Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans

Abstract

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Figure 1. The growth profile, viability profile during course of culture, and the final titer on day 11. (A-C) are data for mAb1 when adding 20 (diamond), 40 (square), 60 (triangle), 80 (cross), and 100 ng/mL (circle) of kifunensine into basal media; (D-F) are data from mAb2 when adding 0.1 (diamond), 0.5 (square), 2 (triangle), 5 μg/mL (circle) of kifunensine into basal media. (A) and (D) are viable cell density at 1 × 10⁵ cells/ mL measured on selected days using Vi-Cell XR (Beckman Coulter, Fullerton, CA). (B) and (E) are viability measured in percent. (C) and (F) are titer of antibody on day 11 after harvest.

Figure 2. Mass spectra of the heavy chain of antibodies with different glycans measured by rpHPLC ESI-MS. Antibodies were expressed with or without kifunensine treatment during cell culture. (A) mAb1 without kifunensine; (B) mAb1 with 100 ng/mL kifunensine; (C) mAb2 without kifunensine; (D) mAb2 with 5 μg/mL kifunensine.

Figure 3. General scheme of the approach to make Man5 antibodies.

Figure 4. High mannose glycoform distribution before and after enzymatic trimming using Aspergillus saitoi α-1,2-mannosidase.

Figure 5. ADCC activity of mAb1 using peripheral blood mononuclear cells (PBMCs) from healthy donors as effector cells. (A) Percentage of ADCC measured at different concentrations of mAb1 for different levels of high mannose, 100% complex-afucosyl (AF), and complex-fucosylated (Ref) glycoforms. The data are shown as an average from duplicate experiments. (B) EC₅₀ of the different glycoforms extracted from (A), and then normalized to reference.

Figure 6. Binding affinity of mAb1 with different glycoforms toward Fcγ receptors. All data are EC₅₀ values, normalized to reference. (A) Binding affinity toward FcγRIA; (B) binding affinity toward FcγRIIA and FcγRIIB with two allotypes (H131 and R131); (C) binding affinity toward FcγRIIIA with two allotypes (F158 and V158).

Figure 7. Complement-dependent cytotoxicity (CDC) of mAb1 with different glycoforms as indicated. EC₅₀ of each data set was determined, and reported on this plot after normalizing to the EC₅₀ of the reference glycoform.

Figure 8. Pharmacokinetic profiles of mAb variants in Harlan athymic nude mice. The two groups conducted in this study were mAb2 with > 99% Man5 (solid black circle) and mAb2 with > 99% Man8/9 (solid gray triangle). The complex-fucosylated profile (open squares) was from a separate study conducted in a similar fashion to the current study. Mice were injected with single I.V. dose of 10 mg/kg mAbs with specific glycoform, and serum samples were collected in the indicated time points up to 28 d. Triplicate sample set was collected for each time point. Average values are reported and the error bars represent standard deviation. The data was fit to a 2 compartment model for the calculation of pharmacokinetic parameters illustrated in Table 1.

Figure 9. Glycan analysis showing high mannose glycoform distribution for samples collected at the different time points of the PK study. The antibodies were first purified from serum, and then subsequently analyzed using rpHPLC ESI-MS method. (A) Glycan profile of mAb2 for mice injected with 100% Man5 glycoform; (B) glycan profile for mAb2 for mice injected with 100% Man8/9 glycoform.

Figure 10. Glycan profile of mAb2 after incubation for indicated time period with mouse serum in vitro. The antibodies were spiked into mouse serum, and then incubated at 37°C to investigate the presence of mannosidase activity in commercially available mouse serum. The antibodies were then purified from serum and analyzed using rpHPLC-ESI MS. (A) Mouse serum spiked with mAb2 with 100% Man5; (B) mouse serum spiked with mAb2 with 100% Man8/9.