合治疗方面，我们已经证明，转化生长因子-β抗体的治疗效果可以通过联合亚治疗剂量的免疫调节化疗药物来增强，从而产生明显的生存益处。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细胞标记物CD79a在未成熟髓系细胞促进转移的作用中的新功能。2.开发工具来识别和量化非规范的转化生长因子-β信号。最近来自其他实验室的数据表明，通过非典型机制的转化生长因子-β信号转导与转化生长因子-β的肿瘤促进作用有关，而典型信号转导是肿瘤抑制作用的关键。因此，非规范的转化生长因子-β信号通路的激活可能有助于转化生长因子-β从肿瘤抑制因子向肿瘤促进剂的转变。非规范的信号机制包括形成包含转化生长因子-βSmad(Smad2/3)和BMP Smads(Smad1/5/8)的“混合Smad”信号复合体。原位邻近结扎(Duolink)是一种新技术，它使用成对的抗体来检测和定量冰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复合体的抗体，并成功地开发了这项技术的第一个光明领域的应用，用于福尔马林固定的石蜡包埋组织，从而应用于存档的临床标本。我们已经证明，混合的Smad复合体在胚胎组织中高表达，但在正常成人组织中不表达，并在疾病状态下重新激活。我们正在应用这种方法来观察在临床前和临床乳腺癌材料中癌症进展的不同阶段非典型信号的开始。我们还在筛选我们的转移性乳腺癌小组，以确定非规范的转化生长因子-β信号是否可以作为有用的预测生物标志物用于转化生长因子-β拮抗剂的治疗。3.转化生长因子-β亚型在肿瘤发生中的作用在体外，有三种具有非常相似活性的转化生长因子-β亚型。相关的临床数据和文献证据表明，尽管转化生长因子-β1主要与不良预后有关，但转化生长因子-β3实际上可能与转化生长因子-β1相反，并与良好的预后相关。为了解决这个问题，我们正在与XOMA公司合作，评估不同异构体选择性的治疗性抗转化生长因子-β抗体在我们的转移模型中的相对有效性。同时，我们正在对不同模型的转化生长因子-β亚型表达谱进行详细的表征。通过这种方法，我们希望产生更好的转化生长因子-β靶向治疗方法。

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.