Engineered antibody Fc variants with enhanced effector function.
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ABSTRACT:
SUBMITTER: Lazar GA
PROVIDER: S-EPMC1389705 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibodies mediate effector functions through Fcγ receptor (FcγR) interactions and complement activation, causing cytokine release, degranulation, phagocytosis, and cell death. They are often undesired for development of therapeutic antibodies where only antigen binding or neutralization would be ideal. Effector elimination has been successful with extensive mutagenesis, but these approaches can potentially lead to manufacturability and immunogenicity issues. By switching the native glycosylation site from position 297 to 298, we created alternative antibody glycosylation variants in the receptor interaction interface as a novel strategy to eliminate the effector functions. The engineered glycosylation site at Asn298 was confirmed by SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB code 6X3I). The lead NNAS mutant (S298N/T299A/Y300S) shows no detectable binding to mouse or human FcγRs by surface plasmon resonance analyses. The effector functions of the mutant are completely eliminated when measured in antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. In vivo, the NNAS mutant made on an antibody against a human lymphocyte antigen does not deplete T cells or B cells in transgenic mice, in contrast to wild-type antibody. Structural study confirms the successful glycosylation switch to the engineered Asn298 site. The engineered glycosylation would clash with approaching FcγRs based on reported Fc-FcγR co-crystal structures. In addition, the NNAS mutants of multiple antibodies retain binding to antigens and neonatal Fc receptor, exhibit comparable purification yields and thermal stability, and display normal circulation half-life in mice and non-human primate. Our work provides a novel approach for generating therapeutic antibodies devoid of any ADCC and CDC activities with potentially lower immunogenicity.
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Project description:Monoclonal antibody (mAb) therapy has rapidly changed the field of cancer therapy. In 1997, the CD20-targeting mAb rituximab was the first mAb to be approved by the U.S. Food and Drug Administration (FDA) for treatment of cancer. Within two decades, dozens of mAbs entered the clinic for treatment of several hematological cancers and solid tumors, and numerous more are under clinical investigation. The success of mAbs as cancer therapeutics lies in their ability to induce various cytotoxic machineries against specific targets. These cytotoxic machineries include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which are all mediated via the fragment crystallizable (Fc) domain of mAbs. In this review article, we will outline the novel approaches of engineering these Fc domains of mAbs to enhance their Fc-effector function and thereby their anti-tumor potency, with specific focus to summarize their (pre-) clinical status for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM).
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Project description:Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.
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