Application of strain-promoted azide-alkyne cycloaddition and tetrazine ligation to targeted fc-drug conjugates

Article abstract of DOI:10.1021/bc300052u

We have previously described an approach whereby antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In the p

Full text of DOI:10.1021/bc300052u

Communication
pubs.acs.org/bc
Application of Strain-Promoted Azide−Alkyne Cycloaddition and
Tetrazine Ligation to Targeted Fc-Drug Conjugates
Joshua D. Thomas,† Huiting Cui,‡ Patrick, J. North,‡ Thomas Hofer,‡ Christoph Rader,‡
and Terrence R. Burke, Jr.*,†
‡
†Chemical Biology Laboratory and Experimental Transplantation and Immunology Branch, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Frederick, Maryland, United States
S
* Supporting Information
ABSTRACT: We have previously described an approach
whereby antibody Fc fragments harboring a single C-terminal
selenocysteine residue (Fc-Sec) are directed against a variety
of targets by changing the peptide or small molecule to which
they are conjugated. In the present work, we describe
methodology for improving the efficacy of these Fc-Sec
conjugates by incorporating cytotoxic drugs. The Fc-Sec
protein is first programmed to target specific tumor cell types by attachment of a bifunctional linker that contains a “clickable”
handle (e.g., cyclobutane or cyclooctyne) in addition to a tumor cell-binding peptide or small molecule. Following Fc-Sec
conjugation, a cytotoxic warhead is then attached by cycloaddition reactions of tetrazine or azide-containing linker. To validate
this approach, we used a model system in which folic acid (FA) is the targeting moiety and a disulfide-linked biotin moiety serves
as a cytotoxic drug surrogate. We demonstrated successful targeting of Fc-Sec proteins to folate-receptor expressing tumor cells.
Tetrazine ligation was found to be an efficient method for biotin “arming” of the folate-targeted Fc-Sec proteins. We also report
novel bioconjugation methodologies that use [4 + 2] cycloaddition reactions between tetrazines and cyclooctynes.
(five Fc-fusion proteins are currently on the market5),
he therapeutic value of monoclonal antibodies (mAbs) in
Tcancer therapy is well established. One reason for the introducing specificity for each new target requires its own
protein engineering project. We have previously described an
alternative approach for grafting antigen-binding sites onto Fc
proteins that does not rely on genetic engineering.8,9 This
involves initial protein engineering to introduce a single
selenocysteine (Sec) residue into the C-terminus of an IgG
Fc fragment (Fc-Sec). A tumor cell-binding peptide or small
molecule can then be attached using standard protein alkylation
methodologies (e.g., via maleimide-containing linkers) to
generate conjugates having a 1:1 ratio of targeting agent to
protein. This approach allows a single generic Fc-Sec protein to
be directed against a wide variety of targets by simply changing
the peptide or small molecule to which it is conjugated.
Although targeted Fc-Sec proteins may be therapeutically
valuable in their own right, their efficacy may be improved by
incorporating cytotoxic drugs. Several drug conjugate linker
systems are designed to release their cargos under acidic (e.g.,
hydrazones) or reductive (e.g., disulfides) conditions. However,
because Fc-Sec-conjugates are prepared under just such
conditions, the use of these linker modalities is potentially
contraindicated. In order to avoid the loss of drug cargo during
conjugation to Fc-Sec proteins (Figure 1, Route A), we are
examining two-step “program and arm” approaches (Figure 1,
Route B). Here, the Fc-Sec proteins are first programmed to
success of mAbs is that they intrinsically exhibit many ideal
drug properties, including high target specificity and affinity and
long circulatory half-life. However, therapeutic mAbs often lack
the potency and tissue penetration (particularly against solid
tumors) required to eradicate target cell populations.1,2
A
common strategy for improving antibody potency involves
attaching powerful cytotoxic drugs that are too poisonous to be
safely administered as single agents.3 Antibody−drug con-
jugates (ADCs) function as “smart bombs” that allow the toxin
to bypass healthy tissue and hit only the targets of interest. The
remaining problem with mAbs of poor solid tumor penetration
has spurred research into alternative antibody-like proteins that
retain high affinity and selectivity for tumor cells but are smaller
than full-size antibodies.4 Therapeutic proteins based on
antibody Fc (fragment crystallizable) fragments are attractive
examples of this latter class, since they often exhibit serum half-
lives close to those of full-size antibodies, yet they are much
smaller in size.5 An additional property of Fc proteins is that
they can recruit elements of the immune system to attack
targets of interest via interaction with immune effector cells
(antibody-dependent cellular cytotoxicity, ADCC) and comple-
ment (complement-dependent cytotoxicity, CDC).6
Once they are separated from their antigen binding (Fab)
regions, Fc proteins lose their ability to attack specific cell
populations. One method for restoring antigen-recognition to
the Fc moiety involves genetically fusing tumor cell-binding
peptides or proteins.7 While this can be an effective strategy
Received: February 7, 2012
Revised: September 12, 2012
© XXXX American Chemical Society
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Figure 1. Two methods of producing Fc-Sec drug conjugates: route A, a one-step approach that may result in loss of drug cargo; and route B, a two-
step program and arm protocol that preserves drug cargo.
Scheme 2. Synthesis of TL Dieneophiles 11 and 8
Figure 2. Examples of readily prepared cyclooctynes (1 and 2) and the
synthetically more challenging 3. Rate constants refer to cycloaddition
reactions with benzyl azide in CD3CN.18−20
target specific tumor cell types by attachment of bifunctional
linkers that contain “clickable” handles (e.g., cyclobutane or
cyclooctyne) in addition to tumor cell-binding peptides or
small molecules. These linkers are conjugated to Fc-Sec
proteins using previously established conditions (Figure 1).8
Following appropriate Fc-Sec conjugation, a tetrazine or azide-
containing linker can be used to attach a cytotoxic warhead by
means of cycloaddition reactions. Our current paper presents a
model version of this “program and arm” concept in which folic
acid (FA) serves as the targeting moiety, and a disulfide-linked
Scheme 1. Synthesis of the Water-Soluble Cyclooctyne Derivative 6
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Figure 3. Bifunctional FA conjugates 18a−d for Fc-Sec programming.
Scheme 3. [4 + 2] Cycloaddition Reactions of Free 6 and 2 with Tetrazines 12 and 15, Respectively
biotin group is employed as a cytotoxic drug surrogate. We also
report the use of [4 + 2] cycloaddition reactions between
tetrazines and cyclooctynes as novel bioconjugation method-
ologies.
functionality could be introduced following Fc-Sec alkylation
(Figure 1, Route B).
Chemistries used to attach drug cargos should be both mild
and chemoselective in order to preserve the well-defined
Since most previously prepared FA−drug conjugates rely on
disulfide linkages for intracellular drug release,10 we chose this
type of linkage for our Fc-Sec conjugates, although it was not
clear whether disulfides would survive the reductive conditions
previously established for Fc-Sec conjugation.8 We found that,
in the absence of Fc-Sec protein, a model disulfide was unstable
under anticipated coupling conditions (25 equiv DTT at pH
5.2, 80 min, data not shown). This indicated that undesired
reductive cleavage may occur if a disulfide-containing linker
were used to alkylate Fc-Sec. Therefore, an alternative approach
to Fc-Sec modification was examined in which sensitive
stoichiometry and residue specificity of Fc-Sec conjugates.8,9
A
number of bioorthogonal “click” reactions that meet these
requirements have been reported recently.11 Among these, the
most widely utilized click reactions employ Cu(I)-catalyzed
azide−alkyne [3 + 2] cycloaddition (CuAAC) chemistries.12
However, because it can be difficult to remove cytotoxic Cu
from the conjugated products,13,14 CuAAC may not be
appropriate for constructing Fc-Sec drug conjugates. Therefore,
we evaluated strain-promoted azide−alkyne cycloaddition
(SPAAC)15 and tetrazine ligation (TL)16 as metal-free
alternatives to CuAAC.
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Figure 4. Fc-Sec was programmed with either 18d or biotin iodoacetamide to afford biotinylated constructs 19 and 20, respectively. Additional
programming experiments were conducted with 18a, 18c, and 18b to afford “clickable” proteins 21−23.
Figure 5. Biotinylation reagents 24a and 24b used to “arm” Fc-Sec proteins 21−23. (A) SPAAC reaction of Fc-Sec protein 21 (5 μg/mL) with
biotin azide 24a. Reactions were conducted at room temperature (3 h) then overnight (4 °C) in PBS, pH 7.4. (B) SPAAC reaction of 21 with 24a.
Protein 21 was mixed with 10-, 50-, and 100-fold excess 24a as described above and then incubated with FR(+) HeLa cells. The cells were stained
with PE-coupled avidin and then analyzed by flow cytometry. The upper limit of biotinylation is defined as the mean fluorescence intensity (MFI)
exhibited by 19. Biotinylation efficiency is expressed as 100(MFI25/MFI19).
The synthetic complexity of many reactive cyclooctyne-
containing reagents can be a major limitation to their use in
SPAAC bioconjugations. This complexity often arises from the
difficulty of introducing the required electron-withdrawing
groups adjacent to the alkyne moiety.17 Although synthetic
routes have improved in recent years, many of the most reactive
cyclooctynes still require lengthy and low-yield procedures.15
For this reason, our attention was drawn to cyclooctyne 1,18
which affords a good balance between synthetic accessibility (3
steps, 47% overall yield for 1 versus 8 steps, ∼25% overall yield
for 319) and reactivity (relative reactivity compared to similar
reagents: 3 > 1 > 2,20 Figure 2). Cyclooctyne 1 was prepared
according to literature procedures18 and converted to the
water-soluble piperazine salt 6 in 4 steps (Scheme 1). Although
piperazine derivatives of 1 have not been reported, tertiary
amines are well-known hydrophilic modifiers that can often
improve the water solubility of liphophilic compounds.21,22
Conversion to 6 improved the aqueous solubility of the
cyclooctyne and rendered it as an easily handled solid.
alternative to SPPAC.23 Tetrazine-based [4 + 2] cycloadditions
are generally much faster than SPAAC reactions. They also
utilize coupling partners (i.e., dienes and dienophiles) that are
usually either commercially available or more synthetically
accessible than cyclooctynes.24 This is exemplified by the
dieneophile 8 used in the present work, which is both easily
synthesized (Scheme 2) and an efficient TL coupling partner.25
Strained alkenes, such as norbornene, trans-cyclooctene, and
cyclobutane, have traditionally been the dienophiles of choice
for TL strategies.15 Although other potentially suitable alkenes
and alkynes, such as cyclooctyne, have been reported,26,27 these
have not yet been applied in a bioconjugation context. In order
to evaluate the performance of cyclooctynes in side-by-side
fashion with an established TL dieneophile such as 8, we
included the known cyclooctyne 2 in the present study. The
synthesis of 2 proceeded smoothly according to literature
procedures,20 with the exception that the reported NaOMe-
mediated bromide elimination in 9 to give 10 proved to be
inefficient (Scheme 2). In contrast, the use of DBU at elevated
temperature14 afforded 10 in good yield. Subsequent hydrolysis
gave 2. Cyclooctyne 2 was converted to the corresponding p-
Although TL has appeared as a bioconjugation strategy only
relatively recently,16 it is quickly becoming an attractive
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smoothly to afford a mixture of 16 and 17 in 79% combined
yield (Scheme 3). The ability of 2 and 6 to undergo [4 + 2]
cycloaddition reactions with tetrazines in nonprotein-bound
forms indicated that they would likely retain their reactivity
once they were bound to Fc-Sec protein.
Bifunctional FA-containing constructs 18a−c, employing
maleimide for Fc-Sec alkylation and clickable functionality for
drug attachment, were prepared using a previously described
PEG-SU-Lys-Lys-maleimide linker (Figure 3) (see Supporting
Information).28 The biotin-containing derivative 18d was also
prepared for use as a control in Fc-Sec alkylation and click
experiments. In order to show that Fc-Sec programming was
possible using 18a−d, Fc-Sec was first alkylated with 18d to
afford 19 (Figure 4). Conjugate 19 was subsequently incubated
with FA receptor-expressing (FR+) HeLa cells and analyzed by
flow cytometry (Figure S1). For comparison, Fc-Sec was also
alkylated using commercially available biotin-iodoacetamide to
give the Fc-Sec-biotin adduct 20. It was found that 19 showed
strong and selective binding to (FR+) cells (Figure S1A),
whereas 20 failed to show binding (Figure S1B). Attempted
alkylation of Fc-Stop8 (a nearly identical protein in which the
C-terminal Sec residue is missing) with 18d resulted in only
∼10% biotinylation relative to the same reaction with Fc-Sec
(Figure S1C). These results demonstrate that alkylation of the
Fc protein under our optimized conjugation conditions8 is very
limited unless the protein harbors a Sec residue. This data also
serves as indirect evidence that alkylation of Fc-Sec proteins
occurs primarily at the C-terminal Sec residue and not at hinge
cysteines.
Having demonstrated successful programming with 18d, we
next examined SPAAC and TL chemistries for “arming” Fc-Sec
proteins (21−23) with either biotin-azide 24a29 or biotin-
tetrazine 24b (Figure 5).29 The cycloaddition products (25−
28) produced by these reactions were then analyzed by flow
cytometry to determine the extent of biotinylation as described
above for 19. Since conjugation occurs only at the single Sec
residue, 19 consists of a 1:1:1 ratio of biotin/FA/Fc-Sec.
Consequently, it represents the maximum level of biotinylation
that can be achieved by the two-step approach shown in Figure
5 and could serve as a positive control for evaluating the
efficiency of the SPAAC and TL reactions.
The SPAAC reaction of 21 with 24a was the least efficient
method for arming Fc-Sec (Figure 5B). At the same reactant
stoichiometry as that used to prepare 19 (1:10 protein/small
molecule modifier), the SPAAC reaction gave only 5% of the
biotin load as was obtained for 19 (Figure 5B). At a 50-fold
excess of 24a, the biotin load increased to approximately 15%
of maximum. However, it did not improve substantially at
higher concentrations of 24a (Figure 5B). The reaction of 22
with 24a was not examined. However, it would be expected to
be even less efficient, based on the previously determined
reaction rates of benzyl azide with 118 and 2.20
Of the three reagents tested with 24b in TL reactions,
reagent 21 was the least reactive (Figure 6). Although this
result was not unexpected given the electron deficient character
of 1,26 it is noteworthy when compared to the analogous
SPAAC reaction with 24a (Figure 5B). In this latter case,
approximately the same levels of biotinylation were obtained
with SPAAC as with TL, but at a 5-fold lower concentration of
biotinylation reagent 24b (Figure 5). Protein 22 was much
more effective reacting with 24b as compared with 21.
Differences in steric crowding around the alkyne moieties in
6 and 2 may be partly responsible for the observed differences
Figure 6. (A) TL reactions of proteins 21−23 with biotin tetrazine
24b to generate 26−28, respectively. All reactions included 10-fold
excess 24b and were conducted at room temperature (3 h), then
overnight (4 °C) in PBS, pH 7.4. (B and C) Reaction products 26−28
were first incubated at the indicated concentrations (5 μg/mL data
shown in B) with FR(+) HeLa cells and then analyzed by flow
cytometry as described above. The upper limit of biotinylation defined
as MFI exhibited by 19. (B) Biotinylation efficiency expressed as
100(MFI26, 27, or 28/MFI19). (C) Maximum fluorescence signal defined
as MFI of 19 at 10 μg/mL. All MFI data shown in C was normalized
to MFI of 19 at 10 μg/mL.
nitrophenyl ester 11 prior to final incorporation into a folate-
containing scaffold (Figure 3).
Before evaluating TL reactions with Fc-Sec constructs
containing alkynes 2 and 6, their reactivities with tetrazines in
free form were evaluated (Scheme 3). Reaction of 6 with
tetrazine 12 resulted in a 28% combined yield of regioisomers
13 and 14, while the reaction between 2 and 15 proceeded
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in reactivity between 21 and 22.26 Biotinylation efficiencies
with the cyclobutane construct 23 approached 80% of the
maximum, which made 23 the most reactive TL coupling
partner tested. This is an important finding, since the
dieneophile used in 23 (i.e., compound 8) was the simplest
to prepare. It should also be noted that although fused
cyclobutane−norborene compounds, such as 8, have been used
previously in bioconjugation contexts,25,30 this is the first
example of their use in protein modification.
The reactivity ranking observed in our initial experiments
(23 > 22 > 21) was confirmed in subsequent titration binding
experiments with 19 and 26−28 (Figure 6C). Among the three
TL conjugates, 28 consistently gave higher MFI across the
concentration range studied. These results suggest that the
differences in biotin load observed in 26−28 accurately reflect
differences in biotinylation efficiency of the various TL
reactions and are not artifacts of the flow cytometry protocol
used for their analysis.
In conclusion, reported herein is methodology whereby Fc-
Sec proteins can be sequentially programmed to target cancer
cells and then armed with cytotoxic payload using SPAAC or
TL chemistries. This “program and arm” approach should be
more compatible with cleaveable linkers, such as disulfides and
hydrazones that may not otherwise survive the acidic, reducing
conditions previously used to prepare Fc-Sec conjugates. Our
work demonstrates for the first time that cyclooctynes can be
effective dieneophiles in TL bioconjugation contexts. We are
currently using these approaches to prepare Fc-Sec conjugates
that are armed with cytotoxic drugs.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Materials and methods including detailed synthetic procedures,
Fc-Sec programming, arming of Fc-Sec-folate and flow
cytometry are included. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tburke@helix.nih.gov.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Intramural Research Program
of the NIH, Center for Cancer Research, Frederick National
Laboratory for Cancer Research and the National Cancer
Institute, National Institutes of Health. The content of this
publication does not necessarily reflect the views or policies of
the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government.
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