ANTI-CD20 ANTIBODY THERAPY OF NHL-- MECHANISM OF ACTION

尽管积极的化疗，几乎没有改善已观察到低级别B细胞NHL患者的生存。利妥昔单抗是一种抗CD 20 mAb，可诱导50-60%的滤泡性NHL患者缓解。作用机制尚不清楚，但可能包括通过人IgG 1恒定区赋予的抗体依赖性细胞介导的细胞毒性（ADCC）增强免疫效应机制。然而，我们已经观察到1）仅50%的患者响应，和2）仅40%的初始响应患者响应于再治疗，和3）没有预测响应的免疫功能的相关性，和4）初步数据证明抗体在体外对一些人肿瘤细胞系的直接抗增殖作用，和5）这些直接作用大于用其它抗CD 20 mAb观察到的作用。我们假设这些对mAb的直接作用（阻断增殖和诱导凋亡）是治疗效果的原因，而这些直接作用的丧失有助于获得性耐药。因此，我们将通过开发用于测试抗CD 20抗体对原代人淋巴瘤细胞的抗增殖作用的方法来测试我们的假设，然后确定这些作用的存在或不存在是否预测临床活性。本建议的目的是：通过评价对以下方面的影响，开发对体外抗CD 20介导作用敏感或耐药的B细胞淋巴瘤系中诱导的细胞信号传导早期事件的替代指标：（a）细胞增殖和凋亡;（B）CD 20抗原重新分布到不溶性膜部分中;（c）通过Src家族激酶的酪氨酸磷酸化;（d）钙动员;（e）bcl-2家族蛋白的表达/调节。二.评估接受利妥昔单抗治疗患者的原发性NHL细胞的这些细胞信号传导事件，并与观察到的临床敏感性或耐药性发展相关。(a)来自早期利妥昔单抗临床试验的冷冻保存的NHL细胞的分析;（B）来自FHCRC方案#1344的患者的肿瘤活检的前瞻性分析。三.确定mAb FC区在NOD/SCID小鼠中的B细胞NHL系异种移植物中介导抗CD 20 mAb的抗增殖作用中的作用。

Despite active chemotherapy, virtually no improvement has been observed in the survival of patients with low grade B cell NHL. Rituximab, a anti-CD20 mAb induces remissions in 50-60% of patients with follicular NHL. The mechanism of action is not known but likely includes augmented immune effector mechanisms through antibody dependent cell mediated cytotoxicity (ADCC) conferred by the human IgG1 constant region. However, we have observed 1) only 50% of patients respond, and 2) only 40% of initially responding patients respond to re-treatment, and 3) there are no correlates of immune function that predict response, and 4) preliminary data demonstrates direct anti- proliferative effects of the antibody in vitro on some human tumor cell lines, and 5) these direct effects are greater than that observed with other anti-CD20 mAbs. We hypothesize that these direct effects on this mAb (block of proliferation and induction of apoptosis) are responsible for the therapeutic effects and that the loss of these direct effects contributes to acquired resistance. We will therefore test our hypothesis by developing methods for testing the anti-proliferative effects of anti-CD20 antibody on primary human lymphoma cells and then determine whether the presence or absence of these effects predict clinical activity. The Aims of this proposal are to: I. Develop surrogate measures of early events in cell signaling induced in B cell lymphoma lines that are sensitive or resistant to in vitro anti-CD20 mediated effects by evaluating effect on (a) cell proliferation and apoptosis; (b) re-distribution of the CD20 antigen into a insoluble membrane fraction; (c) tyrosine phosphorylation through Src family kinases; (d) calcium mobilization; (e) expression/regulation of bcl-2 family proteins. II. Evaluate primary NHL cells from patients treated with Rituximab for these cell signaling events and correlate with observed clinical sensitivity or development of resistance. (a) analysis of cryopreserved NHL cells from earlier Rituximab clinical trials; (b) prospective analysis of tumor biopsies from patients on FHCRC protocol #1344. III. Determine the role of the mAb FC region in mediating anti- proliferative effects of anti-CD20 mAbs in xenografts of B cell NHL lines in NOD/SCID mice.