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. 2010 Mar-Apr;2(2):181-9.
doi: 10.4161/mabs.2.2.11158.

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Gregory L Moore  1 , Hsing Chen, Sher Karki, Greg A Lazar
Affiliations

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Gregory L Moore et al. MAbs. 2010 Mar-Apr.

Abstract

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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Figures

Figure 1
Figure 1
Cartoon representation of human IgG1 antibody Fc from Protein Data Bank record 1E4K with positions at which substitution modulates C1q binding affinity highlighted as space-filling spheres. The putative C1q binding center (D270, K322, P329 and P331) is colored orange. Residues S267, H268 and S324 are indicated in blue. Oligosaccharides are represented as sticks. (A) Full Fc. (B) CH2 domain only.
Figure 2
Figure 2
Fc engineering generates variant anti-CD20 antibodies with enhanced binding affinity for C1q and enhanced cytotoxicity of CD20+ Ramos cells. (A and B) CDC activity of Fc variant anti-CD20 mAbs against opsonized Ramos cells using human complement. Antibody-dependent % lysis was measured at multiple antibody concentrations by Alamar Blue-based detection (mean ± SE of duplicate wells). EC50s are listed in Table 1. (C) SPR sensorgrams for native IgG1 (black) and variant EFT (green) are shown. C1q concentrations range from 100 nM to 6.25 nM by 2-fold serial dilution. (D) Correlation between the fold improvements in C1q affinity as determined by SPR (Table 1) and CDC activity (Table 1, A and B).
Figure 3
Figure 3
Fc variants enhance CDC, ADCC and ADCP. (A and B) CDC activity of Fc variant anti-CD20 mAbs against opsonized Ramos cells using human complement. Antibody-dependent % lysis was measured at multiple antibody concentrations by Alamar Blue-based detection (mean ± SE of duplicate wells). (C and D) ADCC activity of Fc variant anti-CD20 mAbs against Ramos cells using human PBMCs (FcγRIIa genotype was H131/R131, FcγRIIIa genotype was V158/F158). Antibody-dependent % cytotoxicity was measured at multiple antibody concentrations by lactate dehydrogenase release (mean ± SE of triplicate wells). (E and F) Phagocytosis activity of Fc variant anti-CD20 mAbs against Ramos cells using purified human monocytederived macrophages. Antibody-dependent % phagocytosis was measured at multiple antibody concentrations by flow cytometry (mean ± SE of triplicate wells). Macrophages for this experiment were H131/R131 FcγRIIa and V158/F158 FcγRIIIa genotype. Ctrl + DE for ADCC (C and D) and ADCP (E and F) experiments represents an isotype control anti-RSV antibody with DE substitutions. For all of the data shown, fold improvements in EC50 and maximal lysis relative to native IgG1 are listed in Table 2.
Figure 4
Figure 4
EFT + AE variant enhances CDC in anti-CD19 and anti-CD40 mAbs. CDC activity of variant and IgG1 versions of humanized anti-CD19 and anti-CD40 antibodies was tested against Ramos cells using human complement. Antibody-dependent % lysis was measured at multiple antibody concentrations by Alamar Blue-based detection (mean ± SE of duplicate wells).
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