Antibody-CpG conjugates for the treatment of B cell lymphoma

描述（申请人提供）：抗CD 20单克隆抗体（mAb）如利妥昔单抗已成为治疗B细胞非霍奇金淋巴瘤（NHL）的基石，但仅部分有效，因为大多数患者最终复发并保持“不可治愈”。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性细胞毒性（ADCC）、补体依赖性细胞毒性（CDC）和促凋亡功能的mAb，但这些改善在很大程度上是递增的，并且不会产生能够改变肿瘤微环境和邻近宿主免疫细胞以有利于免疫介导的肿瘤破坏的mAb。我们假设，通过将这些精确的肿瘤靶向载体与免疫调节物质连接，在肿瘤微环境中释放，可以提高抗淋巴瘤mAb的疗效。我们的实验室现已收集了一组独特的细胞系模型和抗CD 20试剂，包括表达人CD 20的鼠淋巴瘤、人CD 20转基因小鼠和抗鼠CD 20 mAb，首次允许在免疫活性宿主中进行抗CD 20治疗的详细建模。Toll样受体9（TLR 9）激动剂CpG寡脱氧核苷酸是用于增强抗淋巴瘤免疫的主要候选物，因为直接瘤内注射在动物和临床研究中都显示出有希望的抗肿瘤作用。为了在体内将CpG靶向所有肿瘤部位，我们采用了我们最近描述的抗体缀合技术，以实现CpG与抗CD 20抗体利妥昔单抗的直接化学连接。值得注意的是，抗体-CpG缀合物能够从100%携带高度侵袭性、利妥昔单抗抗性人CD 20 + B细胞淋巴瘤的小鼠中可再现地根除已建立的肿瘤。因此，我们提出以下目的来表征针对B细胞淋巴瘤的抗体-CpG缀合物的活性、作用机制和临床相关的翻译原理：目的1。评价新型利妥昔单抗-CpG偶联物在携带表达人CD 20的2种不同鼠淋巴瘤的同基因免疫活性小鼠中的疗效。目标2.比较含有功能不同的A、B和C类CpG寡脱氧核苷酸的利妥昔单抗缀合物的体内功效。目标3：确定利妥昔单抗-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垂死淋巴瘤细胞释放的肿瘤抗原的二次适应性T细胞应答。目标4。测试利妥昔单抗-CpG偶联物抗人B细胞淋巴瘤异种移植物和在免疫缺陷小鼠中生长的原代人淋巴瘤样本的作用。目标5。探索针对替代性B细胞淋巴瘤表面抗原（例如CD 19）的抗体-CpG缀合物，这些抗原在抗体结合后内化，进一步拓宽了这种方法的适用性。

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.