The inhibitory potencies of monoclonal antibodies to the macrophage adhesion molecule sialoadhesin are greatly increased following PEGylation

Abstract

PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.

Figure 1

Anion exchange chromatograms of unconjugated SER-4 mAb (top) and modified SER-4 mAb following PEGylation in PBS pH 7.4 with initial PEG 5 kDa/SER-4 molar ratio of 30:1 and incubation for the different times indicated.

Figure 2

SER-4 mAb was conjugated to PEG 20 kDa (3 h in PBS pH 7.4 at room temperature) and PEG conjugates were purified by anion exchange chromatography (A). Fractions were analyzed by SDS-PAGE under nonreducing and reducing conditions to determine the degree of attachment and location of PEG on heavy and light chains. Sizes of molecular markers are shown. Under nonreducing conditions (B), SDS-PAGE stained with Coomassie blue shows 3 different degree of PEGylation. Under reducing conditions (C), arrows show heavy (H) chains of SER-4 at ~50 kDa and light (L) chains of SER-4 at 25 kDa. The band running between the 62 and 83 kDa markers in all tracks is a contaminant protein that was also present in the buffer-only lane (not shown).

Figure 3

Binding activity of PEGylated mAbs. Conjugates of SER-4 (A,C) and of 3D6 (B,D) were tested at several concentrations for their ability to competitively inhibit the binding of biotin-labeled SER-4 and 3D6, respectively, to Sn-coated microtiter plates. Panels A and B: PEGylation of both mAbs with 1 (open triangles) or 2 (open squares) molecules of PEG 5 kDa does not affect the binding activity of mAbs. Panels C and D: Both SER-4 and 3D6 modified with 2 (open squares) molecules of PEG 20 kDa show a slight reduction of binding activity when compared to unconjugated mAbs (solid circles). * indicates a statistically significant difference in binding inhibition compared to unconjugated mAbs (Tukey test, P < 0.001). Each data point represents the mean ± SD (n = 3 in duplicate wells).

Figure 4

Inhibitory potency of PEGylated SER4 and 3D6 mAbs. A truncated form of Sn (domains 1 to 3) fused to the Fc region of human IgG1 was coated on wells at a concentration of 2 μg/mL. Wells were then preincubated with serially diluted unconjugated mAbs or PEG conjugates prior to the addition of human RBC. A preincubation of wells with SER-4 carrying 2 molecules of PEG 20 kDa reduced binding by about 75% (A). In the case of 3D6, a preincubation of wells with 3D6–2PEG 5 kDa resulted in ~75% inhibition, and a complete inhibition of RBC binding was seen with 3D6 carrying either 1 or 2 molecules of PEG 20 kDa (B). Each data point represents the mean ± SD (n = 3 in duplicate wells).

Figure 5

Inhibitory potency of PEGylated SER4 and 3D6 mAbs used in combination. A truncated form of Sn (domains 1 to 3) was coated on wells at a concentration of 2 μg/mL. Wells were then preincubated with serially diluted unconjugated mAbs or PEG conjugates in combination prior to the addition of human RBC. A total of sixteen combinations of SER-4-PEG and 3D6-PEG were compared to the parent SER-4 and 3D6 combination in RBC binding assays and concentrations inhibiting 50% of RBC binding (IC₅₀) were compared. All combinations of PEGylated antibodies were better inhibitors than the mixture of unlabeled SER-4 and 3D6. Bar represents the mean values of IC₅₀ ± SD (n = 3 in duplicate wells).