Immunotherapy for Malignant Mesothelioma and Lung Cancer

1. 我们目前的研究重点是针对肿瘤分化抗原间皮素（mesothelin）的免疫治疗。间皮素在胸膜、心包和腹膜的正常间皮细胞上表达，但在几种人类肿瘤中，尤其是间皮瘤、卵巢癌、肺癌和胰腺腺癌中高度表达。这种间皮素的差异表达使其成为肿瘤特异性治疗的有吸引力的候选者。我们现在的工作重点是利用不同的方法将其用于间皮瘤的治疗。其中包括抗间皮素免疫毒素（LMB-100）、抗间皮素药物偶联物（BAY 94-9343）、间皮素疫苗（CRS-207）和过继t细胞治疗（TC-210、TC-510）。我们目前正在开发靶向间皮素的过继细胞疗法，使用嵌合抗原受体(CAR)-T细胞。临床开发中的大多数抗间皮素抗体靶向间皮素的膜远端区域，这可能部分解释了临床上抗间皮素CAR-T细胞治疗缺乏活性的原因。为了提高CAR-T细胞的抗肿瘤活性，我们提出了一种新的方法，即开发靶向肿瘤细胞表面附近表位的抗间皮素CAR-T细胞。我的合作者Mitchell Ho博士已经鉴定出一种高亲和力的兔单克隆抗体（YP218），特异于III区，该区位于靠近肿瘤细胞表面的间皮素c端。我们在动物模型中测试了hYP218 CAR-T构建体对几种表达间皮素的细胞系的细胞毒性和抗肿瘤作用。临床前研究表明，它增加了肿瘤杀伤(1)。这种结构目前正处于临床开发阶段。我们正在研究hYP218 CAR - T细胞在结直肠癌和胃癌临床前模型中的功效，以便我们可以将其转化为这些恶性肿瘤的临床试验。与此同时，我们正致力于进一步提高CAR-T细胞疗法的疗效。我们已经证明，在临床前模型中，幼稚T细胞或干细胞记忆T细胞比散装T细胞具有更好的持久性。我们正在研究肿瘤微环境在CAR - t细胞疗效和持久性中的作用。到目前为止，大多数研究都是在免疫功能低下的宿主中进行的。我们正在开发具有免疫功能的小鼠肿瘤模型来实现这些目标。同时，我们正在进行t细胞受体融合构建（TRuCs）的临床试验。与其他结构体不同，TRuCs自然地结合到天然TCR复合体中，从而充分发挥TCR驱动的T细胞激活、效应功能和调控的潜力。TC-210（也称为gavocabtagene autoeucel或gavo-cel）的一期试验最近已经完成。gavoo -cel在实体瘤中显示出良好的疗效，毒性可控（Hassan等人，Nature Medicine，出版中）。我们现在正在进行加沃细胞的二期试验。t细胞衰竭是过继细胞治疗活性有限的主要原因。为了克服PD-1/PD-L1介导的t细胞衰竭，一种新的TRuC构建物TC-510被开发出来。这是一个间皮素导向的TRuC，包含一个PD-1xCD28受体开关。它具有克服PD-L1/PD-L2介导的免疫抑制的细胞内在机制，并将这种抑制功能转化为共刺激信号。我们正在治疗TC-510的一期研究患者。LMB-100是一种由抗间皮素Fv组成的免疫毒素，它与强效细菌毒素假单胞菌外毒素a的截断形式相连，假单胞菌外毒素a已被去免疫以降低其抗原性。我们最近完成了LMB-100的I期试验，并确定了其安全性和最大耐受剂量（MTD），发表在《癌症》杂志(2)上。目前，我们正在评估最近结束的LMB-100联合免疫检查点抑制剂Pembrolizumab治疗间皮瘤患者的II期研究结果。由于LMB-100在患者肿瘤中引起全身性炎症和免疫细胞浸润增加，我们假设瘤内注射LMB-100会导致炎症和免疫细胞浸润增加。使用检查点抑制剂会进一步增加肿瘤细胞的杀伤。我们目前正在间皮瘤患者中进行一项1期临床试验，在第1天和第4天瘤内给予LMB-100，然后在21天周期的第2天静脉注射CTLA-4检查点抑制剂ipilimumab。患者将接受2个周期的LMB-100联合伊匹单抗治疗，然后单独接受2个周期的伊匹单抗治疗。肿瘤活检将在每次给药LMB-100之前进行，以评估肿瘤免疫微环境的变化。该试验正在招募患者。我们之前的研究表明，DNA修复基因的种系突变增加了胸膜间皮瘤患者对铂治疗的敏感性，并提高了患者的总生存率(3)。目前，我的实验室正在研究DNA修复基因的种系突变，这种突变可能易患间皮瘤并影响临床结果。我们正在招募携带这种突变的患者及其亲属进行长期随访研究，以进行早期癌症检测和预防。在实验室，我们专注于开发人类间皮瘤的体外和体内模型。我们从患者的腹水和胸水中建立了几个早期传代肿瘤细胞系。我们已经评估了这些细胞系的形态和遗传特征，并正在使用它们来研究体外药物敏感性。此外，我们已经建立了一个人源性间皮瘤异种移植肿瘤模型，该模型使用来自患者的肿瘤细胞和来自健康供体的人pbmc进行体内研究。由于pmc人源化小鼠模型中移植物抗宿主病（GVHD）的发展限制了抗肿瘤疗效持续时间的评估，我们开发了一种同源免疫能力小鼠模型。由于免疫毒素LMB-100能特异性靶向人间皮素，我们利用编码hMSLN膜结合片段的hMSLN表达载体转染PD-L1阳性小鼠肺腺癌细胞系，建立了表达人间皮素免疫活性的同基因小鼠肿瘤模型。这些细胞系被用来发展肿瘤。我们研究了LMB-100联合抗pd1抗体在两种模型中的作用，并观察到肿瘤消退。我们的研究结果发表在《科学转化医学》(4)上。这些模型对于评估间皮瘤的新型治疗药物和抗肿瘤疗效的机制研究至关重要。其他正在进行的实验室研究主要集中在了解间皮瘤肿瘤免疫微环境和抗间皮瘤靶向药物治疗后的变化。2. 免疫疗法治疗肺癌。目前，我们正在对先前治疗失败的肺腺癌患者进行抗pd - l1单克隆抗体MSB0010718C的临床试验。我们的实验室最近发现约25%的转移性肺腺癌患者高表达间皮素。这些肿瘤中的间皮素表达与KRAS突变和野生型EGFR状态高度相关，并且独立地与不良预后相关。我们的假设是，K-RAS突变肺癌患者可以从间皮素定向治疗中获益。间皮素导向疗法治疗肺癌的临床试验即将开启。我们的实验室也在研究免疫检查点在恶性间皮瘤中的作用，以便针对这一途径的药物可以用于治疗间皮瘤。我们目前正在进行LMB-100联合派姆单抗治疗的NSCLC患者的II期试验。

1. Exploiting mesothelin for mesothelioma therapy and related translational research Our current studies are focused on using immunotherapy directed against the tumor differentiation antigen mesothelin, which is expressed on normal mesothelial cells lining the pleura, pericardium, and peritoneum, but is highly expressed in several human tumors especially mesothelioma, ovarian cancer, lung cancer and pancreatic adenocarcinomas. This differential expression of mesothelin makes it an attractive candidate for tumor specific therapy. Our efforts are now focused on exploiting it for mesothelioma therapy using different approaches. These include anti mesothelin immunotoxin (LMB-100), an anti-mesothelin drug conjugate (BAY 94-9343), mesothelin vaccine (CRS-207) and adoptive T-cell therapy (TC-210, TC-510). We are currently developing mesothelin-targeting adoptive cellular therapy, using chimeric antigen receptor (CAR)-T cells. Majority of the anti-mesothelin antibodies in clinical development target the membrane distal region of mesothelin, that could partly account for the lack of activity of anti-mesothelin CAR-T cell therapy in the clinic. To improve CAR-T cell anti-tumor activity, we propose a new approach i.e. developing anti-mesothelin CAR-T cells that target an epitope close to the surface of tumor cells. My collaborator, Dr. Mitchell Ho, has identified a high affinity rabbit monoclonal antibody (YP218) specific for region III, which is located at the C-terminal end of mesothelin close to the tumor cell surface. We have tested the cytotoxicity of the hYP218 CAR-T construct on several mesothelin expressing cell lines and anti-tumor effect in animal models. The pre-clinical studies have shown increased tumor killing (1). This construct is now under clinical development. We are studying the efficacy of hYP218 CAR T cells in colorectal and gastric carcinoma pre-clinical models so that we can translate it to clinical trials of these malignancies. Along side we are working on further improving the efficacy of CAR-T cell therapy. We have shown that naive or stem cell memory T cells have better persistence than bulk T cells in pre-clinical models. We are studying the role of tumor microenvironment in CAR T-cell efficacy and persistence. Most studies so far have been done in immunocompromised hosts. We are developing immunocompetent mice tumor models to accomplish these goals. Parallelly, we are conducting a clinical trial of a T-cell receptor fusion construct (TRuCs). Unlike other constructs, TRuCs are naturally incorporated into the native TCR complex, thus exploiting the full potential of TCR-driven T cell activation, effector function, and regulation. The phase 1 trial of TC-210 (also known as gavocabtagene autoleucel or gavo-cel) has recently been completed. Gavo-cel has shown promising efficacy in solid tumor with manageable toxicity (Hassan et al., Nature Medicine, in press). We are now conducting the phase 2 trial of gavo-cel. T-cell exhaustion is a major reason for limited activity of adoptive cellular therapies. To overcome PD-1/PD-L1 mediated T-cell exhaustion, a new TRuC construct, TC-510, has been developed. This is a mesothelin directed TRuC that contains a PD-1xCD28 receptor switch. It has cell intrinsic mechanism to overcome PD-L1/PD-L2 mediated immunosuppression and turns this inhibitory function into a costimulatory signal. We are treating patients on Phase 1 study of TC-510. LMB-100 is an immunotoxin consisting of the anti-mesothelin Fv linked to a truncated form of the potent bacterial toxin, Pseudomonas exotoxin A, which has been de-immunized to decrease its antigenicity. We have recently completed the phase I trial of LMB-100 and established its safety and maximum tolerated dose (MTD), published in Cancer (2). Currently, we are evaluating the results of a recently concluded a phase II study of LMB-100 in patients with mesothelioma, in combination with immune checkpoint inhibitor Pembrolizumab. Since LMB-100 causes systemic inflammation and increase in immune cell infiltration in patient tumors, we hypothesized that intra- tumoral injection of LMB-100 would lead to increased inflammation and immune cell infiltration. Administering checkpoint inhibitors would further increase tumor-cell killing. We are presently conducting a phase 1 clinical trial in patients with mesothelioma where they are intratumorally administered LMB-100 on days 1 and 4 followed by CTLA-4 checkpoint inhibitor, ipilimumab, given i.v. on day 2 of a 21-day cycle. Patients will receive 2 cycles of LMB-100 plus ipilimumab, followed by 2 cycles of ipilimumab alone. Tumor biopsies will be performed prior to each administration of LMB-100, to evaluate changes in the tumor immune microenvironment. The trial is recruiting patients. We have previously shown that germline mutations in DNA repair genes increases sensitivity to platinum therapy and improves overall survival in patients with pleural mesothelioma (3). Currently, my laboratory is studying germline mutations in DNA repair genes that could predispose to mesothelioma and influence clinical outcome. We are enrolling patients and their relatives harboring such mutations for a long term follow up study, for early cancer detection and prevention. In the laboratory, we have focused on developing in-vitro and in-vivo models of human mesothelioma. We have established several early passage tumor cell lines from ascites and pleural fluid of patients. We have evaluated the morphological and genetic characteristics of these cell lines and are using them to study in-vitro drug sensitivity. Additionally, we have established a humanized mesothelioma xenograft tumor model with patient derived tumor cells and human PBMCs from healthy donor for in-vivo studies. As the development of Graft Versus Host Disease (GVHD) in the PBMC-humanized mouse model limits assessment of duration of anti-tumor efficacy, we have developed a syngeneic immunocompetent mouse model. Because the immunotoxin LMB-100 can target human mesothelin specifically, we established a human mesothelin expressing immunocompetent syngeneic mouse tumor model by transfecting PD-L1 positive mouse lung adenocarcinoma cell line with a hMSLN expressing vector encoding the membrane bound fragment of hMSLN. These cell lines were used to develop tumor. We have studied the effect of LMB-100 in combination with anti-PD1 antibody in both the models and have seen tumor regression. We have published our findings in Science Translational Medicine (4). These models are essential to evaluate novel therapeutic agents for mesothelioma and for the mechanistic studies of anti-tumor efficacy. Other ongoing laboratory studies are focused on understanding the mesothelioma tumor immune micro-environment and changes following treatment with anti-mesothelin targeted agents. 2. Immunotherapy to treat lung cancers. We are currently conducting clinical trial of the anti-PD-L1 monoclonal antibody MSB0010718C in patients with lung adenocarcinoma who have failed prior therapies. Our laboratory has recently shown that about 25% of patients with metastatic lung adenocarcinoma highly express mesothelin. Mesothelin expression in these tumors is highly associated with KRAS mutations and wild type EGFR status and is, independently, associated with poor prognosis. Our hypothesis is that patients with K-RAS mutant lung cancer can benefit from mesothelin directed therapies. Clinical trials of mesothelin directed therapies for treating lung cancer are about to open. Our laboratory is also studying the role of immune checkpoints in malignant mesothelioma so that drugs targeting this pathway can be exploited for treating mesothelioma. We are currently conducting a Phase II trial of NSCLC patients treated with LMB-100 in combination with pembrolizumab.