Proactively Reducing Anti-Drug Antibodies via Immunomodulatory Bioconjugation

Article abstract of DOI:10.1002/anie.201814275

Although PEGylation reduces the immunogenicity of protein drugs to some extent, its limitations for highly immunogenic biotherapeutics have been demonstrated. Herein, a proactive strategy to alleviate the development of anti-drug antibodies (ADAs) against protein drugs by immunomodulatory bioconjugation is reported. Rapamycin was conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The conjugated rapamycin can be released from the bioconjugate and prevent immune responses once the bioconjugate is uptaken by antigen-presenting cells. The immunomodulatory bioconjugate significantly reduced the titers of ADAs compared with a PEGylated protein. The inhibition of immune responses was specific to the conjugated antigen, avoiding systemic immune suppression and the risk of increased susceptibility to infections. The reported approach breaks the limitations of PEGylation by the proactive prevention of ADAs.

Full text of DOI:10.1002/anie.201814275

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Accepted Article
Title: Proactively Reducing Anti-Drug Antibodies via
Immunomodulatory Bioconjugation
Authors: Peng Zhang, Priyesh Jain, Caroline Tsao, Kan Wu, and
Shaoyi Jiang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814275
Angew. Chem. 10.1002/ange.201814275
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10.1002/anie.201814275
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Proactively Reducing Anti-Drug Antibodies via
Immunomodulatory Bioconjugation
Peng Zhang, Priyesh Jain, Caroline Tsao, Kan Wu and Shaoyi Jiang*
Abstract: Although PEGylation reduces the immunogenicity of
protein drugs to some extent due to its “stealth effect”, more and
more reports demonstrated its limitations for highly immunogenic
biotherapeutics. Here we report a proactive strategy to alleviate the
development of anti-drug antibodies (ADAs) against protein drugs by
immunomodulatory bioconjugation. An immunomodulator rapamycin
was conjugated to a PEGylated protein therapeutic via a cleavable
disulfide linker. When circulating in blood, the bioconjugate will play
its therapeutic role as a PEGylated drug while the conjugated
rapamycin can be released and prevent immune responses once the
bioconjugate is uptaken by antigen presenting cells. In vitro and in
vivo results showed that the immunomodulatory bioconjugate
significantly reduced the titers of ADAs compared with a PEGylated
protein. Importantly, the inhibition of immune responses was specific
to the conjugated antigen, avoiding systemic immune suppression
and the risk of increased susceptibility to infections. The reported
immunomodulatory bioconjugation approach breaks the limitations of
PEGylation by the proactive prevention of ADAs.
Pegaspargase with reported incidence rates of up to 24%.[7]
Recently, the US Food and Drug Administration (FDA) has
advocated that the biopharmaceutical industry take a proactive
risk-based approach to mitigate unwanted immunogenicity of the
biologic therapeutics.[8] An effective approach to improve
PEGylation to further reduce ADAs is apparently desired.
Anti-drug antibodies (ADAs) remain as one of the major
obstacles that limit the widespread applications of biologic
therapeutics.[1]
Although
naturally
derived
biologic
macromolecules, including enzymes, antibodies, hormones,
cytokines and nucleic acids, comprise a broad reservoir of drug
candidates with exceptional medicinal potency and specificity,
they carry an inherent risk of eliciting ADAs that can adversely
affect the efficacy and safety of the treatment.[1-2] Virtually all
biologics can elicit an ADA response, even fully human growth
factors and human antibodies.[3] The generated ADAs may
negatively
affect
treatments
by
neutralizing
drug
pharmacological activity, expediting blood clearance or causing
hypersensitivity
anaphylaxis).[4]
reactions
(including
life-threatening
Various approaches have been developed to reduce the
immunogenicity of biologic therapeutics.[5] Among which, the
conjugation of polyethylene glycol (PEG) (known as PEGylation)
has successfully brought a number of PEGylated therapeutics
into market.[5c] PEGylation has improved the pharmacokinetics
and immunogenicity of biologic macromolecules by sterically
shielding their antigenic epitopes from immune system
recognition. However, more and more reports demonstrated its
limitations for highly immunogenic biotherapeutics.[6] For
example, Pegloticase (PEGylated uricase) induces ADAs in
∼90% of the refractory chronic gout patients, resulting in a
reduction in drug effectiveness and increased risk of infusion
reactions.[4b, 4c] Similarly, clinical hypersensitivity occurred in a
subset of acute lymphoblastic leukemia patients received
Scheme 1. a) Schematic showing of the immunomodulatory
protein bioconjugate structure. b) When uptaken by dendritic
cells, rapamycin released from the bioconjugate inhibits dendritic
cell maturation and prevents ADA generation.
Traditional methods, e.g., PEGylation, passively reduce
ADAs by physically hiding the underlying protein cargos from
immune recognition. However, such passive strategies may
suffer from particular limitations, such as material
immunogenicity and insufficient protein surface epitope
coverage.[6a] A nanogel encapsulation strategy was proposed to
overcome the concerns of ADAs by sterically covering the whole
protein surface using zwitterionic polymers [5d]. However, as a
trade-off, the hydrogel coating strategy can only be applied to
enzymes with small substrates. As an alternative, a proactive
strategy that endows PEGylated therapeutics with the ability to
modulate the immune system is a new effective approach.
Rapamycin, a commonly used immunosuppressant to prevent
organ transplant rejections, has been shown to inhibit dendritic
cell (DC) maturation and induce tolerogenic DCs.[9] Rapamycin
encapsulated PLGA nanoparticles have been used to reduce
[*]
Dr. P. Zhang, Dr. P. Jain, C. Tsao, K. Wu, Prof. S. Jiang
Department of Chemical Engineering, University of Washington,
Seattle, WA, 98195 (USA)
E-mail: sjiang@uw.edu
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201814275
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ADAs of biotherapeutics, and human clinical trials are currently
ongoing.[10] Herein, we report an immunomodulatory
bioconjugation chemistry to proactively mitigate ADA responses,
by conjugating rapamycin to PEGylated therapeutic proteins.
When circulating in blood, the bioconjugate will play its
therapeutic role as normal PEGylated drugs; while the
conjugated rapamycin can be released and prevent immune
responses once the bioconjugate is uptaken by antigen
presenting cells. The conjugation chemistry ensures the co-
delivery of the immunomodulator and the protein, avoiding
potential systemic immune suppression and related
complications.
Rapamycin is a macrocyclic polyketide whose biological
activities are dependent on the binding of the left-hand portion of
the molecule, from C8 to C31, to FKBP12 (FK-506 binding
protein) and the subsequent formation of a tertiary complex with
mTOR (mammalian target-of-rapamycin) protein.[11] Modification
of the 42-OH position has led to a series of derivatives with good
activity.[12] Thus, the 42-OH position was selected in our study to
introduce the reactive moiety for bioconjugations. Rapamycin
42-hemisuccinate was firstly synthesized via lipase-catalyzed
regioselective esterification following a published procedure.[12a]
The carboxyl group was then activated and conjugated to PEG
molecules (10 kDa) through a disulfide linker, denoted as PEG-
ss-rapa. The successful conjugation was verified by both NMR
(Fig. S2) and UV-Vis spectra (Fig. 1a). A rapamycin substitution
degree of 87% was calculated from the UV absorption intensity.
The disulfide bond was introduced as the environmental
triggering mechanism to release the immunostimulators
intracellularly. As shown in Fig. 1a, when treated with
dithiothreitol (DTT), the characteristic absorption peak of
rapamycin around 280 nm rapidly disappeared, indicating its
release from the conjugate.
PEG-ss-rapa was subsequently conjugated to uricase to
test its performance both in vitro and in vivo. Uricase derived
from Candida sp. was selected as the model protein in this study
due to its strong immunogenicity. To perform the protein
conjugation, the carboxyl end-group of PEG-ss-rapa was
activated as N-hydroxysuccinimide (NHS) ester, and then
bioconjugation was completed by reacting with the protein lysine
residues. The successful protein conjugation was revealed by
UV-Vis spectra (Fig. 1b), as the bioconjugate (denoted as Ur-
PEG-ss-rapa) showed an 11-fold stronger absorption at 280 nm
compared with the native uricase. Based on the UV absorptions,
the conjugation density was calculated to be 18.6 rapamycin
molecules with a total of 21.4 PEG polymer chains per uricase
tetramer. A PEGylated uricase (Ur-PEG, 22 PEGs per protein
tetramer) was also prepared using 10 kDa PEG-NHS for the
purpose of comparison. GPC analysis showed that Ur-PEG-ss-
rapa possessed a similar hydrodynamic size as the Ur-PEG
bioconjugate at a similar polymer conjugation density (Fig. 1c).
Both uricase bioconjugate showed residue activities of > 95%
with respect to the native enzyme. The in vivo circulation
behavior of Ur-PEG-ss-rapa was evaluated in a rat model. As
shown in Fig. 1d, no significant difference was observed
between the serum concentration-time curves of Ur-PEG-ss-
rapa and the regular PEGylated uricase, indicating that the
conjugation of rapamycin did not sacrifice its circulation half-life.
Rapamycin is known to inhibit dendritic cell maturation,
which is considered as the key mechanism for immune tolerance
induction against specific antigens.[9c] We incubated the Ur-PEG-
ss-rapa bioconjugates with mouse dendritic cells (DC 2.4) to
examine its influence on dendritic cell activation. The dendritic
cells were firstly incubated with the bioconjugates for 24 h,
followed by exposure to lipopolysaccharides (LPS) for another
24 h. The expression of two biomarkers, CD80 and CD86, was
examined by flow cytometry. As shown in Fig. 2a, the
costimulatory molecules on DCs in the Ur-PEG group were all
up-regulated compared with the untreated immature DC control,
showing the sign of activation. The Ur-PEG-ss-rapa treated
group exhibited lower surface expression of CD86 compared
with immature control cells, which is consistent with previous
reports regarding rapamycin treated DCs.[9c] Similarly, the up-
regulation of CD80 was also reduced compared with Ur-PEG
treated cells. Normalization of independent experiments by the
conversion of flow data to the percentage of the MFI for the
indicated group, relative to the MFI for immature control
confirmed that the decreases in CD80 and CD86 on Ur-PEG-ss-
rapa treated DCs were statistically significant (Fig. 2b).
Collectively, the low expression level of costimulatory molecules
CD80 and CD86 in the Ur-PEG-ss-rapa group proved the
biological activity of the conjugated rapamycin derivative.
The ADA generation against Ur-PEG-ss-rapa bioconjugate
was revealed by measuring both protein and bioconjugate
specific antibodies after three weekly intravenous (I.V.)
injections in a rat model. An uricase-PEG-rapamycin (denoted
as Ur-PEG-rapa) bioconjugate without cleavable disulfide bond
was included for comparison. As shown in Fig. 3a, the native
uricase stimulated a robust antibody response due to its strong
immunogenicity, while regular PEGylation reduced anti-uricase
by about 6-fold. By contrast, Ur-PEG-ss-rapa bioconjugate
Figure 1. a) UV-Vis spectra of the PEG-ss-rapa polymer before
and after DTT treatment (in PBS), in comparison with the
spectrum of rapamycin (DMSO solution). b) UV-Vis spectra of
the native uricase and bioconjugate, at a protein concentration
of 2 mg/ml. c) Gel-permeation chromatography (GPC) traces of
uricase and the bioconjugates. d) Circulatory profiles of uricase
and bioconjugates (n=6). The serum uricase activity was
measured after one intravenous injection in rats.
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10.1002/anie.201814275
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Figure 2. Ur-PEG-ss-rapa inhibited dendritic cell maturation. DC 2.4 cells were treated by the bioconjugates for 24 h and stimulated
by LPS. The control cells were neither treated by bioconjugates nor the LPS. The upregulation of CD80 and CD 86 was analyzed by
flowcytometry. a) Histograms and b) averaged mean fluorescence intensity (MFI) of the samples (n=3). Student’s t test was chosen
to compare two small sets of quantitative data, *p<0.05, **p<0.01.
showed a similar trend as that of the anti-uricase antibody (Fig.
3b). The non-releasing Ur-PEG-rapa bioconjugate showed a 3-
fold less anti-bioconjugate titer compared with regular
PEGylated uricase, while the cleavable Ur-PEG-ss-rapa reduced
the titer by 34-fold to a negligible level. As a classic passive
strategy, PEGylation reduced ADAs generation by the “stealth”
effect; nonetheless, the effectiveness is limited by the
achievable protein conjugation density. In addition, the
immunogenic protein carrier amplified the immunogenicity of the
haptenic PEG molecules, which results in the generation of anti-
bioconjugate ADAs.[6a] The combination of the proactive
mechanism with traditional PEGylation breaks the limitation of
the passive strategy, as the released drug actively modulates
the immune activity. In order to inhibit ADAs, the protein/antigen
and rapamycin need to be delivered into the same dendritic cell.
Compared with the linked protein/antigen in the bioconjugate,
Figure 3. Antibody responses measured by enzyme-linked
immunosorbent assay (ELISA). a) Anti-uricase and b) anti-
bioconjugate antibodies after three weekly injections. c) Anti-
OVA antibodies of the groups received three weekly OVA or
OVA/Ur-PEG-ss-rapa co-injections. d) The pre-treatment group
firstly received three weekly administrations of the
immunomodulatory bioconjugates. After two weeks, three
weekly administrations of native uricase were then given. The
antibody responses were compared with the group received
solely three injections of the native uricase. All statistical
analyses were performed using a one-way ANOVA with a
Bonferroni posttest, n=6, *p<0.05, **p<0.01, ***p<0.001.
co-injected free antigens have far less opportunity to be uptaken
by a dendritic cell that also uptakes rapamycin. As a result, only
the immune responses against the conjugated proteins are
suppressed.
The usage of immunosuppressive drug may cause non-
selective immunosuppressive effect, which could result in
increased susceptibility to infections.[13] To examine the
selectivity of the antibody generation inhibition, we co-injected
Ur-PEG-ss-rapa together with ovalbumin, and compared the
anti-ovalbumin antibody titers with the group received ovalbumin
alone. As shown in Fig. 3c, both groups had strong responses
against ovalbumin, with no significant differences in antibody
titers. The similar antibody levels demonstrated that the
conjugated rapamycin suppresses the immune responses in an
antigen-specific manner. As showed in the above in vitro tests,
the conjugated rapamycin inhibits DC maturation, and the
immature DCs are known to induce immune tolerance.[14] To
further test whether the antibody inhibition is a result of antigen-
specific tolerance, we conducted another study by measuring
the antibody response of the native uricase administration post a
pre-treatment regimen of the Ur-PEG-ss-rapa. In this test, the
animals firstly received three dosages of Ur-PEG-ss-rapa as the
pretreatment. Two weeks later, three weekly administrations of
native uricase were given and the antibody titers were examined
only generated a negligible anti-uricase response, which was
35-fold lower than the regular PEGylated uricase. Simply mixing
and co-injection of uricase with PEG-ss-rapa free polymers did
not significantly reduce the anti-uricase response compared with
the native uricase. Other studies also suggested that co-injection
of free rapamycin with protein antigens cannot induce antigen
specific tolerance,[10a,10b] indicating that rapamycin needs to be
co-delivered with the antigen. It should be noted that the Ur-
PEG-rapa bioconjugate showed a similar anti-uricase response
with regular PEGylated uricase, demonstrating that rapamycin
needs to be released as the free form to function as an
immunomodulator. The anti-bioconjugate antibody responses
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J. W. Twisk, B. A. Dijkmans, L. Aarden, G. J. Wolbink, JAMA 2011, 305,
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two weeks post the final injection. As shown in Fig. 3d, the
pretreatment resulted in a 51-fold lower anti-uricase IgG titer
compared with the control group, demonstrating a persistent
antigen-specific tolerance. As a benefit, the rapamycin-antigen
conjugation strategy may also have the potential to be used as a
treatment for autoimmune disease in addition to the reduction of
ADAs.
In conclusion, we report a proactive strategy to reduce ADAs
of the PEGylated biologic drugs via immunomodulatory
bioconjugation chemistry. The rapamycin derivative was
conjugated to PEGylated proteins via a cleavable disulfide linker.
In vitro tests showed that the conjugated rapamycin is capable
of inhibiting the maturation of dendritic cells. In vivo studies
demonstrated that rapamycin conjugated via the cleavable linker
effectively mitigates ADAs against PEGylated proteins. Further
studies proved that the inhibition of ADAs was a result of the
antigen-specific immune tolerance induced to the conjugated
protein, which avoided systemic immune suppression and the
risk of increased susceptibility to infections. All of these results
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This work was supported by the Defense Threat Reduction
Agency (HDTRA1-13-1-0044) and the University of Washington.
We are thankful to Dr. K.L. Rock (University of Massachusetts
Medical Center, Worcester, MA) for providing the mouse
dendritic cell line DC2.4 as a generous gift.
Keywords: bioconjugation • immune response • PEGylation •
protein therapeutic • rapamycin
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COMMUNICATION
Peng Zhang, Priyesh Jain, Caroline
Tsao, Kan Wu, Shaoyi Jiang*
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Page No. – Page No.
Proactively Reducing Anti-Drug
Antibodies via Immunomodulatory
Bioconjugation
Immunomodulatory bioconjugation: An immunomodulator rapamycin was
conjugated to a PEGylated protein therapeutic via a cleavable disulfide linker. The
immunomodulatory bioconjugation effectively reduces anti-drug antibody (ADA)
generation by inducing antigen-specific immune tolerance. The reported approach
breaks the limitations of PEGylation by the proactive prevention of ADAs.
This article is protected by copyright. All rights reserved.