SITE-SPECIFIC CONJUGATION OF SCFVS ANTIBODIES TO ...

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12 Δεκ 2012 (πριν από 8 χρόνια και 7 μήνες)

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SITE
-
SPECIFIC CONJUGATION OF
SC
F
VS

ANTIBODIES TO NANOPARTICLES BY
BIOORTHOGONAL STRAIN
-
PROMOTED ALKYNE

NITRONE CYCLOADDITION


Silvia Sommaruga

,
Miriam Colombo

, Serena Mazzucchelli

,
Laura Polito*
,

and Davide Prosperi

*


Universit
à

di
Milano Bicocca
,

Piaz
za della Scienza 2, 20126 Milan
o

(Italy)

*
Istituto di Scienze e Tecnologie Molecolari, CNR Via Fantoli 16/15, 20138 Milano (Italy)



Hybrid multifunctional nanoparticles (MFN), which combine

unique superparamagnetic properties and
fluorescence
emission,

have been envisaged as promising bimodal tracers for

noninvasive diagnosis of cancer
both in vitro and in vivo.

The design of ideal targeted MFN
(TMFN)
needs careful

optimization of fundamental
features including uniform size

and shape, surface c
harge,

o
ptical and magnetic properties

and efficient
functionalization with suitable homing

ligands to improve the signal amplification and target

selectivity toward
malignant cells. When the ligands are

complex molecules, such as proteins, their proper or
ientation

on the
surface of nanoparticles becomes a crucial factor for

maximizing the affinity fo
r their molecular counterparts.

In this work w
e present the potential of a new method based on a single
-
step bioorthogonal reaction,
namely the strain
-
promoted

alkyne
-
nitrone cycloaddition (SPANC), as an efficient variant of the copper
-
free
“click” reaction, for the reliable

surface functionalization of magnetofluorescent nanoparticles with proteins.
This approach has several advantages

compared with other metho
ds: 1) the reaction is fast and versatile, 2) a
complete control on the site of conjugation of

the protein (and consequently on the protein orientation on the
nanoparticle) can be achieved with a precision of a

single aminoacid, 3) the reaction works best
in a
biocompatible environment and 4) the reaction is byproduct
-
free,

thus each step of purification is simple and
efficient.


The method was validated using an scFv antibody fragment

against HER2 tumor marker

with
high target
selectivity and
reduced immunogenicity compared to whole antibodies. ScFv molecule was modified with the
introduction of a N
-
terminal serine residue
(scFv1)
by genetic engineering and produced in
P. pastoris
.

ScFv1
immobiliz
ation on
MFN
leads

to water
-
stable bioengineered

targeted MFN
. P
rotein effectiveness in selectively
targeting HER2 receptor in living cells

was confirmed by
flow cytometry

and
confocal laser

scanning
microscopy.


As

the structural motif of scFv fragments is highly conserved, and other kinds of
nanoparticles can be
modified

identically with the same polymer used herein, this approach is expected to be of general utility and
may become a

universal strategy for the development of a new gener
ation of targeted nanoparticles
.