Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic Polypeptides Minimizes Antidrug Antibody Generation

Abstract

Polymer conjugation is a clinically proven approach to generate long acting protein drugs with decreased immune responses. Although poly(ethylene glycol) (PEG) is one of the most commonly used conjugation partners due to its unstructured conformation, its therapeutic application is limited by its poor biodegradability, propensity to induce an anti-PEG immune response, and the resultant accelerated blood clearance (ABC) effect. Moreover, the prevailing preference of unstructured polymers for protein conjugation still lacks strong animal data support with appropriate control reagents. By using two biodegradable synthetic polypeptides with similar structural compositions (l-P(EG3Glu) and dl-P(EG3Glu)) for site-specific protein modification, in the current study, we systematically investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates. Our results reveal that the conjugate l20K-IFN, interferon (IFN) modified with the helical polypeptide l-P(EG3Glu) shows improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the unstructured polypeptide analogue dl-P(EG3Glu) or PEG. Moreover, l20K-IFN triggered significantly less antidrug and antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a human growth hormone (GH) conjugate series. Subcutaneously infused l20K-GH, GH modified with l-P(EG3Glu), evokes considerably less anti-GH and antipolymer antibodies compared to those modified with dl-P(EG3Glu) or PEG (dl20K-GH or PEG20K-GH). As a result, repeated injections of dl20K-GH or PEG20K-GH, but not l20K-GH, result in a clear ABC effect and significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the helical l20K-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.

Full text

Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic
Polypeptides Minimizes Antidrug Antibody Generation
Yingqin Hou,
†
Yu Zhou,
†
Hao Wang,
†
Jialing Sun,
†
Ruijue Wang,
†
Kai Sheng,
†
Jingsong Yuan,
†
Yali Hu,
‡
Yu Chao,
§
Zhuang Liu,
§
and Hua Lu*
,†
†
Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science and Engineering, Key Laboratory of Polymer
Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing
100871, People’s Republic of China
‡
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, People’s Republic of China
§
Institute of Functional Nano & Soft Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and
Technology, Soochow University, Suzhou, Jiangsu 215123, China
*
SSupporting Information
ABSTRACT: Polymer conjugation is a clinically proven approach to
generate long acting protein drugs with decreased immune responses.
Although poly(ethylene glycol) (PEG) is one of the most commonly
used conjugation partners due to its unstructured conformation, its
therapeutic application is limited by its poor biodegradability,
propensity to induce an anti-PEG immune response, and the resultant
accelerated blood clearance (ABC) effect. Moreover, the prevailing
preference of unstructured polymers for protein conjugation still lacks
strong animal data support with appropriate control reagents. By using
two biodegradable synthetic polypeptides with similar structural
compositions (L-P(EG3Glu) and DL-P(EG3Glu)) for site-specific
protein modification, in the current study, we systematically
investigate the effect of the polymer conformation on the in vivo pharmacological performances of the resulting conjugates.
Our results reveal that the conjugate L20K-IFN, interferon (IFN) modified with the helical polypeptide L-P(EG3Glu) shows
improved binding affinity, in vitro antiproliferative activity, and in vivo efficacy compared to those modified with the
unstructured polypeptide analogue DL-P(EG3Glu) or PEG. Moreover, L20K-IFN triggered significantly less antidrug and
antipolymer antibodies than the other two. Importantly, the unusual findings observed in the IFN series are reproduced in a
human growth hormone (GH) conjugate series. Subcutaneously infused L20K-GH, GH modified with L-P(EG3Glu), evokes
considerably less anti-GH and antipolymer antibodies compared to those modified with DL-P(EG3Glu) or PEG (DL20K-GH or
PEG20K−GH). As a result, repeated injections of DL20K-GH or PEG20K-GH, but not L20K-GH, result in a clear ABC effect and
significantly diminished drug availability in the blood. Meanwhile, immature mouse bone marrow cells incubated with the
helical L20K-GH exhibit decreased drug uptake and secretion of proinflammatory cytokines compared to those treated with one
of the other two GH conjugates bearing unstructured polymers. Taken together, the current study highlights an urgent necessity
to systematically reassess the pros and cons of choosing unstructured polymers for protein conjugation. Furthermore, our results
also lay the foundation for the development of next-generation biohybrid drugs based on helical synthetic polypeptides.
■INTRODUCTION
Therapeutic proteins are important biologics that frequently
exhibit high potency and selectivity. However, their clinical use
has been hampered by their rapid renal clearance, susceptibility
to proteolysis, and strong immunogenicity.
1−3
Particularly, the
generation of antidrug antibodies (ADAs) has been a serious
hurdle for many protein drugs.
4
One proven strategy to
overcome these limitations is to covalently conjugate the
protein of interest to polymers such as poly(ethylene glycol)
(PEG), a process known as PEGylation, which can lead to
significantly increased hydrodynamic volume, in vivo stability,
and circulation half-life.
5−10
However, there is mounting
evidence that PEGylated proteins tend to show poorer binding
affinity and biological activity than their unconjugated
equivalents.
11,12
Furthermore, although one of the initial
purposes of PEGylation is for reduced ADA generation, PEG
is known to elicit anti-PEG antibodies that adversely accelerate
the blood clearance of the PEGylated proteins or nano-
particles, known as the ABC effect. As evidence, reduction in
the therapeutic efficacy of many PEGylated proteins, such as
uricase, asparaginase, and interferon (IFN), has been found to
strongly correlate with the occurrence of the anti-PEG immune
response that they induce.
13
More worrisome is the fact that
Received: August 10, 2018
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the percentage of healthy adults carrying pre-existing anti-PEG
antibodies has increased sharply from 0.2% to 42% during the
past three decades, likely because of their daily exposure to
PEG-containing commodities.
13
Thus, a pressing need in this
field is seeking new polymers beyond PEGylation.
In recent years, researchers have investigated a wide range of
alternative conjugation partners,
14
including zwitterionic
polymers,
15,16
polyglycerol,
17
glycopolymers,
18
and oligo-
EGylated poly(meth)acrylates,
19,20
with varying degrees of
success. Despite the potential of these methods, the lack of
biodegradability has remained a central problem.
5
Synthetic
polypeptides have been increasingly considered as a biode-
gradable and biocompatible alternative to PEG with great
clinical promise.
21−25
There has been evidence that the genetic
fusion of therapeutic proteins/peptides to intrinsically
disordered polypeptides, such as XTEN, PAS, and elastin-like
polypeptides (ELP), can lead to improved pharmacological
performance in vivo.
26−32
We envisage that the chemical
modification of proteins by synthetic polypeptides, which we
call PEPylation, could open up enormous possibilities.
33−35
Particularly, the chemical diversity of synthetic polypeptides
has been greatly expanded by incorporating noncanonical
amino acids via ring-opening polymerization (ROP) of α-
amino acid N-carboxyanhydrides (NCA) and utilizing D-amino
acids.
20
Notably, during the preparation of this manuscript,
Jiang et al. reported the nonspecific grafting of zwitterionic
polypeptides to uricase,
36
which showed extraordinarily low
immunogenicity and outstanding safety profile in vivo. Their
work underscored the exceptional clinical potential of
PEPylation.
When surveying the aforementioned polymers for protein
modification, one can easily draw the conclusion that
unstructured and flexible polymers (e.g., PEG) have long
been the preferred conjugation partners due to their ability to
augment the hydrodynamic volume of the modified protein
and provide an excellent stealth effect that minimizes renal
Scheme 1. Site-Specific Conjugation of Synthetic Polypeptides or PEG To Engineer Therapeutic Proteins (IFN and GH) via
Native Chemical Ligation
Figure 1. Characterization and in vivo pharmacological performances of various IFN conjugates. (A) SDS-PAGE gel, stained by Coomassie blue.
(B) Circular dichroism (CD) spectroscopy. (C) Melting temperature (Tm) measured by thermofluoro assay. (D) In vivo pharmacokinetics (i.v.
injection) of wt-IFN (n= 6), L20K-IFN (n= 6), and DL20K-IFN (n= 6), and PEG20K−IFN (n= 3). (E−F) Tumor growth inhibition curves. BALB/
C-nu mice bearing s.c. OVCAR-3 xenograft (E) or patient-derived xenograft (PDX) tumors (F) were i.v. injected with PBS saline or one of the
IFN-based drugs (n= 7 each); treatments began on day 0, and the black arrows indicate ending of the treatments. The total injection numbers are
six in E and three in F. Data are expressed as mean ±SD. Pvalue is determined by two-way ANOVA (Bonferroni post-test) analysis: *p< 0.05, **p
< 0.01, ***p< 0.001.
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filtration and immune attack. Following the same principle,
elementary amino acids are carefully selected in the design of
XTEN to ensure an unstructured conformation and absence of
helical structures.
26
However, it is surprising that there have
been very few studies that attempt to investigate whether the
conformation, particularly the helix, of the polymer has any
effect on the in vivo performance of the protein that it
modifies. One practical challenge resides in the difficulty of
generating protein conjugates that only differ in the
conformation of the attached polymers to ensure a fair
comparison. We reason that synthetic polypeptides offer an
ideal solution to this problem as their secondary conformations
(e.g., helix and coil) can be easily manipulated by switching the
chirality of the monomers without altering the overall chemical
composition.
37,38
■RESULTS
Synthesis and Characterization of Different IFN−
Polymer Conjugates. Recombinant IFN, an antiviral and
antitumor cytokine, was selected as our first model drug. For a
fair comparison, we synthesized two chemically similar but
conformationally varied polypeptides (Scheme 1).
34,35
Specif-
ically, monomer γ-(2-(2-(2-methoxyethoxy)ethoxy)ethyl L-
glutamate NCA
39
(L-EG3GluNCA) was polymerized by
trimethylsilyl phenylsulfide (PhS-TMS) to yield phenyl
thioester-functionalized L-P(EG3Glu) (Scheme 1). Similarly,
DL-P(EG3Glu) was produced from a racemic mixture of DL-
EG3GluNCA. The molecular weights (MW) of both polymers
were carefully controlled to be ∼20 kDa, in line with many
clinically approved PEG conjugates. Gel permeation chroma-
tography (GPC) indicated that the two polymers had a similar
MW ≈22−23 kDa and narrow dispersity (D̵) below 1.05
(Figure S1). 1H NMR spectroscopy showed that the two
polymers differed in the chemical shift of the α-H due to the
different α-C chirality (Figure S2). As expected, circular
dichroism (CD) spectroscopy revealed that α-helices con-
stituted more than 90% of L-P(EG3Glu), whereas DL-
P(EG3Glu) was unstructured as design (Figure S3). Sub-
sequently, we conjugated each synthetic polypeptide to an IFN
mutant bearing a N-terminal cysteine (Cys-IFN) via native
chemical ligation, thereby forming two PEPylated IFNs
denoted as L20K-IFN and DL20K-IFN (Scheme 1). We also
generated PEG20K-IFN as a positive control by attaching a
thioester-functionalized PEG (MW ≈20 kDa) to IFN via the
same method (Scheme 1 and Figure S4).
All purified IFN conjugates exhibited a narrow size
distribution based on SDS-PAGE analysis (Figure 1A). L20K-
Table 1. In Vitro Binding, Anti-Proliferative Activity, and in Vivo Pharmacokinetics
a
of wt-IFN and Various IFN Conjugates
sample IC50 (pg/mL) KD(nM) elimination half-life (h)
b
AUC0‑t((μg/mL)*h)
c
Vd(mL)
d
CI (mL/h)
wt-IFN 8.5 ±1.4 1.0 0.5 ±0.1 0.4 ±0.1 125 ±21.8
L20k-IFN 36.0 ±1.3 5.8 9.6 ±0.6 15.5 ±2.2 167 ±49 3.2 ±0.6
DL2ok-IFN 160 ±4 19.6 7.8 ±0.3 8.6 ±0.7 239 ±49 5.8 ±0.7
PEG20k-IFN 190 ±10 15.9 9.8 ±1.9 17.0 ±3.0 161 ±25 2.9 ±0.4
a
Dose: 50 μg/rat on IFN base.
b
Elimination half-life: Time points used to calculate t1/2βare 3−12 h (wt IFN), 12−72 h (all conjugates).
c
AUC
calculated by logarithmic trapezoidal rule up to 12 h (wt-IFN),72 h (L20K-IFN, DL20K-IFN, and PEG20K−IFN).
d
Vdcalculated at 12 h after
intravenous injection.
e
Data are expressed as mean ±SD.
Figure 2. In vivo immune responses triggered by IFN conjugates. (A−B) Anti-IFN IgG (A) and IgM (B) contents in the sera measured by ELISA;
the plates were coated with wt-IFN and then incubated with 104-fold (for IgG) or 500-fold (for IgM) prediluted sera in PBS. (C−D) Antipolymer
IgG (C) and IgM (D) contents in the sera immunized with various polymer-IFN conjugates; for each polymer-of-interest, the ELISA plates were
coated with the corresponding polymer-GH conjugate. (E−F) Antipolymer ELISA assays using free DL-P(EG3Glu) (E) or PEG (F) as the
competition agent; sera immunized with DL20K-IFN or PEG20K−IFN (week 4) were prediluted 200-fold and incubated with the corresponding free
polymer at gradient concentrations. Immunization protocol: rats were s.c. infused with L20K-IFN, DL20K-IFN, or PEG20K−IFN at a weekly dose 0.2
mg/kg for 4 weeks; sera were drawn from the rats (n= 3) every week starting from week 0. For ELISA analysis, after sera incubation and washing,
all plates were incubated with antimouse IgG-HRP or IgM-HRP, and analyzed by TMB solution (CWBIO). TWEEN was excluded from the
buffers in all antipolymer ELISA studies. Data are expressed as mean ±SD Pvalue is determined by two-way ANOVA (Bonferroni post-test)
analysis: *p< 0.05, **p< 0.01, ***p< 0.001.
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IFN and DL20K-IFN shared an almost identical apparent MW,
whereas PEG20K-IFN appeared to electrophoresize slightly
slower than its PEPylated counterparts but was still
comparable (Figure 1A). CD spectroscopy suggested that
PEG20K-IFN and DL20K-IFN were similar in helicity as wt-IFN,
whereas L20K-IFN produced a stronger helical signal intensity
(Figure 1B). A thermofluoro assay
39
indicated that L20K-IFN
possessed a higher Tm, and therefore greater thermostability,
than both DL20K-IFN and PEG20k−IFN (Figure 1C). All
conjugates were shown to be significantly more resistant to
proteolysis than wt-IFN in trypsin digestion assays (Figure S5).
Surface plasmon resonance (SPR) found the KDvalues for the
binding of L20K-IFN, DL20K-IFN, and PEG20K−IFN to human
IFNAR2 were 5.8, 19.6, and 15.9 nM, respectively (Table 1
and Figure S6). Thus, L20K-IFN appeared to be ∼3−4 fold
more efficient in its receptor interaction than DL20K-IFN or
PEG20K−IFN. Consistently, an in vitro viability assay
demonstrated that the IC50 values of L20K-IFN, DL20K-IFN,
and PEG20K−IFN against Daudi cells, an IFN-sensitive human
cancer cell line, were 36, 160, and 190 pg/mL, respectively
(Table 1). This implied that L20K-IFN could induce a
significantly more potent antitumor effect than DL20K-IFN or
PEG20K−IFN does.
In Vivo Pharmacological Performances of IFN
Conjugates. We next measured the pharmacokinetic
parameters of the IFN variants in female Sprague−Dawley
rats. As shown in Figure 1D and Table 1, the elimination half-
lives (t1/2β)ofL20K-IFN, DL20K-IFN, and PEG20K−IFN were
9.6, 7.8, and 9.8 h, respectively, all significantly longer than the
0.5 h t1/2βof wt-IFN. Interestingly, L20K-IFN was slightly but
consistently longer-lived than DL20K-IFN (Pvalue < 0.05;
reproducible in at least two independent experiments with
different batches of materials). This was further evidenced by
the greater AUC of L20K-IFN than that of DL20K-IFN (Table 1).
The in vivo efficacy of the conjugates was further evaluated in
two murine models, one bearing OVCAR-3 tumor xenografts
and the other xenografts derived from a prostate cancer patient
(PDX) (see Materials and Methods). In both cases,
administration of L20K-IFN, which carried the helical L-
P(EG3Glu), led to significantly slower tumor growth (Figure
1A). The superior antitumor efficacy was further confirmed by
the reduced tumor cell proliferation activity according to Ki-67
staining (Figure S7). No body weight loss was observed in
either model during the treatment with L20K-IFN, suggesting
that the drug was well tolerated under the regimen that we
employed (Figure S8).
Figure 3. In vivo immune responses triggered by GH conjugates. (A) SDS-PAGE gel analysis. (B) Trypsin degradation curves. (C−D) Anti-GH
IgG (C) and IgM (D) contents in the sera measured by ELISA; the plates were coated with wt-GH and then incubated with 104-fold (for IgG) or
500-fold (for IgM) prediluted sera in PBS. (E−F) Antipolymer IgG (E) and IgM (F) contents in the sera immunized with polymer-GH conjugates;
for each polymer of interest, the ELISA plates were coated with the corresponding polymer−IFN conjugate and then incubated with the 200-fold
prediluted sera. Immunization protocol: rats were s.c. infused with L20K-GH, DL20K-GH, or PEG20K-GH at a weekly dose 0.2 mg/kg for 4 weeks; sera
were drawn from the rats (n= 3) every week starting from week 0. For ELISA analysis, after sera incubation and washing, all plates were incubated
with antimouse IgG-HRP or IgM-HRP, and analyzed by TMB solution (CWBIO). TWEEN was excluded from the buffers in all antipolymer
ELISA studies. (G−I) Blood GH contents at selected time points, measured by ELISA, after the first and third s.c. injections of L20K-GH (G), DL20K-
GH (H), or PEG20K−GH (I). Data are expressed as mean ±SD. Pvalue is determined by two-way ANOVA (Bonferroni post-test) analysis: *p<
0.05, **p< 0.01, ***p< 0.001.
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Antibody Generation Triggered by IFN Conjugates.
To investigate the immune response of the conjugates,
Sprague−Dawley rats were randomly grouped and subcuta-
neously administrated with L20K-IFN, DL20K-IFN, or PEG20K−
IFN at a weekly dose of 0.2 mg/kg. Interestingly, sera from the
mice immunized with L20K-IFN showed significantly lower
levels of anti-IFN IgG and IgM than those receiving DL20K-IFN
or PEG20K−IFN (Figure 2A−B). Serial dilution of sera from
week 4 revealed that L20K-IFN produced ∼50−100 fold lower
anti-IFN IgG and ∼5−10 fold lower IgM titers than those
immunized with DL20K-IFN or PEG20K−IFN (Figure S9). In
addition, injection with DL20K-IFN or PEG20K−IFN appeared
to also induce a detectable amount of antipolymer antibodies,
particularly IgM (Figure 2C−D). The specificity of the
antipolymer antibodies in DL20K-IFN and PEG20K−IFN sera
was further validated by the corresponding polymer competi-
tion (Figure 2E−F). Strikingly, we discovered that L20K-IFN
exhibited almost no detectable effect on the serum level of
antipolymer IgG or IgM in the immunized rats.
Synthesis of and Immune Responses Triggered by
Different GH−Polymer Conjugates. To test whether our
findings observed in the IFN conjugates were also applicable to
other therapeutic proteins, we selected human growth
hormone (GH)
41,42
as our second example and engineered
the protein with a N-terminal cysteine (Cys-GH), similar to
that in Cys-IFN. We next covalently tethered L-P(EG3Glu), DL-
P(EG3Glu), and PEG separately to Cys-GH to generate three
conjugates denoted as L20K-GH, DL20K-GH, and PEG20K−GH,
respectively (Scheme 1 and Figure 3A). Trypsin digestion
revealed that L20K-GH was significantly more resistant to
proteolysis than DL20K-GH and PEG20K-GH (Figure 3B).
Furthermore, injection with L20K-GH provoked substantially
less production of anti-GH IgG and IgM antibodies in rats
from week 2, compared to treatment with DL20K-GH or
PEG20K-GH (Figure 3C−D). Serial dilution of sera from week
4 revealed that L20K-GH produced ∼100 fold lower anti-GH
IgG and ∼20-fold lower IgM titers than those immunized with
DL20K-GH or PEG20K−GH (Figure S10). The same trend was
observed when we measured the levels of antipolymer IgG and
IgM following the immunization (Figure 3E−F and Figure
S11). To examine the ABC effect, we measured the blood
concentration of GH at selected time points after the first and
third injection of each conjugate. The results demonstrated
that infusions of L20K-GH led to very similar blood levels of GH
during the first 12 h and generated almost no ABC effect in 24
h(Figure 3G, statistically insignificant). In sharp contrast, both
DL20K-GH and PEG20K−GH caused a characteristic ABC effect
after the third injection (Figure 3H−I). In fact, our ELISA kit
failed to detect blood GH at 24 h following the administration
of DL20K-GH or PEG20K−GH (Figure 3H−I). As a result, the
AUC0−24h of L20K-GH were comparable after the first and third
injection (100% vs 112%), whereas the AUC0−24h of both
DL20K-GH and PEG20K−GH after the third infusion were only
∼6% of those after the first drug infusion (Table S1).
BMDC Uptake and Activation. During antibody
production, the antigens are usually internalized, fragmented
Figure 4. BMDC internalization and activation. (A) Flow cytometry analysis of BMDC internalization of various FAM-labeled GH conjugates. (B−
D) Flow cytometry analysis of proinflammatory cytokines secretion: IL-6 (B), IFN-γ(C), and TNF (D). Freshly separated naï
ve BMDCs were ex
vivo incubated in 24-well plate (5 ×105cells/well) for 6 days and treated with conjugates for 12 h (A) or 24 h (B−D) at 37 °C. The cytokines in
the medium were measured with CBA Mouse Inflammation kit following manufacturer’s protocol. The PBS-treated BMDCs were served as
controls.
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in lysosome, and displayed on the cell surface by dendritic cells
(DCs) to trigger downstream T cell and B cell response. To
understand the different antibody responses triggered by the
conjugates,wesoughttoexaminetheveryfirst DC
internalization step. For this, we incubated the GH conjugates
with freshly induced immature mouse bone marrow-derived
dendritic cells (BMDCs), which are widely used for the
assessment of antigen presenting.
43
Flow cytometric analysis
found clear evidence for the internalization of DL20K-GH and
PEG20K−GH into BMDCs after 12 h of incubation, whereas
the uptake level of L20K-GH was considerably lower (Figure
4A). Consistently, treatment of BMDCs with L20K-GH resulted
in appreciably less secretion of proinflammatory cytokines,
including interleukine-6 (IL-6, Figure 4B), interferon-γ(IFN-γ,
Figure 4C), and tumor necrosis factor (TNF, Figure 4D),
compared to the other two GH conjugates carrying
unstructured polymers.
■DISCUSSION
The conjugation of polymers to a protein has been
demonstrated to extend its half-time by increasing its
hydrodynamic volume and mitigating the ADA generation.
1
However, the role that the secondary conformation of a
polymer plays in the resultant protein conjugate has been very
rarely investigated, as unstructured polymers have been the
heavily favored choice in past studies. Notably, the
polypeptide−uricase conjugate reported by Jiang focused on
the zwitterionic side chain without studying the secondary
conformation effect.
36
We speculated that peptide-based drugs
and biomaterials covalently modified with α-helical polypep-
tides could exhibit improved proteolytic and thermal stability,
binding, as well as other biological functions over those
conjugated with disordered polymers.
37,44−47
To ascertain
whether this is the case, however, one needs to employ
polymers that only differ in conformation. Gratifyingly,
controlled NCA ROP and chemoselective labeling collabo-
ratively enabled us to generate protein conjugates that shared
almost identical modification sites and MWs, and were
attached to nearly the same number of polypeptides with
highly similar chemical compositions.
34
As a result, the
secondary conformation of the tethered polypeptides became
the only major variable. This was corroborated by the GPC
curves of the polymers and the narrow size distributions of the
resultant conjugates on the SDS-PAGE gel (Figure S1,Figures
1A and 3A). Of note, due to the distinct chemical structures of
PEG and our P(EG3-Glu), the migration of those conjugates in
SDS-PAGE gel might not completely correlate their MWs,
which is often observed for other polymer modified proteins.
Our results found the helical polypeptide-bearing L20K-IFN
to have higher binding affinity and antiproliferative activity in
vitro than DL20K-IFN and PEG20K−IFN, both of which were
attached to unstructured polymers (Table 1). This could be
partially attributed to the less steric hindrance imparted by the
rigid helical polypeptides. Moreover, L20K-IFN exhibited
significant improvement in circulation half-life and in vivo
efficacy compared to DL20K-IFN (Figure 1D). Taken together,
these data suggested that the conjugation of a rigid helical
polypeptide could improve the blood retention of the modified
protein drug without significantly affecting its binding affinity
or potency, thereby offering a viable solution to the well-
known “PEG dilemma”.
11
Some of the greatest controversies of PEGylation include the
insufficient protection of the conjugated proteins from immune
recognition and the generation of anti-PEG antibodies.
13,48
In
the clinic, the anti-IFN neutralizing antibodies has previously
been observed in nonresponding patients and believed to be
the major reason for their development of resistance.
49
In this
regard, it was remarkable that the administration of L20K-IFN
provoked substantially lower production of anti-IFN, as well as
antipolymer IgG and IgM, than DL20K-IFN or PEG20K−IFN
(Figure 2). Importantly, similar results were also obtained from
the GH conjugates, indicating that the benefits we observed
were independent of the modified protein (Figure 3). We also
synthesized a left-handed helical polypeptide D-P(EG3Glu)
(∼23 kDa) and produced two conjugates, D20K-IFN and D20K-
GH (data not shown). We discovered that both D20K-IFN and
D20K-GH, similar to L20K-IFN or L20K-GH, showed almost no
antibody response after repeated administration (data not
shown). The results lent further evidence to the generality of
the helix effect. Moreover, the above study help ruling out the
possibility of D-amino acid-induced antibody production in the
cases of DL20K-IFN and DL20K-GH. Although the exact
mechanistic role of helicity remains insufficiently explored, a
number of reasons may count for the unexpected findings. First
of all, the helical L-P(EG3Glu) seems to provide better
antifouling property than DL-P(EG3Glu) and PEG, and thus
minimizing nonspecific internalization with cells and proteins.
Our initial investigation provided preliminary evidence of
conformation-dependent internalization and activation of
immature BMDCs for those examined protein−polymer
conjugates. In fact, this helical conformation enhanced
antifouling and anticell adhesion was also observed when the
polypeptides were anchored on gold surfaces.
50
Second, helical
polypeptides are well-known more proteolytic stable (Figure
3B) than those unstructured peptidyl analogues, which may
lead to inefficient fragmentation and MHC presentation after
BMDC internalization. More rigorous experimental and
modeling studies are currently ongoing to fully uncover the
molecular mechanism of the unusual helical conformation
effect.
■CONCLUSIONS
In conclusion, we generated synthetic polypeptides that only
differed in conformation and compared their effects on the in
vivo therapeutic and immunological properties of the protein
drugs to which they were conjugated. Compared with the
unstructured DL-P(EG3Glu) or PEG, the covalent attachment
of the helical L-P(EG3Glu) to therapeutic proteins (namely,
IFN and GH) led to substantial improvement in a variety of
pharmacological properties, such as binding affinity, stability,
and in vivo efficacy. Most interestingly, the helical L-
P(EG3Glu)-conjugated IFN and GH elicited a significantly
milder immune response and exhibited a much weaker ABC
effect than those modified with unstructured polymers. Thus,
the helical nonfouling polypeptides that we employed could be
excellent alternatives to PEG for mitigating the antibody
response to repeatedly administrated therapeutic proteins,
though whether similar benefits apply to more immunogenic
foreign proteins requires further validation. Moreover, our
results suggested that the helical conformation of the synthetic
nonfouling polypeptides played an important role in
minimizing/delaying this antibody response. Taken together,
the current study highlighted an urgent necessity to system-
atically reassess the pros and cons of choosing unstructured
polymers for protein conjugation. Furthermore, our results also
ACS Central Science Research Article
DOI: 10.1021/acscentsci.8b00548
ACS Cent. Sci. XXXX, XXX, XXX−XXX
F

lay the foundation for the development of next-generation
biohybrid drugs based on helical synthetic polypeptides.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscents-
ci.8b00548.
GPC curves, 1H NMR, CD spectra, trypsin degradation
curves, SPR binding curves, Ki-67 stained images of
tumor, relative body weight of mice, antibody titer
curves, AUC0−24h of GH-polymer conjugates at first and
third dose (PDF)
■AUTHOR INFORMATION
Corresponding Author
*E-mail: chemhualu@pku.edu.cn.
ORCID
Zhuang Liu: 0000-0002-1629-1039
Hua Lu: 0000-0003-2180-3091
Funding
This work was financially supported by National Key Research
and Development Program of China (2016YFA0201400). We
acknowledge grants from National Natural Science Foundation
of China (21474004 and 21722401). H.L. acknowledges the
startup funding from Youth Thousand-Talents Program of
China.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
We thank Prof. Demin Zhou for sharing the plasmid encoding
the wt-GH, Prof. Wei Wei for the help of BMDC culturing,
and Prof. Richard Lerner, Prof. Xing Chen, Prof. Feng Wang
for helpful discussion.
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