(Received for publication, July 24, 1995)
From the
In this report the V and V
genes of the
anti-T cell receptor (TCR) antibody KJ16, which recognizes the TCR
V
8.1 and V
8.2 regions in mice, were cloned and expressed as a
single-chain antibody (scFv) in Escherichia coli. A 29-kDa
protein was obtained after renaturation from inclusion bodies. The KJ16
scFv had a relative affinity for the native TCR that was slightly
higher than KJ16 Fab fragments. The scFv and Fab fragments of the KJ16
antibody, together with monovalent forms of two other anti-TCR
antibodies, were evaluated as antagonists of the T cell-mediated
recognition of a peptide-class I complex or of a superantigen, Staphylococcus enterotoxin B (SEB) bound to a class II
product. Each of the anti-TCR antibodies was efficient at inhibiting
the recognition of the SEB-class II complex. In contrast, only the
clonotypic antibody, which binds to epitopes on both the V
and
V
regions, inhibited the recognition of peptide-class I complex.
We conclude that the TCR binding site for the SEB-class II ligand
encompasses a larger surface area than the TCR binding site for the
peptide-class I ligand.
Specific recognition of foreign antigens by T cells is mediated
through a T cell receptor (TCR) ()complex composed of the
heterodimer and CD3 subunits. Under normal conditions, T
cells that mature in the thymus undergo a process of selection in which
self-reactive cells are eliminated(1) . Despite this process, a
number of diseases appear to be the result of aberrant T cell activity.
These include autoimmune diseases in which the
receptor
presumably recognizes a self-antigen(2) , transplantation
reactions that involve alloreactive T cells(3) , and
``superantigen''-mediated diseases caused by a hyperactive
immune response when the
-chain of the TCR, expressed by a large
fraction of T cells, binds to the superantigen (4) .
Because
the TCR is the common element in all these diseases, there has been
considerable interest in using the TCR as a specific target to
eliminate or inhibit detrimental T cells(5) . Among the
molecules targeted have been CD3 (6, 7) and regions of
the heterodimer
itself(8, 9, 10, 11, 12) .
In some cases, treatment with anti-TCR antibodies in vivo has
led to specific elimination of target T cell populations in animal
models of human diseases. For example, most T cells involved in
experimental autoimmune encephalomyelitis in mice bear the V
8
domain, and treatment with the V
8-specific monoclonal antibody
F23.1 (8) prevented or reversed the disease(9) .
Another V
8-specific antibody, KJ16 (10) was used to
control graft versus host disease in SCID mice injected with
alloantigenic spleen cells (11) and to control the pathogenesis
of collagen-induced arthritis(12) . Each of these diseases is
suspected to be caused by T cells that expressed primarily V
8
regions.
In addition to their uses in the control of diseases,
monoclonal anti-TCR antibodies have been used on a limited basis to map
the topology of TCR binding
sites(13, 14, 15) . This interest stems from
the fact that resolution of the TCR interactions with either
peptide-MHC or superantigen-MHC at the molecular level remains to be
determined. Nevertheless, several studies have used TCR mutagenesis to
begin to define the regions involved in ligand
binding(16, 17, 18, 19, 20) .
For instance, Hong et al.(20) used transfectants of
chimeric T cell receptors to show that the CDR1 and/or CDR2 regions of
the TCR chain are important for MHC specificity and to help
define the orientation of the TCR relative to its MHC ligand. Other
analyses that mutagenize CDR regions have examined their role in both
peptide-MHC specificity and superantigen reactivity(17) .
The size of the TCR -heterodimer is approximately that of
an antibody Fab fragment, and thus topology mapping (as well as
therapeutic efficacy) might be improved by reducing the size of the
anti-TCR antibody to the minimal possible unit. We have previously
expressed an anti-clonotypic antibody, 1B2, as a single-chain Fv (scFv; (21) ), and in this report we characterize the binding
properties of the scFv from the anti-V
8 antibody
KJ16(10) . KJ16 scFv was expressed as a single-chain in which
the V
was joined to the V
by a 26-amino acid
linker (205s) (22) . KJ16 scFv preparations bound to the native
TCR with a relative affinity that was approximately 1.5-fold higher
than Fab fragments. The biphasic nature of the binding isotherm
suggested that the scFv may exist in monomer/dimer equilibrium; as with
other scFv such as 1B2, the higher affinity component of the observed
binding may be due to the dimeric form(21, 23) .
The three anti-TCR antibodies KJ16, 1B2, and F23.1 were also used to
probe the topology of the TCR binding sites for either peptide-class I
MHC or staphylococcal enterotoxin B (SEB)-class II MHC. KJ16 and F23.1
bind to different determinants on the V8 domain of the
TCR(24) , and the clonotypic 1B2 binds to determinants on both
the V
8 and V
3 regions of the cytotoxic T cell clone
2C(21, 25, 26, 27, 28) .
All three of the antibodies were able to inhibit SEB-class II
recognition, but only the clonotypic antibody inhibited recognition of
peptide-class I. Thus, all three of these antibody epitopes reside
within the superantigen binding site of the TCR. This finding is
consistent with recent reports that the superantigen binding site on
the TCR may extend to the V
domain (29, 30, 31) and that the area on the TCR
required to bind the superantigen-class II complex is larger than the
area required for normal peptide-MHC recognition. Finally, because
intact KJ16 has been shown to be effective in vivo in several
diseases mediated by V
8
T cells, comparisons
among scFv, Fab fragments, and intact antibody for their potential to
target T cell populations in vivo can now be undertaken.
To determine the extinction coefficient at 280 nm for each
antibody, protein concentrations were determined by the bicinchoninic
assay (Pierce) using protein A-purified F23.1 intact antibody as the
standard. From the protein concentrations and A of each sample, the following
were obtained: 1.22 (KJ16 Fab); 1.34 (KJ16 scFv); 0.92 (1B2 Fab);
1.40 (1B2 scFv); 0.83 (F23.1 Fab).
of intact antibodies was assumed to be 1.35(37) .
To detect
direct binding of KJ16 scFv to the V8 region, partially purified
preparations of a single-chain TCR (20 µg/ml) were adsorbed to the
wells of a 96-well plate (Immulon 2) overnight at 4 °C. The
single-chain TCR contained the variable regions of the
and
chains encoded by CTL clone 2C (V
3V
8) linked by a short
peptide (28) . Wells were washed and blocked using
phosphate-buffered saline, pH 7.3, containing 0.1% bovine serum albumin
and 0.1% Tween-20 (PBSBT) for 1 h at room temperature. Serial dilutions
(in PBS) of KJ16 scFv were added for at least 1 h, wells were washed
with PBSBT, and bound scFv was detected with the mouse anti-c-Myc
antibody CT14. CT14 was detected with goat anti-mouse antibodies
conjugated to horseradish peroxidase followed by the addition of
substrate (tetramethylbenzidine; Kirkegaard and Perry Laboratories)
followed by acid. Absorbances at 450 nm were quantitated using a
96-well plate reader.
To determine the
relative affinity of KJ16 scFv for the TCR on CTL 2C, a competition
assay was performed with FITC-labeled KJ16 Fab fragments. KJ16 FITC/Fab
conjugates were generated as described(38) . In brief, an
aliquot of 0.9 mg/ml FITC in PBS was added to 0.5 mg/ml KJ16 Fab
fragments in 0.2 M sodium bicarbonate, pH 8.5, in order to
yield a 50:1 (FITC:antibody) molar ratio. After incubation at room
temperature for 2 h, the solution was dialyzed into PBS, pH 7.3. The
number of FITC bound per antibody was calculated as follows: 3.08
(A
/(A
-
(0.145)A
))(39) . Various concentrations
of scFv KJ16 were added to 6
10
2C cells in the
presence of a constant amount of FITC-labeled Fab. After a 30-min
incubation on ice, the entire mixture (scFv + KJ16 FITC/Fab +
2C cells) was passed through a flow cytometer without washing. The
amount of inhibition was measured by quantitating the fluorescence from
cells by flow cytometry. The concentrations of unlabeled antibody
giving 50% inhibition (IC
) relative to the maximum
fluorescence (in the absence of inhibitor) and the background
fluorescence (in the presence of a large excess of intact antibody)
were used to compare affinities. Inhibition curves were generated for
KJ16 scFv and unlabeled Fab preparations in two independent
experiments. Each concentration was performed in triplicate.
Figure 1:
Schematic of the anti-V
single-chain antibody KJ16. A, cDNA was generated from the
KJ16 hybridoma, and the V
and V
genes were
amplified by polymerase chain reaction using a set of degenerate
primers. The genes were linked by a sequence encoding a 26-amino acid
linker called 205s, and the construct was cloned behind the ompA signal
sequence and includes a carboxyl-terminal peptide from the c-Myc
protein. B, sequence of scFv KJ16 derived from the KJ16
hybridoma. The 205s linker is a modified 205 linker in which a
carboxyl-terminal glycine is replaced with alanine-serine in order to
incorporate an NsiI restriction
enzyme.
Although binding of anti-c-Myc antibody
demonstrated that the recombinant protein was fully translated, it was
unknown whether the protein was functional (i.e. could bind
the TCR ligand). To test this, an ELISA was used to determine if the
scFv could bind to a recombinant V8 domain absorbed to microtiter
wells. The V
8.2 domain was part of a single-chain TCR (scTCR) that
could be readily detected with intact KJ16 antibody, as described
previously by Soo Hoo et al.(28) . The scTCR was
adsorbed to wells, incubated with various dilutions of an unpurified
preparation of refolded KJ16 scFv, and the bound scFv was detected with
anti-c-Myc antibody (Fig. 2). KJ16 scFv bound specifically to
the scTCR, as compared with bovine serum albumin used as a negative
control. This assay could be used to detect activity at dilutions of
the unpurified preparation as high as 1:1000 and thus was used to
routinely monitor HPLC purifications of the KJ16 scFv.
Figure 2:
ELISA to demonstrate KJ16 scFv binds
specifically to a soluble V8 bearing scTCR. Serial dilutions of
crude KJ16 scFv were added to wells adsorbed with scTCR
(
-
) or bovine serum albumin (BSA)
(
-
) at 20 µg/ml for 1 h. After washing with
PBS/bovine serum albumin, bound scFv was detected by adding anti-c-Myc
antibody and incubating for 1 h. The plate was washed, and a secondary
antibody (goat anti-mouse horseradish peroxidase-labeled antibody) was
added. After 1 h and washing, substrate was added, and the absorbance
was monitored at 450 nm.
KJ16 scFv was
purified by HPLC chromatography through a G-200 column under
nondenaturing conditions. The elution profile yielded three major
peaks: one at the column void volume, a second at approximately 30 kDa,
and a third at the total column volume (Fig. 3A). A
V8-specific ELISA showed that the 30-kDa peak contained the
majority of the activity (data not shown). Reduced SDS-PAGE gels (Fig. 3B) and Western blot analysis with the anti-c-Myc
antibody (Fig. 3C) were used to further characterize
each peak. A major overexpressed protein of 35 kDa was present in the
refolded preparations. This protein was positive by Western blotting
with the anti-c-Myc antibody, confirming that it represented the
recombinant KJ16 scFv. Since the first peak runs at the exclusion
volume and contains a significant amount of anti-c-Myc reactive
material, it likely contains covalent or noncovalent aggregates of the
scFv protein. The second peak eluted at the expected size of a scFv
monomer, and it contained an
35-kDa species as detected by
SDS-PAGE and Western blots (Fig. 3, B and C).
The third peak eluted at the total column volume, and it did not
contain detectable protein by SDS-PAGE and Western blot analysis; it
likely consists of proteolysed material and protease inhibitors from
the refolding buffer.
Figure 3:
HPLC gel filtration profile, SDS-PAGE
analysis, and Western blot of refolded KJ16 scFv. A, KJ16 scFv
was refolded by dilution into TKC buffer and concentrated; a sample was
injected onto a Superdex G-200 column equilibrated in
phosphate-buffered saline, pH 7.3; and the eluent was monitored for
absorbance at 280 nm. Fractions were collected and analyzed by
SDS-PAGE. B, SDS-PAGE of peak fractions obtained by size
exclusion HPLC. Samples were electrophoresed through a 10%
polyacrylamide gel under reducing conditions, and proteins were
visualized by staining with Coomassie Blue. Pre-HPLC sample
represents TKC-refolded preparation before HPLC purification. Peak 1,
which elutes in the column void volume, is likely covalent and
noncovalent aggregates of scFv protein. Peak 2, which elutes at the
expected size of an scFv monomer, is composed of two monomeric forms of
KJ16 scFv. Fraction 2a contains a 28-kDa species, which could represent
a proteolysed form of scFv (referred to as KJ16 scFv`), fraction 2c
contains a 29-kDa species, which is consistent with the calculated mass
of the KJ16 scFv monomer (28,932), assuming a cleaved signal sequence.
The last peak to elute is at the total column volume and likely
consists of proteolysed material and protease inhibitors from the
refolding buffer. C, an SDS-PAGE gel similar to that shown in B was blotted onto nitrocellulose, scFv was detected with the
anti-c-Myc antibody CT14, and bound CT14 was detected with goat
anti-mouse antibodies conjugated to horseradish peroxidase. Both the
28- and 29-kDa species shown in fractions 2a, 2b, and 2c react with
anti-c-Myc antibody. Since the 10-amino acid c-Myc tag is encoded at
the carboxyl terminus, the reactivity of the 28-kDa species with the
anti-c-Myc antibody CT14 suggests that the truncated scFv` was cleaved
at its NH terminus.
Closer inspection of the fractions that were
contained within the second peak revealed minor size heterogeneity
among the fractions (Fig. 3, B and C); all
three different KJ16 scFv preparations that have been examined yielded
identical results. SDS-PAGE analysis of reduced fractions from peak 2
showed that it contained two proteins with similar molecular mass. Mass
spectrometry analysis indicated that the major protein in fraction 2a
had a mass of 28,262 and that of fraction 2c had a mass of 29,030.
Although the two proteins eluted as a single peak, the smaller 28-kDa
species eluted before the larger 29-kDa species. The apparent
discrepancy in the order of elution could be due to differences in the
shape of the two forms, but we have not explored this possibility
further. The 29,030 mass of the later species is consistent with the
predicted molecular mass of the KJ16 scFv monomer (28,932), assuming a
cleaved signal sequence. Thus, this species will herein be referred to
as KJ16 scFv. Since the amount of the 28-kDa species increased over the
lifetime of stored inclusion bodies (data not shown), the 28-kDa
species could represent a proteolysed form of KJ16 scFv. This species
will be referred to as KJ16 scFv`. The observed reactivity in a Western
blot with the anti-c-Myc antibody (Fig. 3C) may suggest
that the COOH terminus is intact and that the NH terminus
has been cleaved. If this is the case, then the scFv` form may not fold
properly; this possibility is consistent with a shape that differs from
the scFv and thus with its increased size exclusion. Without further
optimization of expression, the estimated yield of HPLC-purified KJ16
scFv from a 1-liter equivalent of fermentation is approximately 300
µg.
Figure 4:
Binding of a cell surface T cell receptor
by KJ16 scFv. A, flow cytometry histogram of 2C cells
incubated for 30 min at 4 °C in the presence or absence of KJ16
scFv. Bound scFv is detected by the anti-c-Myc antibody, CT14, followed
by a goat anti-mouse FITC-labeled antibody. B, inhibition of
binding by FITC-labeled KJ16 Fab fragments with purified scFv or Fab
fragments. 6 10
2C cells were incubated 30 min at 4
°C with FITC-labeled KJ16 Fab fragments and various concentrations
of KJ16 scFv (
), KJ16 scFv` (
), and unlabeled Fab
fragments (
). A relative affinity of the scFv was determined by
comparing the concentrations required to inhibit 50% of the
FITC-labeled Fab fragments from binding the 2C TCR
(IC
).
Analysis of the binding curves
suggests that the molar concentration of scFv required to inhibit 50%
of the FITC/Fab from binding the 2C TCR was 1.5-fold less than
that of unlabeled Fab fragments, indicating that the scFv had a higher
binding affinity. However, the binding curve of the scFv appeared to be
contain two components: one that resembled that of the Fab fragments,
and a second component that appeared to be derived from a higher
affinity species. Although we have not resolved a dimeric form by size
filtration, it is possible that the higher affinity component
represents a dimer that exists in equilibrium with monomer. Such dimers
can be observed, especially after concentration, in other scFv
antibodies such as 1B2(21, 23) . Nevertheless, the
results show that the recombinant scFv can fold to an active form that
binds the cell surface TCR at least as well as Fab fragments.
To examine recognition of SEB-class II ligand,
the human class II cell line Daudi was used together
with SEB as a target in a cytotoxicity assay with CTL clone 2C.
Recognition of SEB-class II was inhibited by all forms of the KJ16,
1B2, and F23.1 antibodies (Fig. 5A). The inhibitory
potential of each antibody directly correlated with their binding
affinity (Table 1). However, the KJ16 scFv was approximately
5-fold less effective than KJ16 Fab fragments, a finding that would not
be expected from the nearly equal binding affinities of these
preparations. There are at least two possible explanations for this
finding. First, the scFv is less effective because it is smaller and
does not sterically prevent TCR binding as well as Fab fragments.
Alternatively, the scFv is less stable than Fab fragments during the
course of the 4-h cytotoxicity assay at 37 °C. The latter
possibility may be supported by our previous finding that 1B2 scFv also
appeared to be approximately 5-fold less effective at inhibiting
cytolysis than would be expected from its affinity. These possibilities
are currently being examined as they have direct relevance to the use
of these agents in vivo.
Figure 5:
Inhibition of recognition by T cell clone
2C with different forms of three anti-T cell receptor antibodies.
Inhibition of 2C-mediated lysis of Cr-labeled target cells
was used to evaluate the inhibitory properties of KJ16 (scFv (
)
and Fab (
)), 1B2 (scFv (
) and Fab (
)), and F23.1
(Fab (
)). KJ16 and F23.1 are antibodies that recognize different
epitopes on V
8, and 1B2 is clonotypic for the 2C TCR. A,
inhibition of lysis of the MHC class II target cell Daudi pulsed with
10 µg/ml SEB. B, inhibition of lysis of the MHC class I
targets T2/L
-pulsed with 1 nM QL9 peptide. 2C
cells were incubated with antibodies for 30 min at room temperature
before the addition of
Cr-labeled target cells at an
effector:target ratio of 5:1. Percentage of inhibition was calculated
by normalizing values to the amount of specific
Cr release
that occurred in the absence of inhibiting antibody (21.5 ± 3.4%
for SEB-Daudi and 19.8 ± 1.1% for QL9/T2/L
). All
titrations were performed in triplicate, and a separate inhibition
experiment yielded similar results.
In contrast to results with the ligand
SEB-class II, inhibition of peptide-class I recognition was observed
only with fragments derived from the clonotypic antibody 1B2 (Fig. 5B and Table 1). Because 1B2 binds to
determinants on both the V and V
chains, this result was not
unexpected since recognition of peptide-MHC is predicted to involve
both V
and V
regions of the TCR. On the other hand,
recognition of peptide-MHC is not predicted to involve the region on
the face of the
-chain that contains the KJ16 and F23.1
epitopes(42) .
There have been many reports that use antibodies to map the
topology of proteins. Most of these studies have used intact antibodies
to examine surface-accessible epitopes on the protein antigen (e.g. 13, 24, 34). In principle, the smaller size of a scFv (30
kDa) compared with Fab fragments (
60 kDa) and intact antibodies
(150 kDa) may provide better resolution in topology mapping of
proteins. As it becomes more routine to produce and purify scFvs, these
recombinant antibodies can also be more readily prepared than Fab
fragments for studies that examine protein structure. In this report,
we have produced and characterized a scFv that recognizes an epitope on
the V
8 chain of the TCR. This epitope includes residue 16 in the
framework region of the V
8.2 domain(43) , a residue that
has recently been shown to reside on the outer face on the crystal of a
V
8.2 dimer(44) . Another antibody, F23.1, has been shown
to interact with residue 60 of the V
8.2 chain(45) . This
residue is located closer to the CDRs of the
-chain than the KJ16
epitope, but both of these antibodies and their monovalent fragments
are capable of cross-inhibition(24) . (
)Antibody 1B2
is a clonotypic antibody as defined by the fact that it only reacts
with the TCR of CTL 2C and not other T cells. This antibody has been
shown to bind determinants on both the V
and V
regions of the
TCR from
2C(21, 25, 26, 27, 28) .
1B2 binding is not blocked by binding of F23.1 and KJ16,
further evidence that this epitope is located distal to the
V
framework regions recognized by the latter antibodies.
The
role of antibody affinity, epitope, and size in the ability to inhibit
TCR recognition of two different ligands were examined. One ligand was
the classical peptide-MHC complex that is recognized by T cells, and
the other ligand was a complex of the superantigen SEB and a class II
product. Numerous studies have shown that the V chain is primarily
involved in the recognition of the latter complex (reviewed in (4) ), while both the
and
chains are involved in
peptide-MHC recognition (reviewed in (42) ). Our results showed
that all three antibodies were capable of blocking the recognition of
the SEB-class II ligand. The finding extended to both scFv antibodies
KJ16 and 1B2, suggesting that the scFv approach should be useful in
examining the topology of protein binding sites. The observation that
the V
8-specific antibody fragments inhibit SEB-class II
recognition was not surprising given that this region of the TCR has
been predicted to be involved in superantigen recognition. However, the
even greater inhibition with 1B2 antibody fragments suggested that the
TCR binding site for SEB-class II extends over an area that includes
the V
region. Recently, a completely different approach was used
to also implicate the
chain in this
recognition(29, 30) . Our results are consistent with
this notion and the possibility that a significantly larger surface
area of the TCR is involved in SEB-class II binding compared with
peptide-class I binding.
Single-chain antibodies have now been generated against a variety of cell surface molecules with the goal of using them for specific cell targeting in vivo (reviewed in (46) ). For the delivery of toxic agents to tumor cells, it seems likely that the affinity of the scFv will be a key factor in the effectiveness of the agent. In this respect it is notable that the scFv of KJ16 appears to have an affinity that is very similar to the Fab fragments. Like scFv preparations from other antibodies, there also appear to be other forms of the scFv. The KJ16 scFv` ( Fig. 3and Fig. 4) appears to be a proteolytic fragment of the full-length scFv, and it has reduced or negligible binding activity. The binding studies with the KJ16 scFv (Fig. 4B) also suggested that there may be dimeric or multimeric forms that yield higher apparent affinities then the monovalent scFv. These forms have now been detected in many scFv preparations(21, 23, 47) .
In comparison with the use of scFv antibodies to deliver toxic agents to cells, the ability of anti-TCR scFv to directly control T cells in vivo may be even less predictable. This stems from the nature of the interaction of the anti-TCR antibody with the T cell. Recently, Janeway has suggested that the mechanism of antagonism by anti-TCR antibodies may involve not only steric inhibition of ligand binding but direct conformational effects induced in the TCR(15, 48) . The latter effect could result in signals that either activate or anergize a T cell. Our data cannot distinguish between these possibilities in the inhibition of cytolytic activity (Fig. 5, Table 1), but the proposition that anti-TCR antibodies could act simply by signaling the T cell through a monovalent interaction will have some implications in the selection of anti-TCR antibodies for use in vivo. For example, one might have selected an antibody that had the highest possible affinity as an appropriate candidate for scFv engineering and subsequent therapeutic use. However, the fact that T cells undergo negative and positive selection in the thymus and that antibodies to different epitopes may influence these processes in unpredictable ways suggests that it may be necessary to compare scFv and Fab fragments from antibodies that recognize different epitopes on the same TCR. The in vivo effects of KJ16 scFv and 1B2 scFv can now be studied in light of the in vitro effects reported here.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34924[GenBank].