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

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

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

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

BrdU uptake and real-time RT-PCR analysis.

<p>(A) The percentage of osteoblasts positive for BrdU at 2 weeks of age. n = 4. (B, C) Real-time RT-PCR analysis. B, Expression of <i>Col1a1</i>, <i>Spp1</i>, <i>Ibsp</i>, and <i>Bglap2</i> was determined using RNA from whole bones of femurs and tibiae in wild-type (wt), tg1, and tg2 mice at 4 weeks of age. n = 5–8. *P<0.05 and **P<0.01 vs. wild-type mice. C, Expression of <i>Dmp1</i>, <i>Mepe</i>, <i>Phex</i>, <i>Fgf23</i>, <i>Sost</i>, <i>Atf4</i>, <i>Tnfsf11</i>, <i>Tnfrsf11b</i> was determined using RNA from the osteocyte fraction of femurs and tibiae in wild-type and tg2 mice at 12 weeks of age. n = 5. **P<0.01 vs. wild-type mice. The values of the wild-type mice were defined as 1, and relative levels are shown in B and C.</p

X-ray and histological analyses of <i>Sp7</i> transgenic mice.

<p>(A) X-ray analysis of the femurs of a wild-type (wt), tg1, and tg2 mouse at 6 weeks of age. (B–G) Histological appearance of bone in transgenic mice. Longitudinal sections through the distal part (B–D) and diaphysis (E–G) of femurs in a wild-type (B, E), tg1 (C, F), and tg2 (D, G) mouse at 6 weeks of age. The sections were stained with H–E. (H–J) Polarized microscopic examination of the diaphyses of femurs from wild-type (H), tg1 (I), and tg2 (J) mice at 6 months of age. (K–T) TEM images of osteoblasts (K, L) and osteocytes (M, N), canalicular staining of tibiae (O, P), and SEM images of preosteocytes (Q, S) and young osteocytes (R, T) of wild-type (K, M, O, Q, R) and tg2 (L, N, P, S, T) mice at 10 weeks of age. In Q–T, bone and osteoid in cortical bone were dissolved and preosteocytes and young osteocytes just beneath the osteoblast layer in the endosteum were observed by SEM. Bars: (B–D) 500 μm; (E–J) 50 μm; (K–N), 1 μm; (O–T), 10 μm.</p

Positive regulation of <i>Sp7</i> promoter by SP7.

<p>(A, B) Real-time RT-PCR analysis. A, Endogenous <i>Sp7</i> expression in wild-type and tg2 mice at 18 weeks of age. RNA was prepared from tibiae and femurs, in which hematopoietic cells were flushed out. *P<0.05 vs. wild-type mice. n = 4–5. B, Osteoprogenitors isolated from wild-type calvaria using collagen gel were infected with adenovirus expressing EGFP or <i>Sp7</i> at 6×10⁶ pfu/well, and endogenous <i>Sp7</i> expression was examined. **p<0.001 vs. EGFP-expressing cells. The level in wild-type mice or EGFP-expressing cells was set at 1, and relative values are shown. (C, D) Reporter assays using the <i>Sp7</i> promoter. C, The reporter activity using the 1.8-kb <i>Sp7</i> promoter-luciferase (Luc) construct and the deletion constructs. Reporter assays were performed using 293T cells with (closed columns) or without (open columns) the <i>Sp7</i> expression vector. The lines in the schematic diagram of the <i>Sp7</i> promoter indicate putative SP1-binding sites. D, The reporter activity using the 1.8-kb <i>Sp7</i> promoter in ATDC5 cells infected with adenovirus expressing EGFP or sh-<i>Sp7</i> at 6×10⁶ pfu/well. *p<0.05 and **p<0.001 vs. control. (E) ChIP assay. DNA before immunoprecipitation (input) and after immunoprecipitation with anti-SP7 antibody or anti-IgG antibody was amplified by PCR using primers that amplify the region containing the proximal 170 bp of <i>Sp7</i> promoter.</p

Osteoblast differentiation <i>in vitro</i>.

<p>(A–D) Real-time RT-PCR analysis. A, Induction of <i>Sp7</i> expression by RUNX2 in <i>Runx2</i>^−/− calvarial cells. The cells isolated using collagen gel were infected with EGFP or <i>Runx2</i>-EGFP expressing adenovirus (6×10⁶ pfu/well). RNA samples were obtained at 6, 12, 24 and 48 hours after infection. Expression levels before infection (0) were defined as 1 and relatives levels are shown. <i>Runx2</i>^−/− calvarial cells express an aberrant transcript of <i>Runx2</i> at a low level. n = 3. B, <i>Sp7</i> expression after the infection of adenovirus carrying EGFP or <i>Sp7</i>. C, <i>Sp7</i> expression after the infection of adenovirus carrying EGFP or sh-<i>Sp7</i>. Adenoviruses were used at 3×10⁶ pfu/well or 6×10⁶ pfu/well as indicated. D, <i>Alpl</i>, <i>Col1a1</i>, and <i>Bglap2</i> expressions were examined at 4 days or 9 days of culture after the infection. Adenoviruses were used at 3×10⁶ pfu/well. The levels in cells, which were infected with EGFP-expressing adenovirus at 3×10⁶ pfu/well, at 4 days of culture were defined as 1, and relative levels are shown. *p<0.05 and **p<0.001 vs. EGFP. n = 4. (E) Von Kossa staining in primary osteoblasts infected with an adenovirus carrying EGFP, <i>Sp7</i>, sh-<i>Sp7</i>, or <i>Runx2</i>. Three independent experiments were performed and representative data are shown in A–E.</p