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Facile modulation of antibody fucosylation with small molecule fucostatin inhibitors and co-crystal structure with GDP-mannose 4,6-dehydratase John G. Allen, Mirna Mujacic, Michael J Frohn, Alex J. Pickrell, Paul Kodama, Dhanashri Bagal, Tisha San Miguel, E. Allen Sickmier, Steve Osgood, Aleksander Swietlow, Vivian Li, John B. Jordan, Ki Won Kim, Anne-Marie C. Rousseau, Yong-Jae Kim, Seb Caille, Mike Achmatowicz, Oliver Thiel, Christopher H. Fotsch, Pranhitha Reddy, and John D McCarter ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00460 • Publication Date (Web): 19 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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ACS Chemical Biology

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Figure 1. Fucostatins and the ADCC response. Figure 1 38x22mm (600 x 600 DPI)


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Figure 2. 6,6,6-Trifluorofucose (1) is proposed to utilize the fucose salvage pathway to form GDP-6,6,6trifluorofucose (2D) and inhibit GMD in the de novo pathway. Figure 2 143x191mm (300 x 300 DPI)



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Figure 3. Compounds prepared by synthesis or referenced in the text. A indicates the corresponding phosphonate, B the phosphonate analog of the GDP sugar, C the sugar phosphate, D the GDP sugar. Figure 3 69x18mm (300 x 300 DPI)


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Figure 4 43x30mm (600 x 600 DPI)



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Figure 4. a) Dose-response inhibition of cell surface fucosylation by 1 and, b) rescue of cell surface fucosylation by L-fucose and L-fucose per-O-acetate. Addition of 20 µM 1 to cell culture medium results in 14.0 ± 3.3% cell surface fucosylation. The y-axis represents normalized LCA lectin binding, error bars represent the standard deviation. Figure 4 41x28mm (600 x 600 DPI)


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Figure 5. Metabolite data for fucosylation inhibitors. a) Log(AUC) of metabolites observed by SRM-MS in lysate from cells treated with 1, 5, 7 and 8; b) Relative concentrations of GDP-mannose (orange bars) and GDP-fucose (blue bars) observed by SRM-MS in lysate from cells treated with 1, 5, 7 and 8, and normalized to 100 %; c) Chromatographic data for metabolites observed by SRM-MS in cells treated with 1 and arbitrarily normalized to 100% intensity; d) Derived SPR dissociation curves for 2D (red, KD 11 µM), 5B (blue, KD 9 µM), GDP-mannose (green, KD 51 µM), GDP-fucose (black, KD 35 µM). Figure 5 29x14mm (600 x 600 DPI)



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Figure 6. GDP-fucose (PDB 5IN5) and 2D (PDB 5IN4) co-crystallized with GMD. a) Four molecules of 2D bind to the tetramer at the interface of a dimer pair (arrows); b) GDP-fucose (magenta) and 2D (white) overlaid in the GMD binding pocket; c) 2D in the binding pocket, partial space-filling model. Figure 6 39x39mm (600 x 600 DPI)


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Figure 7. Protein production in the presence of fucosylation inhibitors and efficacy. a) 1 was added on day 0 of production at the levels indicated on the x-axis to an anti-TR-2 antibody-expressing CHO cell-line grown under fed-batch production conditions. Glycan analysis was performed on the produced antibodies (circle = percent fucosylated glycans, square = percent afucosylated glycans, triangle = percent high mannose glycans; open circle = percent glycans with incorporated 6,6,6-trifluorofucose); b) Cumulative viable cell density, titer and specific productivity obtained under increasing concentrations of 1 in two different cell lines (each data point represents an average of duplicates); c) The impact of repeated additions of 5 on anti-TR-2 antibody-expressing CHO cell culture performance using medium exchanges initiated on day 3. Compound 5 was added to the cultures daily at the levels indicated on the x-axis; d) Cumulative viable cell density, titer and specific productivity of anti-TR-2 antibody-expressing CHO cell culture obtained in the presence of different 5 concentrations (each data point represents an average of duplicates); e) Anti-MSLN mAb antibodies produced in the presence of varying amounts of 1 and with varying levels of fucosylated glycans (filled circles) were purified and assayed in a calcein-based ADCC assay with cells with either high (N87; black bars) or medium (CAPAN2; gray bars) MSLN expression. N87 cells were incubated with NK cells from a high affinity FcγRIIIa donor, CAPAN2 cells were incubated with NK cells from a low affinity FcγRIIIa donor. EC50 values represent the amounts of anti-MSLN mAb needed to lyse 50% of target cells at indicated anti-MSLN mAb fucosylation levels; f) CHO cells expressing anti-TR-2 mAb were grown under fed-batch production conditions. Compound 1 was added to the cells on day 0 at 20 µM concentration. Production cultures were also dosed with 1 mM fucose on either day 0 or day 6. Glycan profile of the produced anti-TR2 mAb antibodies was measured (black bars = percent fucosylated glycans, gray bars = percent afucosylated glycans, white bars = percent high mannose). Each bar represents and average of duplicates; g) HILIC Asn297 glycan analysis of Ab produced in the presence of 250 µM 1 showing 98% afucosylated glycan and 0.7% incorporation. Figure 7 39x11mm (600 x 600 DPI)



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Table 1 19x2mm (300 x 300 DPI)


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Facile modulation of antibody fucosylation with small molecule fucostatin inhibitors and co-crystal structure with GDP-mannose 4,6-dehydratase John G. Allen,a* Mirna Mujacic,b Michael J. Frohn,a Alex J. Pickrell,a Paul Kodama,b Dhanashri Bagal,a Tisha San Miguel,a E. Allen Sickmier,a Steve Osgood,c Aleksander Swietlow,c Vivian Li,a John B. Jordan,a Ki-Won Kim,d Anne-Marie C. Rousseau,e Yong-Jae Kim,a Seb Caille,f Mike Achmatowicz,f Oliver Thiel,f Christopher H. Fotsch,a Pranhitha Reddy,b and John D. McCartera a

Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320

b

Process Development – Drug Substance Technologies, Amgen Inc., 1201 Amgen Court W., Seattle, WA,

98119 c

Process Development – Attribute Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA

91320 d

e

Cardiometabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320

Therapeutic Innovations Unit, Amgen Inc., 1201 Amgen Court W., Seattle, WA, 98119

f

Process Development – Drug Substance Technologies, Amgen Inc., One Amgen Center Drive, Thousand

Oaks, CA 91320 ABSTRACT The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumor, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-Oacetate and 6,6,6-trifluorofucose (fucostatin I) were identified that reduced the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray co-crystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (< 1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent—a key advantage in the production of therapeutic antibodies especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

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Therapeutic IgG1 antibodies bearing Asn297 glycans with reduced levels of fucosylation or with no fucosylation have been shown to exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) due to increased binding of the Fc region to FcγRIIIa of effector cells (Fig. 1). Increased ADCC has been associated with increased in vivo efficacy in animal models and in the clinic.1,2 Fucosylation levels are therefore an important product attribute to control in the production of therapeutic antibodies.3 Antibody fucosylation may be modulated to an extent by changing cellular growth conditions, by selection of clones that express antibodies with lower fucosylation levels, by use of engineered cell lines in which key enzymes involved in protein fucosylation are knocked out, or by inclusion of chemical inhibitors of fucosylation.4,5 Some glycosidase inhibitors (including castanospermine,6 swainsonine7 and Nbutyldeoxynojirimycin8) can result in significant changes in protein glycans but their effects are multiple and extensive. The α-mannosidase inhibitor kifunensine can reduce antibody fucosylation more specifically in cell culture but also increases high mannose glycans, an undesirable side effect since this often results in faster clearance of antibodies in vivo.9 More potent and specific inhibitors of fucosylation are desirable. Selective chemical inhibition of fucosylation has the potential advantages of impacting only fucosylation without altering other glycan, protein, or cell growth attributes, allowing fucosylation levels to be varied depending on how much inhibitor is added, and offering the convenience of working with existing cell lines without further genetic modification. Some metabolic inhibitors of fucosylation in cells10,11 and other metabolic glycosylation inhibitors12,13,14 have been described, however, reported potencies are typically in the micromolar range. In addition to weak potency, many glycosylation inhibitors carry the risk of or are actually intended to undergo incorporation into protein glycans which is undesirable for a therapeutic antibody,15,16,17 and especially for biosimilar antibodies where the molecule is intended to substantially match the attributes of the original product. Despite weak potency and significant incorporation (ranging from < 1% to almost 100%), fucosylation inhibitors have themselves shown potential as therapeutics. For example, 2-deoxy-2-fluoro-L-fucose (2FF) or 2-deoxy-D-galactose18 have demonstrated therapeutic efficacy in animal models of sickle cell disease,19 tumor progression,11 and arthritis.18 6,6,6-Trifluorofucose per-O-acetate (1) and 6,6,6-trifluorofucose (fucostatin I, 2) were initially designed as possible mechanism-based metabolic inhibitors of fucosyltransferases.20 It was later determined that the probable target of inhibition is GDP-mannose 4,6-dehydratase (GMD), binding to which was demonstrated by surface plasmon resonance (SPR) and X-ray crystallography. Inhibition of fucosylation in cell culture by 1 was the most potent of any reported small molecule inhibitor of fucosylation. Incorporation of the 6,6,6-trifluorofucose moiety of 1 in recombinantly expressed antibodies occurred at low (< 1%) levels. Such incorporation of a non-native sugar in antibody glycans was completely eliminated through the rational design of the phosphonate analog, fucostatin II (5). In the case of 1, the per-O-acetylated sugar is proposed to diffuse into cells as a prodrug that once deacetylated is then utilized in the fucose salvage pathway to form the C-1 phosphate 2C and corresponding GDP sugar 2D (Fig. 2). While 2D is a poor substrate for fucosyltransferases it is nevertheless incorporated at low levels into fucosylated proteins through the action of these enzymes. In contrast, the phosphonate analog 6B uniquely does not lead to the incorporation of any non-native sugar in antibody glycans, a key advantage for a fucosylation inhibitor to be used in the production of therapeutic antibodies and especially of biosimilars. Herein is described the identification through selective reaction monitoring-mass spectrometry (SRM-MS) of salvage pathway metabolites and de novo pathway metabolic precursors that 2 ACS Paragon Plus Environment

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elucidate the mechanism of action of these cell active inhibitors of fucosylation. Co-crystallization of 2D with GMD, metabolite studies, and rescue of antibody fucosylation by addition of L-fucose during protein production led to the identification of GMD as the likely principal target of these inhibitors. RESULTS AND DISCUSSION Preparation of fucosylation inhibitors. The compounds discussed herein are shown in Fig. 3. The trifluoromethyl group of the fucose-based inhibitors was introduced by alkylation followed by stereoselective acetal or ketone reduction. The phosphonate group was introduced by Wittig or HornerEmmons alkylation followed by stereoselective dehydroxylation or separation of the anomeric mixture. GDP-derivatives were made by chemoenzymatic methods or by alkylation with guanosine 5'monophosphomorpholidate 4-morpholine-N,N'-dicyclohexylcarboxamidine salt. The per-O-acetylated trifluoromethyl sugar 1 is proposed to diffuse into cells as a prodrug that once deacetylated is then utilized in the fucose salvage pathway to form the C-1 phosphate 2C and corresponding GDP sugar 2D (Fig. 2). Deacetylation of 1 results in fucosylation inhibitor 2 (fucostatin I) that displays similar fucosylation inhibition activity to 1 and has the additional advantage of high aqueous solubility. It was surmised that upon addition to cell culture medium, 2 would enter the cell by active transport similar to fucose itself.21 Compound 2 exists as an interconverting mixture of hemiacetals (ratio 59:47:44:37 in MeOH-d4 at room temperature).22 Detailed procedures for the synthesis of compounds used in these studies are provided in Supporting Information or have been reported.22,23,24 Inhibition and rescue of cell surface fucosylation. Compounds were initially assessed for their ability to inhibit protein fucosylation in a FACS assay of cell surface fucosylation using a FITC- Lens culinaris A (LCA) lectin-antibody conjugate (Fig. 4a). Both L-fucose and L-fucose per-O-acetate reduced the inhibition by 1 in a dose-dependent manner presumably by competition of GDP-fucose produced through the salvage pathway with 2D. Significantly, D-fucose, which is not a substrate for fucose transporters nor presumably a substrate for fucose kinase or other enzymes in the salvage pathway failed to rescue inhibition by 1 (Fig. 4b). Characterization of salvage pathway metabolites is consistent with the observed inhibition of cell surface fucosylation. Upon addition to Chinese hamster ovary (CHO) cell culture medium, 1 diffuses into cells, is de-esterified, and is converted by fucose kinase into the C-1 phosphate (2C) and subsequently into the GDP derivative (2D) by GDP-β-L-fucose pyrophosphorylase (GFPP) as both of these metabolites were identified by SRM-MS in cell lysates (Fig. 5a). The GDP derivative 2D was prepared synthetically by chemo-enzymatic methods and was shown to bind GMD by SPR with a KD value of 11 µM. Inhibition of GMD is consistent with the increase in GDP-mannose (Fig. 5b), the decrease of GDP-4-keto-6-deoxy-mannose (data not shown), and the decrease of GDP-fucose in the cell. Addition of 1 to cells expressing proteins led to inhibition of protein fucosylation and to low levels of 6,6,6-trifluorofucose incorporation into glycans of expressed proteins in place of fucose (vide infra). The phosphonate prodrug 5 that was designed not to incorporate into expressed proteins was also tested. 5 was deprotected in the cell and gave rise to both phosphonate (5A) and phosphonate GDP metabolites (5B). 5B was found to bind to immobilized GMD by SPR. Phosphonate prodrug 5 inhibited protein and cell surface fucosylation but importantly did not incorporate into expressed protein. The corresponding non-native α-anomer phosphonate 6 was inactive in the cell surface assay as expected since it is unlikely that deprotected 6 would be a substrate for GFPP in order to produce 6B. 3 ACS Paragon Plus Environment


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The β-trifluoromethylphosphonate 7 formed both phosphonate (7A) and phosphonate GDP metabolites (7B) but failed to inhibit fucosylation, likely due to lack of GMD inhibition as indicated by the absence of GDP-mannose accumulation. Unexpectedly, the corresponding non-physiological α-anomer αtrifluoromethylphosphonate 8, was a relatively potent inhibitor of cell surface and protein fucosylation. Glycans of protein produced in the presence of 8 showed low ( 40 µM there was some impact on cumulative growth, viability and productivity (Fig. 7c and d). As expected, target cell lysis was increased with afucosylated or reduced fucosylated IgG1 molecules (Fig. 7e) particularly in combination with natural killer (NK) cells isolated from a human donor with low affinity FcγRIIIa receptor (V158F allele). Anti-MSLN mAb was produced with decreasing levels of fucosylation by addition of increasing concentrations of 1. The antibodies were purified and used to target NK cells from either high or low affinity FcγRIIIa receptor human donors to N87 (gastric carcinoma) or CAPAN2 (pancreatic adenocarcinoma) cells. N87 cells have a high expression level of the MSLN antigen and were targeted with high affinity NK cells (black bars). CAPAN2 cells have a lower expression level of the MSLN antigen and were targeted with low affinity NK cells (grey bars) representing a more challenging disease model. In the N87/high affinity system, unmodified anti-MSLN mAb cleared the target cells with EC50 ~0.05 µg/mL. In the CAPAN2/low affinity system, similar efficacy was achieved with 80% fucosylated anti-MSLN antibody. The 80% fucosylated antibody resulted from a single 2 µM dose of inhibitor 1 on day 0 of a 10 day production run.

Table 1. Phosphate and phosphonate metabolite data.

FITC-LCA Cmpd IC50 (µM)a

GMD SPR KD (µM)

GDPMetabolites Mannose Identified Accumulation

Fucosylation Inhibition in Glycan

Glycan Incorporation (% of total glycan)b

1

0.87±0.71

11 for 2D

Yes

C, D

Yes

1.1

2

1.8±1.3

11 for 2D

Yes

C, D

Yes

0.6



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3

4.7±1.9

NTc

NT

NT

Yes

11

4

6.6±5.5

NT

NT

NT

Yes

70

5

18.1±6.5

9 for 5B

Yes

A, B

Yes

0

6

>100

NT

NT

NT

NT

NA

7

>100

NT

No

A, B, C

NT

NA

8

7.0±7.0

NT

Yes

A, B, C

Yes

0.4

2FF

>100

NT

NT

NT

NT

0.211

a

FITC-LCA binding to cell surface fucosylated glycan±sd. bIncorporation of inhibitor was dependent on dose and production conditions, the highest level of observed incorporation is reported. cNot tested.

Discussion. 6,6,6-Trifluorofucose per-O-acetate (1) was found to be a potent inhibitor of protein fucosylation as measured by FITC-LCA assay of cell surface glycoproteins (Table 1). 1 also inhibited the fucosylation of glycans of recombinantly expressed antibodies as determined by HILIC analysis. Metabolite identification from the lysate of treated cells showed 1 was metabolized via the fucose salvage pathway as shown by formation of the 1-phosphate metabolite (2C) and the GDP-sugar (2D) metabolite. The increase in intracellular GDP-mannose and decrease in GDP-fucose suggests the principal mode of action is inhibition of GMD, an enzyme positioned between these two precursors of fucosylated protein biosynthesis in the biosynthetic pathway. Indeed 2D bound GMD (KD 11 µM, SPR) and was cocrystallized in the GMD allosteric site.32 The surprisingly potent inhibition of fucosylation by 1 or 2 initially suggested the possibility that 2D might form a covalent adduct with GMD upon oxidation and elimination.33,34,35 There is precedent for a covalent mechanism of inhibition by 6,6,6,-trifluoromethyl glycosides with other dehydratases. However neither the biophysical nor the crystallographic data were consistent with covalent inhibition of GMD by 1 or 2. Prolonged incubation of 2 or 2D with GMD did not result in detectable covalent adduct formation by mass spectrometry (data not shown) nor was electron density in the crystal structure consistent with such a covalent adduct. The high potency of 1 in cells and low dissociation constant of the corresponding GDP derivative 2D was instead rationalized by interactions revealed in the 2D-GMD co-crystal structure. Overlap of bound 2D with bound GDP-fucose was nearly complete with the exception of deeper penetration of the trifluoromethyl group into a pocket lined with alanine and leucine. The resulting additional Van der Waals interactions likely drive the reduced dissociation constant for 2D relative to GDP-fucose (KD 11 vs. 35 µM respectively). The activity of 1 is remarkable compared to related fluorinated analogs. 6,6-Difluoro- (3) and 6-monofluorofucose (4) were less potent in the cell surface assay and exhibited much higher incorporation in protein glycans. 2-Fluorofucose (2FF) is reported11 to inhibit both GMD (~50 µM) and FUT8 (~200 µM) as its GDP derivative and did not inhibit cell surface fucosylation up to 100 µM. These comparisons underscore the importance of the trifluoromethyl group to both potent inhibition of GMD and to minimal incorporation of the inhibitor in protein glycans.


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Fucosylation of recombinantly expressed antibodies was similarly reduced (or abolished at higher concentrations of inhibitor). Antibodies produced in the presence of increasing concentrations of 1 showed decreased levels of fucosylation and correspondingly increased activity in a calcein cell lysis assay (Fig. 7e). Using antibody produced in the presence of 2 µM 1 (~20% afucosylated), high MSLN expressing N87 or medium MSLN expressing CAPAN2 cells were efficiently (EC50 < 0.1 µg/mL) lysed by natural killer (NK) cells from either high or low affinity FcγRIIIa healthy human donors respectively. The combination of medium antigen expressing CAPAN2 cells with low FcγRIIIa affinity NK cells represents a particular challenge for effective target cell killing. Patients with high and low affinity FcγRIIIa receptor alleles show different responses to ADCC therapeutics, with patients harboring low affinity FcγRIIIa receptor responding poorly.36 One advantage of afucosylated therapeutic antibodies is improved responses across broad patient populations.37,38 HILIC analysis of antibodies expressed in the presence of 1 also showed low levels of incorporation of 6,6,6-trifluorofucose in place of fucose (Fig. 7g). No other aspects of cell growth including cumulative viable cell density, antibody titer, and specific productivity in the expression system are affected by treatment with 1. The low levels of incorporation (40 µM 5 due to decreases of cell viability. The non-physiological α-anomeric stereoisomer 6 was inactive in the cell surface assay and was not further evaluated in cells or in antibody production. Although 5 effectively blocked fucosylation, importantly with no detectable incorporation, 5 was approximately ten-fold less potent than 1 in the cell surface fucosylation assay (FITC-LCA IC50 18.1±6.5 µM). The analysis of the GMD-1 co-crystal structure identified additional beneficial contacts formed by the trifluoromethyl group and building the trifluoromethyl group of 1 into the phosphonate inhibitors was considered as a strategy to improve potency. Unfortunately, compound 7, the trifluoromethyl analog of 5, was inactive even though 7 did result in the anticipated GDP metabolite 7B. Lack of inhibition of fucosylation suggests that 7B might be a poor ligand of GMD, the combination of both exo-anomeric methylene and 5-trifluoromethyl perhaps being too dissimilar from native GDP-fucose for effective inhibition of GMD. Alternatively, 7B may be too unstable in cells over the course of several days’ incubation to accumulate a concentration sufficient for effective inhibition of fucosylation. Further mechanistic characterization of 7B could not be completed due to chemical instability. Unexpectedly, the non-physiological α-isomer of 5, compound 8 resulted in relatively potent inhibition of fucosylation in the cell surface assay (FITC-LCA IC50 7.0±7.0 µM) and of the fucosylation of expressed and purified antibodies. An analysis of metabolites from cells treated with 8 found deprotected 8A along with the GDP derivative 8B. However, in addition to the phosphonate metabolite 8A, the phosphate metabolite 2C identical to that formed in cells treated with 1 or 2, was also detected. Similarly, low levels (< 1%) of incorporation of 6,6,6-trifuorofucose into recombinantly expressed antibodies were observed. Rigorous purification and characterization of 8 eliminated the possibility of contamination by 2, and prolonged stirring in water, phosphate buffered saline or cell-free medium did not hydrolyze the phosphonate. One other possible explanation of these results is that the phosphonate group of 8 is hydrolyzed in the cell to the hemiacetal 2. There is some precedent for enzyme-catalyzed C(sp3)-C(sp3) bond cleavage of 2-hydroxyethylphosphonate to hydroxymethylphosphonate25,27 and cleavage of Cglycosides in bacteria,26 although similar processes are not known in mammalian systems. Direct observation or reconstitution of this reaction using for example α-L-fucosidases would provide much needed support of this interesting hypothesis, and would rule out the possibility of an artifact in the assays or analysis. Similar phosphate metabolites derived from apparent phosphonate cleavage were observed to a much lower extent in cells treated with 7. If a hydrolysis mechanism could be considered, the alpha-L anomer 8 might be more prone than the beta-L anomer 7 to eject the axial leaving group in 8 rather than the equatorial leaving group in 7 (due to the anti-periplanar lone pair in the developing oxocarbenium of 8) in such a reaction. Significantly while there are at least 39 α-L-fucosidases there are no known β-Lfucosidases.41 Similar intracellular hydrolysis of the fucose α-isomer 6 was not detectable in these studies since this would lead only to naturally-present fucose, fucose-1-phosphate, and fucose-GDP. Additional study of the apparent possible phosphonate hydrolysis reaction and its dependence on anomeric configuration is needed. Conclusion. 6,6,6-Trifluorofucose (1, fucostatin I) and per-O-acetate prodrugs thereof were found to be potent inhibitors of antibody fucosylation. The molecular mechanism of inhibition of fucosylation was 8 ACS Paragon Plus Environment

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elucidated by the co-crystal structure of GDP-trifluorofucose bound to the allosteric site of GMD, cellular and inhibitor metabolite studies, and L-fucose competition. Fucostatin I resulted in a small amount of incorporation (441.6), GDP-mannose (604.01->441.6), 2C 1-phosphate metabolite (297->78.63), 2D (642.3->361.7), 5A 1-phosphonate metabolite (240.96->136.7) and 5B (586.16->361.7). SPR measurements on GMD. His6 Tagged chinese hamster GMD(12-372) was expressed in BL21 Star e. coli cells in Terrific Broth Complete (4 x 1 L) at 20 °C for 20 h and pelletized by centrifugation and frozen. The cell pellets were thawed and suspended in 10 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, Complete Protease Inhibitor Tablets (Roche) and 25 mM imidazole, then lysed by microfludizer on ice. The lysate was cleared by centrifugation, and the supernatant was captured by batch-binding on Ni-NTA Superflow resin (QIAGEN) at 4 °C for 2 hours. The captured protein was eluted with 10 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, Complete Protease Inhibitor Tablets (Roche) and 250 mM imidazole. The eluted His-TEV-Chinese hamster GMD(∆11) was buffer-exchanged into 10 mM HEPES, pH 7.5, 500 mM NaCl, and then purified by Superdex 200 column (GE Healthcare). SPR binding analysis was performed on a Biacore T200 SPR (GE Healthcare). Briefly, purified hamster GMD was immobilized onto the surface of a CM5 sensor chip using a standard amine coupling protocol. The sensor chip surface was activated with a 6 min injection (10 µL/min) of EDC/NHS. The protein sample was exposed to the activated surface for a period of 10 minutes in 10 mM sodium acetate, pH 5.5 at a concentration of 50 µg mL-1. Lastly, a blocking step was performed with ethanolamine for 5 min. An immobilization level of approximately 15000 RU was obtained. Solutions of 2D, 5B, GDP-mannose, GDP-fucose, 2, fucose-1-phosphate, trifluoromethylfucose-1phosphate (2C), and fucose-1-phosphonate (5A) were injected over the surface using a series of 2x dilutions at top concentrations of 250 µM in 25 mM MOPS, pH 7.0, 100 mM NaCl, 5 mM EDTA, 10 mM DTT, 100 µM NAD+, and 0.005% P20. Sensorgram analysis was performed using Scrubber (BioLogic Software, Campbell, Australia) using steady-state analysis.


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Crystallographic methods. Recombinant human GMD residues 23-372, prepared as Chinese hamster GMD for SPR measurements was used in initial sitting drop vapor diffusion crystallization trials. The protein buffer contained 10 mM Hepes pH 7.5, 500 mM NaCl with added ligands 2D and NADPH at 1mM concentration each. Sitting drops were set up in Axygen 96 well plates against PEG suite, JCSG II, and Morpheus. The drops consisted of 50 nL protein solution at 15 mg mL−1 added to 50 nL precipitant over a reservoir containing 100 uL precipitant solution. Crystal hits were detected in various conditions at 293 K. The best crystals were from PEG suite E2 condition with 0.2 M potassium fluoride and 20% PEG 3350. The crystals were briefly transferred to a cryoprotectant consisting of 0.2 M potassium fluoride, 20% PEG 3350, and 20% glycerol and flash frozen in liquid nitrogen. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Data was processed and scaled using HKL2000.42 The crystals belong to the space group P21 with approximate unit cell dimensions of a =63 Å, b = 140 Å, c = 86 Å, β=106. Molecular replacement was performed with Phaser,43 using a GMDS structure (PDB entry 1T2A) as a search model. The structure was refined using Phenix refine,44 and the model with ligand was built with Coot.45 For crystallization of the natural inhibitor GDP-fucose, the protein buffer contained 15 mg mL-1 protein in 10 mM Hepes pH 7.5, 500 mM NaCl with 10 mM GDP-fucose and 1.0 mM NADPH. It was crystallized by the same procedure using 0.1 M MES pH 5.0, 10% PEG 6K, and a final cryoprotectant containing 0.1 M MES pH 5.0, 10% PEG6K, and 20% glycerol. The data were collected on a Rigaku FR-E SuperBright™ home source and a Saturn 92 detector. Data processing, molecular replacement, refinement and model building followed the same procedure as for 2D. The crystals belong to the space group P21 with approximate unit cell dimensions of a =63 Å, b = 140 Å, c = 86 Å, β=106. Data collection and refinement statistics are available in Supplementary Table 1. Cell-lines, media and production assays. Two Chinese hamster ovary cell-lines producing either antiTR-2 mAb or anti-mesothelin mAb were used for cell culture evaluations. Cells were grown in suspension cultures. Production assays were carried out using in-house developed chemically-defined media. In fed-batch production assays, antibody-producing cells were inoculated at a 1:5 ratio into chemically-defined base production medium in duplicate wells of a deep 24-well plate (Axygen, Union City, CA). 1000x stock solutions of 1 and 5 were prepared in DMSO (Sigma Aldrich, St. Louis, MO) as the solvent. 2 was dissolved in water. Compounds or vehicle controls (i.e. DMSO or water only) were added to the production cultures on day 0 at the levels indicated in Fig. 7. Some cultures also received a 1 mM fucose (Sigma Aldrich, St. Louis, MO) dose on either day 0 or day 6 of the production assay. Production cultures were placed in a shaking incubator with the following set-points: 36 °C, 5% CO2, 225 rpm. Cell density and viability were measured by Guava easyCyte flow cytometer (Miliopore, Billerica, MA) on days 3, 6, 8, and 10. Glucose was also measured on the same days using a 96-well plate-based assay employing a chromogenic reagent (MedTest DX, Canton, MI). Productions were fed with a chemically-defined feed medium and glucose on days 3, 6, and 8. All cultures were harvested on day 10 via centrifugation at 1000 rpm for 10 minutes. Produced antibodies were purified from the spent medium fractions and were subjected to titer and glycan analyses. In a 10-day mock perfusion assay, cells were diluted 1:5 into a chemically-defined base medium in duplicate wells of a deep 24-well plate. Productions were placed into a shaking incubator with the following set-points: 36 °C, 5% CO2, 225 rpm. Mock perfusion was initiated on day 3 when the cells were spun down at 1000 rpm for 5 minutes, and 25% of each spent culture medium was exchanged with the equivalent volume of fresh chemically-defined perfusion medium. Medium exchange percentages 11 ACS Paragon Plus Environment


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increased over the course of the assay and were as follows: 40% on days 4-8 and 50% on day 9. Using a 1000x stock solution, DMSO-dissolved 5 was added to the cells at 0-100 µM doses on a daily basis starting on day 0. The concentration of the inhibitor at any give time-point after day 3 was the sum of the amount of inhibitor present in the leftover spent medium and the daily bolus amount added. Starting on day 3 and continuing on a daily basis until day 10 harvest, viable cell density and viability were analyzed using the easyCyte flow cytometer. Glucose was also monitored on days 3-10 via a plate-based chromogenic assay and was maintained in production cultures at 12 g L-1. Antibodies were purified from the spent medium fractions collected on days 3-10 and were subjected to titer and glycan analyses. Antibody Titer. Antibody titer was measured by loading filtered cell culture supernatants over a POROS A/20 Protein A column (Applied Biosystems, Carlsbad, CA) equilibrated with 20 mM Tris, 150 nM NaCl, pH 7.0 buffer. Antibody elution was performed with 220 mM acetic acid, 150 nM NaCl, pH 2.6 buffer at a mobile phase flow rate of 4.0 mL min-1. Eluted antibodies were detected at a wavelength of 280 nm. Antibody concentration was determined based on a standard curve with a reference antibody standard. Titer was normalized to the values in the cells that received vehicle only. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay. Anti-mesothelin mAb antibodies were produced in cells supplemented with increasing amounts of 1. The ability of produced antimesothelin mAb antibodies to induce a cytotoxic response was measured in a 4-hour calcein ADCC assay with high (N87) and medium (CAPAN2) target coverage cells and natural killer cells with low and high affinity FcγRIIIa receptor alleles collected from healthy donors. The effector to target cell ratio of 10:1 was used. Target cells were labeled with 10 µM calcein, and percent specific lysis was calculated using spontaneous release and 100% lysis control. EC50 values were calculated based on the specific lysis curves. Glycan Analysis. For glycan analysis, enzymatically released N-linked glycans from protein A-purified antibodies were labeled with 2-aminobenzoic acid (2-AA) and separated by HILIC (hydrophilic interaction liquid chromatography) in-line with a fluorescence detector. The separation was performed using a Waters Acquity UPLC (Waters, Milford, MA). In-line MS, using an ion trap mass spectrometer (LTQ; Thermo Scientific, Waltham, MA) in a positive mode, was incorporated to accommodate mass determination of species. Glycans were injected and bound to the column in high organic conditions and were then eluted with an increasing gradient of an aqueous ammonium formate buffer. Fast separation times were achieved using a 1.7 µM small particle column format (Acquity UPLC BEH Glycan Column, 2.1 x 100 mm; Waters, Milford, MA). Accession Codes. Crystallographic data for GDP-fucose (PDB 5IN5) and 2D (PDB 5IN4) cocrystallized with GMD are available from the PDB. ASSOCIATED CONTENT Supporting information Available: This material is available free of charge via the Internet. AUTHOR INFORMATION Corresponding author *E-mail: [email protected]. 12 ACS Paragon Plus Environment

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Notes The authors were all employees of Amgen, Inc. at the time of the work and hold Amgen stock and/or other long term incentives. ACKNOWLEDGEMENTS The authors thank P. Schnier for consultation on SRM-MS studies, L. Zalameda for early cell surface assay measurements, M.-C. Lo and H. Eastwood for preparation and purification of 2D, and R. Hungate for support and guidance. The authors thank R. Senaiar, M. Bushaboina, R. Bhat, R. Majeti, S. Gondu, and S. Reddy of Aurigene, India for preparation of lots of 1, 2, 7 and 8, and I. Campuzano for determination of HRMS. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. REFERENCES 1

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