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Clinical and Diagnostic Laboratory Immunology, September 1998, p. 636-644, Vol. 5, No. 5
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Selection of Recombinant, Library-Derived Antibody Fragments
against p24 for Human Immunodeficiency Virus Type 1 Diagnostics
Hans J. W.
de
Haard,1,*
Bert
Kazemier,1
Marck J. M.
Koolen,1
Liekle J.
Nijholt,1
Rob H.
Meloen,2
Bob
van
Gemen,1
Hennie R.
Hoogenboom,3 and
Jan-Willem
Arends3,4
Biosciences Research Unit, Organon Teknika,
Boxtel,1
Department of Molecular
Recognition, Institute for Animal Science and Health (ID-DLO),
Lelystad,2 and
CESAME, Department of
Pathology, University Hospital,3 and
Maastricht University,4 Maastricht, The
Netherlands
Received 4 February 1998/Returned for modification 29 April
1998/Accepted 22 June 1998
 |
ABSTRACT |
By application of combinatorial library technology, we generated
the first recombinant antibody fragments directed against the major
capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A
library of single-chain Fv fragments (scFvs) was constructed by using
the antibody variable-region (V) genes of B cells derived from the
spleen of a viral lysate-immunized mouse. Antibodies were selected by
panning or by enrichment with biotinylated antigen, yielding four
different families of antibody fragments. The different types of scFvs
were characterized by affinity measurements, by antigen recognition on
Western blots, and by pepscan analysis. The epitope of one of the scFvs
is located near the residues involved in CypA binding, thereby making
it an attractive candidate for therapeutic applications. Comparison of
the V gene sequence of this scFV with that of a previously described
monoclonal antibody reactive against this immunodominant epitope
revealed the usage of the identical combination of VH and
V
regions. Thus, this is one of the rare examples in
which the original combination in a library-derived antibody fragment
was retrieved. After appropriate affinity and format improvements, the
best of our recombinant scFvs may form the basis for a sensitive p24
assay as a measure of viral load. In addition, anti-p24 scFvs could be
expressed as intracellular antibodies (intrabodies) to aid in the
treatment of HIV infections.
| |
INTRODUCTION |
Human immunodeficiency virus (HIV)
infection is diagnosed by detecting virus-specific antibodies (Abs), or
the virus itself, by means of p24 antigen (Ag) detection or by
quantitative amplification procedures such as PCR (38) or
nucleic acid sequence-based amplification (62) or by
coculturing and subsequent virus detection procedures. During a
diagnostic window of 6 to 8 weeks after infection, Abs to HIV are
undetectable, and alternative diagnostic methods would help to reduce
the residual risk of transfusion transmission of HIV. Recently, the
Food and Drug Administration recommended the implementation of p24 Ag
tests in donor screening (20). The p24 capsid protein forms
the viral core containing the single-stranded RNA genome and is
abundantly present in the virus particle. Besides the structural role
of the protein in forming the core of the mature virion, the molecule
is essential during viral assembly; it plays a pivotal role in viral
penetration or uncoating or both, a function which may be mediated by
binding of p24 to the human cellular proline rotamase cyclophilin A
(4, 41, 60). With current enzyme-linked immunosorbent assays
(ELISAs), the presence of p24 Ag may be assessed 5 to 14 days earlier
than could an Ab response measured by anti-HIV type 1 (anti-HIV-1) or
anti-HIV-2 enzyme immunoassays (8, 9, 66). In addition, the
capsid protein may be considered a marker for virus replication
(3, 26, 65), and its detection in an extremely sensitive
immunoassay would offer a cheap and generally applicable alternative to
PCR-based assays for the diagnosis of reactivation during treatment of
HIV-1-infected patients with (combinations of) nucleoside reverse
transcriptase inhibitors or protease inhibitors (19, 59,
64). When reactivation, as the result of the evolution of
drug-resistant HIV mutants, is detected, treatment may be changed to
other drugs. Rapid and sensitive assays that can carefully detect the
presence of p24 in serum are therefore crucial for early detection and
monitoring of viral replication (66).
The sensitivity and specificity of the presently used anti-p24
immunoassays are limited by the affinity of the monoclonal Abs (MAbs)
used for capturing and/or detection of the Ag, although by signal
amplification in combination with heat denaturation, the sensitivity
can be increased to the level obtained by PCR (6). The
availability of the Ab genes in recombinant anti-p24 Abs allows the
improvement of affinity by mutagenesis methods, as well as the
engineering of avidity, thereby helping to improve the sensitivity of
early virus detection. During in vivo maturation, the obtained
affinities are limited by the off-rate, i.e., the rate at which the
Ab-Ag complex dissociates. The off-rate of in vivo-matured Abs is on
the order of 10
3 to 104 s1,
which permits endocytosis of membrane-bound Ab-Ag complexes on B cells
(21). However, in vitro maturation with phage display allows
the selection of Abs with lower off-rates, leading to affinities in the
picomolar range (1, 57). Besides their obvious diagnostic application, it may also be possible to use anti-p24 single-chain Fv
fragments (scFvs) for therapy. By expression with a retention signal
for the endoplasmic reticulum, the scFvs may interfere with virus
assembly in the infected cells, as was demonstrated with anti-gp120
(12) and anti-Tat (45) Abs. For intracellular expression, even murine Abs might be applied in humans: a human anti-mouse antibody (HAMA) response cannot be induced, since the fragments are shielded from the immune system.
In this context, we aimed to isolate anti-p24 scFvs from a phage Ab
library made from a mouse immunized with HIV-1 viral lysate. Affinities
were determined with surface plasmon resonance and compared with those
of MAbs currently used in p24 assays. The specificity of the selected
Ab fragments was determined by ELISA, Western blotting, and pepscan
analysis. One of the recombinant Ab fragments recognized an epitope
which was also detected by a previously described and sequenced murine
MAb. Both the recombinant Ab and the hybridoma-derived Ab have nearly
identical heavy and light chain variable regions, and, as such, the
selected scFv D2 is one of the first examples in which the original
combination of both VH and VL was maintained,
in spite of the very low probability of finding this in a combinatorial
library (23).
These newly described anti-p24 recombinant scFvs offer a starting point
for Ab affinity maturation and engineering (for reviews, see
30, 67, and 68) and may be useful as
intracellular Abs (intrabodies) for HIV therapy.
| |
MATERIALS AND METHODS |
Library construction.
A BALB/c mouse was immunized with 100 µg of protein from a viral lysate, which had been prepared from
infected H9 cells (52) and purified by ultracentrifugation,
plus Freund's complete adjuvant. After 6 weeks, a booster with 100 µg of lysate proteins plus incomplete adjuvant was administered.
Three days later, the animal was sacrificed and the spleen was removed.
Upon extraction of the spleen, 20 µg of total RNA was isolated
(13) and 5 µg of this was transcribed into random-primed
cDNA.
The immunoglobulin G1 (IgG1)-derived VH- and
V-encoding DNA fragments as well as the linker fragment
(the latter was cloned in pUC19, by using synthetic oligonucleotides
which encoded the 15-residue linker described by Huston and colleagues
[33]) were obtained by amplification (Amplitaq;
Perkin-Elmer Cetus) with the primers (14) listed in Table
1. The fragments were purified from an
agarose gel with the QIAEX kit (Qiagen, Hilden, Germany) and assembled
in splicing by overlap extension-PCR (32).
The assembled scFv-encoding DNA fragments were also purified from the
gel, digested with SfiI and NotI (Pharmacia,
Lund, Sweden), and cloned into the phagemid vector pV1, which is
identical to pHEN1 (31) but with a FLAG-tag (53)
instead of a Myc-tag. Transformations were performed by electroporation
in JM101 (17). The library consisted of 107
clones, of which 60 to 70% contained a complete insert as was deduced
from PCR analysis.
Selection of libraries.
The rescue of phagemid particles was
performed as described before (43). The capsid protein p24,
expressed as full-length product in Escherichia coli under
control of the inducible lac promoter (constructed at
Organon Teknika [3a]) and encoding the HIV-1 strain RF
sequence, was affinity purified with MAb 39B (generated at Organon
Teknika [unpublished data]). A six-well tissue culture plate (Costar,
Cambridge, Mass.) was coated with Ag at a concentration of 3 µg/ml in
50 mM NaHCO3 (pH 9.6). Selection, washing, and elution were
performed as described elsewhere (43).
For the construction of a light chain shuffling library of clone D2,
the V
H segment was amplified on plasmid DNA. The
V
H fragment
was recombined with the linkers and the
corresponding kappa light
chain fragments, initially used for the
generation of the library,
yielding the shuffling library containing
10
6 clones. Selection was performed with p24 biotinylated
with
N-hydroxy-succinimide-biotin
(Pierce, Rockford, Ill.);
phage Ag complexes were captured with
streptavidin-coated paramagnetic
beads (Promega, Madison, Wis.).
Screening and sequencing of clones.
Soluble scFv was
produced from individual clones by growth in V-shaped microtiter plates
in 2*TYE medium as described before (43). The described
method was used for constructs transformed in the nonsuppressor strain
TOP F' as well as in the suppressor strain JM101. Suppression is
incomplete and results in the leakage of free scFv in the culture
medium, while the expression of gene III makes the cellular membranes
more permeable (5), leading to higher concentrations of Ab
fragments in the medium.
Microtiter plates were coated with 3 µg of recombinant p24 per ml
(see above) or 5 µg of viral lysate per ml in 50 mM
NaHCO
3 (pH 9.6) for 16 h at room temperature. The
plates were blocked
for 2 h at room temperature with bovine serum
albumin (BSA; 0.2%
[wt/vol] in 0.1 M Tris [pH 7.4]-30 mM KI). The
culture supernatant
was diluted fivefold in sample diluent
(phosphate-buffered saline
[PBS], 20% normal goat serum, 1.1%
Triton X-100). After incubation
at room temperature for 2 h, the
plates were washed four times
with PBS-Tween 20 (0.05%), and bound
scFv was detected with a
mixture of a 1/4,000 dilution of anti-FLAG Ab
M2 (Kodak IBI, New
Haven, Conn.) and a 1/2,000 dilution of anti-mouse
Ab-horseradish
peroxidase conjugate (DAKO, Glostrup, Denmark).
Following a 1-h
incubation and washing as before, staining was
performed with
tetramethylbenzidine and ureaperoxide as the substrate
and stopped
by adding an equal volume of 1 M
H
2SO
4; the optical density was
measured at 450 nm.
Clones giving positive signals and with a unique
BstNI
fingerprint (New England Biolabs, Beverly, Mass.) were analyzed by
sequencing with the T7 sequencing kit (Pharmacia) with the M13rev
primer, the geneIII primer, and two primers located in the linker
(linkfwd and linkrev) (Table
1). Plasmid DNA was purified with
the
QIAGEN kit (Qiagen).
Characterization of scFvs.
For the preparation of
periplasmic fractions by the borate-buffered saline shock procedure
(58), clones obtained by transfection to the nonsuppressor
strain TOP F' were cultured on a 50-ml scale and induced as described
previously (43). The fractions were analyzed on 15%
polyacrylamide gels and then blotted onto nitrocellulose. After
blocking with a skim milk solution (5% [wt/vol] in Tris-buffered saline [TBS]), the scFv was detected with anti-FLAG M2 diluted 1/4,000 in TBS-0.05% Tween (TBST). After a 2-h incubation period, the
anti-FLAG was detected with anti-mouse Ab-horseradish peroxidase and
the ECL detection kit (Amersham, Buckinghamshire, United Kingdom).
For the purification of scFv, refolding was used in combination
with affinity chromatography on p24 columns. The pelleted
cells from a
50-ml culture were resuspended in 8 ml of an 8 M
urea solution (in PBS)
and sonicated. The mixture was rotated
head-over-head for 30 min, and
insoluble material was removed
(centrifugation for 30 min at
13,000 ×
g). The supernatant was
dialyzed against PBS
with four buffer changes. Insoluble proteins
were removed by
centrifugation, and after passage through a 0.2-µm-pore-size
filter,
the flowthrough was immediately loaded on a p24 column
(bed volume, 0.3 ml). The column material was prepared by coupling
8.4 mg of protein to
1 g of Tresyl Sepharose in accordance with
the supplier's
instructions (Pierce). Washing and elution were
performed as in the
panning procedure. The yield was determined
by measuring the optical
density at 280 nm, assuming that an scFv
has a molar extinction
coefficient (
E2801%) of 14.3 (
27).
To establish the valencies of the scFvs, the molecular weights of the
Ag binding molecules were determined by gel filtration
(
27)
on a Superdex 75HR column (Pharmacia), which was calibrated
with a
mixture of BSA, chymotrypsin, ovalbumin, and RNase A (Pharmacia).
As a
control, an anti-human chorionic gonadotropin (anti-hCG)
scFv selected
from a murine immune library was used (unpublished
data). Either 20%
of a periplasmic fraction from a 50-ml culture
or affinity-purified
scFv was injected onto a calibrated column.
Fractions (0.5 ml) were
collected and analyzed on Western blots.
Epitope mapping was performed with the pepscan method (
22).
Overlapping decapeptides of p24 from HIV strain ANT70, coupled
to a
solid support and obtained from the Department of Molecular
Recognition
of the Institute for Animal Science and Health (IDO-DLO),
were tested
with the scFv. The periplasmic fraction of clone D2
was diluted
100-fold in Super Q buffer (PBS containing 5% [vol/vol]
horse serum,
5% [wt/vol] BSA, and 1% [vol/vol] Tween 20) to a
concentration of
80 ng/ml. After 16 h of incubation at 4°C, scFv
bound to
peptides on the solid support was detected as described
above.
Affinity measurements in solution.
The affinities measured
by the "in solution" method were determined with surface plasmon
resonance on BIAcore (Biacore AB, Uppsala, Sweden) in accordance with
the suggested procedures described in the BIAevaluation software. A
high-density p24-coated (3,777 Response Units [RUs] of immobilized
Ag) CM chip (Biacore AB, Uppsala, Sweden) was prepared by covalent
coupling as described in the supplier's recommendations. A fixed
amount of scFv or MAb, yielding a signal of between 1,000 and 1,500 RUs
when injected onto the p24 chip, was mixed with a variable amount of Ag
in a volume of 60 µl by using HEPES-buffered saline (10 mM HEPES, 3.4 mM EDTA, 150 mM NaCl, 0.05% surfactant P20 [pH 7.4]) as the dilution
buffer. After a 1-h period, needed to achieve equilibrium, the samples were injected. For the quantification of free Ab, a standard curve was
prepared by injection of a dilution series of the tested Ab on the same
flow cell (37).
Nucleotide sequence accession numbers.
The GenBank accession
numbers for the sequences of the antibodies reported in this study are
as follows: D2 VH, AF083186; A2 VH, AF081541;
A3 VH, AF081545; A5 VH, AF081543; D1 VH, AF083189; D2 VL, AF083188; A2
VL, AF081542; A3 VL, AF083185; A5
VL, AF081544; D1 VL, AF083187.
| |
RESULTS |
Library construction and selections.
Our major aim was
to evaluate the library technology as a substitute for hybridoma
technology for the generation of anti-HIV-1 Abs, which might be useful
for in vitro diagnostics. Therefore, we immunized a mouse with a lysate
prepared from HIV-1-infected H9 cells (52). After
amplification of the V genes of the murine spleen B cells and cloning
in the phagemid vector pVI, a library containing 107 clones
was obtained. Seventy-five percent of the clones expressed scFvs, as
was determined by PCR screening and by inspecting the morphology of the
colonies: clones which express gene III (fusions) are usually visible
as flat colonies, while those having an interrupted frame exhibit a
more solid appearance (14a).
The library was subjected to four rounds of panning on
recombinant p24. Four rounds of panning produced a 100-fold enrichment
in the number of eluted phages, indicating the presence of Ag-specific
phage Abs. The scFvs produced by 48 individual clones after round
4 were analyzed by ELISA (
43). Seventy percent of the tested
scFvs produced high signals on viral lysate and p24 Ag, whereas
no
responses were found on BSA. After 24 clones were DNA fingerprinted
by
BstNI digestion (
43), two different patterns were
identified;
five clones representing the two patterns were selected for
sequence
analysis and further binding analysis. All five clones
analyzed
had minor differences in sequences and were classified as scFv
D2 (Fig.
1).
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FIG. 1.
Deduced amino acid sequences of the variable heavy and
light chain regions and germ line origin of the p24 Abs derived from
the murine library. Identical residues are indicated by dashes, while
different residues are indicated by uppercase characters and
primer-encoded differences are indicated by lowercase characters.
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|
Immune libraries usually contain a very diverse set of Ag-specific Abs
(
14,
44); thus, we modified our method of selection
to
retrieve phage Abs other than the ones found before. Selections
were
repeated with soluble (biotinylated) recombinant p24 (Sol-p24)
and with
p24-coated magnetic particles (Bead-p24). After four
rounds, enrichment
factors of 100 and 10,000 were found for Sol-p24
and Bead-p24,
respectively. Concomitant with this enrichment in
the absolute number
of eluted phages, we found an enrichment in
the fraction of clones
producing scFv, as determined by colony
morphology. The percentage of
scFv-producing clones increased
from 50 to 75% of the clones before
selection and after round
1 to more than 99% after round 4. At this
stage, 8 of 11 clones
of Sol-p24 and 12 of 12 clones of Bead-p24
contained a complete
insert, whereas
BstNI fingerprinting
revealed four different but
closely related clones for Sol-p24 and only
one type for Bead-p24.
The four Sol-p24 scFv clones reacted positively
on p24 and negatively
on BSA, while the Bead-p24 clones gave high-level
responses on
p24 and BSA. Obviously, panning with the beads resulted in
the
isolation of an scFv specific for the blocking reagent BSA, which
was also used for blocking the microtiter plates, which explains
the
positive signals in the p24 ELISA. The selection of BSA-specific
clones
can be explained by the presence of this Ag in the viral
lysate used
for immunization and its use for blocking the beads
and the microtiter
wells. We chose three different p24 binders
of Sol-p24 and the Bead-p24
BSA binder for sequencing and further
characterization. For Sol-p24,
the clones designated A2, A3, and
A5 were used; for Bead-p24, the clone
used was named D1.
Characterization of isolated scFvs.
Figure 1 shows the deduced
amino acid sequences of the VH and VL gene
products of the clones derived from panning the murine library with
Ag-coated polystyrene plates (D2) and with biotinylated Ag (A2, A3, and
A5) together with the most related mouse germ line analogues (V
regions). The sequence of the BSA binding antibody (D1) selected with
the beads is also included.
The four anti-p24 scFvs use highly homologous V
H gene
segments. They belong to class IIA according to Kabat's classification
(
36) and use two germ line gene segments only. Clones A2 and
D2 and clones A3 and A5 can be grouped in two classes depending
on the
germ line used. D2 has a divergent and shorter CDR3 than
clone A2 and
thus is clonally not related. The light chain of
clone D2 could be
matched adequately with a germ line segment
and is very different from
the A2 light chain. The kappa light
chains of the three antibodies A2,
A3, and A5 use a nearly identical
V
gene segment,
derived from the germ line segment coded
k2/MMIG27 (for classification
and the codes used in Fig.
1, see
"Germline gene directories of the
mouse" homepage (
http://www.ibt.unam.mx/~almagro/V_mice.html)).
The specificities of the scFvs were determined by Western blot analysis
(Fig.
2). Periplasmic fractions
containing scFv were
used for the detection of viral and recombinant
p24. Two different
recognition patterns were obtained. Abs A2, A3, and
A5 reacted
with (the reduced form of) p24, in a viral lysate, and as a
recombinant
product (for example, the blot incubated with scFv A3 is
shown
in Fig.
2A). ScFv D2 recognizes p24, but it also detects the
viral
Gag polyprotein p55 (Fig.
2A, left panel, lane 1) from which the
matrix protein p17, the nucleocapsid p7, and the capsid protein
p24 are
proteolytically processed. Another product with a molecular
mass of
approximately 40 kDa is visible. This protein seems also
to be
recognized by polyclonal serum (Fig.
2B) obtained from the
mouse that
was used for the construction of the library. The p24
dimer, which is
the predominant form of the native protein as
was observed by gel
filtration (results not shown), might also
be detected with mouse serum
(Fig.
2B, left panel, band between
p55 and the 40-kDa derivative).
Indeed, the native p24 protein
can form a complex of oligomers in
solution, including dimers,
tetramers, dodecamers, spheres, fibers, and
tubes (
18). An intense
reactivity of the original murine
polyclonal serum against p24
and its precursors can be observed, while
the mouse seems to have
a lower Ab titer against the envelope protein
gp120. Thus, the
original library was biased in its immune response
towards p24,
which is probably caused by the abundance of the antigen
in the
viral lysate used for immunization (
10,
63).
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FIG. 2.
Western blot detection of p24 by scFvs and mouse
polyclonal Abs (A) Detection with scFv D2 (left panel) and A3 (right
panel). Viral lysate (lanes 1), BSA as a negative control (lanes 2),
and purified recombinant p24 (lanes 3) were analyzed on a 12% gel. The
band indicated with an asterisk represents the dimer of p24, present in
the recombinant and in the viral product. (B) Blot analysis of viral
lysate with polyclonal serum (lanes labeled +) obtained from the mouse
used for library construction (left blot made with an 8% gel and right
one made with a 12% gel). Amido black-stained markers (lanes labeled
M) are shown. The Gag precursor p55 and its derivative p24, the
envelope protein gp120, and the polymerase p31 are indicated with
arrows. The slow-migrating protein indicated by the unlabeled arrow at
the top of the left blot is probably the envelope precursor gp160. The
other marked protein (bottom unlabeled arrow) is a cleavage product of
Gag, which is also recognized by scFv D2 (Fig. 2A).
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|
D2 is a diabody.
After performing the sequence analysis of
clone D2, selected on immobilized p24, we noticed that it contained a
linker consisting of 2 amino acids (GlySer) rather than 15 amino acids
[(Gly4Ser)3]. As suggested in the literature
(29), shortened linkers may lead to the formation of dimeric
molecules with two functional binding sites, the so-called
"diabodies." The shorter linker in clone D2 might therefore be
responsible for the formation of a bivalent scFv molecule, and this
type of molecule might be strongly enriched over the other anti-p24
scFvs by its greater avidity in the panning selection. We generated a
number of D2 derivatives and characterized their behavior.
From a light chain shuffling library, we selected a derivative of clone
D2 that had an identical light chain and a complete
15-amino-acid
linker; the clone was designated D2/15. We examined
the molecular
weight of the antibody fragments present in the
periplasmic space of
the producing bacteria by gel filtration
on a calibrated Superdex 75HR
column (
27); the obtained chromatogram
of clone D2 is shown
in Fig.
3A. The scFv with a
two-amino-acid
linker emerged from the column as a single peak with a
molecular
mass of 45 kDa, as could be deduced from Western blot
analysis
with the fractions collected during gel filtration (Fig.
3B).
Both of the scFvs, D2/15 and a control anti-hCG Ab fragment with
a
15-residue linker, gave two peaks: the dimeric fraction at 45
kDa and a
monomeric fraction at 25 kDa.
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FIG. 3.
Gel filtration of periplasmic fractions on Superdex 75HR
and analysis of the collected fractions on a Western blot with
anti-FLAG M2. (A) Chromatogram, obtained by plotting
A280 against the fraction number, illustrating
the separation of the periplasmic proteins from clone D2. The retention
times of the marker proteins are indicated with arrows. (B) Western
blot analysis (15% gel) of the collected fractions (fraction number is
shown above the blots) obtained from gel filtration of anti-p24 clones
and of an anti-hCG scFv-producing clone. Two peak fractions can be
discriminated in the scFvs having a complete linker (the derivative
D2/15 and the anti-hCG): a main peak of the monomer (fraction 20;
molecular mass, 25 kDa) and a dimer peak (fraction 17; molecular mass,
45 kDa). scFv of clone D2 emerged from the column in a single peak
corresponding to the dimeric product.
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|
By chain shuffling the light chain of the D2 diabody, clone D2/15 was
isolated by using an excess of Ag during selection.
By increasing the
stringency of selection, i.e., decreasing the
Ag concentration from 100 nM to 100 pM, the monomeric clone D2/15
was completely lost and the
selected population was overtaken
by (avid) diabodies. We retrieved no
D2 derivatives with other
light chains, not even the one of clone A2,
in spite of its highly
related V
H gene, as was also
reported by others (
56). However,
it should be emphasized
that the diabody's CDR3 sequence is completely
different from the CDR3
sequence of clone A2 in length as well
as in sequence. Due to the close
proximity of H3 to the light
chain variable-region loops, this may
determine the pairing light
chain. In the course of the shuffling
experiment, we isolated
one clone, D2/L3mut, which appeared to have a
15-residue linker
but still produced predominantly dimeric scFvs.
Sequencing revealed
a single mutation from the D2 sequence, of residue
W (shown in
boldface type in Fig.
1) to Y, in the third hypervariable
region
of the light chain. Since this region of the Ab is in close
contact
with the V
H region, the mutation may destabilize
the V
H-V
L association,
thereby promoting
dissociation and intermolecular pairing (
56).
Affinity measurements.
For affinity measurement, the scFvs
were purified by affinity chromatography on Ag columns. The scFv
fragments were refolded after denaturation of all cell proteins with
urea by subsequent dialysis against PBS. Although the efficiency of
refolding is limited, the yield of functional scFv is much higher than
that in periplasmic fractions obtained by the osmotic shock procedure (58). The purity was analyzed on a Coomassie blue-stained
gel (data not shown).
All purified scFvs were analyzed by gel filtration chromatography, as
was already performed with the periplasmic fraction
of clone D2 (Fig.
3); the purified product of the D2 diabody contains
dimers only, while
the analogue with the complete linker, D2/15,
is predominantly
monomeric. Clones A3 and A5 produce monomeric
scFv fragments only
(Table
2).
We compared the affinities of the library-derived scFvs with the
binding strength of hybridoma-produced MAbs. We chose to
measure the
kinetics of binding by surface plasmon resonance on
a BIAcore machine.
When using Ag immobilized on Sensorchips, we
always observed biphasic
dissociations, probably caused by the
oligomerization of p24
(
18), resulting in a heterogeneous coat.
The abnormal
sensorgrams were also noted before by others (
25).
Therefore, the affinity was determined in solution (
47), by
injecting a preestablished equilibrium mixture of a fixed amount
of Ab
and a variable amount of Ag on a sensorchip with a high-density
coat of
Ag. Under mass transfer limiting conditions, the amount
of free Ab
(Ab
free) is proportional to the binding rate. Subsequently,
the
Kd is obtained by plotting the concentration
of Ab
free (or
the fraction
Ab
free/Ab
0; Ab
0 is the
concentration of Ab when no
Ag has been added) against the total
concentration of Ag. In Fig.
4, the
binding curves derived from the MAbs and scFvs are shown;
Table
2 gives
the corresponding
Kds. The affinities of MAbs
34A
and 39B are threefold higher than those of scFvs A3 and A5, both
selected on biotinylated Ag, which in turn have slightly higher
affinities than that of MAb 5B4. The D2 diabody, selected on
immobilized
p24 via panning, has a much lower affinity (micromolar
range).
The monomeric version of D2, D2/15, has a threefold-lower
affinity
than that of its dimeric counterpart, demonstrating that the
effect
of avidity on the apparent affinity determined in this assay is
limited. Finally, clone D2/L3mut, which expressed mainly dimeric
scFv
(as gel filtration of affinity-purified scFv revealed [results
not
shown]), had an affinity approaching that of clone D2.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 4.
Percentage of Abfree as a function of Ag
concentration (logarithmic scale). The amount of Abfree
(i.e., not bound to soluble Ag) was determined by surface plasmon
resonance and related to the input amount of Ab (no Ag added). The
Kd was derived from the plots at 50% binding.
|
|
Epitope recognition.
Pepscan analysis (22) was used
in the identification of the epitopes of the scFvs. Although all
recombinant fragments recognized denatured Ag on Western blots,
only the diabody type D2 gave a good response. The results obtained
with the overlapping decapeptides based on the sequence of HIV-1
subtype O strain ANT70 and the scFv-containing periplasmic fraction are
shown in Fig. 5A. The Ab reacted
with the peptides starting with the sequence INEEAVEEWDRTH and
ending with EWDRTHPPPVGP, thereby having in common the sequence EWDRTH.
This sequence is also recognized by serum Abs from an HIV-1 patient
(Fig. 5B), indicating that the immune system of the mouse recognizes
the same epitope on the immunized Ag as that of humans after virus
infection. It should be noted that the p24 used for immunization and
selection is derived from strain IIIB, while the pepscan is based on
strain ANT70. For the A2, A3, and A5 Abs, the pepscan based on the
ANT70 and the IIIB sequences did not reveal any unique epitopes
(results not shown), indicating that these Abs may recognize a
conformation-sensitive epitope.
View larger version (32K):
[in this window]
[in a new window]
|
FIG. 5.
Pepscan results obtained with decapeptides of p24 of
HIV-1 strain ANT70 with scFv D2 (A) and serum Abs of an HIV-1 patient
(B). The numbering on the horizontal axis corresponds with the
amino-terminal amino acid of the peptide: the first peptide starts with
a Q at  3 relative to the cleavage site of p17 and p24, and the last
peptide (no. 240) ends with an A located at 3 relative to the
cleavage site of p2 with p7. On the vertical axis, the optical density
at 450 nm obtained for each peptide in ELISA has been plotted. The
common sequence recognized by the human serum and scFv D2, starting
with peptide 76 and ending with peptide 82, is indicated in panel B by
solid bars.
|
|
Inspection of the HIV-1 B-cell epitope database (Los Alamos) showed us
that several MAbs that share the epitope with scFv
D2 have been found.
In one of these, hybridoma BB128
(
61),
the V gene
sequence contains a nearly identical heavy chain variable
region, with
a remarkably short H3 loop, and, even more surprisingly,
a light chain
variable region with only one residue difference
in the germ
line-encoded region from the sequence of D2 (Fig.
1).
| |
DISCUSSION |
Isolation of different sets of anti-p24 Abs dependent on the
selection procedure.
When we used the combinatorial library
technology for the generation of MAbs, we found that the selection
method determines to a great extent what type of Ab will be found. By
using Ag-coated polystyrene, the high Ag density probably favors the
selection of scFvs which are avid, exemplified by the selection of the
D2 clone, while selection with soluble Ag leads to the isolation of
monomeric Ab fragments produced by clones A2, A3, and A5. In the course
of the assembly reaction between VH, linker, and
VL to generate scFvs with 15-residue linkers
(14), mispriming may infrequently lead to short-linker
versions of VH and VL as reported previously
(42). In the resulting Abs with a 5- or 10-residue linker,
structural constraints prevent the intramolecular combination of
VH and VL. Instead, the product forms an
intermolecular pair with another VH-VL fusion
product, yielding a bivalent scFv molecule, the so-called diabody
(29, 44). The D2 diabody is heavily dependent on avidity for
its selection; when a selection procedure with soluble p24 is used, the
avidity effect is less pronounced and other clones (A3 and A5) are
selected on the basis of affinity. The avidity effect may be more
pronounced also by the intrinsic aggregation behavior of p24, which
upon coating may lead to epitopes that are spatially ordered such that
they are more prone to cross-linking by scFv Abs. Alternatively, the
epitope recognized by clones A3 and A5 might be shielded or modified on
p24 adsorbed onto polystyrene, although this hypothesis was not
supported by the high ELISA signals. Our data indicate that selections
should be carefully fine-tuned to retrieve the desired Abs. To obtain a
wider panel of anti-p24 Abs, the various selection procedures used here
may be altered or extended, for example, by using epitope-shielding Abs
as described elsewhere (16).
The affinities of the Abs were determined with an in-solution method.
The multimerization behavior of p24 complicated the
affinity
determination by direct kinetic measurement, while the
alternative
assay gave reliable data with only a minor effect
of avidity. The
affinities of the monomeric scFvs A3 and A5 found
by selection with
soluble Ag are in the nanomolar range and are
slightly better than the
affinity of one of the studied MAbs,
which was taken from a panel of
hybridoma-produced MAbs that are
currently used in diagnostic assays.
Their affinity is still threefold
lower than of the best MAbs tested,
but this might be due to the
avidity of the MAbs. Indeed, a threefold
difference in affinity
was also found between the dimeric and monomeric
versions of clone
D2.
The present study showed that Ab fragments obtained from the murine
library have affinities comparable to those of their hybridoma-derived
analogues. For obtaining high-affinity Abs useful as diagnostic
reagents, avidity may be recruited, for example, by making diabodies
(
29), scFv trimers (
39), or tetramers (for a
review, see reference
51). The affinities of the
best Abs selected, A3 and A5, may
be improved, for example, by targeted
mutagenesis (
1,
30,
57). Finally, additional alternative
selections should be employed
to further explore the diversity of the
murine phage library that
was constructed.
D2 recognizes an immunodominant epitope closely located at the CypA
binding site.
All selected scFvs recognized linear epitopes of
p24, as was concluded from Western blot studies. p24 expressed in
E. coli as well as Ag, derived from cultured virus and
presented in a reduced form was reactive on Western blots. Pepscan
analysis with overlapping decapeptides yielded the epitope of the scFv
produced by clone D2. The three-dimensional structure of the
amino-terminal core domain of the HIV-1 capsid protein is known
(24); it reveals the presence of an arrowhead-shaped domain,
with seven helices and two 
hairpins, and an exposed partially
ordered loop involved in binding cyclophilin A. Recently, the binding
domain was analyzed in more detail by crystallization of a peptide
fragment of p24 with cyclophilin A (69), indicating the
direct interaction of residues Ala 88, Gly 89, Pro 90, and Ile 91 with
residues from the cellular enzyme. The epitope of D2 is located within
the carboxy-terminal part of helix IV, ending at His 84 (or Leu in
strain IIIB), and it is separated by three residues from the residues
interacting with cyclophilin A.
The interaction of p24 with cyclophilin A appears to result in the
packaging of approximately 200 copies of CypA into each
HIV-1 virion.
Although the function of CypA remains unclear, virions
lacking the
enzyme are known to be poorly infectious (
7). Cyclosporins
capable of binding to cyclophilin A are effective in inhibiting
virus
replication, as was reported before (
4,
41,
60).
Agents that
interfere with the CypA binding function of p24 may
be useful in
reducing viral load; application of our scFv D2 as
intracellularly
expressed Ab may therefore prevent binding of
the enzyme in infected
cells, rendering the progeny virus less
infectious or noninfectious, as
was done successfully with anti-Tat
(
45) and anti-gp120
(
12) Abs.
Retained VH-VL pairing.
Several
epitopes have been mapped within the CypA loop region, which by its
exposed orientation is accessible for reaction with Abs. Other MAbs
that recognize the same epitope have been reported (28, 34,
48-50, 54, 55). In addition, the structure of a complex between
an anti-p24 Ab and its Ag was determined with an Ab with nearly
identical epitope recognition as that of Ab D2 (46).
Moreover, the epitope is also reactive with human Abs, as indicated by
our pepscan with serum from an HIV-1-infected patient and as was also
concluded in a study in which the peptide was incorporated in a
diagnostic assay (35).
Despite the recognition of the same epitope by the Abs mentioned above,
their heavy chain V genes have little homology to
each other, except
that they both use a CDR3 of the same size
and a nearly identical CDR1.
The most striking resemblance exists
between scFv D2 and anti-p24 MAb
BB128
(
61), which have almost
identical V
H
and V
sequences (differences of six and two
amino acid
residues, respectively). Clones isolated from a V
light
chain shuffling library were found to have the original
MAb light chain
sequence, including the tyrosine (replacing the
tryptophan present in
clone D2), indicating a favorable selection
for original pairings. It
has been argued that the changes of
retrieving the original pairing
from a combinatorial library are
very low, as the result of RNA
isolation (
67), and that it is
particularly difficult to
retrieve the original light chain (
2).
During the early days
of combinatorial library construction with
the bacteriophage lambda
system, an original V
H-V
L combination
identical
to the one found in a hybridoma antibody was reported
(
11).
However, the identity was less pronounced than that with
our p24 scFv:
in the partially sequenced V
H, containing CDR2,
FR3, and a
part of FR2, there were eight mismatches present and
the light chain
comprised four differences. Moreover, the V
H could
pair
with a different V
L, as was also found by Barbas and
colleagues
(
2). Considering the results from the light chain
shuffling
library, the D2 V
H is strictly monogamous, i.e.,
it can pair with
its original light chain only. When a mouse has
10
6 different B cells, a library with at least
10
12 clones must be made to have a chance to identify an
original
V
H-V
L combination. After immunization,
the number of anti-p24-expressing
B lymphocytes will be larger, but
still a rather huge library
would be needed to find original
combinations. One of the reasons
that we isolated the original clone
may be because of a high titer
of these Abs, since pepscan analysis
with the polyclonal mouse
serum gave a major response on the peptides
containing the EWDRTH
sequence (data not shown).
Diagnostic applications.
The selected anti-p24 Abs could be
used in a sensitive p24 Ag assay in which HIV-1 infection could be
detected in an early phase (20). The correlation between
affinity and sensitivity in ELISA has been clearly demonstrated
(15, 40). The affinities of the selected scFvs approach the
values of MAbs, which can detect 5 to 10 pg of p24 per ml of serum,
when combined in a capture assay. An exception is scFv D2, with its
micromolar level of affinity. It has been reported that the closely
related MAb BB128 is a synergistic Ab, meaning that its affinity is
enhanced dramatically (up to 1,000-fold) when combined with MAbs, which
recognize epitopes on the opposite side of the Ag. In vitro affinity
maturation (1, 30, 57) of the Abs might improve the
sensitivity to a level comparable with that of PCR detection
(6). The format of the Abs could be adapted to their
application; for instance, a high-affinity monovalent fragment might be
useful for capturing Ag, while a multivalent scFv might be more
suitable for the sensitive detection of high-density Ag arrested on a
solid surface. Therefore, the application of engineered Ab fragments
should be very useful for the development of new generations of
diagnostics.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Functional
Biomolecules, Unilever Research Laboratorium Vlaardingen, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands. Phone: (31)-10-4606175. Fax:
(31)-10-4605383. E-mail: HANS-DE.HAARD{at}UNILEVER.COM.
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Clinical and Diagnostic Laboratory Immunology, September 1998, p. 636-644, Vol. 5, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
