(Received for publication, June 27, 1994; and in revised form, January 9, 1995)
From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody.
Polyclonal antisera
with high titer neutralizing activity against human GM-CSF were
developed in BALB/c mice. Purified immunoglobulins were prepared and
used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which
demonstrated biological antagonist activity against GM-CSF-dependent
cellular proliferation. RNA was extracted from spleen cells of mice
with biologically active anti-anti-GM-CSF, cDNA synthesized, and
polymerase chain reaction performed with primers specific for murine
light chain V regions. Polymerase chain reaction products were
cloned into the pDAB
vector and an expression library
developed. This was screened with anti-GM-CSF neutralizing mAb 126.213,
and several binding clones isolated. One clone (23.2) which inhibited
126.213 binding to GM-CSF was sequenced revealing a murine
light
chain of subgroup III. Comparison of the 23.2 sequence with the human
GM-CSF sequence revealed only weak sequence similarity of specific
complementarity determining regions (CDRs) with human GM-CSF.
Structural analysis revealed potential mimicry of specific amino acids
in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and
C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound
by 126.213, specifically antagonized GM-CSF binding to cells and
blocked GM-CSF bioactivity. These studies indicate the feasibility of
using recombinant antibody libraries as sources of interaction site
analogs.
Development of small molecular mimics of larger, polypeptide ligands is one approach to pharmacophore design. Several strategies are available for the development of such mimics, including the use of small oligopeptide analogs derived from native sequence(1, 2, 3, 4, 5) , development of peptidic and non-peptidic analogs based on molecular structure data(6, 7) , and analysis of alternative ligands(8) . Alternative ligands that bind to the same site as the native ligand provide the opportunity to investigate structural and chemical constraints for binding in the setting of diverse backbone geometries. This has the potential to identify critical contact residues based on similar structural and chemical characteristics between the diverse ligands.
Prior studies have investigated a
monoclonal antibody (mAb)(), 87.92.6, which mimicked a
neutralizing epitope on the reovirus type 3
hemagglutinin(9, 10, 11, 12) .
87.92.6 was bound both by a reovirus type 3 neutralizing mAb and the
reovirus type 3 receptor. Sequence similarity between 87.92.6 light
chain second complementarity determining region (CDR II) and the
reovirus type 3 hemagglutinin (13) allowed the development of
synthetic peptides and peptidomimetics which bound both the
neutralizing mAb and the reovirus type 3 receptor. These peptides and
peptidomimetics also demonstrated biological activity on reovirus type
3 receptor bearing cells. The use of anti-receptor mAbs as a source of
sequence-structural information to aid in peptide design has allowed
the development of similar biologically active peptides in several
systems, including the platelet fibrinogen receptor(14) , the
thyroid-stimulating hormone receptor(15) , and epitopes on the
human immunodeficiency virus (16) and hepatitis B surface
antigen(17) .
Recombinant antibodies have been developed
which are expressed in bacteria (18, 19) or on the
surface of filamentous bacteriophage (20, 21, 22, 23) . The advantages of
recombinant approaches to antibody development include the ability to
rapidly screen thousands of clones simultaneously, the potential to
detect binding moities poorly represented in the initial repertoire,
and the potential to express isolated variable regions. While intact
mAbs contain both light and heavy chain variable regions (V and V
, respectively), recombinant antibodies can be
developed which express both V
and V
, or
V
or V
alone. This limits the potential
interaction sites of the recombinant antibody, allowing more precise
delineation of critical interaction regions.
Here we describe the
development of a recombinant light chain library in Escherichia
coli derived from mice immunized with polyclonal anti-GM-CSF. This
library was screened with a previously described neutralizing
anti-GM-CSF mAb 126.213 (24) which inhibits GM-CSF binding to
HL-60 cells, neutralizes GM-CSF induced colony formation, and competes
with the chain of the GM-CSF receptor for GM-CSF
binding(25) . Screening with radioiodinated 126.213 yielded
several binding clones, including one that inhibited
immunoprecipitation of GM-CSF by 126.213. Comparison of the recombinant
V
sequence with the human GM-CSF sequence revealed only
weak similarity with GM-CSF, but structural analysis suggested mimicry
of residues on the B and C helices of GM-CSF by a site chiefly made up
of the CDR I region of 23.2. A synthetic peptide corresponding to the
CDR I was bound by the neutralizing anti-GM-CSF mAb and specifically
inhibited GM-CSF binding and the growth of GM-CSF-dependent cells.
These studies suggest a structural basis for recombinant antibody
mimicry of a predominately helical molecule (human GM-CSF), demonstrate
a bioactive peptide analog of a GM-CSF site implicated in receptor
binding, and indicate the feasibility of using recombinant antibody
libraries as sources of interaction site analogs.
Figure 1: Biological activity of antisera. Proliferation of the human GM-CSF-dependent cell line MO7E was performed as noted under ``Materials and Methods'' in the presence of varying dilutions of murine anti-GM-CSF (following the fifth boost) and murine anti-anti-GM-CSF (following the ninth boost). Counts/min incorporated ± the standard deviation of triplicate wells is shown for various dilutions of antisera. In similar experiments, the inhibition induced by anti-GM-CSF titered out at 1:20,000 to-:100,000 dilutions.
For reverse transcription, 10-20 µg of RNA in 10 µl
was utilized to synthesize cDNA primed with random hexamers in the
following reaction mixture: 3 µl of Maloney murine leukemia virus
reverse transcriptase with 6 µl of 5 reverse transcriptase
buffer, 1.5 µl of RNase inhibitor, and 3 µl of 0.1 M dithiothreitol (all from Life Technologies, Inc.), 3 µl (100
pmol) of random hexamers (from Pharmacia LKB Biotechnol), and 1 µl
of 40 mM dNTPs (10 mM in each dNTP,
from Boehringer Mannheim, GmbH W., Germany). Following a 10-min
preincubation at 25 °C, the reaction was carried out for 1 h at 42
°C, then 95 °C for 5 min followed by storage at -20
°C until use.
For PCR amplification, the oligonucleotide primers
3315 and 5591 listed in Table 1were employed at 0.5
nM/ml final concentrations. The relative position of these
primers on Ig cDNA is shown in Fig. 2. The PCR mixture (100
µl) consisted of 10 µl of PCR primers, 16 µl of dNTPs
(final concentration 200 µM in each dNTP), 10 µl of
PCR buffer (10
; Perkin-Elmer Cetus), 61.5 µl of
dH
O, 2 µl cDNA, and 1.2 units of Taq polymerase (Perkin-Elmer Cetus). Amplification was carried out in
a Programmable Thermal Cycler (MJ Research, Watertown, MA). The
amplification program was 94 °C for 3 min followed by five cycles
of 94 °C for 60 s, 52 °C for 60 s, 72 °C for 60 s; followed
by 25 cycles of 94 °C for 60 s, 52 °C for 90 s, and 72 °C
for 120 s. Following 30 cycles, the temperature was held at 72 °C
for 7 min. Positive amplification was determined by agarose gel
electrophoresis. The PCR products were cloned into the pDAB
plasmid, which is of utility for protein expression as has been
published previously (31, 32) . PCR products and
plasmid DNA were cut with the appropriate endonucleases and plasmid DNA
was treated with calf intestinal phosphatase (Boehringer Mannheim),
followed by ligation using 1 unit of T4 DNA ligase overnight at 16
°C. Ligation mixtures were transformed into E. coli DH5
competent cells as described by the manufacturer.
Figure 2:
Library screening. A, first round
library screening was carried out on 30 filters lifted from 30 LB/amp
plates representing a total of 15,000-20,000 colonies. A
representative filter is shown here. B, second round screening
of one positive clone (clone 23.2) replated and probed with
I-126.213. Compare with panel C, second round
screening of a control clone with an irrelevant V
region. D, third round screening of clone 23.2. Compare with panel
E, E. coli transformed with pDAB
alone.
For some experiments, lysates were prepared of bacteria expressing
the recombinant antibody fragments. Lysates of E. coli XL1
Blue cells (Stratagene, La Jolla, CA) were prepared either from
unmanipulated bacteria or E. coli transformed with pDAB alone, or the various V
regions ligated into
pDAB
. Colonies were grown overnight in LB/Amp, and 500
µl used to seed 5-l cultures grown to
0.6 A
units in Superbroth (Cell Center, University of Pennsylvania),
then induced with 1 mM IPTG for for 4-12 h. The cells
were centrifuged (10,000 revolutions/min for 30 min) and the pellets
dissolved in 2 ml of lysis buffer (10 mM Tris-HCl pH 8.0, 100
mM NaCl, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and aprotinin diluted 1:100 from a
concentration of 2.1 mg/ml, all from Sigma). These cells were sonicated
for 45 s on ice and clarified by centrifugation (11,000
g for 15 min at 4 °C) and the supernatant (lysate) used
as sources of V
fragments.
The PCR products were ethanol precipitated
(to remove residual primer DNA) and digested with appropriate
restriction endonucleases (XbaI and EcoRI). These
were ligated into similarly restricted, alkaline phosphatase-treated
pDAB. Following ligation, the reaction products were
transformed into E. coli DH5
cells and plated onto 30
LB/amp plates. This V
C
library was then
screened after induction with IPTG with radioiodinated neutralizing mAb
126.213, which specifically neutralizes GM-CSF activity. Thirty filters
containing 500-1,000 colonies each were screened in this manner.
A representative filter is shown in Fig. 2A. Based on
the observed binding of
I-126.213 to colonies we picked
30 reactive colonies. These were expanded and replated and rescreened
using fresh
I-126.213 and a control mAb (ID6) specific
for HIV-1 gp120(33) . Approximately 50% of the filters were
bound by
I-126.213 but not by
I-ID6
following the second round of screening (see Fig. 2, B and C, for representative filters screened with
I-126.213). Most of these were bound by
I-126.213 in subsequent rounds of screening (Fig. 2, D and E). Ten colonies which were
consistently bound by
I-126.213 but not
I-ID6 in subsequent assays were selected for further
characterization.
Figure 3:
Characterization of rAb V
regions. A, Western blot analysis of rAb fragments. E.
coli transformed with various plasmids were induced or left
uninduced, lysates prepared, and Western blotting performed with
126.213 as the primary antibody as noted under ``Materials and
Methods.'' Lanes were as follows: 1, clone 23.2
uninduced; 2, clone 23.2 induced; 3, clone 5.1
uninduced; 4, clone 5.1 induced; 5, pDAB
alone uninduced; 6, pDAB
alone induced; 7, 300 ng of GM-CSF (positive control). Molecular weight
markers are indicated. The arrow indicates the band
specifically induced. B, inhibition of immunoprecipitation by
23.2. Immunoprecipitation of
I-GM-CSF was performed as
noted under ``Materials and Methods.'' Lysates of E. coli expressing 23.2 or control (irrelevant clone) were prepared,
protein quantified, and 400 µg used to inhibit immunoprecipitation.
Inhibitors were added as follows: 1, pDAB
alone
induced; 2, 300 ng of GM-CSF; 3, clone 25.1
uninduced; 4, clone 25.1 induced; 5, clone 23.2
uninduced; 6, clone 23.2 induced; 7,
C
molecular weight markers.
The
neutralizing mAb 126.213 specifically immunoprecipitates I-GM-CSF. This assay allowed investigation of the ability
of various rAb V
C
regions to compete with
I-GM-CSF binding to 126.213. Of the 10 rAb
V
C
regions screened, only one (clone 23.2)
reproducibly inhibited immunoprecipitation by 126.213 (Fig. 3B). Inhibition with the lysates from bacteria
transformed with 23.2 reproducibly inhibited immunoprecipitation on
multiple experiments (Fig. 3B and data not shown).
Inhibition was much greater for IPTG-induced cell lysates compared with
uninduced lysates. Clone 23.2 was selected for further
characterization.
Figure 4:
Inhibition of I-GM-CSF
binding to HL-60 cells by 23.2. The binding assay was performed as
noted under ``Materials and Methods'' using 2
10
HL-60 cells, in the presence or absence of increasing
amounts of 23.2 or control (pUC18) lysates. The counts/min (cpm) bound ± standard error of replicate
determinations for two lysate preparations are
shown.
Figure 5:
Nucleic acid and derived amino acid
sequences of clone 23.2. Sequencing was performed by double-stranded
DNA sequencing with Taq polymerase, as described previously,
using both the PCR primers and primers derived from the pDAB plasmid. FR, framework; codon numbering (above the
sequence) is according to Kabat et al.(28) with codon
one corresponding to the first amino acid residue of the FR1 region.
Leader peptide sequence is not shown.
The intact 23.2 sequence and the individual CDR sequences were compared with the human GM-CSF sequence using the Bestfit, Gap, Wordsearch, and Segments programs of the Wisconsin package(41) . Several regions of weak sequence similarity were noted which involved CDR regions of 23.2. Prior studies of 126.213 used murine/human chimeric forms of GM-CSF to map interaction sites(24) . These studies suggested that residues 77-83 were critical for 126.213 binding to GM-CSF. We noted weak homology of the CDR I and CDR II with this epitope. An additional region of weak sequence similarity was also seen between amino acids 54-61 of GM-CSF and the CDR III of 23.2. Interestingly, amino acids 54-61 (on the B helix of GM-CSF) lie immediately adjacent to amino acids 77-83 (on the C helix) in the crystal structure of GM-CSF(42) . However, the weak sequence similarity seen here indicated that the mimicry of GM-CSF by 23.2 might be better accounted for on a structural level.
Alternative models were also generated by searching the
crystallographic data base for loops of the same size as the CDR I
region. The spatially conserved Cartesian positions at the
NH- and COOH-terminal regions of CDR I were held fixed in
the search procedure. A Cartesian distance matrix was constructed for
combinations of the residues on the NH
- and COOH-terminal
regions of the CDR I and compared to a precalculated Cartesian distance
matrix data base of high resolution protein structures(46) .
The 20 best matches were examined using the program Insight II and
appropriate choices were made based upon similarities in chiralities of
side chains at the junctures of the CDR I loop. The choice was spliced
into the template using the program Insight II. Two alternative models
were constructed using this approach. The one involved splicing a loop
identified in the immunoglobulin Fc fragment 1Fc2 (Model 2).
The other involved the heavy chain CDR I of 50.1 (Model 3). It
is well known that light chains can adopt heavy chain conformations in
the absence of heavy chain(47) . The CDR I of the heavy chain
50.1 was spliced into the template. The alternative structures were
mutated to the 23.2 sequence and the structures energy minimized. These
models are presented in Fig. 6.
Figure 6:
Structural basis for mimicry. The
structure of GM-CSF was determined from coordinates derived from the
crystal structure (J. M. LaLonde, K. Swaminathan, and D. Voet,
manuscript in preparation), displayed on the MacImdad program
(Molecular Applications Group, Palo Alto, CA) on a Macintosh Quadra 950
computer. The 23.2 V models were derived as described under
``Results.'' The GM-CSF view is directed at the B and C
helices, while the 23.2 models' view is directed at the CDR I
region. Specific residues implicated in mimicry are indicated. The
models are further discussed in the text.
Prior studies investigating
the epitope on GM-CSF recognized by 126.213 by mutagenesis (24) implicated residues 77-83, located on the C helix of
GM-CSF. Peptide mapping studies of this antibody suggest recognition by
the B and C helices as well as an epitope representing the first
strand, which are all structurally adjacent. (
)Analysis of
the 23.2 models suggests a structural basis for mimicry of this site.
This is shown in Fig. 6. All three models center our attention
on residues Thr
, Glu
, and Lys63 of
GM-CSF. The proximity of Lys
and Glu
suggest
a charge-charge interaction. In all three models, these 2 residues are
mimicked by Arg
and Asp
of 23.2. For Models 1
and 3, the mimicry suggests similar orientations of 23.2, while for
Model 2 the structure is rotated
90°. The other GM-CSF
residues mimicked include: Thr
mimicked by Ser
in Models 1 and 3, and by Thr
in Model 2; Lys
mimicked by Arg
in Models 1 and 3, and Lys
in Model 2; Thr
and Ser
mimicked by
Ser
in Models 1 and 3, and by Ser
and
Tyr
in Model 2; Lys
mimicked by Lys
in Models 1 and 3, and by Ser
in Model 2; and
Glu
mimicked by Ser
and Ser
in
Model 1, and by Ser
in Model 3. Thus, while sequence
similarity between GM-CSF and 23.2 is quite low, structural similarity
is suggested centered on the B and C helices of GM-CSF and the 23.2 CDR
I.
Figure 7:
Binding of 126.213 to synthetic peptides
derived from the 23.2 sequence. Binding was performed by ELISA assay as
described under ``Materials and Methods.'' The values shown
are A nm binding to the peptides at the
concentration noted minus A
nm binding to
BSA-coated control plates. Results are compared for 126.213 versus an isotype matched control mAb (D1.H3) specific for a peptide
derived from the hamster
-adrenergic receptor. The mean ±
S.D. of triplicate wells is shown for increasing amounts of purified
126.213 added. A and B, binding to CDR I peptide. C and D, binding to the CDR II peptide. E and F, binding to the CDR III peptide. G and H, binding to the control peptide. The mAbs used were: 126.213
in A, C, E, and G and D1.H3 in B, D, F, and H.
The ability of these peptides to
compete with GM-CSF for binding to HL-60 cells was examined using a
radioreceptor assay. HL-60 cells were preincubated with peptides prior
to the addition of I-GM-CSF and specific binding
determined in the presence of excess unlabeled GM-CSF. A representative
experiment is shown in Fig. 8. Increasing amounts of CDR I
peptide were able to specifically inhibit GM-CSF binding in a
dose-dependent manner, while CDR II and CDR III peptides did not
demonstrate any specific binding inhibition. Thus, the CDR I peptide
antagonizes
I-GM-CSF binding to HL-60 cells, suggesting
interaction of this peptide with the GM-CSFR.
Figure 8:
Inhibition of I-GM-CSF
binding to HL-60 cells by CDR peptides. The radioreceptor assay was
performed as noted under ``Materials and Methods,'' using
10
HL-60 cells. The cells were preincubated with peptides
at varying dilutions for 60 min at room temperature prior to the
addition of
I-GM-CSF. The specific proportion of cpm
bound was determined by subtracting the proportion of cpm bound under
identical conditions in the presence of saturating amounts (50
nM) of unlabeled GM-CSF. The standard deviation of this assay
was
10% on multiple determinations. The percent inhibition of
binding is shown versus increasing amounts of
peptides.
The bioactivity of
these peptides was assessed by their effect on GM-CSF-dependent
cellular proliferation. This was compared with their effect on
interleukin-2-dependent proliferation by the CTLL cell line, to control
for nonspecific toxic effects. The results are shown in Fig. 9.
At the concentrations used, none of the peptides were toxic to CTLL
cells with the exception of the CDR III peptide at 2 mg/ml. The CDR II
peptide had no inhibitory effect on either cell line. In contrast, the
CDR III and CDR I peptides inhibited GM-CSF-dependent cellular
proliferation. For the CDR III peptide, the IC was
1
mg/ml (approximately 400 µM), while for the CDR I peptide,
it was
50 µg/ml (approximately 21 µM). These data
indicate that the CDR I peptide is a specific antagonist of
GM-CSF-dependent cellular proliferation in a micromolar concentration
range.
Figure 9: Inhibition of GM-CSF-dependent cell proliferation by peptides. The proliferation assay was performed as noted under ``Materials and Methods'' on AML193 cells (GM-CSF-dependent) and CTLL cells (IL-2-dependent), in the presence or absence of increasing amounts of peptides as noted. Results from two experiments are combined, with the mean ± standard error percent inhibition of proliferation shown versus increasing peptide concentration. A, CDR I peptide; B, CDR II peptide; C, CDR III peptide.
GM-CSF
binding and bioactivity have been analyzed at a molecular level.
Mutagenesis studies implicate the first (A) helix in binding of GM-CSF
to the high affinity GM-CSFR/
complex, but not to
the low affinity receptor (GM-CSFR
alone)(55, 58, 59) . This is illustrated most
strikingly by studies using mutants of residue Glu
of
GM-CSF, which inhibit binding of GM-CSF to the low affinity receptor,
but display little activity in inhibiting binding to the high affinity
receptor(58) . Based on these experiments, it has been proposed
that the first
helix of GM-CSF is responsible for binding to
(59) . Murine and human GM-CSF display species
specificity and are not cross-reactive. As the substitutions are
scattered throughout the molecule, it was possible to swap regions of
murine and human GM-CSF to locate sites critical for receptor
interaction(37) . These studies indicated a critical role for
amino acids 21-31 and 77-94 in mediating the activity of
human GM-CSF, suggesting that the second site may be involved in
binding to the GM-CSFR
. However, other potential GM-CSFR
interaction sites have also been suggested in mutagenesis
studies(60, 61, 62) , mapping of neutralizing
mAbs(24, 63, 64, 65) , and synthetic
peptide studies(48, 63, 66) . Thus, in spite
of considerable study, the GM-CSFR
interaction site(s) on GM-CSF
remain incompletely characterized.
Recent studies from our group
have used synthetic peptides, anti-peptide antisera, and neutralizing
mAbs to map epitopes on GM-CSF critical for bioactivity. The major findings were: a peptide derived from the sequence of
the A helix (residues 17-31) and antibodies to this peptide
inhibited GM-CSFdependent cellular proliferation; a peptide comprising
portions of the B and C helices (residues 54-78) was recognized
by two neutralizing monoclonal antibodies (including 126.213) and
exhibited biological antagonist activity. Other peptides were also
bound by 126.213 corresponding to residues 78-99 and 31-54,
but were not specific antagonists of GM-CSF bioactivity. These three
peptides together constitute a ``face'' on GM-CSF centered on
the B and C helices and opposite the A helix. Together with the prior
studies noted above, these studies suggest two binding sites on GM-CSF
important in receptor binding: the A helix which likely interacts with
, and the opposite face centered on the B and C
helices which we propose interacts with the GM-CSFR
. The ability
of synthetic peptides corresponding to these epitopes to specifically
inhibit GM-CSF bioactivity strongly supports their role in receptor
interaction.
In
prior studies, we described the molecular basis for antibody mimicry of
a viral
hemagglutinin(9, 10, 11, 12) . Other
groups have applied this technology to platelet fibrinogen
receptor(14) , the thyroid-stimulating hormone
receptor(15) , and epitopes on the hepatitis B surface
antigen(17) . Monoclonal antibodies were utilized in these
studies as mimics and to derive sequence information. The studies
presented here are the first to suggest that recombinant antibodies can
be similarly employed to develop alternative ligands. The prior studies
of antibody mimicry in general described mimicry of structures either
known or predicted to represent reverse turns. As antibody CDRs are
generally reverse turns, the ability of antibody CDRs to mimic other
reverse turn regions does not necessarily imply that CDRs can mimic
amino acid residues presented by other diverse backbone geometries. The
epitopes involved in this study are largely helical in nature. In
spite of this, molecular modeling of this epitope suggests a structural
basis for mimicry as noted above. This indicates that antibody mimicry
of amino acid arrays on helical regions can be understood on a
molecular-structural level. The application of recombinant antibody
technology to development of such mimics should broaden the
applicability of alternative ligand development in the analysis of
active site structures.