Introducing Glycolinkers for the Functionalization of Cytotoxic Drugs and Applications in Antibody–Drug Conjugation Chemistry

Article abstract of DOI:10.1002/cmdc.201600372

Antibody–drug conjugates (ADCs) are promising alternatives to naked antibodies for selective drug-delivery applications and treatment of diseases such as cancer. Construction of ADCs relies upon site-selective, efficient and mild conjugation technologies.

Full text of DOI:10.1002/cmdc.201600372

DOI: 10.1002/cmdc.201600372
Communications
Introducing Glycolinkers for the Functionalization of
Cytotoxic Drugs and Applications in Antibody–Drug
Conjugation Chemistry
Filip S. Ekholm,[a, b] Henna Pynnçnen,[a] Anja Vilkman,[a] Virve Pitkꢀnen,[a] Jari Helin,[a]
Juhani Saarinen,[a] and Tero Satomaa*[a]
Antibody–drug conjugates (ADCs) are promising alternatives
to naked antibodies for selective drug-delivery applications
and treatment of diseases such as cancer. Construction of
ADCs relies upon site-selective, efficient and mild conjugation
technologies. The choice of a chemical linker is especially im-
portant, as it affects the overall properties of the ADC. We en-
visioned that hydrophilic bifunctional chemical linkers based
on carbohydrates would be a useful class of derivatization
agents for the construction of linker–drug conjugates and
ADCs. Herein we describe the synthesis of carbohydrate-based
derivatization agents, glycolinker–drug conjugates featuring
the tubulin inhibitor monomethyl auristatin E and an ADC
based on an anti-EGFR antibody. In addition, an initial in vitro
cytotoxicity evaluation of the individual components and the
ADC is provided against EGFR-positive cancer cells.
properties of the ADCs. In modern ADCs, hydrophobic cytotox-
ic agents are almost exclusively used. The combination of hy-
drophobic cytotoxic agents and the commonly used relatively
hydrophobic chemical linkers is suboptimal, and the addition
of multiple drug–linker moieties of this kind to an antibody
may be devastating with regard to the biocompatibility and
pharmaceutical efficacy of the ADC. These problems are re-
flected in the design of ADCs, which tend to focus on the in-
corporation of a limited amount of drugs per antibody (typical-
ly 2–4 drugs per antibody). Furthermore, problems associated
with the overexpression of efflux pump proteins which are ca-
pable of removing hydrophobic cytotoxic molecules from the
intracellular environment has been encountered with multi-
drug-resistant cancer cells.[6]
To overcome these challenges, an increasing amount of
effort has been invested in the design of hydrophilic chemical
linkers that may counteract these issues.[7] We envisioned that
carbohydrate-based linkers would be a viable option for ADCs,
as carbohydrates are well known for their hydrophilic nature
and biocompatibility. In contrast to the previously reported
polyethylene glycol (PEG)[7a] and sulfonate-linker-based strate-
gies,[7b] the carbohydrate backbone offers further structural var-
iation possibilities through modification of the hydroxy groups
which might be beneficial for fine-tuning of linker properties
and construction of multifunctional linker species. To set up
a suitable synthetic strategy, we decided to investigate which
modification protocols would be applicable for the functionali-
zation of simple sugars and provide cross-linking tools applica-
ble to the construction of ADCs.
Antibody–drug conjugates (ADCs) are an emerging class of cy-
totoxic agents for selective drug delivery applications and
treatment of diseases such as cancer.[1] The large potential
shown by ADCs has not gone unnoticed by pharmaceutical
companies, and there are currently two ADCs on the market
and a growing number in clinical trials.[2] The construction of
ADCs relies on regioselective organic transformations and effi-
cient bioconjugation technologies.[3] The challenges associated
with the chemical aspects of their construction are numerous,
for example: site-selective modification of multifunctional
drugs, site-selective modification of antibodies, development
of robust, benign and highly selective bioconjugation reac-
tions, and optimization of the linker structure in order to ach-
ieve optimal pharmacokinetic and pharmacodynamic proper-
ties in the end-products. While much progress has been ach-
ieved in the past[4] and more recently,[5] the field as whole is
still under constant development.
In the past, reductive amination reactions of carbohydrates
have been developed as a tool for the selective modification
of the reducing end of unprotected mono-, oligo-, and poly-
saccharides.[8] Still today, these robust reactions play a crucial
role in chemical sciences, for example, in the immobilization of
carbohydrates or biomolecules onto surfaces, which has been
a significant contributor (e.g., through microarray experiments)
to the continued advancement of our understanding of biolog-
ical processes.[9] Our initial plan was to exploit this reaction
protocol and turn modified reducing monosaccharides into hy-
drophilic bifunctional derivatization agents (glycolinkers) for
the functionalization of cytotoxic agents. The aldehyde form of
reducing sugars, which is in equilibrium with the hemiacetal
through mutarotation, was viewed as a potential conjugation
site for amine-containing cytotoxic agents. A thorough survey
of bioconjugation protocols was conducted in order to select
an appropriate second functional group for the glycolinkers.
One of the most important aspects of ADC design is the
chemical linker, which has a considerable effect on the overall
[a] Dr. F. S. Ekholm, H. Pynnçnen, A. Vilkman, V. Pitkꢀnen, Dr. J. Helin,
J. Saarinen, T. Satomaa
Glykos Finland Ltd., Viikinkaari 6, 00790 Helsinki (Finland)
E-mail: tero.satomaa@glykos.fi
[b] Dr. F. S. Ekholm
Department of Chemistry, University of Helsinki, PO Box 55, A. I. Virtasen
aukio 1, 00014 Helsinki (Finland)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600372.
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Successful bioconjugation strategies based on maleimide/thiol
chemistry,[10] Staudinger ligation techniques,[11] azide–alkyne cy-
cloaddition reactions,[12] and ketone/aldehyde-amine (oxime)[13]
chemistry have been reported. Because our plans involved the
functionalization of amine-containing cytotoxic agents, the
methods involving oxime chemistry were neglected, and em-
phasis was placed on the design of glycolinkers applicable to
other conjugation strategies.
1,2:3,4-Di-O-isopropylidene-d-galactose was chosen as a suit-
able starting material because it is commercially available and
the free primary hydroxy group can be modified rather easily
by the use of standard synthetic protocols, thereby providing
access to a vast amount of versatile conjugation species. The
chemical structures of, and the short synthetic routes to three
alternatives are provided in Scheme 1. In these model sub-
Scheme 2. Synthesis of MMAE–glycolinker conjugates. Reagents and condi-
tions: a) 2 or 3, NaCNBH3, DIPEA, DMSO, 34% (5), 24% (6).
cell lymphoma.[5a] In addition, 20 ADCs which have proceeded
to clinical trials have relied on auristatin derivatives as their cy-
totoxic agents.[16] The secondary amine in the N-terminal resi-
due in MMAE was modified via a reductive amination reaction
with glycolinkers 2 and 3,[17] thereby providing amphiphilic
MMAE–glycolinker conjugates 5 and 6 in acceptable isolated
yields. Notably, the yields of the presented protocol are similar
to the overall yields obtained in the multistep synthetic se-
quences required for several other linker alternatives.[7,18] Fur-
ther optimization of the reaction conditions was therefore not
attempted at this stage. Instead, focus was placed on the NMR
spectroscopic characterization of MMAE–glycolinker conjugates
5 and 6 (which proved to be challenging).
Scheme 1. Synthesis of bifunctional monosaccharides 1–3. Reagents and
conditions: a) 1. allyl bromide, NaH, DMF, 90%, 2. TFA, H2O, quant.;
b) 1. propargyl bromide, NaH, DMF, 91%, 2. TFA, H2O, quant.; c) 1. TsCl, pyri-
dine, DMF, 81%, 2. NaN3, toluene, 68%, 3. TFA, H2O, quant.
It is well known that proline and other similar amino acids
can populate both the cis and trans isomeric states, in contrast
to other amino acids which are present mostly as the trans
isomer.[19] In the MMAE–glycolinker conjugates, a diastereo-
meric mixture containing the two forms in roughly equal pro-
portions was observed in the NMR spectra. The signal splitting
was not limited to the proline analogue [residue (2)] alone, but
was seen in all of the residues, further complicating assign-
ment. A further indication of the difficulty involved in solving
the complex NMR spectra of MMAE conjugates is the fact that
a full assignment has not been previously reported. While
a complete assignment with accurate coupling constants is dif-
ficult to obtain due to severely overlapping signals, we were
able to locate the chemical shifts for most of the signals of
both diastereomers using a number of one-dimensional (1H,
13C) and two-dimensional (COSY, HSQC, TOCSY, ROESY, and
HMBC) NMR spectroscopic techniques. Even with the two-di-
mensional spectra available, the assignment was not trivial, as
a number of key signals from both diastereomers overlapped
in crowded areas of the spectrum. To the best of our knowl-
edge, the NMR spectroscopic data reported in the Supporting
Information are among the most detailed to date on this im-
portant and widely used class of cytotoxic agents.
strates, an amine-containing drug molecule can be conjugated
to the reducing end, thereby leaving the second functional
group free to react with site-specifically modified antibodies,
for example. Briefly, the terminal alkene in 1 can be used in
thiol-ene reactions and cross-coupling metathesis reactions,[14]
the terminal alkyne in 2 and can be used in azide–alkyne cyclo-
addition reactions,[12] and the terminal azide in 3 can be used
in both azide–alkyne cycloaddition reactions and Staudinger li-
gation protocols.[11,12] Azide–alkyne cycloaddition chemistry is
currently viewed as one of the most promising techniques for
bioconjugation reactions because these functionalities are rare
in naturally occurring biomolecules, in contrast to amines and
thiols, for example.[15] Therefore, we decided to concentrate on
the use of monosaccharides 2 and 3 in this study.
In order to compare the features of the glycolinkers with
other linker alternatives, it was important to use a thoroughly
tested cytotoxic agent. Monomethyl auristatin E (MMAE) was
found to be a suitable choice, especially for ADC purposes.
MMAE is an antineoplastic agent and an antimitotic drug (tu-
bulin inhibitor) composed of five amino acid residues (com-
pound 4, Scheme 2). It is the cytotoxic agent in the approved
ADC brentuximab vedotin (Adcetris), which is used in the treat-
ment of Hodgkin’s lymphoma and systemic anaplastic large-
With the characterization of the MMAE–glycolinker conju-
gates completed, our attention was turned toward the con-
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struction of a model ADC. We chose to use an in-house pro-
duced anti-EGFR antibody (cetuximab) as a model substrate,
because the epidermal growth factor receptor (EGFR) is a clini-
cally validated cancer treatment target in non-small-cell lung
cancer, squamous-cell carcinoma of the head and neck, color-
ectal cancer, and pancreatic cancer. In fact, activation of
growth factors and receptors of the EGFR family is observed in
most human carcinomas.[20] In addition, EGFR is an internalizing
receptor that can effectively transport the drug inside the
tumor cell upon binding of a drug-loaded antibody, and is
therefore a good target for ADC-based therapy.
beling with Alexa Fluor 488 azide followed by spectrophoto-
metric analysis. The results indicated a DBCO-to-antibody ratio
of ~10. The DBCO-modified antibody was reacted with MMAE–
glycolinker 5 by a copper-free strain-promoted alkyne–azide
cycloaddition reaction (SPAAC) to yield the final ADC. The reac-
tion between the DBCO-modified antibody and MMAE–glyco-
linker 5 was found to be complete as indicated by MALDI-TOF
MS analysis of the antibody light chain as well as the Fc
domain (Figure 1). The DAR of the product was found to be
~10 (see Supporting Information for more details).
With access to all of the individual components and the
model ADC, we decided to conduct a preliminary cytotoxic
evaluation of these molecules. MMAE (4) and MMAE–N3–glyco-
linker conjugate 5 were studied in cytotoxicity assays using
the human head-and-neck cancer cell line HSC-2. The MMAE–
glycolinker–ADC was studied in cytotoxicity assays using the
human ovarian cancer cell line SKOV-3 (Figure 2, both cell lines
are EGFR-positive). MMAE was found to be highly cytotoxic to
the cells with a calculated 50% growth-inhibitory concentra-
tion (IC50) of 4 nm.[23] As expected, 5 was less cytotoxic, with an
IC50 value of 12 nm, presumably reflecting decreased cell mem-
brane permeability due to increased hydrophilic character of
the glycolinker-modified drug. This might decrease non-target-
related toxicities caused by drug liberated from the ADC in the
body, which is one of the dose-limiting factors in clinically ap-
plied ADCs.[24] The cytotoxicity of the ADC was in the middle,
with an IC50 value of 9 nm. The successful construction of the
While a number of tools for the site-selective modification of
antibodies have emerged in recent years,[21] we decided that
modification of lysine side chain amino groups would be suffi-
cient for evaluation of the glycolinkers in this study. It is well
known that the modification of lysine side chain amino groups
produces a heterogeneous mixture of products, as similar
modification protocols have been used in the construction of
approved ADCs in the past, for example, in the construction of
trastuzumab emtansine (Kadcyla), which is used in the treat-
ment of metastatic breast cancer and has a drug-to-antibody
ratio (DAR) of 0–8.[22] In the current study, lysine side chain
amino groups were functionalized using a dibenzocyclooctyne
(DBCO)-NHS-ester reagent, which resulted in the successful in-
corporation of DBCO units to the antibody (Scheme 3). The
heterogeneous modified antibody mixture was analyzed by
MALDI-TOF mass spectrometry (light chain analysis) and by la-
Scheme 3. Synthesis of Cetuximab–glycolinker–MMAE conjugates. Reagents and conditions: a) NHS-DBCO, PBS, DMSO, ~10 residues; b) 5, PBS, DMSO, 0 un-
reacted DBCO residues.
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ces). In this study, short synthetic routes leading to modified
galactose substrates were used to create bifunctional glyco-
linkers for the derivatization of amine-containing drugs and
subsequent bioconjugation reactions. The applicability of the
hydrophilic glycolinkers was verified by the construction of
MMAE–glycolinker conjugates and a cetuximab–glycolinker–
MMAE ADC with the considerable drug-to-antibody ratio 10.
The initial cytotoxic evaluation revealed a modest cytotoxicity
for the model ADC and a decreased cytotoxicity for the glyco-
linker–MMAE–conjugate relative to free MMAE. The results of
our studies suggest that carbohydrates may hold a place
within future linker technologies, but more work in this area is
required to determine whether they can surpass the currently
applied linker protocols. The applicability of the presented gly-
colinkers, where the drug is permanently modified by the car-
bohydrate, is likely restricted to compounds that can tolerate
a covalent modification with a stable linker (e.g., auristatins, in-
cluding MMAE, and maytansinoids, used in the construction of
Kadcyla[22]). Inspired by these findings, we have continued with
the development of other types of glycolinkers for other types
of cytotoxic drugs, and further optimization of the associated
reaction conditions and structural properties. The results of
these studies will be reported in due course.
Figure 1. MALDI-TOF MS analysis of the MMAE–(DBCO-N3-glycolinker)–ADC
light chain (above) and Fc domain (below).
Acknowledgements
The authors thank Ms. Anu Karinen for the larger-scale prepara-
tion of conjugate 5. F.S.E. thanks the Walter and Lisi Wahl Foun-
dation for financial support.
Keywords: antibody–drug conjugates
· bioconjugation ·
carbohydrates · cytotoxic activity · structural characterization
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Received: July 21, 2016
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Revised: September 29, 2016
Published online on && &&, 0000
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F. S. Ekholm, H. Pynnçnen, A. Vilkman,
V. Pitkꢀnen, J. Helin, J. Saarinen,
T. Satomaa*
A better tether: Carbohydrates can be
used as chemical derivatization agents
in generating antibody–drug conju-
gates. The potential benefits of such
glycolinkers are their hydrophilicity, rela-
tive ease of structural modification, bio-
compatibility, and their natural occur-
rence. We report new carbohydrate-
based derivatization agents for the
modification of amine-containing drugs
and their applications in antibody–drug
conjugation chemistry.
&& – &&
Introducing Glycolinkers for the
Functionalization of Cytotoxic Drugs
and Applications in Antibody–Drug
Conjugation Chemistry
&
ChemMedChem 2016, 11, 1 – 6
www.chemmedchem.org
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ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!