新型CD20抗体对Rituximab耐受型B淋巴瘤杀伤的分子机理研究

Rationally designed targeted therapies specifically targeted to particular pathways involved in disease pathophysiology could represent the future of personalized medicine for cancer patients. Rituximab, one of the most successful examples of these therapies, is a humanized monoclonal antibody (mAb) targeting human CD20. It has substantially improved outcome for patients with many different types of non-Hodgkin lymphoma (NHL). Despite rituximab has revolutionized the treatment of lymphoma, treatment of patients with refractory or relapsed indolent lymphomas with rituximab was associated with a response rate of only 50% and close to 60% of prior rituximab responding patients will no longer benefit with retreatment with this mAb (acquired resistance). Although rituximab resistance mechanisms have been identified in preclinical studies, no effective regimen has been developed to overcome rituximab resistance in patients. More important, rituximab-resistant (RR) cell lines generated in vitro display cross-resistance when tested against a panel of chemotherapeutic agents because of a block in the cell apoptosis pathway, indicating that inherent tumor heterogeneity and alternative survival pathways developed during drug treatment pose additional challenges to the clinical management of patients with RR lymphoma of different genesis. Two major categories of rituximab resistance mechanisms have been proposed: resistance due to alterations of CD20 expression; and acquired resistance primarily associated with the activation of NF-κB and ERK1/2 pathways and alterations in the expression levels of the anti-apoptotic proteins Bcl-2, and Bcl-xL and the proapoptotic proteins Bax and Bak. Combined, these observations provide a strong rationale for the development of novel therapeutics that are not affected by these molecular changes to successfully treat RR NHL. Recently, several new CD20 mAbs are currently undergoing preclinical and clinical evaluation. Due to no known natural ligand and the phenotype of CD20 knockout mice was surprisingly normal, knowledge about the biology of CD20 is still relatively scarce. Although most CD20 mAbs recognize the larger extracellular loop (only 44 amino acids) of the CD20 molecule. In our previous studies, we have found that Rituximab variant (H102YK)can initiate potent caspase-independent cell death in both Rituxiamb-sensitive and Rituximab-resistance lymphoma.In our present study, we will change the native amino acids at heavy chain 102 position to 18 other amino acids and rational design CD20 molecule to investigate the molecular mechanism of caspase-independent cell death induced by rituximab variants. On that basis, we will study the the killing mechanism of these rituximab variants against RR lymphoma.These studies will give us a better understanding of CD20 molecule,which is helpful to design more effective antibody against B-cell lymphoma.

虽然抗CD20抗体Rituximab已成功用于非霍奇金淋巴瘤的治疗，但是仍有50%的病人产生对该抗体疗法不敏感，大部分的病人在治疗过程中产生复发和对该抗体疗法的耐药。如何提高CD20抗体疗效、扩大其适用范围，是新一代CD20抗体研究和淋巴瘤治疗过程中亟待解决的关键问题。我们在前期研究中发现一株Rituximab单点突变体（H102YK）能够引起强烈的非caspase依赖的细胞死亡，初步证实对Rituximab抵制型肿瘤细胞具有良好的杀伤作用。在本研究中，我们拟将这个突变位点的氨基酸突变为其他十八种氨基酸并对CD20膜分子进行必要的设计与改造。借助超高分辨率的近场光显微镜和基因表达谱芯片,阐明Rituximab突变体诱导非caspase依赖的细胞死亡的分子机理，进而阐述其能够杀死Rituximab抵制型细胞的原因，为后续设计更加有效的新型CD20抗体药物提供指导和理论依据。

虽然CD20抗体Rituximab已经成功应用于肿瘤免疫治疗，但是抗体治疗过程中逐渐产生的耐受极大限制了靶向抗体对B淋巴瘤的临床治疗。因此，如何克服在靶向抗体治疗过程中产生的耐受是一个研究热点。目前，大量的证据表明借助肿瘤细胞的溶酶体诱导的细胞死亡是有效杀伤肿瘤的一种方式。在本研究中，我们发现II型CD20抗体结合靶蛋白后，能够诱导CD20膜蛋白与TNFR1形成共定位，进而诱导ceramide的生成和溶酶体的破裂，导致细胞死亡。进一步的研究表明，CD20抗体诱导的溶酶体介导的细胞死亡能够对Rituximab敏感和耐受的淋巴瘤都表现出较强的杀伤作用。此外抗体我们将广泛在临床使用的I型CD20抗体Rituximab的CDR区引入一个点突变之后，使得I型CD20抗体Rituximab获得了II型CD20抗体诱导细胞死亡的能力，在Rituximab耐受型淋巴瘤细胞和原代淋巴瘤细胞上都能够诱导较强的ceramide和溶酶体依赖的细胞死亡。这些数据表明其能够作为潜在的抗体候选药物用于Rituximab治疗耐受肿瘤的治疗。