(Received for publication, September 16, 1994; and in revised form, December 16, 1994)
From the
Phage display of peptides and proteins has successfully been
employed to produce binding molecules of altered affinity. Little is
known, however, regarding the impact on affinity measurements of
phage-displayed molecules compared to their native freely soluble
configuration. That identical affinities can be obtained was shown by
Scatchard analysis of the native antibody, its single chain derivative
(scFv), and its phage-displayed single chain counterpart for the ligand
digoxin. No significant difference, within one standard deviation, was
detected in affinity for digoxin when the phage-displayed scFv was
compared to either its soluble scFv form or the purified antibody. In
addition, no change in binding specificity was detected, within two
standard deviations, when the binding proteins were challenged with two
commonly cross-reactive compounds (dihydrodigoxin and digitoxin). That
phage-display can be employed for molecules having high binding
affinities (K of 6
10
M) is also shown.
A wide variety of genes including those encoding human growth
hormone(1) , Escherichia coli alkaline
phosphatase(2) , rat anionic trypsin(3) , a library of
protease substrates(4) , elastase inhibitors(5) ,
portions of the human IgE receptor(6) , Staphylococcus
aureus Protein A(7) , a library of zinc finger DNA-binding
proteins(8) , ricin B chain(9) , and a myriad of
peptide, antibody variable domain, and Fab ()libraries (10, 11, 12) have been genetically fused to a
gene encoding one of the coat proteins of the filamentous bacteriophage
(fd or M13) and presented on the phage surface for selection. The phage
containing the genetic material for the molecule displayed on its
surface can be recovered following the specific selection for
appropriate binding or enzymatic activity. Phage display is a powerful
methodology that is capable of speeding the search and isolation of
candidate binding elements by tying together phenotypic positive
selection with ready access to the specific coding DNA sequence. In the
case of antibodies, phage-displayed libraries of antibody fragments can
provide a way to bypass hybridoma technology and even animal
immunization (for review, see (13) ), by tapping directly into
the vast multitude of germline sequences. For phage display to be
successfully exploited, however, it is essential that the molecule
displayed be presented in a manner identical to that of the native
molecule. Antibody heavy and light chain variable regions chosen
randomly from a library of such fragments and displayed on phage as a
single chain antibody (scFv) are selected based on the binding activity
of the displayed scFv. The scFv-binding site must be presented in such
a way that it accurately represents the ligand binding site of the
intact antibody, both with respect to affinity for the ligand and in
the fine specificity of the binding site. If not, the selection of the
scFv using phage display could result in the choice of a binding
protein without the desired characteristics when the scFv is produced
in a soluble form, no longer attached to the phage surface. Numerous
cases of affinity differences between soluble scFv and their respective
whole antibody or Fab
counterparts(14, 15, 16, 17, 18, 19, 20, 21, 22) have
been reported. In many cases these differences can be attributed to the
additive impact on affinity measurements of two binding sites versus the presence of a single site, in antibodies and scFv,
respectively. Additionally, alterations in protein folding of these
molecules have also been invoked to account for the observed
differences; however, there have been few experiments to support this
contention.
The first phage-displayed scFv (11) which was
directed against hen egg white lysozyme showed specificity by a lack of
binding to turkey egg white lysozyme in an enzyme-linked immunosorbant
assay (ELISA); however, no affinity measurements were made on the
displayed scFv. Garrard and coworkers (23) attempted to
separate phage-displayed wild type Fab fragments (K = 3.3 nM) of the humanized 4D5 antibody from
three lower affinity variants (K
between
0.05-1.1 µM). Interestingly, efficient selection of
the higher affinity Fab phage did not correlate strictly with the
affinity of the variants.
Both rat anionic trypsin (3) and bacterial (E. coli) alkaline phosphatase (2) have been displayed as phage enzymes (phage-zymes). In each example, while the catalytic activity of the phage-zyme was unaltered, both proteins demonstrated a reduced affinity for their respective substrates. Since alkaline phosphatase is a dimer in its native conformation, not only is correct folding critical but correct dimerization as well. This additional constraint hampers the interpretation of the alkaline phosphatase results, however, since trypsin is a simpler protein; the difference encountered between native and phage-displayed enzyme may be primarily due to the effect of being phage-bound.
Human growth hormone (hGH) gene III fusion protein was shown to be folded correctly on phage by reactivity with a series of six conformationally sensitive hGH monoclonal antibodies(1) . Mutational variants of hGH were phage displayed and selected for high affinity binding to hGH receptor coated on beads. The affinity of these variant hormones for the receptor was determined after release from the phage surface, and in some cases hGH variants with lower affinities were isolated more frequently than higher affinity variants(24) . Using a similar approach, phage-displayed I-domain derived from leukocyte integrins was shown to bind ligand in an ELISA and react with a panel of conformationally sensitive monoclonal antibodies(25) .
The
widespread use of phage display for selection of peptide/protein
fragments makes essential a controlled comparison of a binding element
expressed on phage to the same binding element in its native
environment. At equilibrium under identical conditions, we have
compared the binding characteristics of a well-defined antibody
directed against the hapten digoxin, its scFv counterpart, and this
same scFv molecule displayed as a gene III fusion on phage. Digoxin, a
cardiac glycoside hapten of approximate molecular dimensions 31
8
9 Å(26) , is very similar in size to that of an
antibody combining site (34
12
7 Å; 27, 28). The
rigid, noncharged structure of digoxin's steroid backbone does
not allow for major conformational changes of the hapten even when
functional groups are substituted at various positions on the multiring
structure. The crystal structure (at 2.7Å) of the high affinity
anti-progesterone Fab` DB3 complexed with the steroid progesterone
showed the ligand (and various progesterone analogs) to be almost
completely buried within the very hydrophobic binding
pocket(29, 30) . The total buried surface area of the
steroid and that of the antibody binding pocket was very close,
241 and
270 Å
, respectively. This close
size approximation and rigidity of the steroid nucleus make possible
the examination of the topographical features of the antibody binding
site by analyzing both fine specificity (with structural analogs) and
affinity for the primary ligand.
The PCR-amplified heavy or light chain
fragment were ligated to the Uni-ZAP XR vector (Stratagene, La Jolla,
CA) at EcoRI-XhoI sites. After phage packing and in vivo excision, the pBluescript phagemids containing the
inserts were isolated and several clones sequenced. To construct the
scFv, the primer mixture of the Recombinant Phage Antibody System from
Pharmacia LKB Biotechnol was used to amplify the V and
V
regions. The carboxyl-terminal of V
was
linked to the amino-terminal of V
by a 15-amino-acid
linker, (Gly
Ser)
. A SfiI and a NotI site were introduced by PCR to the 5` and 3` ends of the
scFv fragment, respectively. The resulting scFv fragment was
subsequently ligated to a pCANTAB 5 (Pharmacia) or a pCANTAB-5 his6
c-myc phagemid (Dr. Greg Winter, MRC, Cambridge, United
Kingdom). This latter phagemid contains a 6-histidine tag and a
c-myc tag followed by an amber stop codon prior to the gene
III protein. The phagemid containing the scFv was transformed into E. coli TG1 cells (supE hsd
5 thi
(lac-proAB) F`
[traD36proAB
lacI
lacZ
M15].
These pCANTAB-5 vectors allow the expression (under the lac promoter)
and transport to the periplasm of the scFv fused to the gene III
protein (g3p) of M13. Upon rescue with a helper phage that carries the
rest of the M13 structural and replication proteins, the phagemid is
packaged as a recombinant M13 phage displaying on its surface one or
more copies of the antibody scFv fusion-g3p along with the native g3p.
The procedures of rescuing phagemid with helper phage M13K07 and the
production of scFv phage antibodies were done as specified in the
Pharmacia kit. Phagemid infectivity titers were based on colony forming
units (cfu) selected on ampicillin containing plates (100 µg/ml).
The final assay volume for
the scFv and the scFv phage was adjusted to 300 µl instead of the
150 µl used for the purifed antibody; this allowed a more efficient
separation of bound from free I-digoxin. Both were
incubated overnight at 4 °C with constant gentle agitation as
specified above. The agitation was that sufficient to keep the scFv
resin evenly suspended throughout the incubation period. The scFv phage
antibody was precipitated with one-fifth volume of 20% polyethylene
glycol and 2.5 M NaCl on ice for 2 h, and then spun at top
speed (2,200
g) in a Sorvall RT 6000B (DuPont) for 1
h. The scFv assay was done in a 0.5-ml microfuge tube, and separation
was accomplished by spinning at top speed (16,000
g)
in an Eppendorf 5415C at 4 °C for 45 min. The radioactivity in the
pellets was counted in a gamma counter as specified. Data were fitted
using a four-parameter logistic curve(32) . Varying dilutions
of purified antibody, scFv, and scFv phage were tested, and affinities
were determined and compared at a dilution of each that gave an
ED
(effective dose at 50%) close to 1 ng/ml. The
association constants were determined according to Scatchard (33) , and assays were routinely done in triplicate.
Figure 1:
Panels A-C, representative
competitive I-digoxin binding curves for the 3H-3H scFv,
the 3H-3H scFv phage, and purified anti-digoxin monoclonal antibody
DIGVIIC12, respectively. Radioimmunoassay was as described under
``Experimental Procedures.'' Panels D-F, Scatchard
analysis of the binding data from the 3H-3H scFv shown in panel
A, the 3H-3H scFv phage shown in panel B, and the
purified antibody shown in panel C,
respectively.
To access the
fine specificity of the binding site, multiple concentrations of two
digoxin analogs with only minor differences in their structure were
allowed to compete with the I-labeled digoxin under the
same assay conditions described under ``Experimental
Procedures.'' The percentage cross-reactivity was calculated by
solving the equation for the line drawn between the actual
concentrations of analog and their digoxin doses read from the standard
curve. The percentage cross-reactivity was determined at the 50%
inflection point of the digoxin standard curve. The structures of
digoxin and the two analogs digitoxin and dihydrodigoxin are given in Fig. 2. Digitoxin is identical to digoxin except it is missing
only one hydroxyl group at carbon-12 of the steroid backbone, while in
dihydrodigoxin only the double bond between carbons 20-22 of the
lactone ring becomes reduced. Table 1gives the mean and standard
deviation of multiple cross-reactivity determinations for the purified
antibody, the 3H-3H scFv displayed on phage, and the 3H-3H scFv using
these two digoxin analogs.
Figure 2: Structures of digoxin (digoxigenin tridigitoxose), dihydrodigoxin, and digitoxin.
Figure 3:
DNA sequence of 3H-3H scFv. Sequencing was
done as described under ``Experimental Procedures.'' The
V region extends from nucleotide 1 to 354, the linker from
355 to 399 (underlined), and the V
from
nucleotide 400 to 720. The nucleotide sequence of the originally cloned
V
and V
from the DIGVIIC12 monoclonal cell
line is the same as the scFv except where designated below the DNA
sequence. The inferred amino acid sequence of the scFv is also given;
with the differences in the originally cloned variable regions given
below (in parenthesis) for comparison. The nucleotide change
at position 380 in the scFv sequence probably resulted from
misincorporation by the AmpliTaq polymerase and led to a glycine to
aspartic acid substitution in the linker; all other changes were due to
degeneracy in the primer mixes used in the scFv
construction.
Within
the V and V
regions of the 3H-3H scFv, there were 17
nucleotide differences from the sequence of the V
and
V
counterparts from the original clones. All 17 of these
discrepancies were caused by the family-specific oligonucleotide primer
mixes used in the construction of the scFv. The primers used to
construct the scFv according to the Pharmacia kit are a family-specific
mixture designed to amplify all murine variable regions. These
differences resulted in two amino acid substitutions in the framework 1
region (positions 1 and 3) and two substitutions in the framework 4
region (positions 113 and 114) of the V
. The V
region
of the 3H-3H scFv shows three substitutions in the framework 1 region
only (positions 3, 4, and 8). The proline at position 8, however, is
probably correct and present in the original cell line but was
introduced by the primer (which coded for glutamine) used in the
original PCR cloning. Proline is found with high frequency in this
position in all seven mouse
chain subgroups(35) . Only
Kabat subgroup V shows 16 out of 324 sequences with glutamine at this
position. An additional base pair change, probably due to
misincorporation by the AmpliTaq polymerase, was found in the 3H-3H
scFv sequence and resulted in an amino acid substitution. This occurred
in the linker region between the V
and V
regions
causing the coded linker sequence to become GGGGSGGGDSGGGGS (an A
instead of a G at base no. 380 causing amino acid change from glycine
to aspartic acid).
The present results demonstrate that the antibody combining
site of the 3H-3H anti-digoxin scFv displayed on the head of M13 phage
is indistinguishable in its hapten binding characteristics from that of
the entire antibody molecule (IgG,
) and the same scFv
expressed without phage. This conclusion is based on the following
observations. First, multiple affinity determinations showed an average
affinity constant of 1.6 ± 0.34
10
liters/mol for the phage displayed binding site, which is not
significantly different at one standard deviation from the affinity
constant determined both for the scFv (1.2
10
liters/mol) and for the purified whole antibody (2.1 ±
0.35
10
liters/mol) studied under the same
conditions. This high affinity for digoxin, a hapten that itself
approximates the antibody combining site in size, suggests extensive
complementarity (multiple contacts) between the binding site and the
hapten. Therefore, any alterations of the uncharged steroid nucleus
should help discriminate these two sites. Second, the reactivity of
these binding sites for the two digoxin analogs, dihydrodigoxin and
digitoxin, also did not differ significantly at two standard
deviations. These two analogs differ from digoxin only slightly, either
the lack of the hydroxyl at carbon-12 or saturation of the double bond
in the lactone ring, yet the conformation of the binding site remains
as discriminatory on the phage as it is in the native structure.
An IgG antibody has two antigen-binding sites/molecule. Since phage have five copies of the gene III protein, scFv-gene III fusions could result in five scFvs displayed per phage. Due to tight control on the promotor, statistically it is more likely that two or less of these gene III proteins are actually displaying scFvs/phage molecule(36) . The affinity constants and analog competitiveness were determined from assays wherein the binding reaction was allowed to reach equilibrium. This was done in order to eliminate any complication of multiple binding partners displayed on a single phage. Antibody selection of phage displayed random peptides or ``epitope libraries'' even under equilibrium binding conditions (37, 38) have shown that the effect of multiple binding partners on a single phage complicates the selection of high affinity ligands. In these instances, all five copies of the gene III coat protein were decorated with a random peptide, making the selection of a ligand, based on affinity for a bivalent antibody, complicated by avidity effects.
Neither the presence of the linker
itself, nor the unintentional aspartic acid residue within it, appears
to have any detrimental effect on the binding functionality of the
site. Although, in previous reports the presence of scFv linkers have
been implicated in the decreased affinity of various soluble scFv as
compared to whole molecule or Fab
fragments(16, 17, 20, 21) .
Primer-induced sequence differences found within the 3H-3H scFv itself
also did not alter the binding affinity or specificity of the site. Two
substitutions were found within the first three amino-terminal residues
of the framework 1 region and two more in the framework 4 region of the
V; in the V
three additional changes were found in the
amino-terminal region of framework 1. In the V
, the proline at
position 8 is most likely the residue within the native antibody since
upon investigation of the mouse V
protein groups, proline is the
most common residue in all 7 V
protein groups. Glutamine in this
position is seen in only 16 of the 324 sequences catalogued in protein
group V(35) . An erronous primer choice in the original PCR
cloning led to the glutamine in position 8 that was confirmed in the
DNA sequencing of the original cDNA clones. Reamplification of the
V
and V
from these clones with the
family-specific primer mix reverted the glutamine in position 8 back to
a proline.
In order to do the I-digoxin binding assay
with the scFv as the binding partner and to keep the same basic assay
format for comparison of all the binding partners, it was necessary to
concentrate the scFv-containing culture supernatant. Digoxin
immunoreactive material could be detected in the culture supernatant
prior to concentration only using the enzyme-amplified detection in an
ELISA format (developed with anti-mouse IgG-horseradish peroxidase).
Protein A was investigated as a method of concentration since by
sequence comparison the 3H-3H scFv was shown to express a V
gene from a V
family, J606(35) , known to
contain members that bind Protein A
alternatively(39, 40) ; however, this binding proved
minimal. Due to the 6-histidine tag on the scFv,
Ni
-NTA resin could be used for concentration and as a
separation step in the assay. With the scFv bound to the surface of the
resin particles, the binding activity could be titered as in solution
provided the resin particles remained evenly
suspended(41, 42) . This allowed a direct comparison
of the 3H-3H scFv and the 3H-3H scFv phage.
The functional qualities of the 3H-3H scFv while displayed on the surface of M13 bacteriophage are native in character and importantly validate the usefulness of the phage display technology for selection of functional molecules from complex mixtures such as libraries. Critical to the selection of compounds from a library displayed on phage is that the qualities chosen during the selection protocols be maintained in the final product. The data presented herein illustrate that phage display of an antibody can provide a protein that mirrors the native antibody with respect to affinity and specificity.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U20617[GenBank].
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