(Received for publication, October 16, 1995; and in revised form, January 12, 1996)
From the
This report describes the construction of leucine zipper-based
dimerization cassettes for the conversion of recombinant monomeric scFv
antibody fragments to bivalent and bispecific dimers. A truncated
murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned
into four scFv fragments previously isolated from a synthetic antibody
phage display library. Cysteine residues flanking the zipper region
were introduced to covalently link dimerized scFv fragments. The
secreted fusion proteins were shown to spontaneously and efficiently
form stable FosFos or Jun
Jun homodimers in the Escherichia coli periplasm at levels comparable to their
monovalent counterparts. The bivalent (scFv)
fragments
performed well in enzyme-linked immunosorbent assay, flowcytometric,
and immunohistochemical analysis. Fos and Jun homodimer
(scFv)
antibodies with different specificities could be
reduced, reshuffled, and reoxidized to form preparations of functional
bispecific (scFv)
Fos
Jun heterodimers. These Fos and
Jun fusion protein cassettes provide a universal basis for the
construction of dimeric scFv antibodies with enhanced avidity or dual
specificity.
Surface display of Fab or single chain Fv (scFv) antibody
fragments on filamentous phage particles in combination with an array
of versatile selection procedures has become a powerful approach to
obtain recombinant molecules with desired specificities and binding
properties from large libraries (reviewed by Winter et al. (1994) and Burton and Barbas(1994)). The Fab and scFv fragments
thus obtained are monovalent, whereas in many in vitro and in vivo applications, multivalency of antibody molecules is a
desirable property. In addition, linking two or more binding sites
efficiently increases the functional avidity of antibody molecules or
results in the construction of antibodies with dual specificities
(Plückthun, 1992). Several approaches have been
employed to generate genetically engineered, multimerized antibody
fragments. Bivalent (and bispecific) (scFv) and (Fab)
fragments have been successfully produced by association of two
molecules through flexible linker polypeptides, chemical cross-linking,
and dimerization domains (reviewed by Holliger and Winter(1993)). In
the latter approach, introduction of amphipathic helices or leucine
zippers was shown to mediate dimerization of scFv or Fab fragments in vivo (Pack and Plückthun, 1992; Pack et al., 1993, 1995; Kostelny et al., 1992). These
efforts have resulted in the production of higher valency antibody
fragments with widely varying physicochemical properties.
In
designing strategies for dimerization of antibody fragments, several
issues need to addressed including stability and homogeneity of the
dimers, resistance to proteolytic cleavage during in vivo assembly, efficient production of preferably soluble protein,
simple engineering steps, and general applicability for the
construction of both bivalent and bispecific recombinant antibodies.
With these issues in mind, we designed dimerization cassettes that
allow the conversion of scFv antibodies from a number of published
phage display libraries to bivalent or bispecific reagents involving a
single cloning step. In this procedure, the flexible and
proteolysis-resistant truncated mouse IgG3 upper hinge region (Pack and
Plückthun, 1992) and either Fos or Jun leucine
zippers were fused to scFv proteins. Two cysteine residues were
engineered in the Fos and Jun zipper domains to produce
disulfide-stabilized homodimers. Using four scFv antibodies previously
isolated from a synthetic phage display library, we show that this
approach results in the efficient in vivo production of
stable, secreted homodimers that retain their specificity as assessed
in a number of assays. Furthermore, exploiting preferential
FosJun heterodimer over Fos
Fos or Jun
Jun homodimer
formation, we show that in vitro reduction, mixing, and
re-oxidation of Fos and Jun scFv antibodies with different
specificities results in the production of bispecific (scFv)
molecules.
Figure 1: Diagram of the Fos and Jun dimerization cassettes cloned into scFv-containing pHEN1 phagemids. Cysteine residues are underlined.
For sandwich ELISAs, 50 µl of 3F 23J (IgG
DNP) bispecific (scFv)
preparation was blocked in
150 µl of 4% MPBS for 15 min before addition to IgG-coated wells.
After a 1-h incubation, plates were washed in PBST, and 200 µl of 1
µg/ml DNP-BSA in MPBS was added to the wells. Following another 1-h
incubation, unbound DNP-BSA was removed by washing in PBST, and bound
DNP-BSA was detected using clone 22 phage antibodies recognizing a
different epitope on DNP than scFv clone 23. Binding of the clone 22
phage antibodies was visualized by incubation with a polyclonal sheep
anti-M13 horseradish peroxidase-conjugated antibody (Pharmacia,
Uppsala, Sweden) as described (de Kruif et al., 1995a). All
incubations were performed at room temperature.
Figure 2:
SDS-PAGE Western blot analysis of
expressed scFv and (scFv) proteins. The upper panel shows the migration of anti-IgG (lane 1) and anti-DNP (lane 2) scFv and their Fos and Jun fusion protein derivatives (lanes 3 and 4, respectively) run under reducing
conditions. In the lower panel, reducing agents were omitted
from the sample SDS buffer.
To investigate the antigen binding potential of the
closely spaced (scFv) bands (Fig. 2), periplasmic
preparations of Fos-dimerized anti-IgG (3F) and Jun-dimerized anti-DNP
(scFv)
(23J) were allowed to bind to DNP-BSA and IgG coated
to paramagnetic beads. After washing, bound proteins were eluted from
the beads and analyzed by non-reducing SDS-PAGE. In control
incubations, no binding of 23J (scFv)
to IgG or 3F
(scFv)
to DNP was observed (Fig. 3, lanes 2 and 6). In contrast, immunoaffinity selection of 3F and
23J scFv dimers on the corresponding antigen-coated beads resulted in
the purification of a set of proteins displaying the same
characteristic banding pattern as their non-purified counterparts (Fig. 3, lanes 1 and 3-5). These results
suggest that all (scFv)
proteins formed in the bacterial
periplasm retained their specific antigen binding capacity.
Figure 3:
SDS-PAGE Western blot analysis of
(scFv) proteins selected for binding to their target
antigen. (scFv)
proteins were incubated with paramagnetic
beads coated with either IgG or DNP. After washing, the beads were
boiled in non-reducing SDS sample buffer, and the resulting protein
mixture was applied to the gel. Lane 1, 3F (scFv)
periplasm; lane 2, 3F (scFv)
selected on
DNP; lane 3, 3F (scFv)
selected on IgG; lane
4, 23J periplasm; lane 5, 23J (scFv)
selected
on DNP; lane 6, 23J (scFv)
selected on
IgG.
Figure 4:
Antigen
specificity of (scFv) antibody fragments. Microtiter plates
were coated with IgG, DNP, or a panel of control antigens including
lysozyme, thyroglobulin, ovalbumin, HMG-box protein, bovine serum
albumin, and milk powder. (scFv)
molecules were allowed to
bind and were detected using the 9E10
antibody.
Figure 5:
Flow cytometric analysis of peripheral
blood leukocytes double stained with anti-CD8 scFv or (scFv)
antibody fragments and a conventional fluorescein
isothiocyanate-conjugated anti-CD8 monoclonal antibody. Peripheral
blood leukocytes were incubated with periplasmic preparations of scFv (middle panel) and (scFv)
(right panel)
secreting bacteria, and bound fragments were detected using the 9E10
monoclonal antibody followed by a goat-anti-mouse phycoerythrin-labeled
polyclonal antibody. As a control, the incubation step with scFv
fragments was omitted (left panel). Only cells with a forward
scatter/side scatter profile corresponding to lymphocytes are shown. Boxed area, CD8+ T cells as detected with a conventional
monoclonal antibody.
Figure 6:
Immunohistochemical staining of COS7 cells
transfected with a cDNA encoding the CD22 chain. COS7 cells were
stained with a periplasmic preparation of bacteria transformed with the
40J construct encoding a Jun dimerized anti-CD22
(scFv)
. Bound antibodies were detected using the 9E10
anti-Myc antibody followed by goat-anti-mouse antibodies coupled to
horse-radish peroxidase.
Figure 7:
Formation of bispecific anti-IgG
anti-DNP (scFv)
fragments in vitro, visualized by
non-denaturing SDS-PAGE and Western blotting. Periplasmic preparations
of scFv clones 3F and 23J were reduced in 2-mercaptoethanol. Reduced
proteins were mixed in a redox buffer followed by dialysis against PBS,
resulting in the generation of bispecific antibody 3F
23J.
Figure 8:
Detection of bispecific anti-IgG
anti-DNP fragments in a sandwich ELISA. IgG-coated plates were
incubated with (scFv)
proteins. After washing, DNP was
added to the wells. Bound DNP was detected using an anti-DNP phage
antibody followed by a horseradish peroxidase-coupled anti-M13
antibody. 3F
23J, anti-DNP
anti-IgG bispecific
(scFv)
fragment; 3F and 23J, anti-IgG and anti-DNP dimers.
-DNP and -(scFv)
, no DNP or scFv protein
added.
We have constructed scFv antibody fragment dimerization cassettes that can be readily introduced in the NotI restriction sites of genes encoding scFvs isolated from a variety of phage display libraries described in the literature (Hoogenboom and Winter, 1992; Nissim et al., 1994; de Kruif et al., 1995a). These cassettes add a truncated, flexible murine IgG3 hinge region and either a Fos or Jun leucine zipper to the scFv proteins. To increase stability of the bivalent antibodies, cysteine residues were incorporated at the N and C termini of each of the leucine zippers, facilitating disulfide bridge formation in the periplasmic space (Crameri and Suter, 1993).
The performance of zipper-linked
(scFv) molecules was assessed using four different scFv
antibodies selected from a synthetic phage display library as starting
material. All scFv-zipper molecules were secreted as soluble proteins
into the periplasmic space, obviating tedious refolding procedures
associated with the formation of insoluble inclusion bodies (Kurucz et al., 1995). In each instance, the level of expression did
not appear to be significantly affected by addition of the hinge and
Fos or Jun zipper regions. In Western blotting under non-denaturing
conditions, periplasmic preparations of Fos or Jun scFv zippers solely
contained dimeric molecules, indicating that the formation of
(scFv)
homodimers from scFv-zipper monomeric precursors is
extremely efficient. The homodimers were resistant to boiling in sample
buffer containing 4% SDS and could only be dissociated to monomers
using reducing agents. We conclude that the monomers in a (scFv)
complex are covalently linked via disulfide bridges connecting
the leucine zippers.
Monomeric scFv molecules were detectable as
single bands in non-reducing SDS-PAGE, consistent with the notion that
proteins secreted into the periplasmic space of Gram-negative bacteria
refold properly with formation of the correct disulfide bonds (Huston et al.(1993) and references therein). In contrast, Fos or Jun
homodimers presented themselves as multiple closely spaced bands in
non-reducing SDS-PAGE. Immunoaffinity purification of (scFv) containing periplasmic preparations on antigen-coated beads
showed that each of the closely spaced bands corresponded to functional
protein retaining the capacity to specifically bind antigen. Others
have also observed multiple scFv bands under non-reducing SDS-PAGE
conditions (Neuberger et al., 1984; Kostelny et al.,
1992; Huston et al., 1993), and it has been suggested that
this results from anomalies associated with SDS binding to unreduced
proteins.
Previously, a tendency of GCN4 zipper-linked
``mini-antibodies'' to display nonspecific binding to
antigens coated to microtiter wells has been noted (Pack et al. 1993). We examined the binding specificities of our bivalent and
bispecific (scFv) fragments in a number of assays,
including ELISA, flow cytometry and immunohistochemistry. In none of
these assays, significant nonspecific binding was observed. A reason
for this apparent discrepancy between GCN4 zippers and Fos/Jun zippers
may be a better shielding of the hydrophobic regions in the latter
and/or the more stable configuration caused by covalently cross-linking
the zipper regions.
Employing the much greater tendency of Fos and
Jun zipper peptides to form heterodimers over homodimers (O'Shea et al., 1989; Kostelny et al., 1992), bivalent Fos
and Jun leucine-zippered (scFv) can be rapidly converted to
bispecific (scFv)
molecules by simple reduction, mixing,
and reoxidation steps. Using this approach, the anti-IgG and anti-DNP
binding activities of two (scFv)
homodimers were shown to
be combined in a single heterodimeric molecule. A major advantage of
this strategy is that only a single straightforward cloning step is
required to produce bispecific antibodies obviating the need for
extensive polymerase chain reaction and cloning efforts (Holliger et al., 1993; Mallender and Voss, 1994; Kurucz et
al., 1995; Mack et al., 1995).
The dimerization system described here may be used to construct phage display libraries of bispecific antibodies. Bispecific antibodies that simultaneously recognize adjacent and non-overlapping epitopes on a target protein have higher avidities than the single chain or Fab antibodies obtained from conventional libraries (Neri et al., 1995). Thus, a Fos-linked scFv with a desirable specificity may be cloned into a phage library of Jun-scFv antibodies, permitting the direct recovery of high avidity bispecific antibodies using stringent selection procedures. We are currently performing experiments to assess the feasibility of this approach.
We show that using cysteine-modified Fos and Jun leucine zipper peptides, scFv antibody fragments isolated from phage display libraries can be simply converted to functional bivalent and bispecific molecules involving only a single cloning step. It is important to note that scFv molecules obtained from phage display libraries have been through a stringent selection for correct expression, transport, and folding in bacterial cells. This explains why these antibodies and the derivatives described in this paper do not appear to suffer from many of the problems associated with bacterially expressed scFvs derived from hybridomas.