.
Angewandte
Communications
DOI: 10.1002/anie.201106775
Protein Nanoconjugation
Site-Specific Conjugation of ScFvs Antibodies to Nanoparticles by
Bioorthogonal Strain-Promoted Alkyne–Nitrone Cycloaddition**
Miriam Colombo, Silvia Sommaruga, Serena Mazzucchelli, Laura Polito, Paolo Verderio,
Patrizia Galeffi, Fabio Corsi, Paolo Tortora, and Davide Prosperi*
Hybrid multifunctional nanoparticles (MFN), which combine
unique superparamagnetic properties and fluorescence emis-
sion, have been envisaged as promising bimodal tracers for
noninvasive diagnosis of cancer both in vitro and in vivo.[1–4]
The design of ideal targeted MFN (TMFN) needs careful
optimization of fundamental features including uniform size
and shape,[5] surface charge,[6] optical and magnetic proper-
ties,[7,8] and efficient functionalization with suitable homing
ligands[9–11] to improve the signal amplification and target
selectivity toward malignant cells. When the ligands are
complex molecules, such as proteins, their proper orientation
on the surface of nanoparticles becomes a crucial factor for
maximizing the affinity for their molecular counterparts.[12–14]
In this context, copper-catalyzed azide-alkyne cycloaddition
(CuAAC), is gaining attention as a versatile strategy for
protein immobilization on iron oxide nanoparticles.[15] How-
ever, CuAAC requires metal catalysts and these are toxic to
cells and unsuited for several kinds of proteins.[16] In light of
this severe limitation, the bioorthogonal conjugation, which
aims at avoiding the use of unsafe promoters, is rapidly
becoming popular.[17–19] The main advantages of this approach
are speed, efficiency, and biocompatibility. The strain-pro-
moted azide–alkyne cycloaddition (SPAAC) modification of
CuAAC, in which the terminal alkyne is replaced by a highly
reactive cyclooctyne, does not require CuI catalysis and has
provided excellent results both in solution and in living
cells.[20–22] The kinetics of the reaction can be improved by
introducing electron-withdrawing substituents adjacent to the
triple bond of the ring-strained cyclooctyne.[23] However,
problems still arise when a site-specific attack to a selected
amino acid residue under physiological conditions is desired.
The attempts so far have mainly followed genetic encoding or
metabolic labeling strategies, in which non-natural amino
acids bearing an alkyne or azide functionality can be
introduced artificially in the peptide sequence.[24,25] However,
these methods suffer from poor generality and involve
complicated and laborious procedures, which are not acces-
sible in most laboratories.
Recently, an elegant variant to SPAAC has been pro-
posed, which replaces the azido functionality with a nitrone
group.[26] Termed strain-promoted azide-nitrone cycloaddi-
tion (SPANC), this reaction was fast and high-yielding with
several molecular species, including peptides,[27] and entire
cells,[28] provided that an accessible nitrone group could be
incorporated in the molecular architecture.
We have recently reported the production of a scFv
variant (scFv800E6) of the anti-HER2 antibody in Pichia
pastoris.[29] There is a growing interest for scFvs due to the
high target selectivity and reduced immunogenicity compared
to whole antibodies.[30] Herein, we explore the potential of the
SPANC reaction for site-specific bioengineering of MFN with
recombinant scFv bioligands and demonstrate the potential of
the resulting targeted MFN (TMFN) in selectively binding to
HER2 breast cancer cell receptors.
In principle, the SPANC reaction seems particularly well
suited for the immobilization of scFv antibodies on nano-
particles, as 1) it prevents homodimer formation, which is
common in thiol-based ligations; 2) the introduction of a
serine at the peptide N-terminus, which can be easily
accomplished by genetic engineering, leads to one single
nitrone species; 3) the conjugation through the N-terminal
residue is expected not to interfere with the affinity toward
HER2. Indeed, N-terminal serine is located sufficiently far
from the antigen-binding site of the peptide. Details on
homology modeling of the N-terminal serine mutant of
scFv800E6 (scFv1) are included in the Supporting Informa-
tion. Serine was inserted during gene amplification by the
polymerase chain reaction (PCR), cloned in pPICZa and
transformed in P. pastoris. ScFv1 was obtained with a C-
terminal c-myc epitope, 6 ꢀ His-tag, and purified by a single-
step purification method on a Ni-NTA agarose affinity
column in 2.5 mgLÀ1 yields.
[*] M. Colombo,[+] P. Verderio, Prof. P. Tortora, Dr. D. Prosperi
NanoBioLab, Dipartimento di Biotecnologie e Bioscienze
Universitꢀ di Milano Bicocca
Piazza della Scienza 2, 20126 Milano (Italy)
E-mail: davide.prosperi@unimib.it
Dr. L. Polito, Dr. D. Prosperi
Istituto di Scienze e Tecnologie Molecolari, CNR
Via Fantoli 16/15, 20138 Milano (Italy)
Dr. S. Sommaruga,[+] Dr. S. Mazzucchelli, Prof. F. Corsi
Dipartimento di Scienze Cliniche “Luigi Sacco”
Universitꢀ di Milano, Ospedale L. Sacco
Via G.B. Grassi 74, 20157 Milano (Italy)
Dr. P. Galeffi
UTAGRI-GEN, ENEA, R. C.Casaccia
Via Anguillarese 301, 00123 Roma (Italy)
[+] These authors contributed equally to this work.
[**] M.C. and S.M. acknowledge the research fellowships of CMENA.
This work was supported by NanoMeDia Project (Regione Lom-
bardia) and “Fondazione Romeo e Enrica Invernizzi”.
Ligand L1 (Scheme 1), containing a 4-dibenzocycloocty-
nol derivatized with a SH-terminated poly(ethylene glycol)
[with a molecular weight of 5000] (PEG5000) carbamate linker,
was synthesized in seven steps according to the procedure
Supporting information for this article is available on the WWW
496
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 496 –499