Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding

Abstract

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

Keywords: ADCC; ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cell-mediated phagocytosis; AUC, area under the curve; CL, clearance rate; CD89; CDC, complement dependent cytotoxicity; Cmax, maximum serum concentration; DSC, differential scanning calorimetry; E:T ratio, effector to target ratio; FCM, flow cytometry; FcRn, neonatal Fc receptor; FcαRI; FcγR, Fc gamma receptor; HER2, human epithelial receptor two; IgA; IgA, immunoglobulin A; IgG, immunoglobulin G; LDH, lactate dehydrogenase; MΦ, macrophage; NK, natural killer; PBMC, peripheral blood mononuclear cell; PK, pharmacokinetics; PMN, polymorphonuclear; SPR, surface plasmon resonance; TAA, tumor associated antigens; T½, half-life; Vss, central compartment volume of distribution; macrophage; monoclonal antibody; neutrophil; tandem; trastuzumab.

Figure 1.

Design of the IgG1/IgA2 tandem Fc fusion. Structural models of antibody formats generated for this study are shown, with IgG1- (left), an IgA2-Fc and hinge domain with IgG Fab domains (middle) and the tandem IgG1/IgA2-Fc format with an IgA2-Fc and hinge fused to an IgG-Fc (right). Structures were generated in Pymol (Schrödinger) using a human IgG1 pdb model and an IgA Fc structure (PDB ID: 1OW0). The DNA and encoded protein sequence of the IgG1/IgA2-Fc linkage is shown with an introduced HindIII site (boxed) highlighted.

Figure 2.

ADCC with different effector cell types. ADCC results are shown using BT474 cells as targets and either NK cell line KC1333 (A), the freshly isolated PMN cells (B) or both the PMN and PBMC cell fractions combined (C). For PMN and PMN plus PBMC experiments, data are the average of 5 different experiments with 5 separate donors. KC1333 data is combined from 3 separate experiments. E:T ratios were 2.5:1 when KC1333 cells were used as effector cells, and 20:1 with PMN and PMN plus PBMC effector cells.

Figure 3.

Visualizing PMN-mediated ADCC of mAb variants via microscopy and FCM. (A) Freshly isolated PMN cells (unlabeled) were incubated with Her2⁺⁺⁺ BT474 cells (green) at an E:T ratio of 10:1 with either IgG isotype control, Her2-IgG1, Her2-IgG1/IgA2 or Her2-IgA2 at a concentration of 6 nM and visualized via fluorescence microscopy. PMN mediated ADCC (B) was assessed using an FCM based 3 color assay to measure target cell killing. An E:T ratio of 10:1 PMN:BT474 cells was used. Data are representative from one donor of 3 tested.

Figure 4.

ADCP of SK-BR-3 and MDA-MB-453 cells with mAb variants. Human monocyte-derived MФ were labeled with celltrace violet and co-cultured with CFSE-labeled Her2 expressing cells at an 8:1 effector: target ratio for 2 h in the presence of the indicated antibodies. Cells were collected and stained with live dead far red, and analyzed by FCM. Dot plots (A) are of live cells in SK-BR-3 cell / MΦ co-cultures in the presence of 650pM IgG isotype control or Her2-IgG1. Images of MФ (purple), SK-BR-3 (green) and double positive cells from the indicated regions of the dot plot were captured using an imaging cytometer. Phagocytosis of SK-BR-3 cells (B) and MDA-MB-453 cells (C) was quantified as the percent double positive cells of CFSE positive cells. Data are mean ± SEM. of experiments performed with MΦ from n ≥ 4 independent donors.

Figure 5.

Pharmacokinetic profiles of mAb variants in BALB/c mice. Serum levels for Her2-IgG1 (black), Her2-IgG1/IgA2 (green), and Her2-IgA2 (red) in BALB/c mice were quantified by ELISA detecting human Fab. Mice were injected with a single IV bolus dose of 2.5 mg/kg. Each data point represents the average serum concentration of the mAb in 4 mice for up to 14 days. Error bars represent standard deviation. The data was fit to a 2 compartment model for the calculation of pharmacokinetic parameters (Table 3).