Antibody-CpG conjugates for the treatment of B cell lymphoma

描述(申请人提供)：抗CD20单抗(单抗)，如利妥昔单抗，已成为B细胞非霍奇金淋巴瘤(NHL)治疗的基石，但只有部分有效，因为大多数患者最终会复发并保持“无法治愈”的状态。虽然目前的基因工程和努力正在产生具有增强的抗体依赖的细胞毒性(ADCC)、补体依赖的细胞毒性(CDC)和促凋亡功能的单抗，但这些改进在很大程度上是渐进的，并且不能导致能够改变肿瘤微环境和邻近宿主免疫细胞的单抗，从而有利于免疫介导的肿瘤杀伤。我们推测，通过将这些精确的肿瘤靶向载体与免疫调节物质联系起来，在肿瘤微环境中释放，可以提高抗淋巴瘤单抗的疗效。我们的实验室现在已经收集了一套独特的细胞系模型和抗CD20试剂，包括表达人CD20的小鼠淋巴瘤、人CD20转基因小鼠和抗鼠CD20单抗，这是首次在免疫活性宿主中建立抗CD20治疗的详细模型。Toll样受体9(TLR9)激动剂CpG寡核苷酸是增强抗淋巴瘤免疫的首选药物，因为直接瘤内注射在动物和临床研究中都显示出良好的抗肿瘤效果。为了将CpG靶向体内所有的肿瘤部位，我们采用了最近描述的抗体偶联技术，实现了CpG与抗CD20抗体利妥昔单抗的直接化学连接。值得注意的是，抗体-CpG结合物能够100%重复性地根除携带高度侵袭性、对利妥昔单抗耐药的人CD20+B细胞淋巴瘤的小鼠已建立的肿瘤。因此，我们提出以下目标，以表征抗体-CpG结合物对B细胞淋巴瘤的活性、作用机制和临床相关的翻译原理：目的1.评价新的利妥昔单抗-CpG结合物在两种不同表达人CD20的小鼠同基因、免疫活性淋巴瘤中的疗效。目的2.比较含有功能不同的A、B、C类CpG寡核苷酸的利妥昔单抗结合物的体内疗效。目的3.探讨利妥昔单抗-CpG在同基因模型中的抗肿瘤作用机制，以及肿瘤消退是否导致针对死亡淋巴瘤细胞释放的肿瘤抗原的二次适应性T细胞反应。目的4.检测美罗华-CpG结合物对人B细胞淋巴瘤异种移植瘤和免疫缺陷小鼠原发人淋巴瘤标本的抑制作用。目的5.探索针对CD19等选择性B细胞淋巴瘤表面抗原的抗体-CpG偶联物，该偶联物可内化于抗体结合，进一步扩大了该方法的适用性。 公共卫生相关性：该项目涉及临床前实验室调查，旨在开发一种新的B细胞非霍奇金淋巴瘤免疫疗法。针对CD20细胞表面抗原的抗体将与免疫刺激CpG寡核苷酸化学偶联，以产生有效的全身治疗。我们将在新的小鼠模型和B细胞淋巴瘤细胞培养中测试这些结合物的有效性和作用机制。

DESCRIPTION (provided by applicant): Anti-CD20 monoclonal antibodies (mAbs) such as rituximab have become a cornerstone in the therapy of B cell non-Hodgkin lymphomas (NHL), but are only partially effective, as most patients eventually relapse and remain "incurable". While current genetic engineering and efforts are yielding mAbs with enhanced antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and pro-apoptotic functions, these improvements are largely incremental, and do not result in mAbs capable of altering the tumor microenvironment and adjacent host immune cells to favor immune- mediated tumor destruction. We hypothesize that the efficacy of anti-lymphoma mAbs can be improved by linking these precise tumor-targeting vehicles to immunomodulatory substances for release within the tumor microenvironment. Our laboratory has now collected a unique set of cell line models and anti-CD20 reagents including murine lymphomas expressing human CD20, human CD20 transgenic mice, and anti-murine CD20 mAbs that for the first time allow detailed modeling of anti-CD20 therapy in immunocompetent hosts. Toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotides are prime candidates for boosting anti-lymphoma immunity, as direct intratumoral injection shows promising anti- tumor effects in both animal and clinical studies. In order to target CpG to all tumor sites in vivo, we adopted an antibody conjugation technique we recently described to achieve direct chemical linkage of CpG to the anti-CD20 antibody rituximab. Remarkably, the antibody-CpG conjugate was able to reproducibly eradicate established tumors from 100% of mice bearing a highly aggressive, rituximab- resistant human CD20+ B cell lymphoma. We thus propose the following aims to characterize the activity, mechanisms of action, and clinically-relevant translational principles for antibody-CpG conjugates against B cell lymphomas: Aim 1. Evaluate the efficacy of novel rituximab-CpG conjugates in syngeneic, immunocompetent mice bearing 2 different murine lymphomas expressing human CD20. Aim 2. Compare the in vivo efficacy of rituximab conjugates containing CpG oligodeoxynucleotides of the functionally distinct A, B, and C classes. Aim 3. Determine the mechanisms of anti-tumor action for rituximab-CpG in syngeneic models, and whether tumor regression leads to secondary adaptive T cell responses against tumor antigens released from dying lymphoma cells. Aim 4. Test rituximab-CpG conjugates against human B cell lymphoma xenografts and primary human lymphoma specimens grown in immunodeficient mice. Aim 5. Explore antibody-CpG conjugates targeting alternative B cell lymphoma surface antigens such as CD19, that internalize upon antibody binding, further broadening the applicability of this approach. PUBLIC HEALTH RELEVANCE: This project involves preclinical laboratory investigations aimed at the development of a new immunotherapy for B cell non-Hodgkin lymphomas. Antibodies targeting the CD20 cell surface antigen are to be chemically conjugated to immunostimulatory CpG oligodeoxynucleotides to yield a potent systemic therapeutic. We will test the efficacy and mechanisms of action for these conjugates in novel mouse models and cell cultures of B cell lymphoma.