Site-Selective Chemoenzymatic Modification on the Core Fucose of an Antibody Enhances Its FcγReceptor Affinity and ADCC Activity

Article abstract of DOI:10.1021/jacs.1c03174

Fc glycosylation profoundly impacts the effector functions of antibodies and often dictates an antibody's pro- or anti-inflammatory activities. It is well established that core fucosylation of the Fc domain N-glycans of an antibody significantly reduces i

Full text of DOI:10.1021/jacs.1c03174

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Article
Site-Selective Chemoenzymatic Modification on the Core Fucose of
an Antibody Enhances Its Fcγ Receptor Affinity and ADCC Activity
Chao Li, Gene Chong, Guanghui Zong, David A. Knorr, Stylianos Bournazos, Asaminew Haile Aytenfisu,
Grace K. Henry, Jeffrey V. Ravetch, Alexander D. MacKerell, Jr., and Lai-Xi Wang*
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ABSTRACT: Fc glycosylation profoundly impacts the effector
functions of antibodies and often dictates an antibody’s pro- or
anti-inflammatory activities. It is well established that core
fucosylation of the Fc domain N-glycans of an antibody
significantly reduces its affinity for FcγRIIIa receptors and
antibody-dependent cellular cytotoxicity (ADCC). Previous
structural studies have suggested that the presence of a core
fucose remarkably decreases the unique and favorable carbohy-
drate−carbohydrate interactions between the Fc and the receptor
N-glycans, leading to reduced affinity. We report here that in
contrast to natural core fucose, special site-specific modification on
the core fucose could dramatically enhance the affinity of an
antibody for FcγRIIIa. The site-selective modification was achieved through an enzymatic transfucosylation with a novel fucosidase
mutant, which was shown to be able to use modified α-fucosyl fluoride as the donor substrate. We found that replacement of the
core L-fucose with 6-azide- or 6-hydroxy-L-fucose (L-galactose) significantly enhanced the antibody’s affinity for FcγRIIIa receptors
and substantially increased the ADCC activity. To understand the mechanism of the modified fucose-mediated affinity enhancement,
we performed molecular dynamics simulations. Our data revealed that the number of glycan contacts between the Fc and the Fc
receptor was increased by the selective core-fucose modifications, showing the importance of unique carbohydrate−carbohydrate
interactions in achieving high FcγRIIIa affinity and ADCC activity of antibodies. Thus, the direct site-selective modification turns the
adverse effect of the core fucose into a favorable force to promote the carbohydrate−carbohydrate interactions.
Fc glycan, but these favorable interactions are significantly
decreased or not existing in the structures of glycosylated
FcγRIIIa complexed with the core-fucosylated IgG-Fc.11,12
Further analysis suggests that the core-fucose residue of the Fc
glycan is oriented toward the second core GlcNAc moiety in
the Asn162-glycan of FcγRIIIa, and as a result, the Asn162
glycan must move away, significantly reducing its interaction
with the Fc glycan. Recent solution NMR and crystallographic
analyses suggest that antibody fucosylation lowers the FcγRIIIa
affinity mainly through limiting the conformations sampled by
the Asn162 glycan instead of disrupting the glycan−glycan
interactions.13 However, it is not clear whether and how
modification of the core-fucose structure could positively
impact the affinity of antibodies to Fcγ receptors. We describe
in the present work a chemoenzymatic method that allows
INTRODUCTION
■
Glycosylation is one of the most important post-translational
modifications to produce fully functional proteins in nature.1,2
Among different types of modifications on glycans, the
attachment of a 1,6-linked fucose to the innermost N-
acetylglucosamine (GlcNAc) moiety of N-glycans, named
core fucosylation, plays a critical role in modulating the
conformations and biological functions of N-glycans and N-
glycoproteins, including therapeutic monoclonal antibodies.3−5
Compelling data have demonstrated that the presence of the
core fucose on the Fc glycan significantly decreases the affinity
of antibodies for FcγIIIa receptor (FcγRIIIa or CD16a),
leading to decreased ADCC activity and decreased anticancer
efficacy in vitro and in vivo.6−11 Human FcγRIIIa itself is a
glycoprotein carrying five N-glycans at the Asn38, Asn45,
Asn74, Asn162, and Asn169 glycosylation sites. Two previous
crystallographic studies on the complexes of recombinant
human FcγRIIIa with afucosylated IgG-Fc or core-fucosylated
IgG-Fc have indicated that in the structure of the FcγRIIIa
complexed with afucosylated IgG-Fc there are extensive
intermolecular carbohydrate−carbohydrate interactions be-
tween the Asn162 glycan of FcγRIIIa and the afucosylated
Received: March 24, 2021
Published: May 12, 2021
https://doi.org/10.1021/jacs.1c03174
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© 2021 American Chemical Society
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Figure 1. Chemoenzymatic approach to site-specific modification at the core fucose of a monoclonal antibody.
Scheme 1. Synthesis of the Selectively Modified α-L-Fucosyl Fluoride Derivatives
introduction of selectively modified fucose analogs at the core
position in intact antibodies using Herceptin as a model
antibody (Figure 1). Using a novel fucoligase mutant that we
have previously discovered,9 we found that the core fucose
could be readily replaced with L-fucose analogs, including D-
arabinose (demethyl-L-fucose), L-galactose (6-hydroxyl-L-fu-
cose), and 6-azido-L-fucose, which could be further derivatized
using a click reaction. Binding studies revealed that replacing
with a D-arabinose, i.e., removal of the methyl group in the L-
fucose, did not change the affinity for FcγRIIIa, suggesting the
methyl group in the core fucose did not impact the antibody−
receptor interactions. However, introduction of an azide group
at the 6 position of the L-fucose resulted in 18-fold and over
100-fold enhancement in the affinity of the antibody to the
high-affinity and low-affinity FcγIIIa receptor alleles, FcγRIIIa-
V158 and FcγRIIIa-F158, respectively. Several other mod-
ifications at the 6 position of L-fucose also led to enhancement
of the antibody’s affinity for FcγIIIa receptors but to a lesser
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Scheme 2. Chemoenzymatic Synthesis of Structurally Well-Defined Core-Fucose-Modified Glycoforms of Herceptin
extent. Cell-based assays confirmed that the 6-azide derivative
had remarkably enhanced ADCC activity compared with the
parent Herceptin. Molecular dynamics (MD) simulations
showed that in contrast to the detrimental effect of the parent
core fucose, the site-specific modification at the 6 position with
an azide or hydroxyl moiety changed the orientation of the
core-fucose moiety and increased the numbers of favorable
carbohydrate−carbohydrate interactions between the Fc and
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the receptor glycans, thereby leading to the enhanced affinity
for the FcγRIIIa receptors.
of the product (calculated for 2, M = 148 778 Da) (Figure S1).
Reduction of the azide group in 2 was achieved by treatment
with tris(2-carboxyethyl)phosphine (TCEP) to give the core 6-
amino-fucosylated Herceptin (3). The 6-triazole-fucosylated
Herceptin (4) was prepared by a copper(I)-catalyzed azide−
alkyne cycloaddition between 2 and propargyl alcohol
(Scheme 2a). We also tested two additional α-fucosyl fluoride
derivatives, the α-L-galactosyl fluoride (12), where a hydroxyl
group was introduced at the 6 position of L-fucose, and the α-
D-arabinosyl fluoride (13), in which the methyl group in L-
fucose was removed with the AlfC-E274A mutant. Again, we
found that the E274A mutant could efficiently use the two α-
glycosyl fluorides as donor substrates for transfucosylation to
provide the core-fucose-modified antibody intermediates (14
and 15), respectively. Extension of the sugar chain using the
EndoS2-D184 M mutant gave the full-length glycoforms (5
and 6) in which the core fucose was specifically replaced with
an L-galactose and D-arabinose, respectively (Scheme 2b).
These results indicated that the AlfC E274A mutant had a
relaxed donor substrate specificity toward various α-fucosyl
fluoride analogs. For the subsequent comparative analysis,
core-fucosylated (G2F) and nonfucosylated (G2) Herceptin
glycoforms carrying the respective biantennary complex-type
N-glycan were prepared following our previously described
procedures.9,17
All of the synthetic antibody glycoforms (1−6) were
characterized by mass spectrometric analysis. The antibodies
were treated with dithiothreitol (DTT) to disconnect the
heavy chain and light chain to give monomeric heavy chains.
MALDI-TOF MS analysis of the monomeric heavy chain
indicated that the glycoengineered antibody heavy chains were
single species with a molecular mass consistent with the
calculated data (Figure S2). In addition, the Fc N-glycans in
the glycoforms (1−6) were released by PNGase F treatment,
and their identities were verified by MALDI-TOF MS analysis
(Figure S3).
Binding Affinity of the Core-Fucose-Modified Glyco-
forms of Herceptin for FcγIIIa Receptors. Fcγ receptor
polymorphism is an important factor for the efficacy of
antibody-based treatment.18 Two polymorphic FcγRIIIa
variants are present in humans in which the amino acid
residue at the 158 position is either a valine (V158) or a
phenylalanine (F158) due to point mutation. The two
FcγRIIIa alleles show significantly different affinity for
antibodies with the V158 allele being a high-affinity FcγRIIIa
with the F158 allele being a low-affinity receptor. Natural killer
(NK) cells expressing the FcγRIIIa-V158 exhibit more potent
NK cell-mediated killing than the F158 variant.19,20 Clinical
studies of Rituximab (an anti-CD20 mAb) and trastuzumab
(Herceptin) for the treatment of non-Hodgkin’s lymphoma
and breast cancer, respectively, have shown that patients with
the high-affinity FcγRIIIa-V158 polymorphism responded to
the treatment with the respective rituximab and trastuzumab
much better than those with the lower affinity FcγRIIIa-F158
polymorphism.21,22 Thus, identifying high-affinity antibody
glycoforms particularly for the low-affinity FcgRIIIa-F158 allele
is clinically relevant.
RESULTS AND DISCUSSION
■
Chemical Synthesis of Selectively Modified α-L-
Fucosyl Fluoride Derivatives. We previously reported that
mutants such as E274A derived from the Lactobacillus casei α-
fucosidase are able to use α-L-fucosyl fluoride as a simple
substrate for core fucosylation.14 To examine if the mutant
enzyme can take modified α-fucosyl fluoride derivatives as
donor substrate for fucosylation, we synthesized several α-
fucosyl fluoride derivatives in which the 6-methyl group was
modified or removed. These included the α-glycosyl fluorides
of 6-azido-L-fucose, L-galactose (i.e., 6-hydroxyl-L-fucose), and
D-arabinose (i.e., the L-fucose derivative in which the methyl
group was removed). Thus, treatment of the per-O-acetylated
sugar with HF in pyridine, followed by de-O-acetylation gave
the corresponding α-glycosyl fluorides (Scheme 1). The
synthetic and selectively modified α-fucosyl fluoride derivatives
1
(8, 12, and 13) were confirmed by H and 13C NMR analysis
(Supporting Information).
Chemoenzymatic Synthesis of Homogeneous Glyco-
forms of Herceptin with Site-Specific Modifications on
the Core Fucose. Using Herceptin (Trastuzumab), a
therapeutic monoclonal antibody used for the treatment of
breast cancer as a model antibody, we developed a two-step
approach for the site-specific modification on the core fucose.
Herceptin was deglycosylated and defucosylated with
endoglycosidase S2 (EndoS2) and the Lactobacillus casei
α1,6-fucosidase (AlfC),15 respectively, to afford the
GlcNAc−Herceptin (7) (Scheme 2). We previously reported
a glycoligase mutant derived from AlfC, AlfC E274A, that can
use α-L-fucosyl fluoride as the donor substrate to transfer a
fucose moiety to a deglycosylated antibody (GlcNAc−Ab) to
restore the core fucose.14,16 However, it was not clear if the
fucosidase mutant could use modified α-fucosyl fluorides as
substrates for transfucosylation. To test this, we incubated the
deglycosylated antibody (7) and the 6-azido-L-fucosyl fluoride
(8) with the AlfC E274A mutant at 30 °C and monitored the
reaction by LC-ESI-MS analysis of the Fc domains released
from IdeS protease digestion of the antibody. We found that
AlfC E274A was able to transfer the 6-azido-fucose from the
glycosyl fluoride (8) to give the 6-AzFucα1,6GlcNAc−
Herceptin (9), which was isolated as a single product in an
86% yield after protein A affinity column chromatography.
Subsequent transfer of a complex type N-glycan using the
complex type N-glycan oxazoline as the donor substrate was
achieved using Endo-S2 D184 M as the glycosynthase,17
affording the fully glycosylated AzFuc−Herceptin (2) in an
excellent yield. As an alternative route, the afucosylated
complex-type glycoform (10) was synthesized by EndoS2-
D184 M-catalyzed glycosylation of the GlcNAc−Herceptin
intermediate (7) with glycan oxazoline 10. Then direct
fucosylation with 6-azido-α-fucosyl fluoride 8 was attempted
with AlfC D274A mutant following our previously reported
procedure.14 However, the direct enzymatic fucose transfer
onto the intact Fc glycan was found to be very slow, and only a
low yield of the desired azido-glycoform (2) was obtained after
using a relatively large amount of enzyme and an excess
amount of the donor substrate (Scheme 2a). The identity of
the azide-tagged antibody (2) was confirmed by LC-ESI-MS
analysis. The deconvolution data of the ESI-MS (found, M =
148 785 Da) were consistent with the expected molecular mass
The affinity of the core-modified Herceptin glycoforms for
two FcγRIIIa alleles, the high-affinity receptor FcγRIIIa-V158
and the low-affinity receptor FcγRIIIa-F158, was assessed by
ELISA assays. We found that different core-fucose modifica-
tions showed significantly different affinities for the FcγIIIa
receptors (Table 1; Figure S4). While all of the glycoforms
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Table 1. Binding Affinity of Core-Fucose-Modified Fc Glycans for the High-Affinity FcγIIIa V158 and Low-Affinity F158
a
Alleles by ELISA
a
FcγRIIIa receptors were coated on 96-well plates. For the V158 allele, binding was tested with Herceptin glycoforms in concentrations ranging
from 250 to 0.016 nM (5-fold serial dilutions). For the F158 allele, binding was tested with Herceptin glycoforms in concentrations ranging from
2000 to 0.128 nM (5-fold serial dilutions).
carrying the natural core fucose demonstrated relatively low
affinity for the FcγIIIa receptors, the 6-azido fucose glycoform
(2) demonstrated 18-fold enhancement in affinity for the high-
affinity receptor FcγRIIIa-V158 and over 100-fold enhance-
ment in affinity for the low-affinity receptor FcγRIIIa-F158.
This remarkable affinity enhancement was unexpected as the
difference was only an attachment of a small azide group at the
6 position of the parent L-fucose. Modification of the 6
position of fucose with an amino, a hydroxyl, and a triazole
group also increased the affinity for the FcγIIIa receptors but to
a lesser extent. Thus, the above 3 modifications resulted in 3−
7-fold increased affinity for the high-affinity receptor
(FcγRIIIa-V158) and about 15-fold-enhanced affinity for the
low-affinity receptor (FcγRIIIa-F158) in comparison with the
parent core-fucosylated glycoform (G2F). Interestingly, the
arabinose glycoform demonstrated essentially the same affinity
as the parent G2F glycoform for both Fcγ receptors (Table 1).
These results suggested that the methyl group itself in the L-
fucose did not impact the antibody’s affinity for the Fc
receptors, as its removal (as shown by its replacement with the
D-arabinose) did not apparently change the affinity. However,
an appropriate modification at the 6 position, such as
introduction of an azide group, could rescue the adverse effect
of core fucose and significantly enhance the antibody’s affinity
for the FcγRIIIa receptors.
line SKBR3 (HER2+) using an engineered stable Jurkat cell
line expressing the F allele of FcγRIIIa as the effector cells.
This activation assay appears to be more sensitive and
quantitative than the conventional in vitro cell killing assays.23
The 6-azido-fucose glycoform (2) exhibited dramatically
enhanced ADCC activity as compared with the commercial
Herceptin and the glycoform carrying unmodified L-fucose
(Figure 2). Its ADCC activity was comparable to the best
optimized Herceptin glycoform (G2) that is afucosylated and
carrying a full-size galactosylated biantennary complex-type N-
glycan.9,24,25 The glycoforms (5 and 3) in which the L-fucose is
modified by attachment of hydroxyl and amino groups,
respectively, also showed significant ADCC activity but to a
lesser extent than that of the 6-azide-modified glycoform (2).
These results are consistent with the binding affinity of the
respective glycoforms for the Fcγ receptors. An exception was
observed for the 6-triazole-modified glycoform (4), which
showed enhanced affinity for the Fcγ receptors but did not
show an apparent increase in ADCC in in this assay. The
opposite was observed for the D-arabinose glycoform (6),
which had similar affinity for Fcγ receptors as the Herceptin
but showed much better ADCC activity than the parent
antibody (Figure 2). An enhanced ADCC activity of the D-
arabinose glycoform (6), which was produced via a metabolic
glycoengineering method, was also observed in a previous
report.26 The reason for the inconsistency between the in vitro
binding affinity for Fcγ receptors and the cellular or in vivo
ADCC activity for some antibody glycoforms was not clear,
which was also observed in previous studies.9
ADCC Activity of the Core-Fucose-Modified Antibody
Glycoforms. The antibody-dependent cellular cytotoxicity
(ADCC) of different Herceptin glycoforms was assessed by a
robust ADCC assay against the human breast carcinoma cell
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180−225° and 225−270° led to conformations in which the
core sugar and its C6 functional group projected out of the Fc−
FcR binding pocket (Figure 3b and 3c). At ϕ = 180−225°, the
hydroxyl groups of the Fc chain A core sugar were oriented
such that they could hydrogen bond with the FcR glycan. In
addition, the azide group in the core azide-fucose was long
enough, compared to the C6 hydroxyl in L-galactose, to
hydrogen bond with FcR Arg155, stabilizing the Fc−FcR
interface through both glycan−glycan and glycan−protein
interactions (Figure 3b) and contributing to its greater affinity.
The orientation of the hydroxyl groups in core sugars with ϕ =
225−270° limited hydrogen-bond contacts with the FcR
glycan, and the C6 functional group was displaced away from
FcR Arg155 (Figure 3c). Finally, the sampling of the ϕ = 0−
45° region with the core azide-fucose led to a number of
hydrogen bonds with the FcR glycan involving the hydroxyls of
GlcNAc1, fucose, as well as the azide moiety (Figure 3d).
These additional interactions led to the increased contacts and
more favorable interaction energies of core azide-fucose over
core galactose that contribute to the more favorable binding
and ADCC activity.
Figure 2. Antibody-dependent cellular cytotoxicity (ADCC) of
different Herceptin glycoforms. ADCC activity was assessed by a
robust ADCC assay against the human breast carcinoma cell line
SKBR3 (HER2+, ATCC) using an engineered stable Jurkat cell line
expressing the F allele of FcγRIIIa as the effector cells. Activation of
the effector cells by the antibodies was probed by measurement of the
luminescence with the Bio-Glo Luciferase assay reagent. Background
luminescence from negative control samples was subtracted, and
signal was expressed as relative luciferase units (RLU).
It has been well established that the attachment of a core
fucose leads to a significant decrease in the affinity of an
antibody for its FcγIIIa receptors and, as a result, remarkably
reduces the ADCC activity of the antibody in vivo. One
explanation of this outcome is that the core fucose interferes
with the favorable carbohydrate−carbohydrate interactions
between the Fc glycan of the antibody and the Asn162 glycan
from the FcγIIIa receptor.11,12 Thus, removal of the core
fucose has become a practical way to enhance the ADCC
activity of therapeutic antibodies such as Herceptin to enhance
their therapeutic efficacy. However, it has not been clear if and
how the core fucose can be modified to modulate the effector
functions of antibodies. One challenge in this pursuit is the
lack of technology in site-specific modification of the core
fucose in intact antibodies. Although attempts have been made
to incorporate modified fucose through metabolic glycoengin-
eering, the sort of modification that can be achieved is
limited.26,29−32 In this study, we established a chemoenzymatic
method that allowed the introduction of selectively modified L-
fucose in an intact antibody and enabled the structure−activity
relationship study of the effects of the modified core fucose on
FcγIIIa receptor binding and ADCC activity. Among the five
selectively modified Herceptin glycoforms, we found that
introduction of an azide group at the 6 position of the L-fucose
resulted in 18-fold and over 100-fold enhancement in the
antibody’s affinity for the high-affinity and low-affinity FcγIIIa
receptor alleles, FcγRIIIa-V158 and FcγRIIIa-F158, respec-
tively. Several other modifications at the 6 position of L-fucose
also led to enhancement of the antibody’s affinity for FcγIIIa
receptors but to a lesser extent. Cell-based assays confirmed
that the 6-azide derivative had remarkably enhanced ADCC
activity compared with the parent Herceptin. These
unexpected experimental results reveal that site-selective
modification of the core fucose, instead of its removal,
constitutes a new molecular approach to modulating the
effector functions of antibodies.
Molecular Dynamics (MD) Simulation Analysis of the
Effect of Core-Fucose Modification on the Antibody−
FcγIIIa Receptor Interactions. To understand the mecha-
nism by which the selected core-fucose modifications alter the
affinity between the antibody and the FcγRIIIa receptor, we
performed MD simulations based on Hamiltonian replica
exchange with solute tempering and biasing potentials
(HREST-BP)27,28 of the Fc−FcγRIIIa receptor complexes for
the glycoforms (1, 2, and 5) and the afucosylated Fc (the G2
Fc glycoform). To obtain information on the extent of
interactions between the Fc chain A and the FcγRIIIa glycans,
the number of contacts between the glycans was determined
based on the distance between the glycan centers of mass,
excluding the core glycan, being <7 Å. Approximately 3−11-
fold increases in the average number of contacts observed in
the simulations occurred in the system with either core
galactose or core azide-fucose and the afucosylated G2 form,
respectively (Table S1, Figure S5). In addition, the interaction
energy showed that the lack of a core fucose led to the most
favorable interaction energies for Fc glycan conformations,
while the presence of the unmodified core fucose gave the least
favorable energies (Figure S6a). The addition of polar groups
at C6 of the parent fucose, as in the case of core galactose and
core azide-fucose glycoforms, expanded the hydrogen-bonding
possibilities, leading to more favorable interaction energies
with the FcR glycan (Figure S6b−d). These results are
consistent with the experimental EC50 values, directly showing
the importance of carbohydrate−carbohydrate interactions in
the binding affinity of the antibody Fc to FcγRIIIa (Table 1).
To understand the molecular contributions to the changes in
the extent of Fc and FcγIIIa receptor interactions, we first
determined the conformations of the core glycans as measured
by the O5′−C1′−O6−C6 dihedral angle ϕ in the core(′)−
GlcNAc1 glycosidic linkage. The conformation of this dihedral
orientation impacted interactions of the Fc glycan with its
environment, offering insights into the molecular interaction
contributing to the relative binding affinities of the antibody Fc
to FcR (Figure 3a). Addition of a polar functional group at C6
in core fucose appears to enable hydrogen bonding between
this group and other glycans, which could facilitate high-energy
transitions about the dihedral angle ϕ. Dihedral angles of ϕ =
Previous crystallographic studies have shown that removal of
the core fucose enables direct contact and hydrogen bonding
between the FcR glycan GlcNAc1/GlcNAc2 monosaccharides
with GlcNAc1 of the Fc chain A glycan.11,33 In addition, a lack
of the core fucose gives Gln295 in Fc chain A access to
hydrogen bonding with the FcR α1,3-branched glycan.11 In
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Figure 3. (A) Distribution of ϕ dihedral angles (O5′−C1′−O6−C6) in the core sugar(′)−GlcNAc1 glycosidic linkage. (B−D) Core azide-fucose of
the Fc chain A glycan linked to GlcNAc1 (carbons in yellow, oxygens in red, and nitrogens in blue) with FcR Arg155 in gray CPK representation
with (B) ϕ = 180−225°, (C) ϕ = 225−270°, and (D) ϕ = 0−45°. Remaining Fc chain A glycan is with carbons in cyan. Fc and FcR proteins are in
cartoon representation, and Fc chain B and FcR glycans carbons are in transparent gray. Hydrogen bonds are indicated by black dashes.
our present MD simulations, interactions between Fc glycan
GlcNAc1 and FcR glycan GlcNAc1 and GlcNAc2 have a ∼2.5-
fold greater likelihood of having interaction energies more
favorable than −10 kcal/mol when the Fc glycan has the core
azide-fucose with ϕ = 0−45° vs core galactose in its preferred
population at ϕ = 180−225° (Figure S6b). Core azide-fucose
with ϕ = 0−45° is the only species in which the core sugar
facilitates interaction of the FcR α1,3 branch with Fc Gln295,
yielding interaction energies more favorable than −5 kcal/mol,
which are more favorable than the same interaction with no
core fucose (Figure S6c and S6d). FcR Lys128 and Fc chain A
Tyr296 were observed to interact in the crystal structures of
Fc−FcR with core afucosylated Fc glycans but not in Fc
glycans with core fucose.11,12 The conformations obtained
from the MD simulations for these species show that systems
with Fc glycans lacking core fucose or carrying the core
galactose have the largest populations of FcR Lys128−Fc
Tyr296 distances of ∼5−8 Å (Figure S5b). Comparison of the
core azide-fucose and L-galactose species show the population
of the shorter interactions to be greater with the latter species.
As the experiments show that the binding affinity of Fc to FcR
is greater when the Fc glycan is engineered with core azide-
fucose compared to galactose, these results suggest that the
FcR Lys128−Fc Tyr296 interaction contributes less to the
binding affinity.
CONCLUSION
■
A chemoenzymatic method is described that permits site-
selective modification on the core fucose of intact antibodies
and enables the investigation of how the modified fucose
affects the antibody’s functions. We have shown that
introduction of a small functional group such as an azide at
the 6 position can have a huge impact on the antibody’s
binding to the FcγRIIIa receptors, leading to 18-fold and 100-
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suspended in CHCl3 and washed with brine and water. The organic
layer was dried and filtered. The filtrate was concentrated, and the
product was purified by silica gel flash chromatography (heptane/
EtOAc = 2:1, v/v) to obtain the azide product as white powder (420
mg, 77%). The purified intermediate was deprotected with 90% TFA
and then acetylated with acetic anhydride in pyridine. The
peracetylated 6-azido-fucose (296 mg, 0.79 mmol) was fluorinated
using HF pyridine (2 mL) and then de-O-acetylated with sodium
methoxide (5 mM) in anhydrous MeOH (10 mL). The reaction
mixture was concentrated, and the residue was dissolved in water and
lyophilized to give the 6-azido-α-L-fucosyl fluoride (8) (157 mg, 64%
in two steps) as a white powder. 1H NMR (D2O, 400 MHz): δ = 5.71
(dd, J1,2 = 2.0 Hz, J1,F = 53.2 Hz, 1H, H-1), 4.20 (dd, J = 4.0, 8.4 Hz,
1H, H-2), 4.02 (d, J = 2.4 Hz, 1H, H-3), 3.89 (d, J = 2.4 Hz, 1H, H-
4), 3.81 (dd, J = 3.6, 11.2 Hz, 1H, H-5), 3.55 (m, 2H, H-6). ESI-MS:
calcd for 6-Az-FucF, M = 207.07 Da; found (m/z), 208.11 [M + H]+.
Synthesis of α-L-Galactosyl Fluoride (12). The synthesis was
performed following the same procedure as the preparation of α-L-
fold enhancement in affinity for the high- and low-affinity
FcγRIIIa (V-158 and F158) alleles, respectively. Cell-based
assays have shown that the 6-azide- and 6-hydroxyl L-fucose-
containing glycoforms demonstrate significantly enhanced
ADCC activity over the antibody carrying the natural core
fucose. These results are unexpected as previously structural
studies indicated that the presence of the natural core fucose
poses steric hindrance to block favorable carbohydrate−
carbohydrate interactions between the Fc and the FcγR N-
glycans, resulting in significantly decreased affinity of the
antibody for the FcγR. Our MD simulations show that the
introduction of an azide or hydroxyl group at the C-6 changes
the orientation of the core fucose to promote favorable
carbohydrate−carbohydrate and carbohydrate−protein inter-
actions, leading to enhanced affinity between antibody Fc and
FcγR receptor. Taken together, the present experimental and
MD simulation studies reveal a new mechanism and a novel
strategy to enhance the antibody’s Fcγ receptor binding and
cellular cytotoxicity.
fucosyl fluoride14 using peracetylated L-galactopyranose as the starting
1
material. H NMR (D2O, 600 MHz): 5.61 (dd, J1,2 = 3.0 Hz, J1,F
=
65.4 Hz, 1 H, H-1), 4.00 (m, 1H, H-2), 3.98 (m, 1H, H-3), 3.79 (m,
1H, H-4), 3.76 (m, 1H, H-5), 3.69−3.63 (m, 2H, H-6a and H-6b). 13
C
NMR (D2O, 125 MHz): 107.75 (C-1), 72.93 (C-5), 68.43 (C-2),
67.51 (C-3), 67.35 (C-4), 60.51 (C-6). ESI-MS: calcd for α-L-GalF,
M = 182.06 Da; found (m/z), 183.09 [M + H]+.
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals, reagents, and solvents
were purchased from Sigma-Aldrich and TCI and unless specially
noted applied in the reaction without further purification. Monoclonal
Herceptin was purchased from Premium Health Services Inc.
(Columbia, MD). Deglycosylated GlcNAc−Herceptin was prepared
using our previously reported method.25 Silica gel (200−425 mesh)
for flash chromatography was purchased from Sigma-Aldrich. Liquid
chromatography electrospray mass spectrometry (LC-ESI-MS) was
performed on an Exactive Plus Orbitrap Mass Spectrometer (Thermo
Scientific) equipped with a C-4 column (XBridge BEH300 C4, 2.1 ×
50 mm, 3.5 μm, Waters) for antibody analysis. Matrix-assisted laser
desorption ionization with a time-of-flight analyzer (MALDI-TOF)
was performed using a Bruker UltrafleXtreme MALDI TOF/TOF
Mass Spectrometer with a dihydroxybenzoic acid/dimethylamide
(DHB/DMA) matrix and used to analyze transfer products including
Synthesis of α-D-Arabinosyl Fluoride (13). The synthesis of 13
was performed following the same procedure as the preparation of α-
L-fucosyl fluoride14 using peracetylated D-arabinopyranose as the
starting material. 1H NMR (D2O, 600 MHz): 5.59 (dd, J1,2 = 3.0 Hz,
J1,F = 57.6 Hz, 1 H, H-1), 3.96 (m, 1H, H-2), 3.94 (m, 1H, H-3),
3.82−3.71 (m, 3H, H-4. H-5a, and H-5b). 13C NMR (D2O, 125
MHz): 107.95 (C-1), 68.42 (C-3), 67.85 (C-2), 67.69 (C-4), 65.01
(C-5). ESI-MS: calcd for α-AraF, M = 152.05 Da; found (m/z),
153.12 [M + H]+.
Synthesis of Core 6-Azido-Fucosylated Herceptin Carrying
a Complex-Type N-Glycan (2). To a mixture of the synthetic α-
monosaccharyl fluoride (55.9 μg, 0.27 μmol) and the GlcNAc−
Herceptin (1 mg, 6.6 nmol) in a buffer (PBS, 150 mM, pH 7.4, 100
μL) was added mutant AlfC E274A (200 μg, 2.0 μL). The solution
was incubated at 30 °C, and the reaction was monitored by LC-ESI-
MS analysis. Additional AlfC E274A and monosaccharide fluoride
were added to drive the reaction to completion, as monitored by LC-
ESI-MS. The mixture was then loaded on a protein A affinity column
(HiTrap Protein A HP, GE Healthcare). After washing, the desired
product was eluted with citrate buffer (30 mM, pH 3.5) and promptly
dialyzed against a buffer (PBS, 150 mM, pH 7.4) at 4 °C. The
solution was concentrated. The quantity of the core-modified
Herceptin was determined with NanoDrop analysis (862 μg, 86%).
To make the fully glycosylated core-modified Herceptin, a solution of
antibody (1 mg, 6.6 nmol) and G2-ox (0.14 mg, 0.26 μmol) was
incubated with Endo-S2 D184 M (20 μg, 0.2 mg/mL) at 30 °C in a
buffer (PBS, 150 mM, pH 7.4, 100 μL) for 2 h. The reaction was
monitored with LC-ESI-MS. The product was purified with a protein
A column. MALDI-TOF-MS analysis: calcd for the heavy chain of 2,
M = 50979 Da; found (m/z), 50971. The engineered glycoforms of
Herceptin were further identified by PNGase F cleavage from the
heavy chain. MALDI-TOF-MS: calcd for the N-glycan of 2, M =
1828.4 Da; found (m/z), 1851.5 [M + Na]+. The core modification of
Herceptin using α-L-GalF and α-AraF was performed in a similar
manner, and the reaction took 6−10 h to completion as monitored by
LC-ESI-MS. The products were purified with protein A chromatog-
raphy.
1
various core-modified N-glycoforms of Herceptin. H and 13C, NMR
spectra were recorded on a 600 MHz spectrometer (Bruker, Tokyo,
Japan) with D2O as the solvent. Endo-S2 D184 M glycosynthase
derived from Streptococcus pyogenes and AlfC E274A fucoligase
derived from Lactobacillus casei were expressed following our
previously described method.14 Fast protein liquid chromatography
(FPLC) was performed using an AKTA Explorer (GE Healthcare)
equipped with a HisTrapTM HP column (1 mL) for protein
purification or a HiTrapTM Protein A HP column (1 mL) for
antibody purification. Protein concentration was determined with
NanoDrop 2000c (Thermo Scientific).
Synthesis of 6-Azido-α-L-fucopyranosyl Fluoride (8). A
solution of L-galactose (500 mg, 2.78 mmol), CuSO4 (1.11 g, 6.93
mmol), and H2SO4 (50 μL) was stirred in acetone (11 mL) at room
temperature for 15 h. Upon completion, the reaction mixture was
neutralized with saturated NaHCO3 and the reaction mixture was
concentrated. The residue was partitioned in CHCl3 and water, and
the organic layer was separated, washed with brine, dried with
anhydrous Na2SO4, and filtered. The filtrate was concentrated to give
the 1,2:3,4-di-O-isopropylidene L-galactopyranose as a white solid
(630 mg, 88%). To a solution of the intermediate (500 mg, 1.92
mmol) in anhydrous pyridine (4 mL) was added TsCl (1 g, 5.26
mmol), and the solution was stirred at room temperature for 12 h.
Then the reaction mixture was diluted with CH2Cl2 and washed with
HCl (10 mM), saturated NaHSO4 solution, and brine, sequentially.
The organic layer was dried and filtered, and the filtrate was
concentrated to give the tosylate derivative as a syrup. The resulting
tosylate ester (760 mg, 1.91 mmol) was dissolved in DMF (13 mL),
and to the solution was added NaN3 (480 mg, 7.38 mol), and the
mixture was stirred under reflux at 115 °C for 15 h. The reaction
mixture was concentrated under reduced pressure. The residue was
Core L-Galactosylated Herceptin (5, 82%). MALDI-TOF-MS:
calcd for the heavy chain of 5, M = 50953 Da; found (m/z) 50944.
MALDI-TOF-MS: calcd for the N-glycan of 5, M = 1803.4 Da; found
(m/z), 1826.4 [M + Na]+.
Core D-Arabinosylated Herceptin (6, 81%). MALDI-TOF-MS:
calcd for the heavy chain of 6, M = 50921 Da; found (m/z) 50914.
MALDI-TOF-MS: calcd for the N-glycan of 6, M = 1771.8 Da; found
(m/z), 1794.8 [M + Na]+.
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Synthesis of Core 6-Amino-fucosylated Herceptin (3). To
the solution of 2 (1 mg, 6.6 nmol) in a buffer (Tris, 100 mM, pH 7.4,
100 μL) was added tris(2-carboxyethyl)phosphine hydrochloride
(TCEP, 50 mM). The reaction was incubated at room temperature
for 2 h and monitored with MALDI-TOF-MS. After completion, the
reaction mixture was buffer exchanged with a PBS buffer (150 mM,
pH 7.4) with a centrifugal filter unit (Amicon Ultra-4, Millipore).34
The product was purified with protein A chromatography as described
above and dialyzed against a PBS buffer (150 mM, pH 7.4) to afford
core 6-amino-fucosylated Herceptin (3) (435 μg, 87%). MALDI-
TOF-MS: calcd for the heavy chain of 3, M = 50952 Da; found (m/z)
50941. MALDI-TOF-MS: calcd for the N-glycan of 3, M = 1802.1
Da; found (m/z), 1825.1 [M + Na]+.
Synthesis of Core 6-Triazole-fucosylated Herceptin (4).
Propargyl alcohol (0.1 μL, 1.8 μmol), CuSO4 (1 μL, 0.5 mM), tris-
hydroxypropyltriazolylmethylamine (THPTA) (2 μL, 0.2 mM), L-
ascorbic acid (5 μL, 0.5 mM), and aminoguanidine hydrochloride (1
μL, 50 μM) were added to a solution of 6-azido-fucosylated
Herceptin (2) (500 μg, 3.3 nmol) in a buffer (PBS, 150 mM, pH
7.4, 100 μL). The reaction was incubated at room temperature
overnight and monitored with LC-ESI-MS. The product was purified
with protein A chromatography as described above and dialyzed
against a PBS buffer (150 mM, pH 7.4) to afford core 6-triazole-
fucosylated Herceptin (4) (423 μg, 84%). MALDI-TOF-MS: calcd
for the heavy chain of 4, M = 51034 Da; found (m/z) 51026. MALDI-
TOF-MS: calcd for the N-glycan of 4, M = 1884.3 Da; found (m/z),
1907.4 [M + Na]+.
ELISA Binding Tests. FcγRIIIA V158 or F158 (0.5 μg/mL, 100
μL/well, Sino Biological) in a buffer (PBS, 150 mM, pH 7.4) was
coated onto a high-binding 96-well plate (Santa Cruz Biotechnology)
at 4 °C overnight. After washing twice with PBS buffer containing
0.5% Tween 20 (PBST, 200 μL/well), 1% Bovine serum albumin
(BSA) in PBS (200 μL/well) was added to block the plate for 2 h.
Subsequently, after being washed twice again, a serial dilution of core-
modified Herceptin in PBST buffer (100 μL/well) was added, and the
plate was incubated for 1 h. For the V158 plate, the concentration of
each Herceptin glycoform ranged from 250 to 0.016 nM (5-fold serial
dilutions). For the F158 plate, the concentration of each Herceptin
glycoform ranged from 2000 to 0.128 nM (5-fold serial dilutions).
After washing five times with PBST buffer, the plate was incubated
with antihuman IgG F(ab′)2 HRP (0.16 μg/mL, 100 μL/well,
Invitrogen) for 1 h. After washing again, 3,3′,5,5′-tetramethylbenzi-
dine (100 μL/well, Thermo Scientific) was added for signal
development. The reaction was stopped by adding sulfuric acid (2
N, 100 μL/well). Absorbance at 450 nm was measured with a
SpectraMax M5 microplate reader (Molecular Devices). EC50 values
were determined by fitting the ELISA binding curves (Figure S4) to
the nonlinear regression dose−response equation in OriginLab 7.5.
ADCC Activity Assay. Anti-HER2 antibody glycoforms were
tested in a robust ADCC assay (PMID 25086226)23 against the
human breast carcinoma cell line SKBR3 (HER2+, ATCC). In brief,
SKBR3 cells were plated 24 h prior to the assay in opaque 96-well flat
bottom plates (Costar) at 104 cells/well. On the day of the assay, each
glycoform was prepared at 5 μg/mL and 3-fold serial dilutions were
made in a separate 96-well plate. Each was then added, in duplicate, to
the SKBR3 cells and incubated for 30 min. The effector cells (Jurkat
cell line expressing the F allele of FcγRIIIA, Promega) were thawed
per the manufacturer’s recommendations and added to the plate at a
concentration of 7.5 × 104 cells/well. The plates were then incubated
for 6 h at 37 °C (5% CO2). Following incubation, the plates were
equilibrated to room temperature for 15 min prior to the addition of
Bio-Glo Luciferase Assay Reagent (Promega). Cells were allowed to
develop at room temperature for 15 min prior to reading on a
SpectraMax Plus spectrophotometer (Molecular Devices). Back-
ground luminescence from negative control samples was subtracted,
and signal was expressed as relative luciferase units.
modified by replacing the C6 methyl group with either a hydroxyl or
an azide group to generate core L-galactose or core azide-L-fucose,
respectively. Force-field parameters for the azide moiety in azide-L-
fucose were generated using CGenFF37,38 to be compatible with the
CHARMM36 additive force field.39,40 Using OpenMM,41 the systems
were energy minimized, followed by molecular dynamics (MD)
simulations at in the NVT ensemble for 0.25 ns at 298 K and then in
the NPT ensemble for 1 ns at 298 K and 1 atm using the
MonteCarloBarostat, to prepare them for Hamiltonian replica
exchange with solute tempering-biasing potential (HREST-BP)
simulations. MD and HREST-BP simulations all employed rigid
constraints for covalent bonds with hydrogen, a 12 Å cutoff for
Lennard−Jones interactions with a switching function to zero from 10
to 12 Å, and the particle mesh Ewald method for calculation of long-
range electrostatic interactions beyond 12 Å.
To enhance the sampling of the glycans, we implemented the
HREST-BP method27,28 in OpenMM. For our application of the
HREST-BP method to the Fc−FcR systems, the potential energy
functions (i.e., the Hamiltonian) for intramolecular dihedral and
nonbonded interactions within the designated solute (the glycans) in
the nth replica were scaled by βn/β0, where β0 is the Boltzmann
constant, kB, × the ground-state temperature T0 (298 K) and βn is kB
× the effective temperature Tn of the nth replica, and the potential
energy functions for intermolecular nonbonded interactions between
solute (glycans) and the designated solvent (proteins and water) were
scaled by β /β0 following the scaling parameters of the REST2
n
Hamiltonian replica exchange method.42 For the flexible glycosidic
linkages between monosaccharides and the glycopeptide linkage
between GlcNAc and Asn162 and Asn297 of the FcR and Fc proteins,
respectively, an additional biasing potential was applied in the form of
a CMAP term.43 The CMAP biasing potentials (bpCMAPs) were
previously computed for the unique pairs of disaccharide and
glycopeptide linkages in the Fc−FcR system.27 The bpCMAPs were
scaled by βn/β0 × λn, with λn as an additional replica-dependent
parameter. Scaling parameters for each replica were obtained from
previously published results27 on the complex biantennary glycan
sequences and were used in this work (Table S2).
Six replicas were run per system in the effective temperature range
of 298−400 K for 40 ns each for a total of 240 ns of sampling time per
system. HREST-BP simulations were run in the NVT ensemble with a
2 fs/time step with a replica exchange attempt made every 2000 time
steps using the Metropolis criterion and data collected from the
ground-state replica every 2000 time steps. The replica walks of the
original ground-state replicas and highest replicas vs time for each
system show a high exchange rate with frequent round trips for the Fc
glycan with core-fucose system (Figure S7). The first 8 ns were not
used in the analysis of the HREST-BP simulations, as the distribution
of contacts between Fc and FcR glycans vs time did not converge
until after 8 ns.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03174.
Supplemental tables and figures; 1H and 13C NMR
spectra of selectively modified α-glycosyl fluorides
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
Lai-Xi Wang − Department of Chemistry and Biochemistry,
University of Maryland, College Park, Maryland 20742,
United States; orcid.org/0000-0003-4293-5819;
Email: wang518@umd.edu
Molecular Dynamics Simulations. The Fc−FcR system with
core-fucosylated FcR glycans and with either core-fucosylated or
afucosylated Fc glycans were set up using CHARMM-GUI35,36 with
PDB ID 3SGK.11 The Fc glycan core L-fucose was structurally
7836
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Article
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potency of a fucose-free monoclonal antibody being developed as an
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Authors
Chao Li − Department of Chemistry and Biochemistry,
University of Maryland, College Park, Maryland 20742,
United States; orcid.org/0000-0002-8090-2017
Gene Chong − Computer Aided Drug Design Center,
Department of Pharmaceutical Sciences, School of Pharmacy,
University of Maryland, Baltimore, Maryland 21201, United
States
Guanghui Zong − Department of Chemistry and
Biochemistry, University of Maryland, College Park,
Maryland 20742, United States; orcid.org/0000-0002-
7335-039X
(8) Kapur, R.; Kustiawan, I.; Vestrheim, A.; Koeleman, C. A.; Visser,
R.; Einarsdottir, H. K.; Porcelijn, L.; Jackson, D.; Kumpel, B.; Deelder,
A. M.; Blank, D.; Skogen, B.; Killie, M. K.; Michaelsen, T. E.; de Haas,
M.; Rispens, T.; van der Schoot, C. E.; Wuhrer, M.; Vidarsson, G. A
prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in
pregnancy. Blood 2014, 123 (4), 471−80.
(9) Li, T.; DiLillo, D. J.; Bournazos, S.; Giddens, J. P.; Ravetch, J. V.;
Wang, L. X. Modulating IgG effector function by Fc glycan
engineering. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (13), 3485−
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(10) Giddens, J. P.; Lomino, J. V.; DiLillo, D. J.; Ravetch, J. V.;
Wang, L. X. Site-selective chemoenzymatic glycoengineering of Fab
and Fc glycans of a therapeutic antibody. Proc. Natl. Acad. Sci. U. S. A.
2018, 115 (47), 12023−12027.
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Waldhauer, I.; Hennig, M.; Ruf, A.; Rufer, A. C.; Stihle, M.; Umana,
P.; Benz, J. Unique carbohydrate-carbohydrate interactions are
required for high affinity binding between FcgammaRIII and
antibodies lacking core fucose. Proc. Natl. Acad. Sci. U. S. A. 2011,
108 (31), 12669−74.
(12) Mizushima, T.; Yagi, H.; Takemoto, E.; Shibata-Koyama, M.;
Isoda, Y.; Iida, S.; Masuda, K.; Satoh, M.; Kato, K. Structural basis for
improved efficacy of therapeutic antibodies on defucosylation of their
Fc glycans. Genes Cells 2011, 16 (11), 1071−80.
(13) Falconer, D. J.; Subedi, G. P.; Marcella, A. M.; Barb, A. W.
Antibody Fucosylation Lowers the FcgammaRIIIa/CD16a Affinity by
Limiting the Conformations Sampled by the N162-Glycan. ACS
Chem. Biol. 2018, 13 (8), 2179−2189.
(14) Li, C.; Zhu, S.; Ma, C.; Wang, L. X. Designer alpha1,6-
Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-
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David A. Knorr − Laboratory of Molecular Genetics and
Immunology, The Rockefeller University, New York 10065,
United States
Stylianos Bournazos − Laboratory of Molecular Genetics and
Immunology, The Rockefeller University, New York 10065,
United States
Asaminew Haile Aytenfisu − Computer Aided Drug Design
Center, Department of Pharmaceutical Sciences, School of
Pharmacy, University of Maryland, Baltimore, Maryland
21201, United States
Grace K. Henry − Department of Chemistry and Biochemistry,
University of Maryland, College Park, Maryland 20742,
United States
Jeffrey V. Ravetch − Laboratory of Molecular Genetics and
Immunology, The Rockefeller University, New York 10065,
United States
Alexander D. MacKerell, Jr. − Computer Aided Drug Design
Center, Department of Pharmaceutical Sciences, School of
Pharmacy, University of Maryland, Baltimore, Maryland
21201, United States; orcid.org/0000-0001-8287-6804
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03174
Notes
The authors declare the following competing financial
interest(s): Lai-Xi Wang is the Founder of GlycoT
Therapeutics, LLC; Alexander D. MacKerell, Jr. is a co-
founder and CSO of SilcsBio LLC. All other authors have no
conflict of interest.
(15) Li, C.; Li, T.; Wang, L. X. Chemoenzymatic Defucosylation of
Therapeutic Antibodies for Enhanced Effector Functions Using
Bacterial alpha-Fucosidases. Methods Mol. Biol. 2018, 1827, 367−380.
(16) Klontz, E. H.; Li, C.; Kihn, K.; Fields, J. K.; Beckett, D.; Snyder,
G. A.; Wintrode, P. L.; Deredge, D.; Wang, L. X.; Sundberg, E. J.
Structure and dynamics of an alpha-fucosidase reveal a mechanism for
highly efficient IgG transfucosylation. Nat. Commun. 2020, 11 (1),
6204.
(17) Li, T.; Tong, X.; Yang, Q.; Giddens, J. P.; Wang, L. X.
Glycosynthase Mutants of Endoglycosidase S2 Show Potent Trans-
glycosylation Activity and Remarkably Relaxed Substrate Specificity
for Antibody Glycosylation Remodeling. J. Biol. Chem. 2016, 291
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(18) Kaifu, T.; Nakamura, A. Polymorphisms of immunoglobulin
receptors and the effects on clinical outcome in cancer immunother-
apy and other immune diseases: a general review. Int. Immunol. 2017,
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ACKNOWLEDGMENTS
■
We thank the members of the Wang lab for technical assistance
and helpful discussions. This work was supported in part by
the National Institutes of Health (NIH grants R01GM096973
and R01AI155716 to L.X.W., R35GM131710 to A.D.M., Jr.,
and R01AI129795 to J.V.R.) and the University of Maryland
Computer-Aided Drug Design Center. The content is solely
the responsibility of the authors and does not necessarily
represent the official views of the NIH.
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