Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans

<p>Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.</p> <p>Dramatic decreases in ADCC activity brought by core-fucosylation (red-colored) of N-glycans attached to anti-HER2 mAbs as illustrated with outline.</p

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities

<div><p>Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain <i>N</i>-glycans at the constant region of the heavy chain (Fc domain), and their <i>N</i>-glycosylation patterns differ during various processes or among cell expression systems. The Fc <i>N</i>-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc <i>N</i>-glycans, we performed a rearrangement of Fc <i>N</i>-glycans from a heterogeneous <i>N</i>-glycosylation pattern to homogeneous <i>N</i>-glycans using chemoenzymatic approaches with two types of endo-β-<i>N</i>-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous <i>N</i>-glycans, another that is used as a glycosynthase to generate homogeneous <i>N</i>-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man_2-3GlcNAc₂), high-mannose type (Man_4-9GlcNAc₂), and complex type (Man₃GlcNAc_3-4) <i>N</i>-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired <i>N</i>-glycans (M3; Man₃GlcNAc₁, G0; GlcNAc₂Man₃GlcNAc₁, G2; Gal₂GlcNAc₂Man₃GlcNAc₁, A2; NeuAc₂Gal₂GlcNAc₂Man₃GlcNAc₁) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous <i>N</i>-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.</p></div

Binding activity for FcγRIIIa of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) using the FcγRIIIa-V158-binding ELISA method.

<p>Binding activity for FcγRIIIa of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) using the FcγRIIIa-V158-binding ELISA method.</p

ENG’ase activity of the anti-Her2 mAbs (a; endoS, b; endoD, c; endoH, d; endoM, e; endoLL).

<p>(Blue bar represents glycopeptides without ENG’ase hydrolysis; red bar represents the remaining glycopeptides with ENG’ase hydrolysis; y-axis indicates each individual glycoform ratio to total glycoform content; % represents total cleaved glycopeptide ratio by ENG’ase hydrolysis.)</p

MALDI QIT-TOF MS spectrum of Bz-labeled glycopeptides from anti-Her2 mAbs produced in silkworm cocoon.

<p>MALDI QIT-TOF MS spectrum of Bz-labeled glycopeptides from anti-Her2 mAbs produced in silkworm cocoon.</p

ADCC reporter gene assay of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) in SKBR-3 (a) and BT474 (b) target cells.

<p>ADCC reporter gene assay of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) in SKBR-3 (a) and BT474 (b) target cells.</p