Expanding the concept of chemically programmable antibodies to RNA aptamers: Chemically programmed biotherapeutics

Article abstract of DOI:10.1002/anie.201001736

(Figure Presented) Aptamer-programmed antibodies: An RNA aptamer was covalently attached to antibody 38C2 (see scheme) to provide an aptamer-programmed antibody with significantly higher affinity and improved pharmacokinetic properties compared to the parent aptamer. This strategy may provide a promising new class of immunotherapeutics that derive their specificity from nucleic acids.

Full text of DOI:10.1002/anie.201001736

Communications
DOI: 10.1002/anie.201001736
RNA-Aptamers
Expanding the Concept of Chemically Programmable Antibodies to
RNA Aptamers: Chemically Programmed Biotherapeutics**
Ulrich Wuellner, Julia I. Gavrilyuk, and Carlos F. Barbas, III*
Analysis of the catalytic antibody 38C2, which efficiently
catalyzes aldol and related reactions through an enamine
mechanism, has proven key to development of chemically
programmable antibodies (cpAbs).[1] In 38C2 and related
antibody aldolases, an exceptionally nucleophilic lysine
residue located on the heavy chain variable domain is critical
for activity. This lysine residue can be selectively and
covalently labeled with 1,3 diketone- or b-lactam-equipped
ligands such as small molecules or peptides to reprogram the
binding specificity of the antibody (Figure 1).[2] Thus, in
contrast to classic monoclonal antibodies that acquire their
To date, this approach has used small molecules and
peptides to direct targeting. However, other classes of
therapeutically active molecules, such as aptamers, should
benefit from antibody conjugation. Aptamers are structured
nucleic acid ligands often selected using the “systematic
evolution of ligands by exponential enrichment” (SELEX)
procedure.[3] Although aptamers are a promising class of
therapeutics because of their excellent binding and inhibitory
properties,[4] only a single aptamer, which targets vascular
endothelial growth factor (VEGF), is an approved drug.[5]
For in vivo applications, aptamers suffer from low
chemical stability (these molecules are readily degraded by
nucleases in serum)[6] and poor pharmacokinetic properties
(circulatory half lives are on the order of several minutes).[7]
Nuclease resistance can be enhanced significantly by incor-
porating 2’ ribose modified nucleobases; 2’-O-methyl modi-
fied oligonucleotides have acceptable serum stabilities.[8]
Other oligonucleotide modifications are also being explored
to solve this difficult problem.[9]
To date, most strategies aimed at enhancing the pharma-
cokinetic properties of aptamers have focused on covalent
attachment of ligands such as polyethylene glycol (PEG) to
reduce renal clearance.[11] In one study, conjugation of a 40 kD
PEG to an aptamer increased the circulatory half-life from
several minutes to 23 h.[11d] Data from a phase I clinical trail
with PEGylated aptamer ARC1779 indicate that the circu-
latory half-life is 2 h in humans.[12] The extent and site of
PEGylation must be evaluated for each aptamer since not all
aptamers tolerate chemical conjugation to PEG molecules
above a certain size.[13] Antibody programming provides an
attractive alternative to current strategies for extending
aptamer half-lifes. By attaching an aptamer to the chemically
programmable antibody, the therapeutically valuable binding
specificity of the aptamer should be combined with the
bivalency, the long in vivo half-life and effector functions of
the antibody.
In order to explore the potential of aptamer-based
programming of antibodies, we synthesized the b-lactam-
based heterobifunctional linker 3 (Scheme 1) with a reactive
maleimide moiety for attachment to a thiol-modified apta-
mer.[2d] In contrast to commercially available linkers like
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbox-
ylate (SMCC), the b-lactam-containing linker 3 leads to site-
specific labeling of both variable domains of the antibody as
previously shown with other targeting molecules.[2d,e] The
synthetic scheme for antibody–aptamer conjugation is out-
lined in Scheme 2. For our proof of concept experiments, we
chose the thiol-modified anti-VEGF aptamer ARC245 since
this aptamer is fully 2-O-methyl modified, highly nuclease
resistant, and its binding and inhibitory properties are well
Figure 1. Chemical reactivity of catalytic antibody 38C2 and active site
structure showing enamine formation with a unique nucleophilic lysine
residue (Lys93) on the heavy chain of the antibody.[1r] This chemistry
can be used to specifically attach targeting molecules (TMs) to the
antibody.
specificity through biology (gene rearrangement and hyper-
mutation), cpAbs acquire their specificity through chemistry.
Conjugation to the antibody equips the small-molecule or
peptide ligand with the pharmacokinetic properties of the
antibody and antibody effector functions mediated by the
antibody Fc domain. Several studies have shown that this
strategy increases the therapeutic efficacy of peptides and
small molecules by several orders of magnitude in animal
models.[2a,c,f,h] Currently, four peptide-based cpAbs are in
human clinical trails (www.clinicaltrials.gov).
[*] Dr. U. Wuellner, Dr. J. I. Gavrilyuk, Prof. Dr. C. F. Barbas, III
Departments of Chemistry and Molecular Biology and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-784-2583
E-mail: carlos@scripps.edu
Homepage: http://www.scripps.edu/mb/barbas
[**] We thank the NIH (R01 CA104045) and The Skaggs Institute for
Chemical Biology for funding.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001736.
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Figure 2. Representative SDS acrylamide gel after conjugation of anti-
body and aptamer. Lane 1, unmodified 38C2; lane 2, human 38C2
(hu38C2) after conjugation to aptamer; lane 3, mouse 38C2 (38C2)
after conjugation to ARC245; lanes 1’, 2’, and 3’, samples as in first
three lanes under reducing conditions.
Scheme 1. Synthesis of heterobifunctional linker 3.
placement ELISA.[14] In this assay, either the unlabeled
ARC245 or hucp38C2-ARC245 competed for binding to
surface-coated VEGF165 with biotinylated ARC245. Data
from this assay is shown in Figure 3. hucp38C2–ARC245
showed a 60-fold lower EC50 value than the aptamer alone
(0.69 nm vs. 41 nm). The affinities of aptamer and aptamer–
Figure 3. Competitive ELISA with increasing amounts of either
*
unmodified ARC245 ( ) or hucp38C2–ARC245 (&) incubated with
20 nm biotinylated ARC245. Biotinylated ARC245 was detected with
streptavidin–horseradish peroxidase and percent displacement was
plotted.
Scheme 2. Irreversible programming of aldolase antibody 38C2 with
aptamer ARC245.
cpAb were determined by the method of Oroz et al.[14] In this
experiment, the affinity of ARC245 was determined to be
1.8 nm and the aptamer–cpAb had a 30-fold higher affinity of
66 pM (Supporting Information). This result shows that the
effective affinity of the hucp38C2–ARC245 conjugate was
increased significantly relative to the monovalent aptamer
ARC245. Other reports indicate that bivalent aptamers have
a higher functional affinity than their monovalent counter-
parts.[15]
The therapeutic target of ARC245 is VEGF, a pro-
angiogenic factor excreted by many tumors that stimulates
blood vessel growth, helping to maintain a supply of oxygen
and nutrients to rapidly dividing tumor cells.[16]
characterized.[11d] Linker 3 was reacted with the aptamer and
purified. Mouse cp38C2 and human 38C2 (hucp38C2) were
then reacted with the lactam portion to yield an irreversible
linkage.
The antibody and ARC245-conjugated antibodies were
analyzed by gel electrophoresis as shown in Figure 2. The
reactive lysine is located on the heavy chain of 38C2;
consistent with this, we observed that only the heavy chain
band was shifted to a higher molecular weight under reducing
conditions indicative of site-specific labeling as demonstrated
previously for other conjugates.[1c,o,r] Labeling was complete as
determined by loss of aldolase activity (Supporting Informa-
tion).
ARC245 is a potent anti-VEGF antagonist that prevents
VEGF from binding to the VEGF receptor.[11d] To study the
biological activity of cp38C2–ARC245, we performed cell
migration assays using human umbilical cord endothelial cells
In order to evaluate the binding properties of the
ARC245–cpAbs, we performed a previously described dis-
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Communications
(HUVEC) in a transwell assay format. Cells were seeded into
increased the circulatory half-life of the aptamer; this should
translate into less frequent dosing regimens for aptamer
drugs.
the top chamber of 8 mm transwell plates and allowed to
migrate at 378C for 4 h towards the bottom chamber to which
10 ngmLÀ1 VEGF was added.
In summary, we demonstrated for the first time site-
specific conjugation of an aptamer to the aldolase antibody
38C2 to produce an aptamer-programmed cpAb. Conjugation
of the VEGF-targeting aptamer ARC245 to the well-charac-
terized chemically programmable antibody 38C2 resulted in a
biologically active aptamer–antibody conjugate that had
significantly increased functional affinity and a much longer
circulatory half-life than the free aptamer. The aptamer–
cpAb strategy developed here should be readily transferable
to other aptamers. Aptamer-based cpAbs of the type devel-
oped here represent a promising new class of aptamer
immunotherapeutics that combine the favorable character-
istics of aptamers with those of antibodies. This approach
might also be applicable to chemically programmed vac-
cines.[18]
As shown in Figure 4, addition of 50 nm cp38C2–ARC245
conjugate potently inhibited VEGF-mediated cell migration,
demonstrating that the biological activity of ARC245 was
retained after antibody conjugation. It is well established that
compounds that inhibit VEGF-mediated cell migration in a
transwell assay also block proangiogenic signaling in vivo
provided they demonstrate favorable pharmacokinetic prop-
erties.[17]
Received: March 23, 2010
Revised: May 27, 2010
Published online: July 19, 2010
Keywords: aldol reaction · angiogenesis · aptamers ·
.
bioconjugation · enamines
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