ABSTRACT

Current combination therapies elicit high response rates in B cell malignancies, often using CD20 antibodies as the backbone of therapy. However, many patients eventually relapse or develop progressive disease. Therefore, novel CD20 antibodies combining multiple effector mechanisms were generated. To study whether neutrophil-mediated destruction of B cell malignancies can be added to the arsenal of effector mechanisms, we chimerized a panel of five previously described murine CD20 antibodies to the human IgG1, IgA1 and IgA2 isotype. Of this panel, we assessed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and direct cell death induction capacity and studied the efficacy in two different in vivo mouse models. IgA antibodies outperformed IgG1 antibodies in neutrophil-mediated killing in vitro, both against CD20-expressing cell lines and primary patient material. In these assays, we observed loss of CD19 with both IgA and IgG antibodies. Therefore, we established a novel method to improve the assessment of B-cell depletion by CD20 antibodies by including CD24 as a stable cell marker. Subsequently, we demonstrated that only IgA antibodies were able to reduce B cell numbers in this context. Additionally, IgA antibodies showed efficacy in both an intraperitoneal tumor model with EL4 cells expressing huCD20 and in an adoptive transfer model with huCD20-expressing B cells. Taken together, we show that IgA, like IgG, can induce ADCC and CDC, but additionally triggers neutrophils to kill (malignant) B cells. We conclude that antibodies of the IgA isotype offer an attractive repertoire of effector mechanisms for the treatment of CD20-expressing malignancies.

摘要

当前的联合疗法通常使用CD20抗体作为治疗的主干，在B细胞恶性肿瘤中引起高应答率。但是，许多患者最终会复发或发展为疾病。因此，产生了结合多种效应子机制的新型CD20抗体。为了研究嗜中性粒细胞介导的B细胞恶性肿瘤的破坏是否可以添加到效应器机制中，我们对五种先前描述的针对人IgG1，IgA1和IgA2同种型的鼠CD20抗体进行了嵌合。在该小组中，我们评估了体外抗体依赖性细胞介导的细胞毒性（ADCC），补体依赖性细胞毒性（CDC）和直接细胞死亡诱导能力，并研究了两种不同体内的功效鼠标模型。在中性粒细胞介导的体外杀伤中， IgA抗体的性能优于IgG1抗体，既针对表达CD20的细胞系，又针对主要患者的材料。在这些测定中，我们观察到IgA和IgG抗体均会导致CD19丢失。因此，我们建立了一种新方法，通过将CD24作为稳定的细胞标志物来改善CD20抗体对B细胞耗竭的评估。随后，我们证明了在这种情况下，只有IgA抗体能够减少B细胞数量。此外，IgA抗体在表达huCD20的EL4细胞的腹膜内肿瘤模型和表达huCD20的B细胞的过继转移模型中均显示出功效。两者合计，我们显示IgA，如IgG，可以诱导ADCC和CDC，但还触发嗜中性粒细胞杀死（恶性）B细胞。