Molecular Targeted Photoimmunotherapy for Bladder Cancer

膀胱癌的分子靶向光免疫治疗

• 批准号: 8763537
• 负责人: Piyush Agarwal
• 金额: $ 28.15万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763537
• 关键词: Accounting; Adverse effects; Antibodies; Apoptosis; Attention; Binding; Biological Assay; Bladder; Bladder Neoplasm; Calmette-Guerin Bacillus; Cancer cell line; Catheters; Cell Culture Techniques; Cell Death; Cell Line; Cell surface; Cells; Cessation of life; Characteristics; Clinical; Colon Carcinoma; Contracture; Cost of Illness; Cutaneous; Cystoscopes; Data; Death Rate; Diagnosis; Disease; Dose; Ensure; Epidermal Growth Factor Receptor; Equipment; Exclusion; FGFR3 gene; Fibroblast Growth Factor Receptors; Flow Cytometry; Future; Goals; Growth; HCT116 Cells; Heterogeneity; High Pressure Liquid Chromatography; Human; Immunofluorescence Immunologic; Immunotherapy; In Vitro; Injection of therapeutic agent; Life; Light; Low Prevalence; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Methods; Mitomycins; Molecular; Molecular Target; Monoclonal Antibodies; Mus; Muscle; Mutation; Mutation Analysis; Necrosis; Nude Mice; Operative Surgical Procedures; PIK3CA gene; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phase I Clinical Trials; Photochemotherapy; Photosensitization; Positive Lymph Node; Prevalence; Property; Publishing; Recurrence; Relative (related person); Research; SW620; Secure; Signal Pathway; Signal Transduction; Testing; Therapeutic; Thymidine; Transurethral Resection; Tyrosine Kinase Inhibitor; United States; Update; Urothelial Cell; Woman; Work; Xenograft Model; antibody conjugate; cancer cell; carcinogenesis; colon cancer cell line; cytotoxic; cytotoxicity; flexibility; humanized monoclonal antibodies; immunoreactivity; improved; in vivo; in vivo Model; interest; men; molecular imaging; novel; novel strategies; panitumumab; pre-clinical; prevent; receptor; receptor expression; standard of care; success; therapy development; tumor; uptake; urinary; urologic

Purpose: Non-muscle-invasive bladder cancer has few available treatments. The mainstay of treatment is surgery with a transurethral resection of the bladder tumor but the recurrence rate is as high as 75%. Mitomycin C is given peri-operatively to reduce the recurrence rate and when disease is high grade, Bacillus Calmette-Guerin (BCG) is used to prevent both recurrence and progression. However, these treatments often fail and when successful, they work through a poorly understood mechanism. Therefore, attention has been directed towards molecular alterations in bladder cancer to identify novel targets. Mutation analysis of 145 urothelial tumors found that FGFR3 and PIK3CA mutations were most commonly found in low grade non-muscle-invasive tumor (Sjodahl et al, 2011). In fact, the FGFR3 mutation is found in up to 80% in low-grade bladder tumors and up to 50% of invasive tumors. In addition, over-expression or amplification of EGFR is seen in the majority of urothelial tumors (Rotterud et al, 2005). However, targeted therapy with the monoclonal antibodies has been disappointing in bladder cancer. Multiple studies have been conducted with EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors without any significant clinical benefit despite encouraging pre-clinical results (Black et al, 2012). Recently, an FGFR3 monoclonal antibody was effective but primarily in invasive bladder cancer cell lines instead of the low grade bladder cancer where mutations in FGFR3 are more commonly found (Qing et al, 2009). Despite having these well-defined targets in non-muscle-invasive bladder cancer, there has been little success with targeted therapy alone. Recently, colleagues from the molecular imaging branch demonstrated successfully that humanized monoclonal antibodies could be used to target infrared light activated compounds selectively into colon cancer cells. Upon introduction of infrared light, these compounds become activated to induce cell death (Mitsunaga et al, 2011). These antibody-infrared light activated drug conjugates are able to capitalize on the targetable property of the antibodies but rely on the cytotoxicity of the drug conjugated to the antibody and not the antibody itself. Given the prevalence of EGFR and FGFR3 mutations in non-muscle-invasive bladder cancer and the disappointing results with standard targeted therapy, we postulate that antibody-infrared light activated drug conjugates will have significant activity in inducing cell death selectively in bladder cancer cells. This is novel as an infrared light could be attached to existing urologic equipment such as a urinary catheter or flexible cystoscope and be introduced into the bladder to activate such conjugates instilled into the bladder should pre-clinical work suggest a benefit of these agents in bladder cancer. We will further investigate molecular targeted photoimmunotherapy in bladder cancer by specifically targeting EGFR, FGFR3, and other related targets. Methods: Initially, bladder cancer cell lines rich in EGFR (UM-UC5, UM-UC9, and RT-4) and FGFR3 (UM-UC1, RT-4, RT-112, and UM-UC14) will be grown in cell culture with appropriate corresponding positive/negative control cell lines for these two receptors. For example, two colon cancer cell lines can be used for these controls in reference to EGFR: SW620 (EGFR negative) and HCT116 (EGFR positive) (Yang et al, 2007). Cell-surface immunofluorescence for all of these cell lines can be done through receptor specific antibodies by flow cytometry to verify and establish cell surface levels of EGFR and FGFR3 respectively. Once receptor expression has been confirmed for these cell lines, we will identify cell lines with the following relative characteristics to study further with our antibody-drug conjugates in conjunction with positive and negative controls: EGFR++/FGFR3++, EGFR--/FGFR3++, EGFR++/FGFR3--, and EGFR--/FGFR3--. In parallel to this cell culture and flow cytometry work, we will conjugate the infrared light-activated cytotoxic compound, IR-700 (purchased from Li-Cor Bioscience), to our monoclonal antibodies directed against EGFR and FGFR3. These monoclonal antibodies are panitumumab (purchased from Amgen), directed against human EGFR, and R3mab (to be donated by Genentech), directed against FGFR3. The purity of the conjugates will then be confirmed with size-exclusion HPLC and SDS-PAGE to ensure that no detectable monoclonal antibody aggregates are noted. Finally, the immunoreactivity of the mAb-IR700 conjugate will be confirmed through the use of a blocking assay where the monoclonal antibody is given first and then the mAb-IR700 is administered to see if any binding of the conjugate occurs. Using the various different cell lines and the pure antibody-drug conjugates, we will then proceed to assess target-specific cell death. We will look at dose-dependent relationships of conjugate and photoimmunotherapy and their impact on cell death (PI-FACS) and growth inhibition (thymidine uptake) in the various cell lines. Depending on this pre-clinical, in vitro work, we will expand these studies into a xenograft model by inducing EGFR and FGFR expressing tumors in vivo in nude mice. Successful data in this scenario, may introduce the possibility of a phase I clinical trial in the future. Update: We have successfully treated UMUC-5 and TCC-SUP cells with our Panitumumab-IR700 conjugate. This conjugate results in minimal cell death when activated by infrared light in normal urothelial cells (HUC-SV). Also, no cell death noted in the EGFR negative cell line, BalB3T3. We are now expanding cells for injection in mice for an in vivo model and we expect to see promising results. We have also successfully secured a MTA with genentech to study their FGFR3 mAb, R3mab.

目的：非肌肉侵入性膀胱癌几乎没有可用的治疗方法。治疗的主要手术是膀胱肿瘤的尿道切除术，但复发率高达75％。丝霉素C进行围手术期以降低复发率，而当疾病是高级时，Calmetles Calmette-Guerin（BCG）用于防止复发和进展。但是，这些治疗方法常常失败，并且在成功的情况下，它们是通过知之甚少的机制来工作的。因此，注意力集中在膀胱癌中的分子改变，以鉴定新的靶标。对145种尿路上皮肿瘤的突变分析发现，在低级非肌肉侵入性肿瘤中，FGFR3和PIK3CA突变最常发现（Sjodahl等，2011）。实际上，在低级膀胱肿瘤中发现了多达80％的FGFR3突变，高达50％的浸润性肿瘤。另外，在大多数尿路上皮肿瘤中都可以看到EGFR的过表达或扩增（Rotterud等，2005）。但是，用单克隆抗体的靶向治疗对膀胱癌令人失望。尽管鼓励了临床前结果，但已使用EGFR靶向的单克隆抗体和酪氨酸激酶抑制剂进行了多项研究（Black等，2012）。最近，FGFR3单克隆抗体是有效的，但主要在浸润性膀胱癌细胞系中而不是低级膀胱癌中，其中FGFR3中的突变更常见（Qing等，2009）。尽管在非肌肉侵入性膀胱癌中拥有这些定义明确的靶标，但仅针对靶向疗法而言，几乎没有成功。最近，来自分子成像分支的同事成功证明了人源化的单克隆抗体可用于选择性地靶向红外活性化合物，以选择性地靶向结肠癌细胞。引入红外光后，这些化合物被激活以诱导细胞死亡（Mitsunaga等，2011）。这些抗体 - 含光活化的药物缀合物能够利用抗体的目标特性，但依赖于与抗体共轭的药物的细胞毒性，而不是抗体本身。鉴于非肌肉侵入性膀胱癌中EGFR和FGFR3突变的患病率以及标准靶向疗法的令人失望的结果，我们假设抗体含量的光活化的药物共轭物在选择性地诱导细胞死亡的膀胱癌细胞中具有显着的活性。这是新颖的，因为红外光可以连接到现有的泌尿外科设备，例如尿道导管或柔性膀胱镜，并被引入膀胱中，以激活灌输到膀胱的这种结合物，应表明这些药物在膀胱癌中有好处。我们将通过专门针对EGFR，FGFR3和其他相关靶标，进一步研究膀胱癌中的分子靶向摄影疗法。方法：最初，将在细胞培养中生长富含EGFR（UM-UC5，UM-UC9和RT-4）和FGFR3（UM-UC1，RT-4，RT-112和UM-IC14）的膀胱癌细胞系，在细胞培养中，具有适当的相应的阳性/阴性对照细胞系的细胞培养中。例如，参考EGFR：SW620（EGFR负）和HCT116（EGFR阳性），可以将两种结肠癌细胞系用于这些对照（Yang等，2007）。所有这些细胞系的细胞表面免疫荧光可以通过流式细胞仪分别通过受体特异性抗体来验证和建立EGFR和FGFR3的细胞表面水平。一旦确认了这些细胞系的受体表达，我们将鉴定具有以下相对特征的细胞系，以与抗体 - 药物结合物以及阳性和阴性对照结合进行进一步研究：EGFR ++/FGFR3 ++，EGFR - /fgfr3 ++，EGFR ++/FGFR ++/FGFR3---和EGFR3--和EGFR3-/FGFR3-/fgfr3--/fgfr3---/fgfr3---/fgfr3----/fgfr3----/fgfr3----与这种细胞培养和流式细胞仪工作并行，我们将将红外光激活的细胞毒性化合物IR-700（从LI-COR Bioscience购买）与针对EGFR和FGFR3的单克隆抗体。这些单克隆抗体是针对人EGFR的panitumumab（从Amger购买）和针对FGFR3的R3MAB（由Genentech捐赠）。然后，将使用尺寸排斥HPLC和SDS-PAGE确认结合物的纯度，以确保未发现可检测到的单克隆抗体聚集体。最后，将通过使用封闭测定法确认MAB-IR700共轭物的免疫反应性，在该测定中首先给出单克隆抗体，然后对MAB-IR700进行施用，以查看是否发生结合结合。然后，我们将使用各种不同的细胞系和纯抗体 - 药物结合物，然后继续评估靶标特异性细胞死亡。我们将研究各种细胞系中结合和摄影疗法的剂量依赖性关系及其对细胞死亡（PI-FACS）和生长抑制（胸苷摄取）的影响。根据这项临床前的体外工作，我们将通过诱导裸鼠体内表达肿瘤的EGFR和FGFR来将这些研究扩展到异种移植模型。在这种情况下，成功的数据可能会引入未来进行I期临床试验的可能性。更新：我们已经通过panitumumab-ir700共轭物成功处理了UMUC-5和TCC-SUP单元。当正常尿路上皮细胞（HUC-SV）激活红外光线时，这种共轭物会导致细胞死亡的最小值。另外，在EGFR负细胞系BALB3T3中没有任何细胞死亡。现在，我们正在扩大体内模型的小鼠注射细胞，并希望看到有希望的结果。我们还成功地使用Genentech获得了MTA，以研究其FGFR3 mAb R3MAB。