Engineered antibody Fc variants with enhanced effector function

Abstract

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

Fig. 1.

Binding of Fc variant Abs to FcγRs measured by competition AlphaScreen. (A) Binding of alemtuzumab Fc variants to human V158 (Left) and F158 (Right) FcγRIIIa. (B) Binding of trastuzumab Fc variants to human V158 (Left) and F158 (Right) FcγRIIIa (n = 2). (C) Binding of trastuzumab Fc variants to human FcγRIIb (n = 2). (D) Binding of alemtuzumab variants to murine FcγRIII (n = 2). Black asterisk, buffer; gray squares, WT; black diamonds, S298A/E333A/K334A; green triangles, S239D; red inverted triangles, I332E; blue diamonds, S239D/I332E; and tan circles, S239D/I332E/A330L. The S298A/E333A/K334A variant was generated in a previous study (14) and is used here as comparison.

Fig. 2.

Affinity of Fc variant trastuzumab Abs for V158 FcγRIIIa measured by competition SPR. (A) Sensorgrams showing binding to an FcγRIIIa surface by unbound S239D/I332E Ab in an Ab/FcγRIIIa equilibrium at increasing receptor concentrations. (B) Plot of normalized initial binding rate vs. log of receptor concentration. Derived K_D values are presented in Table 1. Line colors are as follows: gray, WT; red, I332E; and blue, S239D/I332E.

Fig. 3.

Cell-based ADCC assays of Fc variant Abs. (A) Eu-based detection assays of trastuzumab Abs against SkBr3 breast carcinoma target cells in the presence of human PBMCs allotyped for V/V (Top), V/F (Middle), or F/F (Bottom) 158 FcγRIIIa. (B) LDH-based detection assay of alemtuzumab Abs against DoHH-2 lymphoma target cells in the presence of human PBMCs allotyped for F/F158 FcγRIIIa. (C) LDH-based detection assay of rituximab Abs against WIL2-S lymphoma target cells in the presence of human PBMCs allotyped for F/F158 FcγRIIIa. n = 2 for all assays. Gray squares, WT; black diamonds, S298A/E333A/K334A (14); green triangles, S239D; red inverted triangles, I332E; blue diamonds, S239D/I332E; and tan circles, S239D/I332E/A330L.

Fig. 4.

Cell-based ADCC assay of trastuzumab Fc variants against cell lines expressing varying levels of Her2 receptor. (A) Western blot showing expression levels of Her2 for cell lines SkBr3 (≈10⁶ copies per cell), SkOV3 (≈10⁵ copies per cell), OVCAR3 (≈10⁴ copies per cell), and MCF7 (≈10² to 10³ copies per cell). Specified cancer cells were washed in PBS and resuspended at 1 × 10⁵ cells per ml in lysis buffer. Equivalent amounts of each lysate were loaded per well of a SDS/PAGE gel, followed by Western analysis using 2 μg/ml commercial trastuzumab and 1 μg/ml horseradish peroxidase-conjugated secondary Ab. (B) ADCC of trastuzumab Fc variants against the Her2⁺ cell lines in the presence of human PBMCs (F/F158 FcγRIIIa). Ab concentration was 1 ng/ml, and lysis was measured by using Eu-based detection. Data were normalized to the minimum and maximum fluorescence signal provided PBMCs alone and Triton X-100, respectively (n = 3). Bar colors are as follows: gray, WT trastuzumab; blue, S239D/I332E; and tan, S239D/I332E/A330L.

Fig. 5.

Role of NK cells and macrophages in mediating enhanced effector function. (A) Cell-based ADCC assay of variant trastuzumab Abs against SkBr3 breast carcinoma target cells in the presence of human F/F158 FcγRIIIa NK cells. Lysis was measured by using LDH-based detection. Gray squares, WT trastuzumab; green triangles, S239D; red inverted triangles, I332E; blue diamonds, S239D/I332E; and tan circles, S239D/I332E/A330L. (B) Dual fluorescence merge image of PKH67-labeled SkBr3 cells (green) and RPE-anti-CD11/anti-CD14-labeled macrophages (red) after a 24-h coculture (3:1 macrophage:SkBr3 ratio) in the presence of 100 ng/ml S239D/I332E. Phagocytosis of green target cells within red-labeled macrophages is detected as yellow in the merge image. (C) ADCP enhancement of Fc variant trastuzumab Ab against SkBr3 target cells, measured using flow cytometry. (D) ADCP enhancement of Fc variant rituximab Abs against WIL2-S target cells. % ADCP represents the number of colabeled cells (macrophage plus target) over the total number of target cells in the population (phagocytosed plus nonphagocytosed) after 10,000 counts. Bar colors are as follows: black, buffer; gray, WT trastuzumab; blue, S239D/I332E; and tan, S239D/I332E/A330L. (n = 2 for all assays.)

Fig. 6.

Cell-based CDC assay of rituximab Fc variants. Lysis of WIL2-S lymphoma target cells in the presence of human complement was measured by using Alamar Blue release (n = 2). Gray squares, WT rituximab; black diamonds, S239D/I332E; and black circles, S239D/I332E/A330L.

Fig. 7.

B cell depletion in macaques. (A) Percent CD20⁺ B cells remaining during treatment with WT and S239D/I332E rituximab Abs. (B) Percent CD40⁺ B cells remaining during treatment. (C) Dose-response of CD20⁺ B cell levels to treatment with S239D/I332E rituximab, acquired at day 5. (D) Percent CD3⁻/CD16⁺ NK cells remaining during treatment. Data reported are group averages of three monkeys per treatment group (n = 3). Symbols and experimentally determined doses are as follows: filled gray squares, WT rituximab (2 μg/kg), open gray squares, WT rituximab (34 μg/kg); and black diamonds, S239D/I332E (2 μg/kg).