(Received for publication, December 10, 1996, and in revised form, March 6, 1997)
From Protein Design Labs, Inc., Mountain View, California 94043
A panel of murine monoclonal antibodies was
generated against the extracellular domain of the human
platelet-derived growth factor (PDGF) receptor (PDGFR
). These
antibodies were assayed for both the ability to inhibit binding of PDGF
BB to PDGFR
+ cells as well as the capacity to
inhibit PDGF BB-mediated mitogenesis. As expected, all antibodies that
could prevent PDGF BB binding also inhibited mitogenesis. However one
antibody (M4TS.11), with no detectable ability to inhibit PDGF BB
binding, was a potent inhibitor of proliferation induced by PDGF BB.
Further characterization indicated that M4TS.11 impaired PDGFR
dimerization, revealing the mechanism by which it prevented PDGF
BB-mediated mitogenesis. Using domain deletion mutants of the
extracellular portion of PDGFR
, the determinant recognized by this
antibody was localized to the fourth extracellular domain of PDGFR
,
indicating that this domain, which is not involved in ligand binding,
actively participates in receptor dimerization and signal transduction. The M4TS.11 antibody could also inhibit PDGF BB-mediated proliferation of responsive cells from both the baboon and the rabbit, indicating the
determinant recognized by the antibody is not limited to humans and
making it possible to use this antibody to evaluate the therapeutic benefit of interfering with PDGF in animal models of human disease.
Platelet-derived growth factor (PDGF)1 is a mitogen and chemoattractant for cells of mesenchymal origin, such as fibroblasts, smooth muscle cells, and glial cells (1-3). PDGF is encoded by two genes, the products of which are designated A and B. The active PDGF molecule is a disulfide-linked dimer of these polypeptides and thus can exist in three forms: the homodimers AA or BB or the heterodimer AB (4).
Studies examining the interaction of PDGF with responsive cells have
revealed the existence of two specific receptors designated and
and encoded by separate genes. Each receptor type is composed of five
extracellular immunoglobulin-like domains attached to an intracellular
tyrosine kinase domain via a transmembrane segment (5). This structural
organization is the prototype for the PDGF receptor family of
protein-tyrosine kinases, which includes stem cell factor receptor,
colony stimulating factor receptor, and Flk-2 (6).
The PDGF dimer stimulates responsive cells by cross-linking two
receptor subunits (5, 6). The different forms of PDGF exhibit different
affinities for the two forms of the PDGF receptor. The PDGFR can
interact with all three forms of PDGF; PDGFR
can only interact with
PDGF BB and AB. This pattern of reactivity dictates that PDGF AA can
signal the cell only through homodimers of PDGFR
, PDGF BB can signal
the cell through homodimers of PDGFR
or PDGFR
or the heterodimer
PDGFR
·PDGFR
, and PDGF AB can stimulate cells through either
homodimers of PDGFR
or the PDGFR
· PDGFR
heterodimer
(7).
As a potent mitogenic and chemotactic agent, PDGF has been implicated as a contributing factor in a number of pathologic conditions that involve the migration and proliferation of PDGF-responsive cells. Such conditions include arteriosclerosis (8), restenosis following coronary bypass surgery or balloon angioplasty (9), nephritis (10), scleroderma (11), and some neoplasias (12). Thus, interfering with the biologic activities of PDGF may be of therapeutic value for one or more of these conditions.
We have generated and characterized a panel of murine monoclonal
antibodies against the extracellular portion of the PDGFR and
examined the ability of these antibodies to inhibit PDGF BB-specific binding and/or induction of mitogenesis. One of these antibodies exhibits no effect on PDGF BB binding to the receptor but does inhibit
PDGF-mediated mitogenesis by impairing receptor dimerization. This
antibody cross-reacts with PDGFR
from other species, making it an
ideal candidate to study the therapeutic potential of an antibody PDGF
antagonist in animal models of human disease.
The Chinese hamster ovary cell
line CHO/dhFr and the rabbit cornea cell line SIRC were
obtained from the American Type Culture Collection. Baboon primary
aortic smooth muscle cells (SMC) were kindly provided by J. Anderson
and S. Hanson at Emory University (Atlanta, GA). Purified PDGF BB was
obtained from Boehringer Mannheim.
A gene encoding the PDGFR, lacking the nucleotides
encoding a portion of the 5
end, was obtained from the American Type Culture Collection. The missing portion of the gene, which included the
secretion signal sequence, was constructed by oligonucleotide synthesis
and used to assemble the complete PDGFR
gene with an XbaI
site at each end. This fragment was inserted into the XbaI site of the plasmid pVk (13) and co-transfected together with plasmid
pVgl (13), which contains a dhfr gene, into
CHO/dhFr
cells using the calcium phosphate method
essentially as described (14). Methotrexate-resistant transfectants
expressing the PDGFR
were identified by indirect immunofluorescence
using a commercially available antibody (Genzyme) and cloned by single
cell sorting using a FACStarPLUS (Becton Dickinson). The
resulting cell line (CHO C4) was responsive to PDGF BB as evidenced by
increased DNA synthesis and cell proliferation in the presence of the
growth factor as demonstrated previously (15).
To produce a soluble form of PDGFR (sPDGFR
), the gene encoding
the protein was truncated immediately before the transmembrane sequence, and a stop codon followed by an XbaI restriction
endonuclease site was inserted. This gene fragment was inserted into
the XbaI site of pVk. The resulting plasmid was
cotransfected as above into CHO/dhFr
cells together with
plasmid pVgl. Transfectants resistant to methotrexate were selected and
used for production of sPDGFR
by growing to confluence in
Dulbecco's modified Eagle's medium plus 10% fetal calf serum, then
replacing this serum-containing medium with protein-free medium and
incubating for an additional 72 h. The exhausted medium was
harvested and passed over a wheat germ agglutinin-Sepharose column. The
column was washed and eluted with
N-acetyl-D-glucosamine. The eluant containing
sPDGFR
was dialyzed and concentrated to approximately 300 µg/ml.
The purified protein was >95% pure based on SDS-polyacrylamide gel
electrophoresis analysis.
A panel of monoclonal antibodies was
generated against the extracellular portion of the human PDGFR by
immunizing outbred Swiss Webster mice with 50 µg of purified
sPDGFR
in RIBI adjuvant (ImmunoChem Research). The mice received
booster immunizations of 50 µg of PDGFR
every 1-2 weeks. Mice
were bled 1 week following each boost, and the sera were tested for
reactivity with sPDGFR
by an ELISA. The mouse exhibiting the highest
serum titer of anti-PDGFR
activity was sacrificed 3 days after
receiving a final boost of 50 µg of sPDGFR
, and hybridomas were
prepared. Hybridoma supernatants were tested for reactivity with
sPDGFR
by an ELISA (see below). Hybridomas that exhibited reactivity
were expanded and cloned. All monoclonal antibodies reactive with
soluble PDGFR
were assayed for reactivity against cell
surface-expressed PDGFR
on the surface of CHO C4 transfectants by
flow cytometry. Only antibodies demonstrating reactivity with cell
surface-expressed PDGFR
were further characterized.
Immulon 1 96-well plates (Dynatech Laboratories Inc.)
were coated with sPDGFR by adding 100 µl of a 0.5 µg/ml solution
of sPDGFR
in phosphate-buffered saline to each well. After an
overnight incubation at 4 C°, 200 µl of phosphate-buffered saline
plus 1% bovine serum albumin and 0.5% Tween 20 were added to each
well to block unoccupied protein binding sites. After a 1-h incubation at room temperature, the wells were washed three times with
phosphate-buffered saline plus 1% Tween 20. Dilutions of purified
antibody or antibody-containing supernatants (100 µl) were added to
each well, and the plate was incubated for 1 h at room
temperature, after which it was washed three times as described above.
One hundred µl of a 1 µg/ml solution of horseradish
peroxidase-conjugated GAMIgG (Tago, Inc.) was added to each well, and
the plates were incubated for 1 additional h at room temperature, after
which they were washed three times. One hundred µl of peroxidase
substrate (Bio-Rad) was added to each well, the plate was incubated for
15-60 min, and absorbance at 415 nm was determined.
CHO C4 cells were harvested, washed twice with cold
Dulbecco's modified Eagle's medium, and resuspended at
106 cells/ml in Dulbecco's modified Eagle's medium. The
assay was carried out in triplicate by incubating 100 µl of the cell
suspension with either no antibody (to determine maximum binding), 5 µg of the indicated antibody, or 100 ng of cold PDGF BB (to saturate specific binding sites and determine nonspecific binding) in 12 × 75 mm polystyrene tubes for 15 min at 4 C°. Each sample received 1.0 ng of 125I-labeled PDGF BB (Amersham Life Science, Inc.)
and was incubated for an additional 60 min at 4 C°. Unbound
125I-labeled PDGF BB was separated from that bound to the
cell by layering the sample over a mixture of 80% dibutylphthalate,
20% olive oil in a Reagiergefä test tube (Sarstedt, Inc.) and
microfuging briefly to pellet the cells through the oil mixture (16).
The tubes were placed in dry ice to freeze the contents, the tip of the
tube containing the cell pellet was cut off into a vial, and radioactivity was determined in a gamma counter.
Antibody neutralization of PDGF BB-mediated
proliferation was assessed using CHO C4 cells. The amount of PDGF BB
that induced 90% maximum proliferation (25-50 ng/ml) as measured by
[3H]thymidine incorporation was selected for use in the
assay. All assays were carried out in triplicate or quadruplicate. The
assay involved preparing a 96-well plate containing 50,000 CHO C4
cells/well in Ham's F-12 plus 10% fetal calf serum. After a 24-h
incubation, the medium was replaced with Ham's F-12 plus 0.1% bovine
serum albumin, and an additional 24-h incubation served to put the
cells into a quiescent state. Varying concentrations of anti-PDGFR or control antibodies were added. Following a 2-h incubation with antibody, PDGF BB was added. The cells were incubated overnight, then 1 µCi of [3H]thymidine was added to each well. The cells
were incubated for an additional 4 h then harvested using a PHD
cell harvester (Cambridge Technology, Inc.).
[3H]Thymidine incorporation in each well was determined
with a scintillation counter. The same assay was used to measure the
inhibitory activity of the antibodies on baboon SMC and the rabbit
cornea cell line SIRC.
Domain deletion mutants
of PDGFR were prepared by cloning the PDGFR
gene fragment (with
various extracellular domains deleted) into the XbaI site of
a version of pVgl that had the IgG1 constant region replaced with the
human lambda constant region cDNA. This allowed production of
fusion proteins that contained varying numbers of the PDGFR
extracellular domains with the human lambda light chain constant domain
at the carboxyl terminus. The mutants included only the first, the
first and second, the first through third, and the first through fourth
extracellular domains of PDGFR
fused to the human lambda light chain
constant domain. The lambda constant domain served as a tag and allowed
the deletion mutants to be captured onto the surface of an ELISA plate.
The five-domain extracellular portion of the human PDGFR
with no
lambda constant domain (sPDGFR
) served as the positive control. The
domain deletion mutants were captured onto an ELISA plate using a goat
anti-human lambda chain antiserum (Tago). Deletion mutant binding was
confirmed using a horseradish peroxidase-conjugated anti-human lambda
antiserum. The sPDGFR
(domains 1-5) was directly coated onto the
plate as described above. Reactivity of the antibodies with the domain deletion mutants was determined by incubating antibody with the plate-bound deletion mutants for 1 h, washing away unbound
antibody, and developing the assay with a horseradish
peroxidase-conjugated GAMIgG.
The dimerization
status of PDGFR on the surface of CHO C4 cells exposed to PDGF BB
was carried out in a manner similar to that described previously
(17).
We generated a panel of
monoclonal antibodies against the extracellular portion of the human
PDGFR and assayed the antibodies both for the ability to inhibit
radiolabeled PDGF BB binding to the receptor as well as the capacity to
inhibit PDGF BB-mediated cell proliferation. The antibodies examined
fell into three categories. Fig. 1 presents the results
for a representative example of each category. Several antibodies, such
as M4TS.15, had little or no effect on PDGF BB binding and likewise did
not inhibit PDGF-mediated proliferation. Another class of antibodies,
represented by M4TS.22, inhibited both PDGF BB binding to PDGFR
and
PDGF BB-mediated proliferation. A single antibody (M4TS.11) exhibited
no inhibitory activity in the PDGF BB binding assay; however, it
did display inhibition of PDGF BB-mediated proliferation. Despite the
difference in the ability to inhibit binding of PDGF BB to cells
expressing PDGFR
, M4TS.11 and M4TS.22 were indistinguishable in
their ability to inhibit PDGF BB-mediated proliferation (Fig.
1B).
M4TS.11 Prevents PDGF BB-mediated Proliferation by Impairing PDGFR
The easily detectable ability of M4TS.11 to
inhibit PDGF BB-mediated proliferation in the absence of the ability to
block ligand interaction with receptor implied that the antibody was influencing an event post-ligand binding that was a prerequisite for
the induction of the mitogenic signal. PDGFR is a receptor tyrosine
kinase whose activity is dependent upon ligand-mediated dimerization
(5). To determine if M4TS.11 influenced the ability of PDGF BB to
induce receptor dimerization, we examined the status of PDGFR
on
cells exposed to PDGF BB in the presence and absence of this antibody.
CHO C4 cells were exposed to 125I-labeled PDGF BB after a
preincubation with no antibody or with M4TS.11 or M4TS.15. The cells
were then treated with bis(sulfosuccinimidyl) suberate (Pierce) to
covalently cross-link the 125I-PDGF BB·PDGFR
complex
(17). These cells were lysed with Nonidet P-40, and lysate aliquots
were subjected to SDS-polyacrylamide gel electrophoresis on a 6% gel.
A 6% gel was selected to allow migration of all cross-linked complexes
into the gel, and this was confirmed by the lack of radioactivity
detected at the top of the gel as well as the ability to recover
between 86 and 94% of the counts loaded from the gel lanes (data not
shown). Density scans of autoradiographs of the gels indicated that
radioactive PDGF BB predominantly migrated in the two areas of the gel
that corresponded to the molecular weight of the
125I-labeled PDGF BB cross-linked to one (monomer) or two
(dimer) PDGFR
molecules (unbound 125I-labeled PDGF BB
runs off the gel). Cells preincubated with M4TS.11 before exposure to
125I-labeled PDGF BB and cross-linking had approximately
50% that of the level of receptor dimer (with a corresponding 100%
increase in receptor monomer) as compared with cells exposed to PDGF BB in the presence M4TS.15 or no antibody (Fig. 2).
Mapping Antibody Reactivity Using PDGFR
To identify the portion of the PDGFR recognized by the
M4TS antibodies, domain deletion mutants of the extracellular portion of PDGFR
were constructed and expressed in a soluble form. The deletion mutants included the first, first and second, first through third, and first through fourth extracellular domains of PDGFR
fused
to the human lambda immunoglobulin constant domain (see "Experimental
Procedures"). Each deletion mutant was tested for reactivity with
M4TS.11, M4TS.15, or M4TS.22. This analysis revealed that M4TS.11
exhibited reactivity only when extracellular domain 4 of the PDGFR
was present (Fig. 3). M4TS.15 required the presence of
extracellular domain 2, whereas reactivity with M4TS.22 was dependent
on the presence of extracellular domain 3 (Fig. 3).
Reactivity of M4TS Antibodies with PDGFR
It was of interest to determine if the M4TS.11 and
M4TS.22 antibodies could react with the PDGFR from species other
than human and if they would function as a PDGF BB antagonist for these species. PDGFR
expression has been reported for a variety of cell
types derived from a number of mammalian species, and human PDGF BB can
induce proliferation in these cells. Baboon SMC (18) and the rabbit
SIRC line (19) express PDGFR
and are responsive to human PDGF BB.
Preliminary indirect immunofluorescence experiments revealed that both
M4TS.11 and M4TS.22 could bind baboon SMC, but only M4TS.11 reacted
with SIRC cells (data not shown). Fig. 4 demonstrates
that preincubation with either M4TS.11 or M4TS.22 significantly
decreased PDGF BB-mediated proliferation of the baboon SMC. M4TS.11,
but not M4TS.22, exhibited a similar effect on the rabbit SIRC cell
line (Fig. 4). The M4TS.11 antibody has also been shown to inhibit PDGF
BB mitogenesis of smooth muscle cells from both the rat and the
pig.2
We have generated a panel of antibodies against the extracellular
domain of the human PDGFR. Characterization of this panel revealed
that antibodies fell into three groups: 1) those that had little or no
effect on ligand binding or PDGF BB-induced proliferation, 2) those
that prevented interaction of the ligand with the receptor and thus
prevented ligand-induced proliferation, and 3) a single antibody,
M4TS.11, that had no detectable effect on ligand binding but was a
potent inhibitor of ligand-induced proliferation.
The ability of M4TS.11 to inhibit mitogenesis in the absence of any
detectable effect on PDGF BB binding to the receptor implied that the
antibody was interfering with an event required for triggering mitogenesis but distinct from ligand binding. Ligand-induced
dimerization of transmembrane tyrosine kinases such as PDGFR is
requisite for transmission of the mitogenic signal (6). Examination of the status of PDGFR
on cells preincubated with M4TS.11 and then exposed to PDGF BB revealed a decrease in the level of receptor dimer
and a corresponding increase in receptor monomer as compared with the
levels observed in cells preincubated with a control antibody (Fig. 2).
This indicates that M4TS.11 impairs the ability of PDGF BB to induce
receptor dimerization, a characteristic that makes it a PDGF BB
antagonist that is equivalent in potency to an antibody such as M4TS.22
that directly inhibits PDGF BB binding.
Deletion mutants allowed us to map the determinant recognized by
M4TS.11, M4TS.15, and M4TS.22 to the fourth, second, and third
extracellular Ig-like domains, respectively. Domain 3 is required for
M4TS.22 binding, indicating that the antibody recognizes a determinant
that either resides in domain 3 or is composed of portions of domain 3 and domain(s) 1 and/or 2. The first three domains of PDGFR are
required to form the ligand binding site (20), thus M4TS.22 reacts with
the receptor at a site near to, or possibly identical with, that part
of PDGFR
that binds PDGF BB. This is consistent with the observation
that M4TS.22 can inhibit PDGF BB-induced proliferation by preventing
the interaction of the ligand with the receptor. M4TS.15, an antibody
that has no effect on PDGF BB binding or proliferation, reacts with
each of the deletion mutants that contains domain 2. Thus, despite the involvement of domain 2 in forming the ligand binding site, the portion
recognized by M4TS.15 is spatially distinct from that which interacts
with PDGF BB. The requirement of domain 4 for reactivity with M4TS.11
indicates that the determinant recognized by this antibody is distinct
from the ligand binding portion of the PDGFR
formed by domains 1-3
and suggests that domain 4, despite being uninvolved in PDGF BB binding
(20), does participate in transmitting the mitogenic signal to the
cell. Each of these antibodies appears very different from the
anti-PDGFR
monoclonal antibody 2A1E2, which inhibits ligand binding
and mitogenesis but reacts with the fifth extracellular domain of
PDGFR
(21).
The mechanism by which M4TS.11 inhibits PDGF BB-induced mitogenesis
appears very similar to that of antibodies that have been described by
Blechman et al. (17, 22) against the stem cell factor
receptor (SCFR). The SCFR, which is the product of the c-kit
proto-oncogene, is a receptor protein kinase that, like PDGFR,
transmits a signal when dimerized by the divalent ligand, SCF (23).
Antibodies that bind to the fourth Ig-like domain of SCFR inhibit
SCF-induced dimerization but have no effect on SCF binding (17, 22).
These findings, coupled with the inability of a domain-four deletion
mutant of SCFR to dimerize in the presence of SCF, have led to the
hypothesis that divalent ligand binding per se is
insufficient for receptor dimerization but rather exposes an intrinsic
dimerization site on the receptor that mediates subsequent dimerization
and thus, signal transmission (17). The behavior of M4TS.11, which
binds to the analogous domain four of the PDGFR
, is consistent with
the above hypothesis and supports the contention that the proposed
mechanism may be a general feature of receptor tyrosine kinase
signaling (17). In addition, the reactivity of M4TS.11 with PDGFR
from a wide variety of species indicates that the determinant
recognized by the antibody is evolutionarily conserved. This is a
characteristic expected of a portion of the molecule essential for
proper function.
The unique ability of M4TS.11 to inhibit PDGF BB-mediated proliferation
while allowing the interaction of this ligand with PDGFR may make it
an especially efficient PDGF BB antagonist in vivo, as local
fluctuations in the concentration of PDGF BB should have no effect on
the binding of antibody to the receptor. An antibody that inhibits PDGF
BB-induced proliferation by blocking ligand binding could theoretically
be displaced by high local concentrations of the ligand, possibly
negating the inhibitory effect and making it a less effective
antagonist than M4TS.11.
The involvement of PDGF in a variety of human disease conditions makes it an attractive target for therapy, and neutralizing the activity of PDGF with an antibody is a viable strategy. Advances over the past decade have made human treatment with monoclonal antibodies a feasible therapeutic approach. The major complication of the human anti-murine antibody response has largely been eliminated by "humanization" techniques, which transform a murine monoclonal antibody into a molecule that is indistinguishable from a human immunoglobulin (24). The excellent safety profile and extended half-life of such antibodies (25) make them ideal candidates for applications such as interfering with the PDGF system. We have successfully humanized both the M4TS.11 and M4TS.22 antibodies and retained all the characteristics of the original murine versions,3 thereby making these antibodies excellent candidates for potential development as therapeutic agents.
The authors thank Johanna Anderson and Dr. Steve Hanson at Emory University for baboon arterial smooth muscle cells, Archana Thakur for assistance in cell sorting, E. Poptic for sharing unpublished data, and Dr. Cary Queen for comments on the manuscript.