Antibody-CpG conjugates for the treatment of B cell lymphoma

描述（由申请人提供）：抗cd20单克隆抗体（mab）如美罗华（rituximab）已成为B细胞非霍奇金淋巴瘤（NHL）治疗的基石，但仅部分有效，因为大多数患者最终复发并且仍然“无法治愈”。虽然目前的基因工程和努力正在产生具有增强抗体依赖性细胞毒性（ADCC）、补体依赖性细胞毒性（CDC）和促凋亡功能的单克隆抗体，但这些改进在很大程度上是渐进式的，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促进免疫介导的肿瘤破坏的单克隆抗体。我们假设，通过将这些精确的肿瘤靶向载体与肿瘤微环境中释放的免疫调节物质连接起来，可以提高抗淋巴瘤单克隆抗体的疗效。我们的实验室现在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转基因小鼠和抗小鼠CD20单克隆抗体，首次允许在免疫功能正常的宿主中进行抗CD20治疗的详细建模。toll样受体9 （TLR9）激动剂CpG寡脱氧核苷酸是增强抗淋巴瘤免疫的主要候选药物，因为直接在肿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。为了将CpG靶向到体内的所有肿瘤部位，我们采用了我们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锁。值得注意的是，抗体- cpg偶联物能够100%地重复根除患有高度侵袭性、利妥昔单抗耐药的人CD20+ B细胞淋巴瘤的小鼠的肿瘤。因此，我们提出以下目标，以表征抗体- cpg偶联物抗B细胞淋巴瘤的活性、作用机制和临床相关的翻译原则：评估新型利妥昔单抗- cpg偶联物对携带2种表达人CD20的不同小鼠淋巴瘤的同基因免疫功能小鼠的疗效。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偶联物的体内疗效。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对死亡淋巴瘤细胞释放的肿瘤抗原的继发性适应性T细胞反应。目标4。测试利妥昔单抗- cpg偶联物对免疫缺陷小鼠生长的人B细胞淋巴瘤异种移植物和原发人淋巴瘤标本的作用。目标5。探索针对替代B细胞淋巴瘤表面抗原（如CD19）的抗体- cpg偶联物，这些抗原在抗体结合后内化，进一步扩大了该方法的适用性。

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.