Journal of Immunological Methods 242 (2000) 193–204
www.elsevier.nl/locate/jim
Protocol
Synthesis of high avidity antibody fragments (scFv multimers)
for cancer imaging
a, a,b
*
Barbara E. Power , Peter J. Hudson
a
CSIRO Health Sciences and Nutrition
, 343
Royal Parade
,
Parkville
,
Victoria
3052,
Australia
b
CRC for Diagnostic Technologies
, 343
Royal Parade
,
Parkville
,
Victoria
3052,
Australia
Received 6 April 2000; accepted 25 April 2000
Abstract
Multivalent antibody fragments (scFv dimers, trimers and tetramers) provide high avidity and ideal pharmacokinetics for
tumour targeting applications. This protocol describes our optimised protocol for high-level bacterial synthesis of soluble
antibody scFv fragments, as either monomers or multimers, using the heat-inducible bacterial expression vector pPOW3. Our
protocol is rapid, which minimizes protein degradation, and utilises inexpensive reagents for cost-effective product synthesis.
The strong, temperature-regulated promoters in pPOW3 provide efficient production of either monomeric or multimeric
single-chain antibody fragments as dictated by the gene construct design. 2000 Elsevier Science B.V. All rights reserved.
Keywords
:
E
.
coli; Heat-induction; Soluble; Antibody; Multimers; scFv
1. Type of research tail such as the octapeptide (FLAG) fused to the
C-terminus of the antibody construct, as described by
(1) This protocol describes our preferred method Power et al. (1992).
for high level synthesis of antibody fragments (scFv (2) Single chain Fv (scFv) fragments provide the
monomers or multimers) in the bacterial periplasm, original affinity of the parent antibody and are
utilising procedures for recovery by either osmotic synthesized efficiently in bacteria since the V and
H
shock or by denaturation and refolding. Purification V domains are tethered together with a polypeptide
L
is achieved by affinity chromatography using a tag linker (reviewed by Hudson and Kortt, 1999). Typi-
cally, scFv monomers (30 kDa) are designed with
the C-terminal end of the V domain (defined as
H
Abbreviations: Ab, antibody; CDR, complementarity determin-
112
Ser using the numbering system of Kabat et al.,
ing region; Fab, antibody fragment produced by proteolysis; Fv,
1991) tethered by a polypeptide linker to the N-
complex of V and V domains; Ig, immunoglobulin; kDa,
HL
kilodalton; Mab, monoclonal antibody; M , molecular mass; PCR,
terminal residue of V or, in inverse orientation, the
r
L
107
polymerase chain reaction; scFv, single chain Fv molecule; V ,
H
C-terminal end of the V domain (defined as Arg )
L
variable region from antibody heavy chain; V , variable region
L
is tethered to the N-terminal residue of V (Malby et
H
from antibody light chain
al., 1993, 1998). Owens and Young (1994) showed
*Corresponding author. Tel.: 161-3-9662-7381; fax: 161-3-
that these small scFv monomers have suitable
9662-7314.
E-mail address
:
barbara.power@hsn.csiro.au (B.E. Power).
tumour imaging properties due to rapid tissue pene-
0022-1759/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0022-1759(00)00201-5