(Received for publication, June 23, 1995)
From the
Recently antibodies with a wide range of binding specificities
have been isolated from large repertoires of antibody fragments
displayed on filamentous phage, including those that are difficult to
raise by immunization. We have used this approach to isolate an
antibody fragment against chicken very low density lipoprotein (VLDL)
receptor. It binds to the receptor with good affinity (K = 2
10
M
) as measured by plasmon surface
resonance, and competes for binding of natural ligands (vitellogenin,
VLDL, and receptor-associated protein). The antibody also binds to
other members of the low density lipoprotein (LDL) receptor family
including rat LDL receptor and human and rat low density lipoprotein
receptor-related protein (LRP/
MR), and it competes for
binding of receptor-associated protein to LRP/
MR.
Moreover, the antibody fragment inhibits infection of human fibroblasts
deficient in LDL-R but expressing LRP/
MR by human
rhinovirus. Binding of the antibody is abolished upon reduction of the
receptors and is strictly Ca
dependent. The phage
antibody thus recognizes the ligand binding site(s) of several members
of the LDL receptor family, in contrast to antibodies produced by
hybridoma technology.
The low density lipoprotein (LDL) ()receptor of
mammals is the prototype of a family of related proteins. Members of
the LDL receptor family have several structural modules in common; (i)
``binding repeats,'' complement-type domains consisting of
40 residues displaying a triple disulfide bond-stabilized
negatively charged surface (head-to-tail combinations of these repeats
are believed to specify ligand interaction); (ii) epidermal growth
factor precursor-type repeats, also containing six cysteines each;
(iii) modules of
50 residues with a consensus tetrapeptide,
Tyr-Trp-Thr-Asp (YWTD); and (iv), in the cytoplasmic region, signals
for receptor internalization via coated pits, containing the consensus
tetrapeptide Asn-Pro-Xaa-Tyr (NPXY).
The LDL
receptor family includes at least 4 proteins; the LDL receptor (LDL-R),
the low density lipoprotein receptor related protein (also termed
LRP/MR), gp330 (also termed megalin), and the very low
density lipoprotein (VLDL) receptor. The LDL receptor (LDL-R) has a
cluster of 7 binding repeats and binds to apolipoprotein B (apoB) and
apolipoprotein E (apoE)(1, 2) .
LRP/
MR is a giant receptor (4525 amino acids)
containing 4 clusters of 2 to 11 binding repeats and has many ligands
including apoE(3) ,
M-proteinase complexes (4, 5) among
others(4, 5, 6, 7, 8, 9, 10, 11, 12) ,
and a 39-kDa intracellular protein (receptor-associated protein or
RAP). RAP binds to LRP/
MR with high affinity,
co-purifies with LRP/
M from liver and
placenta(13, 14) , and competes for binding with all
known LRP/
MR
ligands(4, 7, 8, 9, 15, 16) .
Whereas the majority of the ligands bound by LRP/
MR
fail to be recognized by LDL-R, human rhinoviruses (HRVs) of the minor
receptor group type attach to either of these proteins(8) .
Recently, it was shown that RAP also binds to LDL-R but with much lower
affinity than to LRP/
MR(17) . Gp330 (megalin)
is a membrane glycoprotein (the Heymann nephritis antigen in
rats)(18) , is closely related to LRP/
MR in
structure(19) , and binds to many of the same ligands (except
for
M-proteinase complexes)(20). The VLDL receptor
(VLDL-R) is characterized by a cluster of 8 binding repeats, and binds
VLDL and other apoE containing lipoproteins(21) .
The LDL
receptor family is also present in birds; for example, in the laying
hen the chicken LDL-R (22) and an LRP/MR (23) are expressed in somatic cells, and an
LRP/
MR-like protein (380 kDa) and a receptor (OVR) for
very low density lipoprotein, vitellogenin (VTG)(24) , and
-macroglobulin (25) are expressed in oocytes.
The binding specificity and sequence of OVR indicate that it is the
chicken homologue of VLDL-R(26) .
Although polypeptides of
the LDL receptor family are highly related, and most of the known
receptors bind to apoE, it has proved difficult to map the ligand
binding sites with respect to different ligands. The sites are thought
to comprise the cysteine-rich binding repeats, and to involve
carboxylate residues on the receptor and lysine and arginine residues
on the ligands(2) ; in apoE most of the positive charges are
clustered to one side of the protein(27) . Although LDL-R binds
to few ligands,
LRP/MR(3, 4, 5, 6, 7, 8, 9, 10, 11, 12) ,
gp330(20) , and OVR bind to a wide spectrum of ligands with
high affinity(24, 25, 28) . In
LRP/
MR it appears that the different ligands bind to
different clusters of binding repeats(29, 30) , but
OVR and LDL-R contain only one cluster of binding repeats, and more
subtle differences must therefore dictate their ligand binding
properties.
Mapping of the ligand binding sites has been hampered by
the difficulty of raising blocking antibodies by immunization. Recently
the display of repertoires of antibody fragments on the surface of
filamentous bacteriophage, and the selection of antigen-binding
phage(31) , has provided a means of making antibodies without
immunization(32) . Antibody repertoires can be derived from the
rearranged V-genes of populations of lymphocytes (32, 33) or from V-gene segments rearranged in
vitro(34, 35, 36) . Antibodies with many
different specificities have been isolated from the same repertoire,
including some directed against self-antigens (33) and highly
conserved proteins (36) . Although the binding affinities of
the antibody fragments were often moderate, it has been possible to
obtain antibodies with good binding affinities (K = 10
-10
M
) from very large
repertoires(34) . Here we used a large phage antibody
repertoire to isolate antibody fragments against OVR.
LDL-R and
LRP/MR both serve as receptors for one group (minor
group) of HRVs(8) , the main causative agents of the common
cold. Due to the large number of different serotypes, vaccination is
not possible; therefore, other means of preventing or curing the common
cold are being thought of, including inhibition of virus-specific
enzymes(37) , or blockage of the viral
receptors(38, 39) . In this article we show that viral
infection can be blocked with the single chain antibody fragment
described.
OVR was also expressed
transiently in COS-7 cells(26) . Briefly, COS-7 cells (American
Type Culture Collection) were seeded at a density of 1.5
10
/80-cm
dish and incubated overnight in RPMI
1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin,
100 µg/ml streptomycin, 2 mM glutamine, and 0.05 mM
-mercaptoethanol. The expression vector (pCDMCVR-1) carrying
the full-length cDNA for the chicken OVR was transfected into COS-7
cells by electroporation (20 µg of DNA/dish) using a Bio-Rad Gene
Pulser. Dishes (60-mm diameter) were seeded with 4
10
cells each in standard medium; after 48 h the cells were washed 3
times with PBS and harvested in PBS containing 0.5 mM phenylmethylsulfonyl fluoride and 2.5 µM leupeptin.
Cells were pelleted by centrifugation and detergent extracts with
Triton X-100 were prepared as described(22) .
Figure 7:
Inhibition of viral infection of FH cells.
Human fibroblasts from a patient with familial hypercholesterolemia (FH
cells, deficient in LDL-R, expressing LRP/MR) were
preincubated with scFv7 at various concentrations and infected with
HRV2 at an multiplicity of infection of 10. Progeny virus was
determined by plaque assay and the numbers of infectious particles are
plotted against the amount of competitor added. The mean values of
three experiments are shown, and standard deviations are indicated as errorbars.
Figure 1:
Amino acid
sequence of scFv7 as deduced from the cDNA sequence. The DNA fragment
encoding the heavy and light chain of scFv7 was amplified via
polymerase chain reaction from bacterial colonies; DNA obtained was
subjected to automatic sequencing. Sequences were compared to published
heavy and light chain data. The heavy chain fragment closely resembled
DP-7 of the V1 family, the light chain fragment showed a
high degree of similarity to L12a of the V
1 family.
Complementarity determining regions are shown in bold, whereas
the framework region is in normal lettering. Amino acid residues
different from those present in DP-7 and L12a are indicated in small letters. The peptide (composed of four repeats of GGGS)
linking heavy and light chain fragments is not
shown.
Figure 2:
Western blot of membrane extracts prepared
from organs from various species and from cells transfected with
OVR-expression plasmid. Electrophoresis was performed under nonreducing
conditions on 4.5-12% SDS-polyacrylamide gradient gels. Proteins
were electrophoretically transferred to nitrocellulose. The positions
of marker proteins with molecular sizes of 116 and 200 kDa and run on
the same gels are shown. A: lanes 1 and 3,
Triton X-100 extracts of chicken follicle membranes (5 µg of
protein/lane); lanes 2 and 4, Triton X-100 extracts
of chicken liver membranes (15 µg of protein/lane); lanes
5-7, Triton X-100 extracts of estrogenized rat liver
membranes. Nitrocellulose membranes were incubated with scFv7 at 5
µg/ml (lanes 1, 2, and 5); with polyclonal
antibody against OVR and oocyte specific LRP/MR at 1
µg/ml (lane 3); with polyclonal antibody against chicken
somatic LRP/
MR at 10 µg/ml (lane 4); with
polyclonal antibody against mammalian LDL receptor at 10 µg/ml (lane 6); and with an antiserum against human
LRP/
MR diluted 1/1500 (lane 7). Bound
antibodies were visualized using the chemiluminescence detection kit
from DuPont as described under ``Materials and Methods.''
Exposure time was 30 s for lanes 1-3, and 2 min for lanes 4-7. Position of OVR (
) and somatic chicken
LRP/
MR (
) are shown. B, COS-7 cells
were transiently transfected with the OVR expression plasmid pCDMCVR-1 (lane 1) or vector alone (lane 2), and processed for
immunoblotting following SDS-PAGE under nonreducing conditions as
described under ``Materials and Methods'' (50 µg of
protein/lane). Lane 3 contained 5 µg of solubilized
(Triton X-100) oocyte membrane protein as a control. Immunoblotting was
performed with 5 µg/ml scFv7. Detection of bound antibody was
carried out as described under A, exposure was for 1 min. C, follicle membrane extracts (5 µg of protein/lane) were
separated by SDS-PAGE on 4.5-12% gradient gels under nonreducing (lanes 1 and 3) and reducing conditions (lanes 2 and 4) and electrophoretically transferred to
nitrocellulose. Western blotting was carried out using
I-labeled scFv7 (0.66 µg/ml with a specific activity
of 1.2
10
cpm/µg; lanes 1 and 2) and nitrocellulose strips were exposed for 16 h. For lanes 3 and 4, a polyclonal antipeptide antibody
specific for the carboxyl terminus of OVR was used at 10 µg/ml.
Bound IgG was detected as described under A. Exposure time was
2 min.
In Fig. 2B, we used detergent extracts from COS-7 cells
which had been transiently transfected with a plasmid carrying OVR-cDNA (lane 1) or with a control plasmid (lane
2)(26) . On the Western blot, scFv7 detected a strong band
in lane 1 that comigrated with OVR of an oocyte membrane
extract (lane 3). The weak band seen in the mock-transfected
cells (lane 2) and comigrating with the recombinant OVR
probably represents the endogenous simian VLDL-R. There is an
additional band migrating slightly slower than OVR in lanes 1 and 2. This protein was not further characterized; it
might correspond to the simian LDL-R or to another, still unknown
member of the LDL receptor family. The higher band (500 kDa) seen in lanes 1 and 2 is most likely simian
LRP/MR which is abundantly expressed in COS
cells(57) .
Finally, as shown in Fig. 2C,
the antibody strongly discriminates between non-reduced (lane
1) and reduced (lane 2) receptors (OVR migrating at 95
kDa and somatic LRP/MR migrating at about 500 kDa,
respectively), whereas a control antibody directed against a synthetic
peptide derived from the carboxyl terminus of OVR reacted with both
forms of OVR equally well, but failed to bind to somatic
LRP/
MR (lanes 3 and 4). Note that
OVR migrates with a much higher apparent molecular weight in its
reduced form when compared to the migration of its unreduced form
(compare lanes 3 and 4).
The analyzed scFv7 was
also shown to bind to LRP/M with high affinity and, to
a lesser extent, to LDL-R (Fig. 2A). Affinity constants
for human LRP/
M and bovine LDL-R were therefore also
determined. Immobilization of LRP and LDL-R yielded 2100 and 550 RU,
respectively. scFv7 at concentrations of 2.5
10
to 2
10
M was tested for
binding to the immobilized proteins. scFv7 bound to LRP with an
affinity of K
= 8
10
M
and to LDL-R with K
= 5
10
M
(Fig. 3).
Figure 3:
BIAcore sensograms (RU as a function of
time) of the interaction of scFv7 with immobilized members of the LDL
receptor family. A 45-µl pulse of scFv7 for OVR and a 35-µl
pulse for LRP/MR and LDL-R, respectively, at the
concentrations indicated was passed, with a flow rate of 5 µl/min,
over a sensor chip to which the receptors had been coupled. On/off
rates, affinity constants, and the range of concentration used for the
determinations are indicated. Constants are mean values of
determinations at five different concentrations. All measurements were
carried out in duplicate. Sensograms shown were obtained with a
concentration of scFv7 of 2
10
M for OVR, 4
10
M for LRP, 6
10
M for LDL-R, respectively. An
irrelevant scFv fragment was passed over the same surface and did not
reveal binding to any of the receptors.
As it has been
recently shown that RAP also binds strongly to the VLDL receptor of
mammalian (58) as well as of avian origin(73) , we also
analyzed the kinetics of binding of RAP to chicken VLDL receptors.
Recombinant RAP fused to glutathione S-transferase (GST-RAP) (15) was injected over a Biacore chip coated with OVR at
concentrations of 2 10
to 2
10
M, yielding an affinity constant in the
same range as scFv7 of 3
10
M
(data not shown).
Figure 4:
Competition of ligands for binding of I-scFv7 to chicken OVR (A) and to human
LRP/
MR (B). Purified OVR (1 pmol/well) and
LRP/
MR (250 fmol/well), respectively, were immobilized
on ELISA plates and incubated with 25 nM (1.2
10
cpm/µg)
I-scFv7 together with competitors at
the molar excess indicated. Radioactivity bound was determined in a
-counter and values were plotted against times molar excess of the
competitors. Data points given are the mean of triplicate
experiments.
Figure 5:
Quantification of the Ca
dependence of scFv7 binding to chicken OVR (A and C)
and human LRP/
MR (B and D) by solid
phase assay (for conditions see Fig. 4). The receptors were
coated onto ELISA plates and incubated with
I-scFv in the
presence of EDTA or EGTA at the concentrations indicated. In C and D, wells were incubated with various concentrations
of Ca
ions in the presence of
I-scFv7
and 7.5 mM EDTA. Radioactivity bound was determined using a
-counter and plotted against the concentrations of EDTA and EGTA (A and B) or Ca
ions (C and D).
Figure 6:
Competition of HRV2 binding to OVR by
scFv7 and RAP. Purified OVR (1 pmol/well) was immobilized on ELISA
plates and incubated with S-HRV2 together with competitors
at the concentrations indicated. Radioactivity bound was determined in
a scintillation counter and values are plotted against the
concentrations of competitors added. Data points given are the mean of
triplicate experiments.
The biological functions of the LDL receptor family are
important and diverse. For example, the LDL receptor has a key role in
cholesterol homeostasis in mammals; mutations disrupting its function
leading to severe hypercholesterolemia and premature artheriosclerosis
in man(63) . The LRP/MR is probably involved
in clearing spent proteases and chylomicron remnants from the
circulation (64) , and may also have a role in development, as
mouse embryos with a homozygous knockout for the
LRP/
MR gene are arrested in various stages of
development(59, 65) . OVR appears to have a role in
reproduction mediating growth of oocytes via uptake of the major yolk
precursors VLDL and VTG from coated pits in the plasma
membrane(66, 67) : mutant ``restricted
ovulator'' hens are sterile(68) . As the yolk precursor
proteins comprise about 50% of the total weight of the egg yolk the
endocytic mechanisms mediated by OVR must be highly efficient.
Antibodies that block the binding of multiple ligands to the LDL receptor family should help in dissecting the roles of these receptors and ligands. Although antisera and monoclonal antibodies have been obtained against several members of the LDL-R family, none have been described that efficiently block the binding of ligands. An exception is IgG-C7, a monoclonal antibody against human LDL-R recognizing the first ligand binding domain of the receptor, which inhibits the binding of LDL or apoE-rich lipoproteins(69) . The difficulty in obtaining antibodies with the desired properties by conventional means may be due to the conserved nature of these epitopes of the receptor between different species, or to the presence of ligands in the serum of the animal to be immunized rendering the binding site(s) inaccessible.
We therefore attempted to make blocking antibodies by
phage display technology without immunization, and selecting with pure
chicken OVR from a large (1.5 10
clones) repertoire
of scFv fragments. We succeeded in isolating an antibody fragment
(scFv7) that bound strongly to chicken OVR from membrane extracts from
follicles and COS-7 cells transfected with a plasmid encoding OVR.
ScFv7 appears to bind at the same site as several natural ligands as its binding to OVR is competed with VTG and RAP (Fig. 4A).
As shown by BIAcore, the binding affinity
to OVR appeared to be good (K = 2
10
M
or K
= 5 nM); the slow off-rate (3
10
s
) makes the fragment
particularly suitable for mapping studies. Indeed the affinity constant
is very similar to that of GST-RAP. As well as binding to OVR, scFv7
also binds to chicken, rat, and human LRP/
MR (Fig. 2A and Fig. 3), and, to a much lesser
extent, to rat and bovine LDL-R (Fig. 3), and its binding to
LRP/
MR is competed with RAP (Fig. 4B).
A 100-fold molar excess of recombinant GST failed to inhibit binding of
scFv7 to either receptor (not shown).
The binding of scFv7, like the
natural ligands, is abolished after reduction of the receptors (Fig. 2C) and also requires Ca ions (Fig. 5). EGTA at about 5 mM led to a significant loss
of activity. These observations suggest that scFv7 recognizes a
conformational epitope rather than the primary sequence. However,
unlike the binding of the natural ligands RAP and apoE which depend on
electrostatic interactions, we see no evidence for such interactions
with the antibody; the sequence of the antibody reveals no charge
clusters in the complementarity determining regions or any sequences
resembling those of the natural ligands. Furthermore, the antibody
fragment does not bind to heparin, while RAP is strongly interacting
with this glycan under identical conditions. (
)Thus the scFv
appears to be ``seeing'' the ligand binding site but in a
different manner to the natural ligands; it therefore differs from the
anti-integrin antibodies isolated from antibody repertoires with a
``built-in'' sequence motif from the natural
ligand(70) .
In addition to the ligands mentioned above,
human rhinoviruses have been shown to gain access to the host cell via
members of the LDL-R family(8) . The large number of different
rhinovirus serotypes is divided into two groups dependent on their
binding to either the intercellular adhesion molecule 1 (ICAM-1, major
group) or to members of the LDL-R family (minor group). Monoclonal
antibodies which effectively block infection by major group viruses
have been obtained and were used to identify the major group
receptor(71, 72) . No antibodies blocking infection by
minor group viruses are available due to the presence of both LDL-R and
LRP/MR on the cell surface. These receptors are
immunologically distinct but are both used as minor group receptors.
ScFv7 cannot block infection of wild type fibroblasts with minor group
HRVs but effectively blocks infection of FH cells (human fibroblasts
deficient in LDL receptor synthesis).
The protection is even
stronger in the presence of the anti-myc-antibody 9E10 which
renders the scFv7 bivalent by binding two molecules via the myc-sequence tag which is COOH terminally fused to the
antibody fragment. Based on the dual specificity it is likely that
minor group HRVs recognize a structure or charge pattern equally
present in LDL-R and LRP/MR which might be detectable
using antibodies with a broader specificity and improved affinity
toward LDL-R. Experiments to produce such antibodies are presently
being carried out in our laboratory.