Selective Uptake of Cylindrical Poly(2-Oxazoline) Brush-AntiDEC205 Antibody-OVA Antigen Conjugates into DEC-Positive Dendritic Cells and Subsequent T-Cell Activation

Article abstract of DOI:10.1002/chem.201403942

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well-defined stru

Full text of DOI:10.1002/chem.201403942

DOI: 10.1002/chem.201403942
Communication
&
Cancer Therapy
Selective Uptake of Cylindrical Poly(2-Oxazoline) Brush-
AntiDEC205 Antibody-OVA Antigen Conjugates into DEC-Positive
Dendritic Cells and Subsequent T-Cell Activation
Jasmin Bꢀhler,[a, b] Sabine Gietzen,[a] Anika Reuter,[a, c] Cinja Kappel,[e] Karl Fischer,[a]
Sandra Decker,[a] David Schꢁffel,[d] Kaloian Koynov,[d] Matthias Bros,*[e] Ingrid Tubbe,[e]
Stephan Grabbe,*[c, e] and Manfred Schmidt*[a, b, c]
Cylindrical polymer brushes have become increasingly popu-
lar because of their anisotropic character and the recent results
on shape dependent endocytosis.[9–12] Furthermore, polymeric
Abstract: To achieve specific cell targeting by various re-
ceptors for oligosaccharides or antibodies, a carrier must
brushes may offer a multiplicity of functional groups which
not be taken up by any of the very many different cells
are advantageous for conjugation of biologically active
and needs functional groups prone to clean conjugation
compounds.
chemistry to derive well-defined structures with a high
Several publications report cylindrical brushes with poly(2-
biological specificity. A polymeric nanocarrier is presented
oxazoline) side chains prepared by “grafting from”[13,14] and
that consists of a cylindrical brush polymer with poly-2-ox-
“grafting through”[15–17] techniques. Recently our group pub-
azoline side chains carrying an azide functional group on
lished the synthesis of cylindrical brushes with poly(2-isopro-
each of the many side chain ends. After click conjugation
pyl-2-oxazoline) side chains by grafting through with unprece-
of dye and an anti-DEC205 antibody to the periphery of
dented high main chain degrees of polymerization.[18] All of
the cylindrical brush polymer, antibody-mediated specific
binding and uptake into DEC205+-positive mouse bone
the cylindrical brushes with poly(2-oxazoline) side chains re-
ported to date do not contain functional groups for further
whereas binding and uptake by DEC205ꢀ negative BMDC
conjugation experiments except for one work in which
marrow-derived dendritic cells (BMDC) was observed,
functionalized polymers were prepared though with a main-
and non-DC was essentially absent. Additional conjuga-
chain degree of polymerization as low as only 13.[19] However,
tion of an antigen peptide yielded a multifunctional poly-
azide functionalized linear poly(2-oxazoline)s have been
described.[20–24]
mer structure with a much stronger antigen-specific T-cell
stimulatory capacity of pretreated BMDC than application
To assess the suitability of the cylindrical brushes described
of antigen or polymer–antigen conjugate.
herein to serve as nanocarriers for immuno-therapeutic ap-
proaches, their binding and uptake by DC was analyzed, be-
cause DC represent an important immune cell population. In
The ideal nanocarrier for biomedical applications is not cyto-
toxic, has a size between 10 and 100 nm, and does not form
aggregates in blood serum that are due to strong interactions
with the numerous proteins and enzymes present in the com-
plex biological fluids.[1] Poly(2-oxazoline)s are excellent candi-
dates for this purpose,[2] because they are known for their low
cytotoxicity,[3] biocompatibility,[4,5] stealth behavior,[6,7] and low
protein adsorption from human blood.[8]
their activated state, DC constitute the most potent antigen-
presenting cells of the immune system that are solely able to
initiate primary immune responses.[25] Of the several DC subpo-
pulations known, CD8+ DC that co-express the C-type lectin
receptor DEC205 bear the highest potential to activate cyto-
toxic T lymphocytes.[26] Conjugation of anti-DEC205 with anti-
gen and adjuvant resulted in partial loss of targeting activity,[27]
whereas conjugation of an antigen only was shown to main-
[a] Dr. J. Bꢀhler,+ S. Gietzen,+ Dr. A. Reuter, Dr. K. Fischer, S. Decker,
Dr. M. Schmidt
[d] D. Schꢁffel, Dr. K. Koynov
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
Institute for Physical Chemistry, University of Mainz
Jakob-Welder Weg 11, 55099 Mainz (Germany)
E-mail: mschmidt@uni-mainz.de
[e] C. Kappel, Dr. M. Bros, I. Tubbe, Dr. S. Grabbe
Department of Dermatology
[b] Dr. J. Bꢀhler,+ Dr. M. Schmidt
University Medical Center of the Johannes Gutenberg
University Mainz
Langenbeckstrasse 1, 55131 Mainz (Germany)
E-mail: mbros@uni-mainz.de
Graduate School Materials Science
Staudinger Weg 9, 55128 Mainz (Germany)
[c] Dr. A. Reuter, Dr. S. Grabbe, Dr. M. Schmidt
Max Planck Graduate Center
stephan.grabbe@unimedizin-mainz.de
[+] These authors contributed equally to this work.
Staudinger Weg 9, 55128 Mainz (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403942.
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tain DEC205 receptor mediated binding and uptake of antigen
by this DC subpopulation.[28,29]
Table 1. Characterization of the macromonomers.
Polymer–antibody rather than polymer–antibody fragment
conjugates are less frequently reported.[30–32] Antithymocyte
globuline,[33] polyclonal human immunoglobulin, and monoclo-
nal anti-RAGE antibody[34] were successfully conjugated to
linear flexible hydroxypropylmethacrylate (HPMA) chains with
no or little loss of antibody activity. Also polymeric capsules of
a few mm in size were decorated by a few hundred thousand
humanized A33 monoclonal antibodies were reported to selec-
tively address human colorectal cancer cells.[35,36]
N3-PiPrOx
N3-PEtOx
N3-PEtOx-b-PiPrOx
Mw/Mn (MALDI/GPC)
Mn (MALDI) [gmolꢀ1
1.12/1.13
4707
14800[a]
4946
5292
40
1.20/1.16
3623
13400[a]
3694
4330
33
–/1.1
–
18900*
5869
–
]
Mn (GPC) [gmolꢀ1
]
Mn (1H NMR) [gmolꢀ1
w (MALDI) [gmolꢀ1
Pn
]
M
]
61
[a] GPC with PMMA calibration yields values that are too large.
Herein, the binding and uptake properties of bare cylindrical
brushes were investigated in comparison to cylindrical brushes
conjugated with a DEC205-specific antibody, when co-incubat-
ed with DC.
butions owing to the different block lengths. For further dis-
cussion we will use the 1H NMR-determined molar masses.
All the three azide-functionalized macromonomers were
polymerized in highly concentrated aqueous solutions at 628C.
The IR spectra of all azide end-functionalized brushes show the
characteristic band at 2100 cmꢀ1 (Supporting Information, Fig-
ure S12). After reduction with tris(2-carboxyethyl)phosphine,
the azide bands disappeared (Supporting Information, Fig-
ure S13).
The synthesis is summarized in Scheme 1. The synthesis of
the azide-functionalized poly(2-oxazoline) macromonomers
was performed similar to the poly(2-isopropyl-2-oxazoline)
macromonomer synthesis published recently[18] and is de-
scribed in some detail in the Supporting Information. Three dif-
ferent azide-functionalized macromonomers were synthesized
and characterized by NMR and IR spectroscopy, GPC, and
MALDI-TOF spectrometry (Supporting Information, Figures S1–
S11). The results are summarized in Table 1.
Polymerization of block co-macromonomers leads to core–
shell cylindrical brushes;[37] that is, polymerization of N3-poly(2-
ethyl-2-isopropyl-2-oxazoline) macromonomers yields a core–
shell structure with a core consisting of slightly hydrophobic
poly(2-isopropyl-oxazoline) chains and a corona of hydrophilic
poly(2-ethyl-oxazoline) chains. Representative DLS and SLS
measurements of the cylindrical brushes with N3-poly(2-ethyl-
2-isopropyl-2-oxazoline) side chains are shown in Figure 1 (for
Zimm plots of the other cylindrical bushes, see the Supporting
Table 1 reveals the molar masses determined by MALDI-TOF
and NMR to be similar. The ratio of the 2-ethyl-2-oxazoline
block to 2-isopropyl-2-oxazoline block of the N3-poly(2-ethyl-2-
isopropyl-2-oxazoline) macromonomer was determined by
1H NMR to be 60% to 40%. MALDI-TOF was not measured for
the block co-macromonomer as there would be different distri-
Scheme 1. Synthesis of azide-functionalized poly(2-oxazoline)s.
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Figure 2. AFM image (height) of azide end-functionalized core–shell poly(2-
oxazoline) brush N3-poly(2-ethyl-block-2-isopropyl-2-oxazoline) spin-cast
onto mica from aqueous solution at c=0.1 gLꢀ1
.
Recently our group developed a method that allows the de-
termination of aggregation behavior of nanoparticles in
human blood serum by dynamic light scattering.[38] The corre-
lation function of nanoparticles or polymers in serum solution,
g1(t)mix, should be well-fitted the adequate weighted sum of
known correlation functions measured from the polymer in
isotonic solution, g1(t)P and of undiluted serum, g1(t)s:
g1ðtÞmix ¼ aP g1ðtÞP þ aS g1ðtÞs þ ðaA g1ðtÞAÞ
ð1Þ
Figure 1. a) Zimm plot and b) reciprocal hydrodynamic radius of the azide
end-functionalized cylindrical core–shell brush N3-poly(2-ethyl-block-2-iso-
propyl-2-oxazoline) in methanol with 5 mm added LiBr at 208C.
where aP and aS represent the amplitudes as the only fit
parameters.
In the case of aggregation, a third correlation function de-
scribing the aggregate, g1(t)A, has to be added:
g1ðtÞmix ¼ aP g1ðtÞP þ aS g1ðtÞS þ aA g1ðtÞA
ð2Þ
Table 2. Light scattering characterization including the degree of poly-
merization Pw of the azide end-functionalized brush polymers with
poly(2-oxazoline) side chains.
Figure 3 shows the autocorrelation function of the mixture
of serum and N3-PiPrOx brush (a) as well as the mixture of
serum and N3-PEtOx-b-PiPrOx brush (b). For the latter sample,
the data points of the mixture are well-described by the force
fit with the sum of individual correlation functions of serum
and polymer brush, meaning no or negligible aggregation has
taken place. Similar results were obtained for the N3-PEtOX
brush (data not shown).
Rg
Rh
1=Rg/ Mw (LS)
dn/dc
Pw Mw/Mn
(LS) (GPC)
[nm] [nm] Rh
[gmolꢀ1
]
[cm3 gꢀ1
]
N3-PiPrOx-brush 37.4 26
N3-PEtOx-brush 32.3 21
1.44
1.54
1.44
1.3·106
6.2·105
3.2·106
0.163
0.177
0.177
254 2.2
167 1.7
547 2.4
N3-PEtOx-b-
PiPrOx-brush
60.5 42
In contrast, the autocorrelation function of the mixture of
serum and N3-PiPrOx-brush could not be perfectly fitted by the
force fit with the sum of individual correlation functions of
serum and polymer brush (Eq. (1); Figure 3 black line). A third
correlation function is necessary to achieve a perfect fit
(Eq. (2); Figure 3, gray line) and indicates a significant amount
of aggregates of 360 nm radius to be present, which may be
caused by the higher hydrophobicity of the N3-PiPrOx-brush.
Hydrophobic proteins such as lipoproteins may interact with
the polymer brush, leading to aggregation, although the
extent of aggregation is quite small in view of the “intensity
weighting” of DLS.
Information, Figure S14). The results are summarized in Table 2.
GPC calibrated by PMMA standards revealed polydispersities
Mw/Mn ꢁ2 for all samples, as expected (Supporting Informa-
tion, Figure S11).
AFM (Figure 2; Supporting Information, Figure S15) illus-
trates the expected worm-like structures in qualitative agree-
ment with the results obtained by static and dynamic light
scattering. The N3-poly(2-ethyl-2-oxazoline) brush shows be-
sides worm-like structures a multiplicity of spherical particles
resulting in the much lower molecular weight compared to
the other two brushes.
None of the cylindrical brush polymers exerted inhibitory ef-
fects on the metabolic activity of both human HEK293 and
mouse BMDCs to any cell-type specific extent, that is, the 50%
cell survival concentration was determined to be well above
1 mgmLꢀ1 (Supporting Information, Table S3).
The data above reveal that the N3-EtOx and the N3-PEtOX-b-
PiPrOx brushes do not form aggregates in concentrated blood
serum, which could provoke unwanted uptake by macrophag-
es and negatively influence the circulation time for later in
vivo experiments.
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Figure 3. Autocorrelation functions of a) N3-PiPrOx and b) N3-PEtOX-b-PiPrOx
brushes in human blood serum; gray lines represent the force fits with the
sum of the individual correlation functions of serum and polymer brush ac-
cording to Eq. (1); black lines represent the fits according to Eq. (2) account-
ing for the presence of aggregates; scattering angle 308.
Dibenzocyclooctyne-modified antiDEC205 antibody was con-
jugated to the N3-poly(2-ethyl-2-isopropyl-2-oxazoline) brush
utilizing copper-free 1–3 dipolar cycloaddition chemistry (Sup-
porting Information, Scheme S2).[39] According to UV/Vis spec-
troscopy, approximately 8 to 10 antibodies were bound to one
cylindrical brush polymer (Supporting Information, Figure S17).
No free antibody could be detected in the conjugate by gel
electrophoresis (Supporting Information, Figure S18).
Figure 4. Binding and uptake of N3-poly(2-ethyl-block-2-isopropyl-2-oxazo-
line brush–aDEC205 conjugates by BMDC. Unstimulated BMDC were co-in-
cubated for 4 hours in parallel with Carboxy-Rhodamine110-labeled N3-
PEtOx-b-PiPrOx-brush, unconjugated or conjugated with anti-DEC205 anti-
body (1012 particles per 5ꢃ105 cells). a) BMDCs were double-stained with
DEC205 and CD11c specific antibodies. The dot plot shows the distribution
of the different subpopulations of BMDC co-incubated with anti-DEC205 an-
tibody conjugated polymer. b)–d) Curves show different evaluations of one
experiment and indicate cellular binding of anti-DEC205 antibody conjugat-
ed (thick solid line) and unconjugated polymer (thin dotted line) by the dif-
ferent cell populations. The corresponding MFI are given in brackets:
b) CD11c+DEC205+ (MFI: 152.6 versus 44.6), c) CD11c+DEC205ꢀ: (MFI: 20.1
vs. 19.5), and d) CD11cꢀ non-DCs (MFI: 8.1 vs. 7.2). Graphs are representative
of 10 experiments. e),f) CLSM pictures of the polymer-DEC205 conjugates
taken up by DCs. The polymer is labeled by Carboxyrhodamin (green), the
DEC205 by AF647 (red). The orange color shows superposition of polymer
and DEC205, that is, the intact conjugate. Hoechst 33342 (blue) was utilized
to label cell nuclei. Scale bars 25.3 mm (e) and 10.6 mm (f).
Fluorescence-activated cell sorting (FACS) showed that 29%
of the CD11+ BMDC population co-expressed DEC205, while
33% of the CD11+ BMDC populations lacked DEC205 expres-
sion, and 37% of the cells were CD11cꢀ negative; that is, were
no DCs (Figure 4a). The mean fluorescence intensity (MFI) of
either CD11c+ BMDC population as well as of non-DC co-incu-
bated with N3-PEtOx-b-PiPrOx-brushes for 4 h was rather low,
as shown by the dotted lines in Figure 4b–d. When co-incubat-
ed with the polymer–antibody conjugate, a three-fold higher
MFI was observed for DEC205+CD11c+ BMDC (solid line in Fig-
ure 4b). In contrast, neither DEC205ꢀCD11c+ BMDC nor non-
DC showed any considerable increase in polymer binding
(solid lines in Figure 4c,d). Confocal laser scanning microscopy
(CLSM) images confirmed this result and revealed polymer and
antibody to be co-localized inside of DEC205+CD11c+ BMDCs
(Figure 4e,f). The corresponding FACS data and CLSM pictures
for BMDC co-incubated with unconjugated polymer brushes
are given in the Supporting Information, Figure S21.
ed with native anti-DEC205 antibody at large excess prior to
addition of polymer antibody conjugates (Supporting Informa-
tion, Figure S22).
To demonstrate a potential biological application of the cy-
lindrical brush conjugates, the SIINFEKL-sequence of the OVA–
antigen was additionally conjugated to the cylindrical brush
polymer. As described in detail in the Supporting Information,
Scheme S3, the AF546-labeled C-SGLEQLE-SIINFEKL oligopep-
tide (AG, derived from the ovalbumine antigen) was conjugat-
ed to the N3-poly(2-ethyl-block-2-isopropyl-2-oxazoline cylin-
drical brush (CB) first, followed by conjugation of DBCO-func-
After 4 h, 73% of the DEC205+ BMDCs had engaged conju-
gate. It should be noted that cellular engagement of polymer
antibody conjugates was blocked, if the BMDCs were incubat-
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tionalized anti-DEC205 antibody (sample CB-AG-aDEC205). The
trailer sequence SGLEQLE is known to be enzymatically cleava-
ble by cells[40] and cysteine was added for the conjugation re-
action. On average, the final conjugate contained 17 antigen
fragments and 7.5 anti-DEC205 molecules (Supporting Informa-
tion, Figures S19 and S20).
several tens of antigen and/or adjuvant molecules to yield
a higher quantity of cargo delivered on a per cell base. The ap-
plied click chemistry yields almost quantitative conjugation re-
sults and allows for the attachment of a predictable number of
even different peptide antigens, which may serve to induce
both CD4+ and CD8+ T-cell responses at the same time. Like-
wise, conjugation with different adjuvants that trigger distinct
signaling pathways may serve to exert synergistic effects in
terms of DC activation, and therefore the extent of subsequent
T-cell stimulation.[46]
The suitability of CB-AG-aDEC205 to confer efficient uptake
of antigen into BMDCs was monitored by a proliferation of
peptide (or SIINFEKL) reactive CD8+ T-cells. As shown in
Figure 5, BMDC pre-incubated for 4 h with the CB-AG-aDEC205
conjugate (no. 4) induced much stronger proliferation of sub-
Acknowledgements
Financial support by the DFG graduate school MAINZ (J.B.), by
the Max Planck Graduate Center, Mainz, and by the German
Science Foundation (SFB625, SFB1066) is gratefully acknowl-
edged. CLSM measurements were performed at the Institute
for Molecular Biology, Mainz. Some dye labeling experiments
were conducted by Meike Schinnerer, Institute of Physical
Chemistry, University Mainz, which is gratefully acknowledged.
Figure 5. Antigen-specific CD8+ T-cell stimulatory capacity of BMDCs pre-in-
cubated with the cylindrical brush conjugate CB-AG-aDEC205. In parallel
assays, unstimulated BMDCs (each 5ꢃ105 cells) were incubated for 4 h with
C-SGLEQLE-SIINFEKL peptide (1), with non-conjugated N3-poly(2-ethyl-block-
2-isopropyl-2-oxazoline cylindrical brushes (2), with cylindrical brush-antigen
conjugate (3), and with CB-AG-aDEC205 carrying both aDEC205 and C-
SGLEQLE-SIINFEKL (4), at equimolar concentration of antigen (no. 1, 3, and
4: 1012 peptide molecules) or equal number of cylindrical brush polymer
chains (no. 2 and 4).
Keywords: brush polymers · cancer therapy · dendritic cells ·
nanocarriers · T-cells
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Chem. Eur. J. 2014, 20, 12405 – 12410
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