Akito Natsume

Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities.

Enhancement of multiple effector functions of an antibody may be a promising approach for antibody therapy. We have previously reported that fucose removal from Fc-linked oligosaccharides greatly enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies. Here, we report a unique approach to enhance complement-dependent cytotoxicity (CDC), another important effector function of antitumor antibodies, by using engineered constant region of human IgG1/IgG3 chimeric isotypes. We systematically shuffled constant domains of IgG1 and IgG3 to generate a comprehensive set of mixed chimeric isotypes of anti-CD20 antibodies. Among these, the variant 1133, consisting of the CH1 and the hinge each from IgG1 and the Fc from IgG3, was unexpectedly found to exhibit markedly enhanced CDC that exceeded wild-type levels. However, it lacked protein A-binding capacity, an important feature for the industrial production. To eliminate this deficiency, a portion in COOH-terminal CH3 domain of 1133 was substituted with IgG1, resulting in full recovery of protein A binding without compromising the enhanced CDC and ADCC activities. The CDC-enhancing effect using a chimeric isotype was also shown in CD52 antigen/antibody system. The ADCC activity of the variants was also maximized by the absence of fucose from its carbohydrate structure, a phenomenon that has previously been observed for wild-type antibodies. Enhanced cytotoxicity of a variant was confirmed in a cynomolgus monkey model. These findings suggest that the variant antibodies with IgG1/IgG3 chimeric constant regions and nonfucosylated oligosaccharides that possess dual-enhanced cytotoxic functions may be an improvement for the next generation of therapeutic antitumor antibodies.

Improving effector functions of antibodies for cancer treatment: Enhancing ADCC and CDC

As platforms for therapeutic agents, monoclonal antibodies (MAbs) have already been approved, and several MAbs have demonstrated clinical effectiveness in a variety of malignancies. However, several issues have also been emerging in antibody therapy, such as high cost and insufficient drug action. Recently, to improve MAb activity in humans, effector functions have been subjects of focus, especially antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Extensive efforts have been made to enhance these effector functions of MAbs, and successful approaches have been reported by us and others, wherein the binding activity of MAbs to FcγRIIIa or C1q is increased by introducing amino acid mutations into heavy chain constant regions or through glyco-modification of Fc-linked oligosaccharides. In addition, one of the next approaches to optimizing therapeutic antibodies would be to combine multiple enhancing modifications into a single antibody platform to overcome the diverse mechanisms of clinical resistance of tumor cells. For this aim, we have recently developed a successful combination composed of ADCC-enhancing modification by the fucose depletion from Fc-linked oligosaccharides and CDC-enhancing modification by IgG1 and IgG3 isotype shuffling in heavy chains, which could be of great value for the development of third-generation antibody therapeutics.

Engineered anti‐CD20 antibodies with enhanced complement‐activating capacity mediate potent anti‐lymphoma activity

One of the major issues in current antibody therapy is insufficient efficacy. Various biological factors relating to the host’s immune system or tumor cells have been suggested to reduce the efficacy of anti‐CD20 therapy in B‐cell malignancies. In this study, we characterized the in vitro anti‐lymphoma activity of anti‐CD20 antibodies having a novel engineered heavy chain with enhanced complement‐dependent cytotoxicity (CDC). Anti‐CD20 antibodies having a variant heavy constant region of mixed IgG1/IgG3 isotype, which have previously been found to enhance CDC, were investigated for their in vitro CDC against lymphoma cells and whole blood B‐cell depletion activity. Use of the variant constant region greatly increased the CDC of an anti‐CD20 antibody having variable regions identical to those of rituximab to the level shown by an IgG1 antibody of ofatumumab. Although the whole blood assay showed different cytotoxicity patterns among individual blood donors, the CDC‐enhancing variant of rituximab showed higher activity than the parent IgG1 and consistently showed maximized activity when further combined with antibody‐dependent cellular cytotoxicity (ADCC)‐enhancing modification by fucose removal from Fc‐linked oligosaccharides. In addition, the rituximab variant showed potent CDC against transfectant cells with lower CD20 expression and chronic lymphocytic leukemia–derived cell lines with higher complement regulatory proteins. These findings suggest that CDC enhancement, both alone and in combination with ADCC enhancement, increases the anti‐lymphoma activity of anti‐CD20 antibodies irrespective of individual differences in effector functions, and renders current anti‐CD20 therapy capable of overcoming the potential resistance mechanisms. (Cancer Sci 2009; 100: 2411–2418)

Nonfucosylated anti-CD20 antibody potentially induces apoptosis in lymphoma cells through enhanced interaction with FcγRIIIb on neutrophils

We demonstrate herein the augmentation of rituximab-mediated apoptosis in lymphoma cell lines by cross-linking with recombinant FcgammaRs, which is further enhanced by using a nonfucosylated variant of rituximab having strong FcgammaRIII-binding capacity. Furthermore, we show that neutrophils can serve as physiological cross-linkers that augment anti-CD20-mediated apoptosis, as evidenced by (i) the neutrophil-augmented apoptosis was more profound for the nonfucosylated variant of rituximab and (ii) the mechanism depended on FcgammaRIIIb but not on FcgammaRIIa. Taken together, we suggest a potential anti-tumour mechanism of nonfucosylated anti-CD20 antibody by which antibody molecules are cross-linked through enhanced interaction with FcgammaRIIIb in neutrophils.