Characterization of antibody products obtained through enzymatic and

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Characterization of antibody products obtained through enzymatic and non-enzymatic glycosylation reactions with a glycan oxazoline and preparation of homogeneous antibody-drug conjugate via Fc N-glycan Shino Manabe, Yoshiki Yamaguchi, Kana Matsumoto, Hirobumi Fuchigami, Taiji Kawase, Kenji Hirose, Ai Mitani, Wataru Sumiyoshi, Takashi Kinoshita, Junpei Abe, Masahiro Yasunaga, Yasuhiro Matsumura, and Yukishige Ito Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00132 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019

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Bioconjugate Chemistry

Characterization of antibody products obtained through enzymatic and non-enzymatic glycosylation reactions with a glycan oxazoline and preparation of homogeneous antibody-drug conjugate via Fc N-glycan

Shino Manabe, * Yoshiki Yamaguchi, , * Kana Matsumoto, Hirobumi Fuchigami, Taiji †

† ‡

‡

¶

Kawase, Kenji Hirose, Ai Mitani, Wataru Sumiyoshi, Takashi Kinoshita, Junpei Abe, $

$

#

#

#

†

Masahiro Yasunaga, Yasuhiro Matsumura, and Yukishige Ito ¶

¶

†

Synthetic Cellular Chemistry Laboratory, RIKEN, Hirosawa, Wako, Saitama, 351-0198

†

Japan Structural Glycobiology Team, RIKEN, Hirosawa, Wako, Saitama, 351-0198 Japan

‡

Exploratory Oncology Research & Clinical Trial Center, National Cancer Center,

¶

Kashiwanoha, Kashiwa, Chiba, 277-8577 Japan Nihon Waters KK., Kitashinagawa, Shinagawa, Tokyo, 140-0001 Japan

$

Fushimi Pharmaceutical Co. Ltd. Nakatsu, Marugame, Kagawa, 763-8605 Japan

#

[email protected], [email protected] Tel: +81-48-467-9432; Fax: +81-48-462-4680

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Glycan engineering of antibody has received considerable attention. Although various endo-β-N-acetylglucosaminidase mutants have been developed for glycan remodeling, side-reaction has been reported between glycan oxazoline and amino groups. In this study, we performed a detailed characterization for antibody products obtained through enzymatic and non-enzymatic reactions with the aim of maximizing the efficiency of glycosylation reaction with fewer side products. The reactions were monitored by an ultra-performance liquid chromatography system using an amide-based wide-pore column. The products were characterized by liquid chromatography coupled with tandem mass spectrometry. The side-reactions were suppressed by adding glycan oxazoline in a stepwise manner under slightly acidic conditions. Through combination of azide-carrying glycan transfer reaction under optimized conditions and bio-orthogonal reaction, a potent cytotoxic agent monomethyl auristatin E was site-specifically conjugated at N-glycosylated Asn297 with drug-to-antibody ratio of 4. The prepared antibody-drug conjugate exhibited cytotoxicity against HER2-expressing cells.


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KEYWORDS antibody-drug conjugate, endo-β-N-acetylglucosaminidase, glycan remodeling, LC/MS/MS, wide-pore amide-bond column chromatography



INTRODUCTION Antibody-drug conjugates (ADCs) are a new class of highly potent pharmaceutical drugs designed for targeted therapy, and they are expected to be the next generation of antibody drugs.1, 2 Presently, 4 ADCs have been approved by the FDA, and more than 60 ADCs are in pipeline.3, 4 Cytotoxic drugs are covalently linked to monoclonal antibodies with tumor antigen-specific activity. ADC can bind to antigen-decorated cancer cells and deliver cytotoxic agents to these cells. Most ADCs are believed to be internalized into cells, and cytotoxic agents are released in lysosomes in their active form. Owing to antibody selectivity against cancer, ADCs can expand the narrow therapeutic index of traditional chemotherapeutic agents. Currently, cytotoxic agents are conjugated to therapeutic antibodies at Lys or Cys residues by traditional protein modification reactions. Typically, amide bond formation between activated carboxylic acid and amino group at Lys residues, or Michael reaction between sulfhydryl group and maleimide is employed. Thus, therapeutic antibodies are heterogeneous mixtures originating from different drug-to-antibody ratios (DAR) and conjugation sites. Conjugation-site differences of ADCs lead to different releasing rates of payloads and different stabilities.5, 6 The development of a method to prepare homogeneous ADCs is therefore strongly desired, because homogenous ADCs expand the safety-margin


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and have homogenous efficacy, pharmacokinetics, and pharmacodynamics.7 Furthermore, homogeneity is favorable in terms of regulatory science. Many trials have reported the preparation of homogeneous ADCs.8 Representative examples include the incorporation of non-natural amino acids into an antibody and the subsequent bio-orthogonal reaction for conjugation9-11 and sequence-selective enzymatic conjugation using enzymes such as sortase A.12, 13 In this study, we focus on conserved N-glycan at Asn297 in IgG Fc region. The oligosaccharide is considerably heterogenous owing to its complex biosynthetic process.14 Typically, the biantennary N-glycan is heterogeneous with respect to its galactose, bisecting GlcNAc, and core fucose residues. The major isoform of biantennary glycans is composed of G0, G1 (two isoforms), and G2 isoforms (i.e., 0, 1, and 2 terminal galactose residues, respectively). In addition, a relationship between glycan structure and antibody function has been reported. For instance, the absence of core-fucose enhances antibody-dependent cellular cytotoxicity (ADCC) 100-fold.15 Non-human type glycan structures from host cells cause severe immunogenicity.16 Hence, regulation of Fc N-glycan structure itself is highly important. Furthermore, conjugation of payloads to N-glycan site is ideal, because the Fc N-glycan at Asn297 is located far from the antigen-binding region. Modification of this



glycan will not affect the antigen-binding ability. Based on these aspects, several conjugation methodologies have been reported using N-glycan, including conjugation via carbonyl group generated by oxidation of 1,2-cis diols,17,

18

transfer of modified monosaccharide by

glycosyltransferases,19-21 and metabolic modification.22 Besides monosaccharyl transferases, endo-β-N-acetylglucosaminidase (ENGase) mutants are also useful for site-specific conjugation of oligosaccharides.23 Various ENGase mutants, such as Endo M N175Q,24 Endo CC N180H,25 EndoS D233Q,26 Endo S2 D184M,27 and Endo S2 D184Q,27 have been developed. Combinations of ENGase mutants with reduced hydrolytic activity and the transition-state mimic sugar oxazoline have been used for the transfer of oligosaccharides to functional groups in acceptor molecules.26, 28, 29 However, side reaction between oxazoline and amino group was recently reported.23, 30, 31 In this study, we describe the reaction monitoring of glycan remodeling in a real-time manner by using ultra-performance liquid chromatography (UPLC), the optimization of enzymatic glycan addition, and the precise characterization of side products. After optimization of the reaction, glycan-based homogeneous ADC was prepared and its cytotoxicity was evaluated.


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RESULTS AND DISCUSSION Establishment of monitoring protocol for removal and transfer of glycan in antibody To optimize the glycan transfer reaction without side-reactions, we initially planned to establish a process to monitor glycan-remodeling reactions in a real-time manner (Figure 1). The glycan-remodeling reactions comprise two-steps: glycan removal reaction by ENGase and glycan transfer reaction by ENGase mutant. Heterogeneous Fc glycans were cleaved in advance by EndoS,32 belonging to glycoside hydrolase family 18. This step makes 297Asn to a unique residue Asn with GlcNAc or Fuc-GlcNAc. Then, homogeneous glycan was added by EndoS mutant D233Q with reduced hydrolytic activity. This step uniquely introduces the homogeneous glycan at Asn297 residue.



Figure 1. A schematic diagram of preparation of homogenous glycan-linked antibody-drug conjugate via glycan remodeling using ENGase mutant and glycan oxazoline.

We chose a wide-pore amide-bond stationary phase in UPLC system, which is suitable for analyzing glycan occupancy and heterogeneity of intact glycoproteins. Two therapeutic antibodies, trastuzumab (Herceptin®) and mogamulizumab (Poteligeo®), with or without core-fucose in the Fc N-glycan, respectively, were selected in our research. IgGs were treated with PNGase F or EndoS, and an aliquot of the reactant was directly injected into the UPLC column and detected with intrinsic tryptophan fluorescence. The deglycosylation reaction of mogamulizumab was monitored by changes in retention time (Supporting Information Figure S1). IgGs were well separated depending on the number of glycans (0, 1, and 2) by UPLC analyses. The peak corresponding to antibody with 2 glycans appeared at 7.14–7.63 min, that corresponding to antibody with 1 glycan appeared at 7.34 min, and that corresponding to antibody without glycan appeared at 6.89–6.53 min. Intact mass spectrometry (MS) (Supporting Information Figure S2) showed the corresponding molecular weights, respectively. Thus, the deglycosylation reaction was quantitatively monitored in a real-time manner using this UPLC system. Glycan cleavage by EndoS was found to be completed within 10 min under the condition tested.


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Optimization of glycan transfer reaction conditions Using the above established reaction-monitoring protocol, N-glycan oxazoline 1 was added to glycan-truncated mogamulizumab with EndoS mutants D233Q, D233A, and D233H at pH 7.5 (Figure 2a), and the reaction progress was monitored by UPLC analyses. EndoS D233H was newly prepared in this study. The efficacy of glycan transfer by EndoS mutants was in the order of EndoS D233Q > EndoS D233A > EndoS D233H. However, we noticed that glycan addition was observed even in the absence of EndoS mutant. This presumably occurred as a side-reaction between glycan oxazoline 1 and amino group at Lys residues30, 31. We presumed that the side-reaction between sugar oxazoline 1 and amino group can be suppressed by lowering the nucleophilicity of amino group, i.e. by lowering the pH of the reaction mixture. The reaction was carried out using EndoS D233Q at the same conditions, except for low pH 6.5 (Figure 2b). Besides the reduction of side-reaction as reported previously,23 the reaction efficacy was also reduced. One possible reason for the reduction in efficacy is decomposition of glycan oxazoline under slightly acidic conditions. To overcome the dilemma between side-reaction and possible oxazoline



decomposition, glycan oxazoline was added to the reaction mixture every 15 min at pH 6.5. In addition, the reaction conditions were optimized by changing the concentrations of antibody, EndoS D233Q, and oxazoline 1 (Table 1). When concentration of oxazoline 1 was low (conditions 1-4), the reaction rates were slow. Even concentrations of mogamulizumab and EndoS D233Q were higher, the reaction rates were still low (condition No.3). At condition No.6 (antibody concentration 17 µM; EndoS D233Q concentration 7.5 µM; and oxazoline 1 concentration 0.75 mM), glycan addition resulted in 92 % yield, which was calculated from the UPLC peak area (Figure 2c). Side-reaction of oxazoline to antibody was almost negligible in the absence of EndoS D233Q under this experimental condition. Thus, side-reaction was suppressed by portion-wise addition when compared with the previous study.23


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Figure 2. a) UPLC profiles of EndoS-treated mogamulizumab (GlcNAc-IgG) incubated in the presence of glycan oxazoline 1 and EndoS mutants D233Q, D233H, and D233A, or with glycan oxazoline 1 in the absence of enzyme at pH 7.5. The UPLC profiles are shown for 15 min, 1 h, and 3 h of reaction time. b) UPLC profiles of EndoS-treated mogamulizumab (GlcNAc-mogamulizumab) incubated with sugar oxazoline 1 in the presence of EndoS D233Q or with sugar oxazoline 1 in the absence of enzyme at pH 6.5. The UPLC profiles are shown for 15 min, 1 h, 3 h, and 6 h of reaction time. c) Addition of glycan oxazoline 1 to EndoS-treated mogamulizumab using EndoS D233Q under the optimized condition (No. 6 in Table 1). Glycan oxazoline 1 was added every 15 min, and the reaction was monitored with the UPLC system. The UPLC profiles are shown for 45, 90, and 150 min. A control reaction 11 ACS Paragon Plus Environment


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was conducted without EndoS D233Q.

Table 1: Relationships between concentration of mogamulizumab, EndoS D233Q, oxazoline 1, reaction period, and yields of products.

No.

mogamulizumab (μM)

EndoS

Oxazoline Reaction

D233Q

glycan*

period

(μM)

(mM)

(min)

Yield (%)** IgG (0)

IgG (1)

IgG (2)

1

13

1.8

0.20

150

9.4

36.9

53.6

2

13

5.4

0.20

150

3.7

25.3

71.0

3

40

18

0.20

105***

28.5

47.0

24.4

4

6.7

3.0

0.20

150

7.1

34.2

58.7

5

13

6.0

0.40

150

2.0

18.8

79.2

6

17

7.5

0.75

150

0.4

7.0

92.7

*Oxazoline glycan 1 was added every 15 min. **Yield was calculated based on the peak areas in the UPLC spectra. ***Reaction was terminated owing to slow reaction. Percentage of fully-glycosylated [mogamulizumab (2)], mono-glycosylated [mogamulizumab (1)], and GlcNAc-glycosylated [mogamulizumab (0)]

Characterization of glycan-remodeling antibody products by intact mass analysis To examine the enzymatic and non-enzymatic reactions, intact mass analysis of the antibody products was performed. Mass spectrum showed a single peak for the antibody prepared under the optimized condition No.6 in Table 1 (Figure 3b). The observed mass (150,602 Da) corresponded to the theoretical mass of the antibody with 2 glycans (150,599 Da), suggesting that sequential addition of glycan oxazoline was successful at pH 6.5, with


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fewer side-reactions. In contrast, the antibody treated with excess oxazoline 1 in the absence of EndoS D233Q gave 5 major peaks in the mass spectrum, corresponding to the antibody with 0–4 glycans (Figure 3c).

Figure

3. Deconvoluted ESI-MS spectra of mogamulizumab. a) EndoS-treated

mogamulizumab, b) enzymatically glycan-transferred mogamulizumab under optimized conditions, and c) non-enzymatically glycan-transferred mogamulizumab using excess glycan oxazoline 1.

Peptide mapping of glycan-remodeling antibody products for characterization of non-enzymatic glycosylation To obtain information on the glycosylation site, peptide mapping was performed. The sample was trypsinized into peptide fragments, and the peptide mixtures were subjected 13 ACS Paragon Plus Environment


to LC/MSE analysis (Supporting Information Figure S3). Peak assignment from the fragment ions provided a sequence coverage ratio of 90 % for the antibody heavy chain and 96 % for the light chain. To investigate the glycosylated peptides for each sample, extraction of peaks was conducted with the fragment ion of N-acetylneuraminic acid (Neu5Ac) (m/z = 274.0926) (Figure 4). In the presence of EndoS D233Q, the Neu5Ac fragment ion was predominantly detected in the peptide EEQYNSTYR, which contains Asn297. This result showed that the glycan was selectively incorporated onto Asn297 in the presence of EndoS D233Q. In contrast, multiple peaks with Neu5Ac fragment ion were observed in the antibody reacted without EndoS D233Q, suggesting that glycan oxazoline 1 can nonspecifically react with many sites. The peptides modified with glycan are listed in Supporting Information Figure S4. All the glycan-modified peptides were found to have trypsin-miscleaved site(s), except for two cases: (i) peptides derived from the N-terminals of heavy and light chains having N-terminal amino group and (ii) the presence of Lys-Pro sequence in the peptide, which cannot be cleaved by trypsin. Taken together, it can be speculated that non-specific reaction mostly occurs at Lys amino group and the modification inhibits trypsin cleavage. This is consistent with previous studies reporting the reaction of oxazoline with primary amines.30,31 Then, assignment of MS elevated-energy (MSE) fragment peaks was conducted to confirm


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the presence of glycan. For the peptide containing Asn297 (EEQYNSTYR) derived from the IgG sample treated with glycan oxazoline 1 and EndoS D233Q, a strong peak (m/z = 1392.59) corresponding to the peptide modified with single GlcNAc (Y1 ion) was detected33 (Supporting Information Figure S5). This stable Y1 ion is often observed in MS/MS analysis of N-glycosylated peptide.34-36 From this observation, it is likely that glycan oxazoline 1 was successfully introduced onto GlcNAc residue of Asn297 to form the conventional N-glycan. For nonspecifically glycosylated peptide (FSGSGSGTDFTLKISR) derived from the IgG sample treated with excess oxazoline 1 alone, the corresponding Y1 ion was not detected (Supporting Information Figure S6). This is possibly due to the formation of a relatively unstable linkage between Lys and glycan. The Lys-glycan linkage was cleaved preferentially than the GlcNAc-Asn amide linkage in MSE analysis. Owing to the unstableness of the glycan-peptide linkage, it was impossible to identify the non-specific glycosylation sites solely from the mass spectrometric analysis.



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Figure 4. LC/MSE analysis of glycosylated peptides derived from mogamulizumab. Extracted ion chromatogram (m/z = 274.0926) of peptide mixtures in LC/MSE analysis derived

from

mogamulizumab.

a)

untreated

mogamulizumab,

b)

endoS-treated

mogamulizumab, c) enzymatically glycan-transferred mogamulizumab under optimized conditions, d) non-enzymatically glycan-transferred mogamulizumab using excess glycan oxazoline. Diagnostic NeuAc peak (m/z = 274.0926) was used for identification of glycosylated peptides.

Next, to estimate the non-specific modification ratio in the absence of EndoS mutant, another peptide mapping experiment was conducted using Asp-N. We assumed that non-specific glycan modification at Lys will not prevent the action of Asp-N, which cleaves the N-terminal peptide bond of aspartic acid residue. In this experiment, the sequence coverage ratio was 82 % for the antibody heavy chain and 99 % for the light chain. The non-specific modification ratio of glycan oxazoline was calculated by comparing the peak 16 ACS Paragon Plus Environment

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intensities of glycosylated and non-glycosylated ion pairs (Supporting Information Figure S7). Overall, the modification ratio was low (2 % in average). This result is consistent with the ESI-MS analysis of whole IgG (Figure 5, bottom), which indicated an average number of 2 glycans with 88 Lys residues and 4 N-terminals in a whole antibody. We could not exclude the possibility of other modifications, but this result suggests that non-specific modification is almost limited to Lys and N-terminal amino groups.

Glycan-linked homogeneous ADC preparation Using the above optimized procedures, we planned to develop glycan-based homogeneous ADCs (Figure 1). After heterogeneous Fc glycans were cleaved by EndoS, homogeneous glycan carrying bio-orthogonal function group was added by EndoS D233Q. This step uniquely introduces the homogeneous glycan carrying bio-orthogonal function group only at Asn297 residue. Bio-orthogonal reaction between sugar-functional group and payload gives a homogeneous ADC including glycan structure. The frequently used cytotoxic agent monomethyl auristatin E (MMAE) and cathepsin-cleavable Val-Cit (citrulline) linker with p-aminobenzylcarbamate group 3 were chosen as the components of ADC. After cleavage of C-terminus of Cit by cathepsin,



self-immolative 1,6-elimination reaction released MMAE.4 Polyethylene glycol (PEG) was introduced to enhance pharmacodynamics37. Strained alkyne was used for bio-orthogonal reaction with azide group. Glycan oxazoline 241 was prepared and introduced into EndoS-treated trastuzumab in the presence of EndoS D233Q. MMAE with alkyne conjugated linker was added to the antibody solution to give the desired ADC. These reactions were monitored by UPLC and gave single peaks after each reaction (Figure 5a). In comparison, the traditional conjugation method between maleimide and sulfhydryl group gave multiple peaks in UPLC analyses (Supporting Information Figure S8). MS analysis of IgG samples with dithiothreitol treatment showed homogeneity of products (Figure 5b). The homogeneity of product ADC was also confirmed by ESI-MS analysis at intact protein level (Figure 5d) and by LC/MS analysis (Supporting Information Figure S9). It was confirmed that four MMAE molecules were attached onto single IgG and the side-reactions were negligible.


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Figure 5. a) UPLC profiles of each reaction, b) Deconvoluted ESI-MS spectra of dithiothreitol treated products, c) Deconvoluted ESI-MS spectrum of trastuzumab modified with azide-containing glycans, d) Deconvoluted ESI-MS spectrum of ADC comprising MMAE with linker via glycan. 19 ACS Paragon Plus Environment


Confirmation of antigen-binding capability of prepared ADC To confirm that the conjugation of payload to Fc N-glycan does not affect antigen binding, we assessed the binding of antibody and ADC to HER2-expressing cells using flow cytometry (FCM). Breast cancer cell lines (MCF-7 and SK-BR-3 cells) and gastric cancer cell lines (MKN-45, N-87, and OE-19 cells) were used in this experiment. As shown in Figure 6, peak shift was comparably observed for both trastuzumab and ADC in all HER2-expressing cell lines regardless of the amount of HER2 expressed on the cells. These results showed that conjugation of payload to Fc N-glycan does not affect antigen binding.

Figure 6. Antigen-binding activity of trastuzumab and MMAE-conjugated trastuzumab ADC by flow cytometry. 20 ACS Paragon Plus Environment

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In vitro cytotoxicity of prepared ADC To assess whether HER2 expression level on the cells is positively correlated with the cytocidal effect of ADC, we tested the in vitro cytotoxicity of trastuzumab, MMAE, and ADC (MMAE-conjugated trastuzumab) with low and high HER2-expressing cells (Figure 7). The low HER2-expressing breast cancer cell line MCF-7 and gastric cancer cell line MKN-45 were insensitive to antibody or ADC. Weak cytocidal effect by trastuzumab was observed in high HER2-expressing breast cancer SK-BR-3 or gastric cancer OE-19 and N87 cells owing to the direct cytocidal effect of the monoclonal antibody. The dose-response curve did not reach IC50 under the assay condition tested (up to 20 µg/mL IgG). In contrast, exposure of high HER2-expressing cells to ADC caused a dose-dependent decrease in cell proliferation, and the IC50 values were calculated as 0.3, 0.9, and 1.8 nM (MMAE equivalent) against SK-BR-3, OE-19, and N87 cells, respectively. These results suggest that the cytotoxicity of ADC is dependent on the expression level of HER2 in cells.




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Figure 7. Cytotoxic assay of MMAE, trastuzumab, and MMAE-conjugated trastuzumab ADC in vitro using gastric cancer and breast cancer cell lines. Estimated IC50 (nM) from the dose-response curve is indicated (n=4). N.D.: IC50 could not be determined. Concentration of trastuzumab was indicated to match the protein concentration of corresponding ADC.

The ADC showed significant toxicity against high HER2-expressing cells such as N-87, OE-19, and SK-BR-3 cells. The IC50 of ADC against SK-BR-3 cells was 0.3 nM in vitro, which was the same as that of MMAE. However, the ADC did not show toxicity against low HER2-expressing cells such as MKN-45 and MCF-7 cells. These results showed selective delivery of cytotoxic agents to high HER2-expressing cells.

CONCLUSION In this study, we performed real-time monitoring of glycan remodeling and optimization of glycan transfer reaction. The products of non-enzymatic reaction as well as enzymatic reaction were precisely characterized. We found that stepwise addition of glycan oxazoline at slightly acidic conditions is effective in maximizing the reaction efficiency with reduced side-reactions. Homogeneity of the prepared antibody was more satisfactory than that reported previously.23 This could be because the stepwise addition of oxazoline is effective for maintaining the concentration of active oxazoline. Based on optimization



conditions, homogeneous ADC via glycan with DAR 4 was prepared. The prepared ADC was fully characterized and showed potent cytotoxicity to the cell line expressing high-level of antigens. The limitations of this study is the unstableness of glycan oxazoline under acidic conditions, and relatively large amount of EndoS is needed. To compensate the degradation of glycan oxazoline, stepwise addition of glycan oxazoline is required. One possible solution is to develop a more active enzyme than EndoS mutant. Recently, more reactive EndoS2 mutants, EndoS2 D184M and D184Q, have been reported for glycan-remodeling of antibody.27 Shorter reaction time with less amount of glycan oxazoline will consequently reduce side-reactions. Another solution is to use a chemically stable sialylglycopeptide as a glycan donor.38-40 Since EndoS is specific for Fc N-glycan, our approach for preparation of homogeneous ADC will be applied to some IgGs with Fab glycans.41 Progress in this field is expected to improve the method of homogeneous ADC preparation at Fc N-glycosylation site. We proposed a method to prepare a homogenous ADC via Fc N-glycan that will be effective for cancer therapy. This methodology can be expanded to prepare a variety of ADCs. Other anti-cancer drugs can be conjugated via bio-orthogonal reaction and the linker can be


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modified to enhance the efficacy of ADC in vivo.

EXPERIMENTAL PROCEDURES General All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and TCI (Chuo-ku, Tokyo, Japan), and used without further purification. Solvents were purchased from Kanto Chemicals (Chuo-ku, Tokyo, Japan). Analytical thin-layer chromatography was performed on silica gel 60 F254 plates (Merck-Millipore, Burlington, MA, USA) and visualized by UV fluorescence quenching and 12 Molybdo(VI) phosphoric acid acid/phosphoric acid/sulfuric acid staining. Flash column chromatography was performed on silica gel 60N (spherical, neutral, 40–100 µm, Kanto Co. Chuo-ku, Tokyo, Japan). Yields reported here are isolated yields. H and C NMR spectra were recorded on a JEOL EX 400 spectrometer (400 and 100 1

13

MHz, respectively) at ambient temperatures (23–24 °C) in CDCl . Chemical shifts (d) are 3

reported in ppm relative to internal trimethylsilane (d = 0.00 ppm) in CDCl for H NMR 1

3

spectra. CDCl (d = 77.00 ppm) was used as an internal standard for C NMR spectra. HRMS 13

3

was measured by quadrupole-TOF MS. Trastuzumab (Herceptin®) and mogamulizumab (Poteligeo®) were available from Chugai Pharmaceutical Co. Ltd. (Chuo-ku, Tokyo, Japan)



and Kyowa Hakko Kirin Co. Ltd. (Chiyoda-ku, Tokyo, Japan), respectively. ACQUITY UPLC H-class (Waters Co., Milford, MA, USA) was used for UPLC analyses.

Preparation of recombinant EndoS and its mutants Codon-optimized synthetic gene encoding Streptococcus pyogenes EndoS (E37-K995) was purchased from Genscript and incorporated into the pCold vector (TaKaRa, Kusatsu, Japan) including a tobacco etch virus (TEV) protease cleavage sequence between the hexahistidine tag and EndoS. The expression plasmid was transformed into an Escherichia coli strain SHuffle (New England Biolab, Ipswitch, MA, USA). The transformed cells were cultured in 1 L of Luria-Bertani medium at 37 oC, and the expression was induced for 16 h with 0.5 mM isopropyl β-D-thiogalactoside at 15 oC. The cells were harvested by centrifugation of cell suspension (10,000 ×g for 10 min at 4 °C) in phosphate-buffered saline (PBS; 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) containing 0.3 % (v/v) Bugbuster (Novagen, Madison, WI, USA) and then sonicated. After centrifugation, the supernatant was applied onto a nickel-nitrilotriacetic acid (Ni-NTA) column and eluted with PBS containing 500 mM imidazole. The eluted protein was dialyzed overnight at 4 oC into PBS and concentrated using Amicon Ultra (molecular weight cut off: 10,000,


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Merck-Millipore, Burlington, MA, USA). The EndoS mutants D223Q, D223A, and D223H were prepared in the same manner as wild type. Deglycosylation of IgG with EndoS IgG antibody (20 mg) (mogamulizumab or trastuzumab, 4 mg/mL in 50 mM sodium phosphate buffer, pH 7.4) was incubated with 30 μg of EndoS for 20 h. Deglycosylation was monitored by an ACQUITY UPLC H-class equipped with a hydrophilic interaction chromatographic (HILIC) column (ACQUITY UPLC Glycoprotein BEH Amide Column, 300 Å, 1.7 µm, 2.1 x 150 mm, Waters Co., Milford, MA, USA). The column temperature was set to 80 oC and the flow rate was 0.4 mL/min. The antibody peaks were detected using intrinsic fluorescence of tryptophan residues (excitation wavelength: 280 nm, fluorescence wavelength: 320 nm). The antibody was eluted using a gradient of mobile phases A and B (A: 0.1

%

TFA/0.3

%

hexafluoro-2-propanol/H2O,

B:

0.1

%

TFA/0.3

%

hexafluoro-2-propanol/acetonitrile). After completion of the reaction, the antibody was purified from the reaction mixture using Protein A Sepharose CL-4B (GE healthcare Japan, Hino, Tokyo, Japan). Preparation of azide-carrying oligosaccharide oxazoline 2 Azide-carrying oligosaccharide oxazoline 1 was prepared according to a previous report.42



Azide-containing amine was added to carboxylic acid of sialic acid by PyBOP (1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate) in DMF. Then, Shoda's protocol was employed for oxazoline preparation.43 Each product was purified with reverse-phase HPLC (Mightysil RP-18 GP, 20 ´ 250 mm, KANTO CHEMICAL Co., Chuo-ku, Tokyo, Japan) Glycosylation of IgG with EndoS mutant and oxazoline EndoS-treated IgG dissolved in 50 mM sodium phosphate buffer (pH 7.5 or 6.5) was incubated with EndoS mutant (D223Q, D223A, or D223H) in the presence of glycan oxazoline 1. To examine the pH dependency of the reaction (shown in Figures 2 and 3), EndoS-treated IgG (33 μM) was treated with 4.5 μM of EndoS mutant with 5 mM (fixed concentration) glycan oxazoline 1 or 2. The reaction was monitored with the UPLC system indicated above. The reaction conditions were then optimized as shown in Table 1. The best result was obtained under condition No. 6 in Table 1: EndoS-treated IgG (17 μM) was treated with 7.5 μM of EndoS mutant with 0.75 mM (initial concentration) of glycan oxazoline 1 or 2. During the reaction, glycan oxazoline 1 or 2 (0.75 mM) was repeatedly added every 15 min to maintain glycan oxazoline in the reaction mixture at pH 6.5. After completion of the reaction (150 min), the glycosylated IgG was purified using protein A column.


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Preparation of payload The preparation scheme is presented in Supporting Information Figure S10. To a solution of 9-fluorenylmethyloxycarbonyl Fmoc-Val-Cit-p-aminobenzyl carbamate (PABC)-MMAE (0.34 g)44 in anhydrous DMF (4.7 mL), Et2NH (1.2 mL) was added. After 1 h, the mixture was reduced in vacuo, and the residue was purified by LH20 (GE Healthcare, Hino, Tokyo, Japan, CHCl3:MeOH 1:1). To a solution of the resulting amine (0.24 g) and Fmoc-PEG12-CO2H (IRIS Biotech., Marktredwitz, Germany) dissolved in anhydrous CH2Cl2 (10 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (67 mg, 0.351 mmol), 1-hydroxybenzotriazole (47 mg, 0.351 mmol), and i-Pr2NEt (0.13 mL, 0.702 mmol) were added at 4 °C. After stirring at room temperature overnight, the mixture was purified by LH 20 (GE Healthcare, Hino, Tokyo, Japan, CHCl3:MeOH, 1:1) to give 0.31 g of Fmoc-PEG12-Val-Cit-PABC-MMAE. To a solution of Fmoc-PEG12-Val-Cit-PABC-MMAE (0.31 g, 0.160 mmol) in DMF (1.5 mL), Et2NH (0.3 mL) was added. After 1 h, the mixture was concentrated. The residue was purified by LH20 (CHCl3:MeOH, 1:1) to give 0.27 g of H-PEG12-Val-Cit-PABC-MMAE. To a solution of H-PEG12-Val-Cit-PABC-MMAE (0.27 g) and 11,12-didehydro-γ-oxo dibenz[b,f]azocine-5(6H)-butanoic acid (DBCO-acid)45 (58 mg, 0.234 mmol) in CH2Cl2 (10 mL), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide



hydrochloride (46 mg, 0.240 mmol), HOBt (32 mg, 0.240 mmol), and i-Pr2NEt (83 µL, 0.480 mmol) were added at 4 °C. After stirring at room temperature overnight, the mixture was purified with LH 20 (GE Healthcare, Hino, Tokyo, Japan, CHCl3:MeOH, 1:1) to give the product (0.29 g). Preparation of ADC Coupling of azide-oxazoline glycan 2 to EndoS-treated IgG was performed in the same manner as that using oxazoline glycan 1 and EndoS-treated IgG. The glycosylated IgG with azide group was purified using protein A column. The purified IgG (1.78 mg/mL) was dissolved in 50 mM sodium phosphate buffer (pH 8.0), and 20 equivalents of payload was added to a final concentration of 30 % (v/v) dimethyl sulfoxide (DMSO). The solution was incubated at 25 oC for 16 h and the reaction mixture was diluted 3-fold with water. Then, IgG was purified from the reactant using protein A column. Intact mass measurement Intact mass measurement was performed using Xevo G2-XS QTof or SYNAPT G2-Si HDMS connected with the ACQUITY UPLC I-class FTN system (Waters Co., Milford, MA, USA). MassPREP Micro Desalting Column (2.1×5 mm) (Waters Co., Milford, MA, USA ) was used with gradient elution of two mobile phases (A: 0.1 % (v/v) formic acid in water, B: 0.1 %


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(v/v) formic acid in acetonitrile). The column temperature was set to 80 oC. Electrospray ionization (ESI)-positive mode was applied. Data analysis was conducted using MassLynx (ver. 4.1) and UNIFI 1.9.0 software (Waters Co., Milford, MA, USA). ESI-MS analysis of the antibody samples treated with DTT was performed using a QSTAR ELITE quadrupole-time-of flight mass spectrometer (AB Sciex) equipped with a Nanospray Tip (Humanix, Hiroshima, Japan) as described previously.38 Briefly, the antibody dissolved in 50 mM sodium phosphate buffer (pH 7.4) was treated with 10 mM dithiothreitol for 15 min at 37°C. Then the sample was desalted and directly transferred to the mass spectrometer. Mass spectra were deconvoluted using Analyst QS software (AB Sciex). Peptide mapping The IgG samples (mogamulizumab, trastuzumab, and their derivatives) were dissolved in 50 mM Tris (tris(hydroxymethyl)aminomethane)–HCl (pH 8.5), 30 mM dithiothreitol, and 10 mM ethylenediaminetetraacetic acid (EDTA) at 37 °C for 2 h and then alkylated with 3 mM 2-iodoacetamide. The reactant was dialyzed against 50 mM ammonium bicarbonate. Trypsin/LysC mix mass spec grade solution (Promega, Madison, WI, USA) or endoproteinase Asp-N (FUJIFILM Wako Pure Chemical Co. Chuo-ku, Osaka, Japan) was added to samples at a weight ratio of 1:25 and incubated at 37 °C for 16 h. The resulting



peptides were analyzed by using ACQUITY UPLC system (Waters Co., Milford, MA, USA) coupled with Xevo G2-XS QTof or SYNAPT G2-Si HDMS mass spectrometer (Waters Co., Milford, MA, USA). The peptides were separated by ACQUITY UPLC Peptide BEH300 column (C18, 1.7 µm, 2.1 × 150 mm; Waters Co., Milford, MA, USA) using the following gradient: A: 0.1 % (v/v) formic acid in water, B: 0.1 % (v/v) formic acid in acetonitrile at a flow rate of 0.25 mL/min. The column temperature was set to 60 oC. The MSE data acquisition mode was performed, and data were analyzed using MassLynx (ver. 4.1) (Waters Co., Milford, MA, USA) and UNIFI (ver. 1.9.0) software (Waters Co., Milford, MA, USA). Flow cytometry Binding of trastuzumab and ADC to low or high HER2-expressing cell lines was evaluated by flow cytometry. The cells were dissociated for 30 min at 37 °C in PBS containing 30 mM trisodium citrate dihydrate and 270 mM potassium chloride. Then, the cells were blocked with PBS containing 2 mM EDTA and 0.5 % bovine serum albumin. Trastuzumab and ADC were added at a concentration of 10 µg/mL, and the cells were incubated for 1 h at 4 °C, followed by incubation with 10 µg/mL of goat anti-human Alexa Fluor® 647 (Thermo Fischer, Waltham, MA, USA) for 1 h at 4 °C. The specimens were analyzed using Guava easyCyte™ (Millipore, Burlington, MA, USA). Data analysis was performed with FlowJo 7.6.5


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(FLOWJO, USA). Cell culture SK-BR-3, MCF-7, and NCI-N87 (N87) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). OE-19 cells were obtained from the European Collection of Cell Culture (ECACC, Salisbury, UK). MKN-45 cells were obtained from the Japanese Collection of Research Bioresources (JCRB, Ibaraki, Osaka, Japan). OE-19 and NCI-N87 cells were maintained in Roswell Park Memorial Institute Medium (RPMI) 1640. MKN-45 and MCF-7 cells were maintained in Eagle's minimal essential medium (EMEM). SK-BR-3 was maintained in McCoy’s 5A medium. All media were supplemented with 10 % fetal bovine serum (FBS) and 1 % antibiotics, and the cells were cultured at 37 °C under 5 % CO2. In vitro cytotoxicity assay The cells were seeded in 96-well plates (Corning, Corning, NY, USA) at a density of 5,000 cells/well in growth media containing 10 % FBS and precultured for 24 h. Then, the cells were treated with MMAE, trastuzumab, or ADC at the indicated concentrations and cultured for 72 h. After incubation, cell viability was assessed with Cell Counting Kit–8 (Dojindo Molecular Technologies, Kumamoto, Japan). Relative cell viability was calculated by



dividing the absorbance of each well by the mean absorbance of growth media-treated wells in each plate.

ASSOCIATED CONTENT Supporting Information Supporting information is available free of charge [UPLC spectra, Base peak ion chromatogram of the peptides, LC/MSE analysis of the glycosylated peptides (PDF)]. AUTHOR INFORMATION Corresponding authors *E-mail: [email protected], [email protected] Phone: +81-48-467-9432. Fax: +81-48-462-4680. ORCID Shino Manabe: 0000-0002-2763-1414 Yoshiki Yamaguchi: 0000-0003-0100-5439 Ai Mitani: 0000-0001-8264-4974 Wataru Sumiyoshi: 0000-0002-2855-4529


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Takashi Kinoshita: 0000-0002-3468-002X Junpei Abe: 0000-0003-2108-2570 Masahiro Yasunaga: 0000-0003-3356-0197 Yasuhiro Matsumura: 0000-0003-4331-8177 Yukishige Ito: 0000-0001-6251-7249

Author Contributions S. M. and Y. Y. conducted the research. S. M. provided the original concept for the research and prepared payload. Y. Y. and K. M. developed glycan remodelling reaction and analysis. W. S., A. M. T. K., and J. A. prepared azide-functionalized oligosaccharide oxazoline. J. A. prepared ADC for cytotoxic assay. T. K. and K. H. performed characterization of glycan-remodelled antibody and ADCs. H. F., M. Y., and M. Y. measured cytotoxicity of ADC. Y. I. joined the discussion. S. M., Y. Y., and K. H. discussed the results and wrote the paper.

Disclosure of interest Y. Y., S. M., K. M., H. F., J. A., M. Y., Y. I., and Y. M. declare that they have no competing



Page 36 of 52

interests. T.K. and K.H. performed this work as paid employees of Nihon Waters KK. A. M., W. S., and T. K. performed this work as paid employees of Fushimi Pharmaceutical Co. Ltd.

Acknowledgments We would like to thank Ms. Akemi Takahashi for her technical assistance. We would also like to thank Dr. Kaori Otsuki, Dr. Masaya Usui, and Dr. Aya Abe of the Research Resource Center at the Brain Science Center, RIKEN for MS measurements. Y. M. received support from National Cancer Center Research and Development Fund (26-A-14 for Y.M.). S. M. and Y. Y. received support on engineering network program from RIKEN.

Abbreviations ADC, antibody-drug conjugate; ADCC, antibody-dependent cellular cytotoxicity; DAR, drug-to-antibody

ratio;

DMF,

endo-β-N-acetylglucosaminidase;

N,N-dimethylformamide;

FCM,

flow

ENGase,

cytometry;

Fmoc,

9-fluorenylmethyloxycarbonyl; HILIC, hydrophilic interaction chromatography; HOBt, 1-hydroxybenzotriazole; LC/MS/MS, liquid chromatography tandem mass spectrometry; MMAE,

monomethyl

auristatin

E;

Neu5Ac,

N-acetylneuraminic


acid;

PABC,

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p-aminobenzyl carbamate; PBS; phosphate-buffered saline, UPLC, Ultra-performance liquid chromatography.

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b-N-acetylglucosaminidation


of

Page 41 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


disialo-oligosaccharide. Helv. Chim. Acta. 95, 1928-1936. (44)

Koga, Y., Manabe, S., Aihara, Y., Sato, R., Tsumura, R., Iwafuji, H., Furuya, F.,

Fuchigami, F., Fujiwara, Y., Hisada, Y. et al. (2015) Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts. Int. J. Cancer 137, 1457-1466. (45)

Chadwick, R. C., Van Gyzen, S., Liogier, S., Adronov, A. (2014) Scalable synthesis

of strained cyclooctyne derivatives. Synthesis, 46, 669-677.


Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 42 of 52

Figure 1. R R Glycan oxazoline 1, 2 ENGase

ENGase mutant

R R

R R

trastuzumab (with fucose) mogamulizumab (without fucose) O N

O N H

O

PEG12

N H

N H

O

O O

NH H 2N

HO

OH O

HO AcHN

O

N Me

H N O

N Me

H N

N OMe O

OH

OMe O

3

O HO O

OHO OH HO

HO

OH O

HO AcHN

O

NHAc O HO HO HO

HO

O O O HO O

R O

HO

HO

O HO O OH HO

HO HO HO O

OH O

O HO

ADC (DAR = 4)

OH O N

O O

O

=

N N

N

O

O N O

N H

PEG12

HN

N H

H N

O N H

O

NHAc H 2N

O

O

O

NH

1 R = OH 2 R=

O

R

O HO

HO

Bio-orthogonal reaction

O

H N

O

N3

ACS Paragon Plus Environment

O

O

N Me

H N O

O N Me

H N

N OMe O

OMe O

OH

Page 43 of 52

Figure 2a) a)

+EndoS D233Q, pH 7.5

+EndoS D233H, pH 7.5

+EndoS D233A, pH 7.5

No enzyme

Not analyzed Relative fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


15 min

1h

3h

Retention time (min)



Figure 2b)

b)

+EndoS D233Q, pH 6.5 15 min

Relative fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 44 of 52

No enzyme, pH 6.5

Not analyzed

15 min

1h

1h

3h

3h

6h

time (min) ACS Retention Paragon Plus Environment

6h

Page 45 of 52

Figure 2c) c)

Relative fluorescence intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


+EndoS D233Q, pH 6.5 Oxazoline addition every 15 min

No enzyme, pH 6.5 Oxazoline addition every 15 min

45 min

Not analyzed

45 min

90 min

Not analyzed

90 min

150 min

Retention time (min)


150 min


Figure 3. a) EndoS-treated mogamulizumab

b) mogamulizumab + EndoS D233Q and glycan oxazoline 1 under optimized conditions

c) mogamulizumab + excess glycan oxazoline 1 without enzyme

Mass (Da)


Page 46 of 52

Page 47 of 52

Figure 4. a) mogamulizumab

b) GlcNAc-mogamulizumab (EndoS-treated)

Intensity (count)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


c) GlcNAc-mogamulizumab + excess oxazoline glycan without enzyme

d) GlcNAc-mogamulizumab + oxazoline glycan + EndoS D233Q under optimized conditions EEQYNSTYR +GlcNAc+Disialo-glycan

Retention time (min) Extracted ion chromatogram (m/z = 274.0926) ACS Paragon Plus Environment


Page 48 of 52

Figure 5a), b). a)

b) light chain

Trastuzumab EndoS heavy chain

EndoS D233Q

N3 N3 EndoS D233Q

N3 N3

N3 N3


Page 49 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Figure 6.

Gastric cancer

Breast cancer MCF-7

MKN-45

SK-BR-3

N-87

OE-19

Count

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 50 of 52

HER2 ACS Paragon Plus Environment

― 2ndary only ― Trastuzumab ― Trastuzumab-ADC

Page 51 of 52

Gastric cancer cell line

1.5

Figure 7.

MKN-45 [HER2 low expression]

Relative viability to 0 nM

1.0

― MMAE ― Trastuzumab ― Trastuzumab-ADC MKN-45 IC 50 (nM) : MMAE: 0.3 Trastuzumab:Trastuzumab-ADC: -

0.5

0.0 0 1.5

1

10

100

N-87 [HER2 high expression]

1.0 N-87 IC 50 (nM) : MMAE: 0.4 Trastuzumab:Trastuzumab-ADC: 1.8

0.5

0.0 0 1.5

1

10

100

MKN-45 [HER2 low expression]


1.0

0.5

0.0 0 1.5

1

10

100

OE-19 [HER2 high expression]

OE-19 IC 50 (nM) : MMAE: 0.5 Trastuzumab:Trastuzumab-ADC: 0.9

1

100

1.0

0.5

0.0 0

10

Breast cancer cell line

1.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MCF-7 [HER2 low expression]

1.0

MCF-7 IC 50 (nM) : MMAE: 2.2 Trastuzumab:Trastuzumab-ADC: -

0.5

0.0 0 1.5

1

10

100

SK-BR-3 [HER2 high expression]

1.0 SK-BR-3 IC 50 (nM) : MMAE: 0.3 Trastuzumab:Trastuzumab-ADC: 0.3

0.5

0.0 0

1

10

MMAE (nM eq.) ACS Paragon Plus Environment

100 All groups :n=4


Page 52 of 52

Table of contents

ENGase Removal of heterogeneity and immunogenicity R R ENGase mutant

IgG

Bio-orthogonal reaction R

R

Homogeneous ADC (DAR = 4)


Reaction monitoring and optimization

R R

Characterization of antibody products obtained through enzymatic and

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