Enzyme-Mediated Modification of Single-Domain Antibodies for Imaging Modalities with Different Characteristics

Article abstract of DOI:10.1002/anie.201507596

Antibodies are currently the fastest-growing class of therapeutics. Although naked antibodies have proven valuable as pharmaceutical agents, they have some limitations, such as low tissue penetration and a long circulatory half-life. They have been conjugated to toxic payloads, PEGs, or radioisotopes to increase and optimize their therapeutic efficacy. Although nonspecific conjugation is suitable for most in vitro applications, it has become evident that site specifically modified antibodies may have advantages for in vivo applications. Herein we describe a novel approach in which the antibody fragment is tagged with two handles: One for the introduction of a fluorophore or ¹⁸F isotope, and the second for further modification of the fragment with a PEG moiety or a second antibody fragment to tune its circulatory half-life or its avidity. Such constructs, which recognize Class II MHC products and CD11b, showed high avidity and specificity. They were used to image cancers and could detect small tumors.

Full text of DOI:10.1002/anie.201507596

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International Edition: DOI: 10.1002/anie.201507596
German Edition: DOI: 10.1002/ange.201507596
Cancer Imaging
Enzyme-Mediated Modification of Single-Domain Antibodies for
Imaging Modalities with Different Characteristics
Mohammad Rashidian+, Lu Wang+, Jerre G. Edens, Johanne T. Jacobsen, Intekhab Hossain,
Qifan Wang, Gabriel D. Victora, Neil Vasdev,* Hidde Ploegh,* and Steven H. Liang*
Abstract: Antibodies are currently the fastest-growing class of
therapeutics. Although naked antibodies have proven valuable
as pharmaceutical agents, they have some limitations, such as
low tissue penetration and a long circulatory half-life. They
have been conjugated to toxic payloads, PEGs, or radio-
isotopes to increase and optimize their therapeutic efficacy.
Although nonspecific conjugation is suitable for most in vitro
applications, it has become evident that site specifically
modified antibodies may have advantages for in vivo applica-
tions. Herein we describe a novel approach in which the
antibody fragment is tagged with two handles: one for the
introduction of a fluorophore or 18F isotope, and the second for
further modification of the fragment with a PEG moiety or
a second antibody fragment to tune its circulatory half-life or
its avidity. Such constructs, which recognize Class II MHC
products and CD11b, showed high avidity and specificity. They
were used to image cancers and could detect small tumors.
positron emission tomography (FDG-PET), which distin-
guishes areas of high metabolic activity, such as tumors, from
surrounding tissue with lesser glucose uptake.[4] These meth-
ods do not usually provide information on immune cells in the
tumor microenvironment. There are now tools to track
immune cells, by the use of isotopically labeled anti-CD11b,
anti-Class-II-MHC, and anti-CD8 antibody fragments.[5–7]
The comparatively large size of intact full-sized antibodies
results in a long circulatory half-life, and may also hinder
efficient tissue penetration.[8] These considerations have
driven the search for smaller antibody-derived formats as
alternative imaging tools.[1,6] We generated camelid single-
domain antibody fragments (VHHs) as the smallest antigen-
binding derivatives obtainable from naturally occurring anti-
bodies.[9] VHHs lend themselves to enzymatic modification
and have been used in a variety of applications, including
imaging.[10]
The relationship between the affinity and valency of the
antibodies or their fragments, and their suitability for various
imaging applications has received scant attention.[1,11] The
production of bivalent single-domain antibodies based on
their monovalent equivalents could address issues of avidity,
while retaining desirable properties, such as small size. For
example, the bivalent derivatives of single-domain antibodies
might still be small enough to penetrate tissues, be rapidly
cleared from the circulation, yet benefit from increased
avidity. On the other hand, tuning of the circulatory half-life
could improve the efficiency with which VHHs stain their
targets. The attachment of small PEG groups could be used as
a tool to “tune” the persistence of a VHH in the circulation.
Moreover, PEGylation can decrease the immunogenicity of
VHHs, which is important in cases in which repeated
administration is required; however, in rare cases even the
PEG functionality itself has been suggested to be immuno-
genic.[12]
Structures of VHHs show that their C terminus is
positioned away from the antigen-binding site.[13] We there-
fore chose a chemical approach to link two fully functional
VHHs through their C termini to ensure that their antigen-
binding capacity would not be compromised by modification
of one of the N termini in the resulting fusion, and that the
two binding sites thus created would be truly equivalent,
which would be more difficult to ascertain for genetic fusions.
Four sortase substrates were designed and synthesized for
the production of dimers or PEGylated VHHs (Figure 1). The
substrates either contained two bioorthogonal handles or
a handle and a fluorophore. An Alexa647-labeled substrate, 2,
was designed in such a way that the reaction products could be
used in fluorescence-activated cell sorting (FACS) experi-
T
he success of immunotherapies, such as the application of
monoclonal antibodies against the immune checkpoint inhib-
itors CTLA4 and PD-1 on T cells and PD-L1 on their targets
cannot be viewed separately from the contributions of
myeloid cells, which are often present at the tumor margin,
and express Class II MHC and/or CD11b products. Macro-
phages can affect tumor growth by establishing either
a detrimental or a favorable microenvironment. Thus, the
ability to image myeloid cellsꢀ presence is of diagnostic
relevance and, compared to tumor-specific markers,[1] may be
a more generally applicable approach for detection of tumor
cells.[2,3] A commonly used minimally invasive clinical diag-
nostic approach is the use of 2-[18F]fluoro-2-deoxy-d-glucose
[*] Dr. M. Rashidian,[+] J. G. Edens, Dr. J. T. Jacobsen, I. Hossain,
Dr. G. D. Victora, Prof. Dr. H. Ploegh
Whitehead Institute for Biomedical Research
Cambridge, MA 02142 (USA)
E-mail: ploegh@wi.mit.edu
L. Wang,[+] Q. Wang, Prof. Dr. N. Vasdev, Prof. Dr. S. H. Liang
Division of Nuclear Medicine and Molecular Imaging
Massachusetts General Hospital and
Department of Radiology, Harvard Medical School
Boston, MA 02114 (USA)
E-mail: vasdev.neil@mgh.harvard.edu
liang.steven@mgh.harvard.edu
Prof. Dr. H. Ploegh
Department of Biology, Massachusetts Institute of Technology
Cambridge, MA 02142 (USA)
[+] These authors contributed equally.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507596.
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Figure 1. A) Site-specific labeling of VHHs with sortase, which recognizes the “LPXTG” motif and replaces the G residue with a substrate
containing a NH2-G3 moiety. B) Structures of the substrates containing bioorthogonal functionalities. The structures were confirmed by LC–MS.
ments to estimate relative in vitro binding affinities; a sub-
strate modified with Texas Red, 3, was designed to enable
two-photon microscopy and the estimation of relative in vivo
binding affinities; and a trans-cyclooctene-modified substrate,
4, was produced to enable rapid installation of a 18F-labeled-
tetrazine radioactive tag for positron emission tomography
(PET; 18F t1/2 < 2 h). The dimers were produced accordingly
(Figure 2; see also the Supporting Information).
For DC8 (anti-Class-II), splenocytes were stained with
different concentrations of monomers and dimers and ana-
lyzed by FACS. For DC13 (anti-CD11b), the CD11b+
mutuDC dendritic cell line was used. For Class II MHC and
CD11b, the VHH dimers were found to bind approximately
3.3- and 2.3-fold more strongly than the corresponding
monomers, respectively (Figure 3).
We next evaluated the in vivo binding characteristics of
the VHH dimers versus monomers. Due to the more
abundant expression of Class II MHC molecules on spleno-
cytes as compared to that of CD11b, we explored the in vivo
binding affinity of anti-Class-II dimers. Mice were injected
intravenously (i.v.) with equal amounts of monomers and
dimers (0.25 nmol) of DC8. The mice were euthanized 2 h
postinjection (p.i.), and lymphoid organs were excised for
examination. The monomers and dimers yielded different
staining patterns (Figure 3B). The dimer showed interspersed
yet more pronounced staining than the monomer, thus
corroborating the FACS results.
To render monomers and dimers suitable for immuno-
PET, we produced dimer VHHs by using substrate 4. To
produce the 18F-labeled tetrazine, we applied a method that
was automated in its key steps to minimize operator radiation
exposure in the course of preparation (Figure 4A; see the
Supporting Information). Next, we produced 18F-labeled anti-
Class-II and anti-CD11b dimers and used them for PET
imaging (Figure 4). PET showed that both dimers stained
lymphoid organs (Figure 4; see also movies 1 and 2 in the
Supporting Information). The anti-Class-II-MHC dimer
showed stronger staining of lymphoid organs, particularly
the spleen, as compared to the anti-CD11b dimer (Fig-
ure 4D,F). We attribute this behavior to the fact that in the
spleen there are fewer CD11b+ cells than Class II MHC+ cells,
and less CD11b expressed per cell. We established specificity
by blocking the targets of these VHHs by the introduction of
unlabeled VHHs as competitors prior to imaging. PET
imaging conducted 2 h after the injection of 18F-labeled
VHHs showed effective blocking of the signals in the
lymphoid organs, thus further underlining the specificity of
the signals (Figure 5C; see also movies 3–6). To further
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Figure 2. A,B) Synthesis of VHH derivatives. C,D) Characterization of the products. The LC–MS and SDS-PAGE analysis for DC13 is shown (see
the Supporting Information for DC8). Lane 1 in (C): marker, lane 2: DC13, lane 3: DC13–DBCO, lane 4: DC13–azide–Texas Red, lane 5: DC13–
dimer–Texas Red, lane 6: DC13–N3–Alexa647, lane 7: DC13–dimer–Alexa647. E,F) PEGylation of VHHs and characterization. Lane 1 in (F): marker,
lane 2: DC8–Alexa647–N3, lane 3: DC8–Alexa647–PEG (5 kDa), lane 4: DC8–Alexa647–PEG (10 kDa), lane 5: DC8–Alexa647–PEG (20 kDa). All
materials were purified by size exclusion chromatography. DBCO=dibenzocyclooctyne.
confirm the specificity of the dimers in the absence of
competing VHHs, we imaged MHC IIÀ/À and CD11bÀ/À mice.
We detected no PET signals in lymphoid organs, thus further
confirming the specificity of both dimers (Figure 4E,G; see
also movies 7 and 8).
commensurate increase (Figure 5A,B). We attribute this
observation to the larger size of the dimer as compared to
the monomer, which may result in lower penetration in
tumors, thus counteracting the effect of the higher affinity of
the dimer.
The monomers and dimers did not differ in uptake into
the kidneys and intestine, organs commonly targeted non-
specifically by VHHs in the course of their clearance.[14] These
experiments helped us set the stage to explore the ability of
VHH dimers to image tumors. We imaged two types of
tumors with the developed bivalent VHHs by the engraft-
ment of mice with the B16 melanoma or the pancreatic tumor
cell line panc02. Both dimers readily detected the lymphoid
organs as well as the B16 melanoma tumor (see movies 9 and
10) or the panc02 graft (see movies 11 and 12). Upon excision,
the panc tumors were no more than about 1–2 mm in
diameter, thus showing the method to be sensitive. Post-
mortem biodistribution analysis also correlated well with the
FACS and two-photon results (Figure 5A,B). Although the
dimers stained secondary lymphoid organs more efficiently
than their corresponding monomers, specifically for the anti-
class-II-MHC VHH, the PET signals in the tumors showed no
We examined whether we could manipulate circulatory
half-life to further improve the signal-to-noise ratio. PEGy-
lation of VHHs results in increased circulatory half-life, and
the increase correlates with the size of the attached PEG
group.[15] VHHs sortagged with substrate 2 or 3 were treated
with DBCO-functionalized PEG (5, 10, or 20 kDa) to yield
the final PEGylated VHHs (Figure 2E,F). B6 mice received
5 mg of PEGylated VHH and were euthanized 3 h later,
followed by dissection of the lymph nodes and spleen for
FACS and two-photon microscopy. FACS showed a significant
increase in staining of the PEGylated DC8–Alexa647 versus
the non-PEGylated DC8. We observed an approximately 30,
100, and 220% increase in staining for 5, 10, and 20 kDa PEG-
conjugated DC8, respectively, thus establishing a correlation
between the size of the attached PEG moiety and staining
efficiency (Figure 3D). When we injected DC8–Alexa647–
PEG (20 kDa), which showed the strongest binding in vivo,
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Figure 3. A,B) Dimers were able to stain their targets with increased efficiency as compared to the corresponding monomers. A) Cells were
stained in vitro with the indicated concentrations of Alexa647-labeled VHH monomers or dimers. B) Equal amounts of the DC8 monomer and
DC8 dimer were injected i.v.; the mice were euthanized 2 h p.i., and their spleens were excised for two-photon imaging. C) Equal amounts of DC8
with or without PEG, both equipped with TexasRed were injected i.v. into mice; spleens were excised 3 h p.i. for two-photon imaging. D) Equal
amounts of DC8 with or without PEG, all equipped with Alexa647, were injected i.v. into mice; spleens were excised 3 h p.i., and cells were
harvested and analyzed by FACS. WT=wild type.
into a MHC IIÀ/À mouse, FACS analysis showed no staining in
the spleen (Figure 3D). This result confirmed that PEGyla-
tion does not affect the specificity of the VHHs.
full retention of the biological activity of the fusion partners.
By cytofluorimetry, dimers bind approximately 3 times more
avidly to their targets. Two-photon microscopy confirmed that
bivalent single-domain derivatives labeled with Texas Red
bind more efficiently to their targets. PEGylated fluorescent
VHHs showed improved staining in vivo, with larger PEG
substituents giving stronger signals in FACS. In immuno-PET
experiments, bivalent 18F-labeled VHHs stained lymphoid
organs in vivo more effectively than monomers. 18F-labeled
PEGylated VHHs showed improved staining, whereby PEG
substituents with a higher molecular weight gave stronger
signals. Finally, immuno-PET of two different models of
tumor-bearing mice showed that the DC8 dimer and DC13
dimer detected not only lymphoid organs, as expected, but
also showed the location of ectopic melanoma and pancreatic
tumor grafts. Overall, this study provides further support for
the notion that derivatives of single-domain antibodies are
a valuable addition to the available imaging and radiodiag-
nostic toolbox.
We then explored the PEGylated VHHs for their
suitability for PET imaging. VHH DC8 was PEGylated and
modified with an 18F radionuclide. PEGylated VHHs showed
increased staining of the lymphoid organs, as confirmed by
FACS and two-photon microscopy (Figure 5D). The longer
circulatory half-life of PEGylated VHHs presumably
increases the likelihood of a VHH binding to its target, as
long as target accessibility is not compromised by PEGyla-
tion. As expected, the 18F-labeled DC8–PEG (20 kDa) had
the highest PET signal in blood at the time of imaging (3 h
p.i.; Figure 5D). Although the larger 20 kDa PEG can
significantly increase the staining efficiency in lymphoid
organs, it delays circulatory clearance, a factor to consider
when isotopes with short half-lives, such as 18F or 11C, are
being used. However, VHHs modified with 10 or 5 kDa PEG
still show significantly increased staining efficiency and yet
can be rapidly cleared. When isotopes with longer half-lives,
such as 89Zr or 64Cu, are used to label VHHs, the use of
a 20 kDa PEG moiety should improve staining.
Acknowledgements
In summary, we have developed a method to produce site
specifically PEGylated or bivalent single-domain antibodies
equipped with either a fluorophore or radionuclide (18F) for
different imaging modalities. The reaction conditions enable
M.R. is a Cancer Research Institute Irvington Fellow
supported by the Cancer Research Institute. S.H.L. is the
recipient of an NIH Career Development Award. This
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Figure 4. A) Synthesis of an
18F-labeled tetrazine.
DMF=N,N-dimethylforma-
mide, TEAB=triethylammo-
nium bicarbonate. B) 18F label-
ing of VHH-dimers, VHH mon-
omers, and PEG-functionalized
VHHs. C) Radio-TLC analysis of
the 18F-labeling reactions. D–
I) Use of the 18F-DC8 dimer
and 18F-DC13 dimer to detect
lymphoid organs and reveal
tumors. PET images are shown
in both coronal and sagittal
views for better visualization.
CT images are provided for
better visualization (H1,2).
D,E) PET images of WT (D)
and class II MHCÀ/À mice (E)
2 h after injection of the 18F-
DC8 dimer (see movies S1 and
S7 for a 3D visualization). CV:
cervical lymph nodes, BM:
bone marrow, SP: spleen, GB:
gallbladder, LV: liver, KD: kid-
neys, Ints: intestines, BL: blad-
der, SC: spinal cord. F,G) PET
images of WT (F) and
CD11bÀ/À mice (G) 2 h after
injection of the 18F-DC13 dimer
(see movies S2 and S8 for
a 3D visualization).
I1,2) Tumor-associated
CD11b+ cells (I1) or class II
MHC+ cells (I2) were visual-
ized by using the 18F-DC13
dimer (I1) or the 18F-DC8 dimer
(I2). Arrows point at the
tumors. In (I2), stars show the
lymph nodes (see movies S11
and S12 for a 3D visualization).
PET scale bars have arbitrary
units. Images are representa-
tive for 2–4 mice with similar
results.
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Figure 5. A,B) Postmortem biodistribution of 18F-VHHs in all organs. Mice were injected with the same amount of either 18F-VHH or the 18F-VHH
dimer. The activity in different organs was measured 3 h p.i. C) The VHH monomer can block binding of the 18F-VHH dimer. WT mice were
injected with different amounts of unlabeled DC8, and the 18F-DC8 dimer was injected 20 min later (50 mCi/5 mg). PET standardized uptake values
(SUVs) for spleen were calculated on the basis of PET images acquired 2 h after injection of the 18F-labeled DC8 dimer (see movies S3–S6 for
a 3D visualization). D) WT mice were injected with the same amount of different 18F-DC8 derivatives with or without PEG moieties as indicated.
The activity in different organs was measured 3 h p.i. (see movies S13–S16 for a 3D visualization). LN=lymph nodes.
[7] M. Rashidian, E. J. Keliher, M. Dougan, P. K. Juras, M.
Cavallari, G. R. Wojtkiewicz, J. T. Jacobsen, J. G. Edens,
J. M. J. Tas, G. Victora, R. Weissleder, H. Ploegh, ACS Cent.
Sci. 2015, 1, 142 – 147.
[8] E. C. Dijkers, T. H. Oude Munnink, J. G. Kosterink, A. H.
Brouwers, P. L. Jager, J. R. de Jong, G. A. van Dongen, C. P.
Schrçder, M. N. Lub-de Hooge, E. G. de Vries, Clin. Pharmacol.
Ther. 2010, 87, 586 – 592.
research was funded by NIH R01-AI087879-06 (to H.L.P.),
DP1-GM106409-03 (an NIH Pioneer Award to H.L.P.), and
R01-GM100518-04 (to H.L.P.), and by the Lustgarten Foun-
dation (H.L.P.).
Keywords: enzymes · isotopic labeling · PEGylation ·
positron emission tomography · single-domain antibodies
[9] M. Rashidian, E. J. Keliher, A. M. Bilate, J. N. Duarte, G. R.
Wojtkiewicz, J. T. Jacobsen, J. Cragnolini, L. K. Swee, G. D.
Victora, R. Weissleder, H. L. Ploegh, Proc. Natl. Acad. Sci. USA
2015, 112, 6146 – 6151.
How to cite: Angew. Chem. Int. Ed. 2016, 55, 528–533
Angew. Chem. 2016, 128, 538–543
[10] T. De Meyer, S. Muyldermans, A. Depicker, Trends Biotechnol.
2014, 32, 263 – 270.
[11] P. Holliger, T. Prospero, G. Winter, Proc. Natl. Acad. Sci. USA
1993, 90, 6444 – 6448.
[12] H. Schellekens, W. E. Hennink, V. Brinks, Pharm. Res. 2013, 30,
1729 – 1734.
[13] E. De Genst, K. Silence, K. Decanniere, K. Conrath, R. Loris, J.
Kinne, S. Muyldermans, L. Wyns, Proc. Natl. Acad. Sci. USA
2006, 103, 4586 – 4591.
[14] V. Cortez-Retamozo, M. Lauwereys, G. Hassanzadeh, M.
Gobert, K. Conrath, S. Muyldermans, P. De Baetselier, H.
Revets, Int. J. Cancer J. Int. Cancer 2002, 98, 456 – 462.
[15] Y. Vugmeyster, C. A. Entrican, A. P. Joyce, R. F. Lawrence-
Henderson, B. A. Leary, C. S. Mahoney, H. K. Patel, S. W. Raso,
S. H. Olland, M. Hegen, X. Xu, Bioconjugate Chem. 2012, 23,
1452 – 1462.
[1] T. Olafsen, S. J. Sirk, S. Olma, C. K.-F. Shen, A. M. Wu, Tumor
Biol. 2012, 33, 669 – 677.
[2] E. J. Lipson, W. H. Sharfman, C. G. Drake, I. Wollner, J. M.
Taube, R. A. Anders, H. Xu, S. Yao, A. Pons, L. Chen, D. M.
Pardoll, J. R. Brahmer, S. L. Topalian, Clin. Cancer Res. Off. J.
Am. Assoc. Cancer Res. 2013, 19, 462 – 468.
[3] T. R. Simpson, F. Li, W. Montalvo-Ortiz, M. A. Sepulveda, K.
Bergerhoff, F. Arce, C. Roddie, J. Y. Henry, H. Yagita, J. D.
Wolchok, K. S. Peggs, J. V. Ravetch, J. P. Allison, S. A. Quezada,
J. Exp. Med. 2013, 210, 1695 – 1710.
[4] R. Boellaard et al., Eur. J. Nucl. Med. Mol. Imaging 2010, 37,
181 – 200.
[5] M. Rashidian, E. J. Keliher, A. M. Bilate, J. N. Duarte, G. R.
Wojtkiewicz, J. T. Jacobsen, J. Cragnolini, L. K. Swee, G. D.
Victora, R. Weissleder, H. L. Ploegh, Proc. Natl. Acad. Sci. USA
2015, 112, 6146 – 6151.
[6] R. Tavare, M. N. McCracken, K. A. Zettlitz, S. M. Knowles, F. B.
Salazar, T. Olafsen, O. N. Witte, A. M. Wu, Proc. Natl. Acad. Sci.
USA 2014, 111, 1108 – 1113.
Received: August 14, 2015
Revised: October 1, 2015
Published online: December 2, 2015
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