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

尽管进行了积极的化疗，但低级别B细胞非霍奇金淋巴瘤患者的生存几乎没有改善。抗CD20单抗美罗华可使50-60%的滤泡性非霍奇金淋巴瘤患者缓解。作用机制尚不清楚，但可能包括通过抗体依赖的细胞介导的细胞毒性(ADCC)增强免疫效应机制，该机制由人IgG1恒定区授予。然而，我们观察到1)只有50%的患者有反应，2)只有40%的患者对再次治疗有反应，3)没有免疫功能的相关性来预测应答，4)初步数据表明该抗体在体外对某些人类肿瘤细胞株有直接的抗增殖作用，5)这些直接作用比其他抗CD20单抗更大。我们推测，这些对单抗的直接作用(抑制增殖和诱导凋亡)是治疗效果的原因，而这些直接作用的丧失导致获得性耐药。因此，我们将通过开发测试抗CD20抗体对原代人类淋巴瘤细胞的抗增殖作用的方法来检验我们的假设，然后确定这些影响的存在与否是否预示着临床活动。这一建议的目的是：1.通过评估对(A)细胞增殖和凋亡的影响；(B)将CD20抗原重新分布到不溶于膜的部分；(C)通过Src家族激酶使酪氨酸磷酸化；(D)钙动员；(E)bcl2家族蛋白的表达/调节，建立对体外抗CD20介导的效应敏感或耐药的B细胞淋巴瘤细胞信号转导的替代方法。评估接受利妥昔单抗治疗的患者的原代NHL细胞的这些细胞信号事件，并与观察到的临床敏感性或耐药性的发展相关。(A)来自早期Rituximab临床试验的冷冻保存的NHL细胞的分析；(B)对采用FHCRC#1344方案的患者的肿瘤活检的前瞻性分析。确定抗CD20单抗Fc区在抗CD20单抗抗NOD/SCID小鼠B细胞NHL移植瘤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.