Summary Patients with chronic lymphocytic leukaemia (CLL) treated with a combination of fludarabine, cyclophosphamide and rituximab show a high response rate. However, only a poor response is observed following rituximab monotherapy. The use of chemo‐immunotherapy is often associated with haematological and infectious complications. Thus, an antibody with an enhanced ability to kill CLL cells could lead to better clinical responses to antibody monotherapy and the possibility of lowering drug doses during chemo‐immunotherapy. We generated a chimeric anti‐CD20 monoclonal antibody (mAb), EMAB‐6, which has a low fucose content. Apoptosis and complement activities for EMAB‐6 were similar to those seen for rituximab. By contrast, EMAB‐6 mAb showed improved Fcγ receptor IIIA (FcγRIIIA)/CD16 binding and FcγRIIIA‐dependent effector functions. It induced a higher in vitro antibody‐dependent cellular cytotoxicity against CLL cells and a higher FcγRIIIA‐mediated interleukin‐2 production by FcγRIIIA+ Jurkat cells in the presence of CLL cells at both low and maximally saturating concentrations. Comparative studies between CLL and lymphoma cells coated with EMAB‐6 or rituximab indicated that the difference of efficacy was more pronounced at low doses and when target cells expressed fewer CD20 molecules. Thus, EMAB‐6 mAb represents a promising drug candidate for the treatment of CLL by inducing a strong cytotoxicity against tumour cells that express low CD20 levels.

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BackgroundSo far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.ResultsA human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10−10 M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.ConclusionsAntibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these “best in class” binding parameters, the therapeutic success of this antibody was prevented by the target biology.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FccRIIIA) is well preserved in CD16 þ CD56 dim cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FccRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FccR engagement in CLL patients.

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