An Fc Double-Engineered CD20 Antibody with Enhanced Ability to Trigger Complement-Dependent Cytotoxicity and Antibody-Dependent Cell-Mediated Cytotoxicity

Abstract

Background: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. Methods and Results: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. Conclusion: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils

“…FITC-labeled mouse IgG1 was used as isotype control (Pelicluster). Afucosylated trastuzumab was generated in our laboratory as described before for afucosylated rituximab (42). Briefly, CHO-KI or Lec13 cells were transfected with antibody LC and HC expression constructs using transfection kit V from the Amaxa Nucleofectior System (Lonza, Cologne, Germany).…”

Antibody Isotypes for Tumor Immunotherapy

“…From an economical point of view, these industrial procedures contributed to the prominent role of IgG1 antibodies in the clinic [17]. Importantly, human IgG1 antibodies are often used as backbone for Fc engineering strategies which aim to further improve effector functions, stability, or pharmacokinetic properties of therapeutic antibodies [18,19]. …”

Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies

“…The strong CDC induction of IgG3 antibodies targeting CD20 [ 113 , 114 ] favors the development of IgG1/IgG3 isotype variants, and a CD20-targeting afucosylated IgG1/IgG3 isotype variant, with increased CDC and ADCC levels in vitro [ 115 ]. Applying multiple Fc-enhancing strategies simultaneously also proved beneficial for a nonfucosylated rituximab variant containing the S267E/H268F/S324T/G236A/I332E mutations, which enhanced both ADCC and CDC in vitro [ 116 ].…”