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Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, D-60596 Frankfurt am Main [F. R., M. B., W. W.], and MEMOREC Stoffel GmbH, D-50829 Köln [B. G.], Germany
ABSTRACT
Efficient T-cell activation requires two signals. The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules. One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells. As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells. We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase. A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1–111 (CD86111) of the mature protein. CD86111 produced in the yeast Pichia pastoris and CD86111 expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors. Bacterially expressed CD86111-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.
INTRODUCTION
Human tumors of epithelial origin often overexpress members of the ErbB/EGFR2 related family of receptor tyrosine kinases. This receptor family comprises ErbB/EGFR, ErbB2/HER-2/neu, ErbB3, and ErbB4 (1) . In particular, overexpression of EGFR and ErbB2 has been shown to directly contribute to malignancy (2 , 3) . Because of their aberrant expression on tumor cells and their accessibility from the extracellular space, these receptors are suitable targets for directed tumor therapy. Mabs specific for the extracellular domains of such receptors have been shown to interfere with signal transduction and are under evaluation for their therapeutic application in clinical trials (4) . To enhance their growth inhibitory potential, recombinant single-chain derivatives of such antibodies have been constructed that target a potent cytotoxin to tumor cells expressing the appropriate receptor (5) . In experimental models, this strategy has been applied successfully to reduce tumor load and eliminate metastatic cells (6) . However, treatment with such antibodies and cytotoxic antibody fusion proteins does generally not result in the induction of specific antitumor immunity and cannot prevent possible tumor recurrence if disseminated tumor cells escape cytotoxic therapy. To achieve long-lasting antitumoral effects, immunotherapeutic strategies have been developed that combine antibodies for specific targeting to tumor cells with cytokines or other immunomodulatory activities for the induction of tumor-specific T lymphocytes (7, 8, 9) .
For optimal induction and clonal expansion, T cells require at least two activating signals (10) . The first signal is antigen-specific and is generated by interaction of the T-cell receptor with peptide-bearing MHC molecules. A second, costimulatory signal is provided by binding of CD28, the major costimulatory signal receptor on CD4+ and CD8+ T cells, to its cognate ligands B7-1 (CD80) and B7-2 (CD86), which are expressed on the surface of antigen-presenting cells (11 , 12) . T-cell receptor stimulation without costimulation can result in T-cell anergy or apoptosis. Although many tumor cells express MHC class I molecules and are able to present antigens, most do not provide costimulation, a possible mechanism for tumor cells to evade immune surveillance. Strategies to provide tumor cells with members of the B7 family of costimulatory molecules have led to promising results. In experimental models, rejection of antigenic tumor cells transfected with B7-1 or B7-2 genes has been demonstrated, which also resulted in protection of mice against subsequent challenge with B7-negative wild-type tumors (13, 14, 15, 16) .
We recently developed an alternative approach to specifically localize
costimulatory activity to the surface of tumor cells that does not
require transduction or transfection of the cells with B7 gene
constructs (Fig. 1)
. A soluble fusion
protein was constructed that consisted of the extracellular domain of
the human CD86 molecule (CD86225), for CD28
activation, genetically fused to an ErbB2-specific scFv antibody
fragment for tumor targeting (9)
. Here we have refined
this strategy and have characterized a minimized recombinant CD86
domain (CD86111) that, when expressed in yeast
and bacteria, retains binding to B7 counter receptors. Fusion proteins
containing this CD86 domain and scFv antibody fragments specific for
the human EGFR and ErbB2 molecules specifically localize to the surface
of tumor cells expressing the respective target receptors and, upon
binding to antigen, stimulate proliferation of primary T lymphocytes.
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Cell Lines and Cell Culture Conditions.
CHO and CHO-CTLA-4 cells expressing glycosylphosphatidyl
inositol-anchored human CTLA-4 on the cell surface
(17)
were maintained in MEM
with deoxyribonucleosides
(Life Technologies, Inc., Karlsruhe, Germany), containing 2
mM glutamine, 50 µM ß-mercaptoethanol, 10%
heat-inactivated FBS, and 1 mg/ml G418 (CHO-CTLA-4). Murine renal
carcinoma (Renca) cells stably expressing Escherichia coli
ß-galactosidase and either human ErbB2 (18)
or EGFR
(6)
were cultured in RPMI 1640 supplemented with 10% FBS,
2 mM glutamine, 100 units/ml penicillin, 100
µg/ml streptomycin, 0.25 mg/ml Zeocin, and 0.48 mg/ml G418.
Pichia pastoris GS 115 yeast cells (Invitrogen, Groningen,
the Netherlands) were propagated in buffered glycerol-complex medium
and expression of recombinant proteins was induced in buffered
methanol-complex medium according to the distributor’s
recommendations.
Construction of CD86111 and CD86111-scFv
Expression Plasmids.
A cDNA fragment encoding the IgV domain of human CD86 (amino acid
residues 1–111 of the mature protein; referred to as
CD86111) was derived by PCR using CD86 cDNA as a
template (17)
and the oligonucleotides CD86-sense
(5'-aaaagtcgacgctagcGCTGCTCCTCTG-3') and CD86-IgV-antisense
(5'-aaatctagacgatcgatAAGCACTGACAG-3'; CD86 sequence in upper case),
which introduce SalI and NheI restriction sites
at the 5' end and ClaI and XbaI restriction sites
at the 3' end of the PCR product. The amplified
CD86111 cDNA fragment was digested with
SalI and XbaI and ligated in frame 5' to a
synthetic sequence that encodes the Myc tag and a cluster of six
histidine residues (His tag) in a modified pBluescript
KS+ vector (Stratagene, Heidelberg, Germany; Ref.
17
). For expression in the yeast P. pastoris,
an NheI/NotI CD86111
fragment including Myc and His tags was isolated from the cloning
vector and inserted into the AvrII/NotI-digested
yeast expression vector pPIC9 (Invitrogen), resulting in the plasmid
pPIC9-CD86111 (Fig. 2
A).
CD86111-scFv fusion genes were derived by
isolating the ErbB2-specific scFv(FRP5) and the EGFR-specific
scFv(14E1) single-chain antibody domains as
ClaI/XbaI fragments from plasmids pWW152-5
(5)
and pWW152-14E1 (19)
, and inserting them
between CD86 and Myc/His tag sequences in the pBluescript cloning
vectors. For bacterial expression of recombinant CD86 proteins, plasmid
pSW5 was constructed by replacing the E. coli ompA signal
peptide and synthetic FLAG sequences of the vector pFLAG-1 (IBI
Biochemicals, New Haven, CT) with a modified multiple cloning site.
Vector backbone, Myc/His tag, and CD86111 and
CD86111-scFv fragments were assembled stepwise,
resulting in plasmids pSW5-CD86111 (Fig. 2
A), pSW5-CD86111-5 and
pSW5-CD86111-14E1 (Fig. 3
A). In the yeast and E.
coli expression plasmids, start codons for translation of the gene
products are provided by the vector backbones.
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-factor secretion signal. In
addition, the plasmid contains a functional histidinol dehydrogenase
(HIS4) gene for positive selection in the P.
pastoris HIS4 mutant strain GS115.
pPIC9-CD86111 DNA was linearized by
SalI digestion and used for transformation of P.
pastoris GS115 cells by electroporation.
His4+/methanol-utilization+
(mut+) yeast colonies were isolated on selection
media following established protocols (20)
, and the
phenotype of the resulting clones was confirmed by PCR using AOX1 5'
and 3' primers (Invitrogen). For expression of recombinant CD86111, a single yeast colony was grown to an absorbance at 600 nm of 3 in buffered glycerol-complex medium (pH 8). The medium was then exchanged with methanol-containing buffered methanol-complex medium (pH 8), and protein expression was induced for 72 h at 30°C. Yeast cells were removed by centrifugation at 20,000 x g. Supernatant containing soluble CD86111 protein was passed through a 45 µm filter and applied onto a Ni2+-saturated Chelating Sepharose column (Amersham Pharmacia Biotech, Freiburg, Germany); recombinant protein specifically bound to the column via the COOH-terminal polyhistidine tag was then eluted with PBS containing 250 mM imidazole. Fractions containing the fusion protein were identified by SDS-PAGE and immunoblotting with Mab 9E10 specific for the Myc tag (21) , pooled, concentrated, and dialyzed against PBS. A CD86 protein fragment comprising the complete CD86 extracellular domain (CD86225) was expressed in the yeast P. pastoris as described previously (17) . N-Linked glycosylation of purified CD86111 protein was analyzed in a deglycosylation reaction. In brief, 0.2 µg of the protein were heated to 100°C for 10 min in PBS containing 0.1% SDS. Triton X-100 at a final concentration of 1% and 1 unit of N-Gycosidase F (Boehringer Mannheim, Mannheim, Germany) were added. After incubation for 16 h at 37°C in a total reaction volume of 100 µl, samples were analyzed by SDS-PAGE and immunoblotting with Mab 9E10.
Bacterial Expression of CD86111 and
CD86111-scFv Proteins.
Plasmids pSW5-CD86111,
pSW5-CD86111-5 and pSW5-CD86111-14E1
were transformed into E. coli BL21 (
DE3)
trxB- (22)
. Single colonies were
grown at 37°C to an absorbance at 550 nm of 0.8–1.0 in Luria-Bertani
medium containing 0.6% glucose and 100 µg/ml ampicillin. Expression
of recombinant proteins was induced with 0.2 mM
IPTG for 1 h at room temperature. Cells were harvested by
centrifugation at 5000 x g for 10 min at 4°C, and a
cell pellet from 1 liter of culture was resuspended in 35 ml of PBS
containing 8 M urea. Cells were disrupted in a
French press, and the lysates were incubated for 45 min at room
temperature and clarified by centrifugation at 16,000 x
g for 30 min at 4°C. Recombinant
CD86111, CD86111-scFv(FRP5)
and CD86111-scFv(14E1) proteins were purified
from the supernatants via binding of the polyhistidine tags included in
the molecules to Ni2+-saturated Chelating
Sepharose equilibrated with PBS, 8 M urea and
elution with PBS containing 8 M urea and
250 mM imidazole. Fractions containing
recombinant proteins were identified by SDS-PAGE and immunoblotting
with Mab 9E10, pooled, and dialyzed against 400
mM L-arginine in PBS at
4°C. Finally, L-arginine was removed from the
samples by stepwise dialysis against PBS.
Binding Assays.
The binding of purified CD86111,
CD86111-scFv(FRP5), and
CD86111-scFv(14E1) proteins to the B7 counter receptor
CTLA-4 was determined by FACS analysis using CHO-CTLA-4 cells. A single
cell suspension was prepared by treatment with trypsin, and 5 x
105 CHO-CTLA-4 cells were incubated for 45 min at
4°C with 0.2–1 µg of each of the recombinant CD86 proteins,
followed by incubation with 2 µg of Mab 9E10 and FITC-labeled goat
antimouse IgG (PharMingen, Heidelberg, Germany) for 30 min. Binding
of CD86 proteins was detected using a FACScan (Becton Dickinson,
Heidelberg, Germany). CHO cells served as a control. Similarly, the
binding of CD86111-scFv(FRP5) and
CD86111-scFv(14E1) to their target receptors
ErbB2 and EGFR was determined by FACS analysis using ErbB2- or
EGFR-expressing Renca-lacZ/ErbB2 and Renca-lacZ/EGFR cells.
For visualization of receptor binding by immunofluorescence, Renca-lacZ/EGFR cells were grown on glass coverslips, washed with PBS, and fixed in a 1:1 (v/v) mixture of acetone and methanol for 10 min at -20°C. Cells were incubated with 0.5 µg of CD86111-scFv(14E1) in TTBS [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] supplemented with normal goat serum (1:100) for 1 h at 37°C. Bound CD86 fusion protein was detected by incubation with 0.5 µg of Mab 9E10 followed by 0.5 µg of FITC-conjugated goat antimouse IgG (PharMingen). Each incubation was carried out in TTBS for 45 min at 37°C and followed by washing steps. Cell nuclei were stained with 50 µg/ml propidium iodide (Roth, Karlsruhe, Germany). Coverslips were mounted on glass slides and analyzed under a fluorescence microscope (Zeiss Axiophot; Carl Zeiss, Jena, Germany).
T-Cell Proliferation Assays.
Spleen cells from BALB/c mice were suspended in 900 µl/spleen of PBS
containing 0.5% FBS, and incubated with 100 µl of MACS rat antimouse
CD90 (Thy 1.2) antibody-conjugated colloidal super-paramagnetic beads
(Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at 6°C.
Cells were washed, resuspended in 1 ml of PBS containing 0.5% FBS, and
applied on a LS+-column (Miltenyi Biotech) placed
in a magnetic field. CD90- cells were eliminated
from the column by rinsing with PBS containing 0.5% FBS. Subsequently,
the column was removed from the magnetic field, and
CD90+ T cells were eluted in 5 ml of the same
buffer. The isolated T cells were kept in DMEM medium supplemented with
10% FBS, 2 mM glutamine, 100 units/ml penicillin, 100
µg/ml streptomycin, 50 µM ß-mercaptoethanol, 10
mM HEPES. Flat-bottomed 96-well plates were coated
overnight with the mitogenic antimouse CD3 Mab 2C11 (23)
alone or in combination with purified recombinant GST-ErbB2 fusion
protein (9)
. The plates were washed with PBS, and 1 x 105 splenic T cells were added to each well
with or without the addition of varying concentrations of soluble
CD86111-scFv(FRP5). After incubation at 37°C
for 60 h, the cells were pulsed with 0.25 µCi/well
[3H]thymidine (NEN DuPont, Zaventem, Belgium)
for 12 h. The cultures were then harvested, and the incorporation
of [3H]thymidine was measured with a liquid
scintillation counter (Beckman Instruments, Munich, Germany).
Human PBMCs were isolated by Ficoll gradient centrifugation and depleted of monocytic cells by adherence to a plastic surface, and 2 x 105 of the remaining cells in 200 µl of RPMI medium supplemented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin were added to each well of flat-bottomed 96-well plates precoated with 1.2 µg/well of a recombinant protein comprising the extracellular domain of human EGFR (kindly provided by D. W. Schneider, Berlex Biosciences, Richmond, CA). After incubation for 3, 6, or 8 days in the presence or absence of CD86111-scFv(14E1) fusion protein, the cells were pulsed with 0.5 µCi/well [3H]thymidine (Amersham Pharmacia Biotech) overnight. The cultures were then harvested, and the incorporation of [3H]thymidine was measured as described above.
RESULTS
Construction and Expression of Recombinant CD86111
Proteins.
We constructed yeast and bacterial expression vectors for the
production of a soluble, recombinant CD86 fragment comprising 111
N-terminal amino acids of human CD86 (referred to as
CD86111). This fragment includes the IgV-like but
not the IgC-like domain of the protein (24, 25, 26, 27)
. A cDNA
fragment encoding CD86111 was derived by PCR and
inserted into the yeast vector pPIC9 for expression of
CD86111 as a secreted protein. The resulting
plasmid pPIC9-CD86111 is shown schematically in
Fig. 2
A and encodes under the control of the
methanol-inducible AOX1 promoter the CD86 fragment fused to an
N-terminal -factor secretion signal from yeast, a COOH-terminal Myc
epitope recognized by Mab 9E10, and a polyhistidine cluster, which
facilitates protein purification (17)
.
P. pastoris GS115 cells were transformed with the expression
construct, recombinant clones were isolated on selection media, and the
phenotype of the clones was confirmed by PCR following established
protocols (17)
. Expression of
CD86111 was induced by the addition of methanol,
and recombinant protein was purified from culture supernatants via
Ni2+ affinity chromatography. In SDS-PAGE
analysis, the purified material migrates as a smear of bands ranging
from 
30 to 50 kDa (Fig. 2
B, Lane 3). Mab 9E10
immunoblot analysis confirmed that the smear of bands represented
recombinant CD86111 (Fig. 2
B,
Lane 2; Fig. 2
C, Lanes 1 and
3). Bands of higher apparent molecular mass represented
differentially glycosylated forms of yeast-expressed
CD86111 as indicated by the increased
electrophoretic mobility of the protein upon N-glycosidase F treatment
(Fig. 2
C, Lane 2).
For the generation of unglycosylated CD86111
protein, a bacterial expression system was used. The
CD86111 cDNA fragment and Myc and polyhistidine
sequences were assembled in a pFLAG-1-derived E. coli
expression vector, resulting in the plasmid
pSW5-CD86111 (shown schematically in Fig. 2
A). After induction of cytoplasmic expression in E.
coli cells harboring the plasmid, the cells were lysed under
denaturing conditions, and recombinant CD86111
protein was purified by Ni2+ affinity
chromatography and refolded by dialysis. In SDS-PAGE and immunoblot
analysis, the bacterially expressed, purified
CD86111 protein was detected as a single band of
the expected molecular mass (Fig. 2
C, Lane 4).
Functional Characterization of Recombinant CD86111
Proteins.
Full-length CD86 is expressed as a transmembrane protein and binds to
the B7 counter receptors CD28 and CTLA-4 on the surface of T cells
(24
, 25
, 28)
. To examine the functionality of truncated,
soluble CD86111 protein, the interaction of
recombinant protein with B7 counter receptors was investigated by FACS
analysis using CHO-CTLA-4 cells. These cells stably express the
extracellular domain of CTLA-4 attached to the cell membrane via a
glycosylphosphatidyl inositol anchor (17)
. CHO-CTLA-4
cells were incubated with CD86111 proteins
purified from yeast culture supernatants or bacterial lysates.
Yeast-expressed CD86225 protein, which represents
a soluble fragment of the complete extracellular domain of CD86
(17)
, was included as a control. Specifically bound
protein was detected with Mab 9E10 and FITC-labeled goat antimouse IgG.
Binding of CD86225 as well as truncated
CD86111 expressed in yeast and E. coli
to CHO-CTLA-4 cells but not to CHO control cells could be detected
(Fig. 2
D). These results demonstrated that the IgV-like
domain of CD86 is sufficient to facilitate binding to B7 counter
receptors similar to a CD86 protein containing IgV-like and IgC-like
domains. Thus, glycosylation of the protein is not required because the
bacterially expressed CD86111 protein bound to
CTLA-4 with comparable efficiency.
Construction and Bacterial Expression of Chimeric
CD86111-Antibody Fusion Proteins.
To localize the CD86 domain selectively to the surface of tumor cells,
chimeric fusion proteins were constructed that contained an N-terminal
CD86111 fragment fused to recombinant
single-chain antibody (scFv) fragments with specificity for EGFR and
ErbB2 (Fig. 1)
. cDNA fragments encoding the EGFR-specific scFv(14E1)
and the ErbB2-specific scFv(FRP5) antibody domains were derived from
previously described constructs (5
, 19)
and assembled with
CD86111, Myc, and polyhistidine sequences in the
bacterial expression vector pSW5. The resulting constructs
pSW5-CD86111-5 and
pSW5-CD86111-14E1 are shown schematically in Fig. 3
A. Cytoplasmic expression of
CD86111-scFv proteins was induced in E.
coli cells harboring the plasmids by the addition of IPTG; the
cells were then lysed under denaturing conditions, and recombinant
proteins were purified by Ni2+ affinity
chromatography followed by refolding. After a single round of
purification, the typical yield of proteins was 1 mg for
CD86111-scFv(FRP5) and 3 mg for
CD86111-scFv(14E1) per liter of original
bacterial culture with a purity of 70–90%, as determined by
SDS-PAGE and Coomassie brilliant blue staining (Fig. 3
B).
The identity of chimeric CD86111-scFv proteins
was confirmed by Mab 9E10 immunoblot analysis (Fig. 3
C).
Characterization of CD86111-scFv Binding Specificity.
In the chimeric CD86111-scFv(FRP5) and
CD86111-scFv(14E1) proteins, antibody domains were included as a means
for anchoring and presentation of the CD86 domain on the surface of
cells via binding to the respective target antigen. The functionality
of the scFv domains was examined by FACS analysis using murine
Renca-lacZ/ErbB2 and Renca-lacZ/EGFR cells stably expressing human
ErbB2 and EGFR, respectively (6
, 18)
. The cells were
incubated with purified CD86111-scFv(FRP5) and
CD86111-scFv(14E1) proteins. Cells treated with
CD86111 protein lacking a scFv domain served as a
control. Specifically bound proteins were detected with Mab 9E10 and
FITC-labeled goat antimouse IgG. The results are shown in Fig. 4
. Specific binding of
CD86111-scFv(FRP5) to ErbB2-expressing tumor
cells (Fig. 4
A) and CD86111-scFv(14E1)
binding to EGFR-expressing tumor cells (Fig. 4
B) was
observed. The CD86111 control protein did not
bind to either tumor cell line. Upon treatment of adherent
Renca-lacZ/EGFR cells with CD86111-scFv(14E1) and
subsequent immunofluorescence microscopy, strong membrane staining was
found (Fig. 4
C), whereas Renca-lacZ/ErbB2 control cells
incubated with the EGFR-specific
CD86111-scFv(14E1) and secondary antibodies
displayed only weak nuclear staining (Fig. 4
D), most likely
because of cross-reactivity of Mab 9E10 with endogenous murine c-Myc
(29)
. Binding of the CD86111-scFv
proteins to their target receptors was also confirmed by ELISA
(19)
. The apparent binding affinities of
CD86111-scFv(14E1) to EGFR and
CD86111-scFv(FRP5) to ErbB2, calculated as the
half-maximal saturation value, were 32 and 150
nM, respectively (data not shown).
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, binding of both
CD86111-scFv proteins to CTLA-4-expressing CHO
cells could be demonstrated. These results illustrate that both the B7
domain and the tumor cell recognition domain of the fusion proteins are
functional.
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To test the dependence of CD86111-scFv-mediated
costimulation on the binding to the scFv target antigen, we designed a
simplified in vitro assay. Mitogenic 2C11 anti-CD3 antibody
(23)
and bacterially expressed GST-ErbB2 fusion protein
representing the epitope recognized by scFv(FRP5) (9)
were
immobilized on the surface of tissue culture plates. Splenic T cells
from BALB/c mice were added and incubated for 60 h in the presence
of purified CD86111-scFv(FRP5) protein. Control
cells were grown in the absence of GST-ErbB2, anti-CD3, and/or
CD86111-scFv(FRP5). T-cell proliferation was then
measured by [3H]thymidine incorporation. The
results are shown in Fig. 6
A.
CD86111-scFv(FRP5) alone had no effect on T-cell
growth, whereas stimulation of the cells with anti-CD3 antibody alone
resulted in a basic level of T-cell proliferation, similar to previous
observations (17)
. In the presence of immobilized
anti-CD3 antibody, addition of CD86111-scFv(FRP5)
resulted in markedly enhanced T-cell proliferation. For this effect,
the presence of immobilized GST-ErbB2 was required, indicating that
CD86111-scFv(FRP5) clustered by binding to the
target antigen, but not monovalent
CD86111-scFv(FRP5) in solution, elicits
costimulatory activity and mimics the effect of natural transmembrane
B7 on antigen-presenting cells.
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B, the addition of
CD86111-scFv(14E1) resulted in markedly enhanced
cell proliferation if the cells were exposed to the fusion protein for
6 or more days. DISCUSSION
Efficient activation of T cells requires interaction of the T-cell receptor with peptides presented by MHC molecules and induction of a costimulatory signal by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the surface of the T cells (11 , 12) . Direct expression of B7 molecules on tumor cells can result in the activation of tumor-specific T lymphocytes and induce protective antitumor immunity. Strategies for the presentation of B7 proteins on cancer cells include transduction or infection of the cells with viral vectors (13, 14, 15) and direct injection of B7 plasmid constructs (31) . However, the broad application of such gene therapeutic approaches is limited by the inability of available vectors to selectively target B7 gene expression to the tumor.
The possibility to generate functional recombinant B7 proteins might provide a basis for novel therapeutic concepts. The transmembrane and intracellular domains of CD80 and CD86 are not required for their costimulatory activity. When expressed in mammalian cells, recombinant proteins comprising the extracellular domain of B7 molecules retain the ability to bind to B7 counter receptors on the T-cell surface (32 , 33) . We have previously demonstrated that functional soluble forms of human CD80 (CD80208) and CD86 (CD86225) can also be produced in recombinant form in the yeast P. pastoris. When coated to a plastic surface, such proteins transmit a costimulatory signal and in the presence of immobilized anti-CD3 antibody facilitate the proliferation of primary T cells (17) . Here we have characterized a smaller recombinant fragment of human CD86 that corresponds to amino acid residues 1–111 (CD86111) of the mature protein. The extracellular domains of CD80 and CD86 contain an IgV-like and an IgC-like domain (26) . Whereas for CD80 sequences, both domains appear to be critical for CD28 binding (34) , in the case of CD86, the IgV-like domain contained within CD86111 is sufficient for functional activity (this report and Ref. 27 ). Thus, for CD86225 and CD86111 from yeast or CD86111 produced in bacteria, no obvious differences in the binding to CTLA-4-expressing CHO cells were noted. Likewise, the proteins displayed similar costimulatory activity when immobilized on a plastic surface (Ref. 17 and data not shown). This indicates that previous difficulties in isolating functional recombinant CD86 from E. coli were not attributable to the absence of glycosylation in bacterially expressed protein but most likely were attributable to folding problems, which appear to be more severe for bacterially expressed CD86225 (17) than for the shorter CD86111 molecule analyzed here.
Combining the ability of antibodies to specifically localize to tumor-associated antigens on the surface of cancer cells with the potent costimulatory activity of recombinant B7 molecules might overcome present limitations of B7 gene therapeutic strategies and antibody-based therapies. Here we report the construction of fusion proteins that contain in a single polypeptide chain the costimulatory IgV-like domain of human CD86 and scFv antibody domains for specific tumor targeting. EGFR and the closely related ErbB2 receptor tyrosine kinases were chosen as suitable targets for such molecules on the tumor cell surface because of their enhanced expression on a wide variety of human tumors and their involvement in malignant transformation (2 , 3) . We demonstrated that the bacterially expressed CD86-scFv fusion proteins are bifunctional. They localize specifically to the respective target antigens on the surface of tumor cells and interact with natural B7 counter receptors. Importantly, as shown for the ErbB2-specific CD86-scFv(FRP5) protein, the molecule was able to markedly enhance the proliferation of primary T cells when bound to immobilized tumor antigen, but not in solution. Likewise, the EGFR-specific CD86-scFv(14E1) protein strongly enhanced the proliferation of human PBMCs grown in the presence of immobilized EGFR. This suggests that in an in vivo situation, T-cell activation would be restricted to the tumor site because the multiple contacts between B7 molecules and CD28 required for CD28 clustering and activation (30) could be provided by cell-bound, but not circulating, monovalent fusion protein.
Because of their smaller size, recombinant scFv antibody fragments penetrate tumors more easily than whole antibodies (35) . This will also be of importance for the in vivo application of reagents that link antibodies with costimulatory activity (9 , 36 , 37) . Here we showed that the CD86111 fragment comprising the IgV-like domain of human CD86 is sufficient to provide tumor-specific scFv antibody fragments with costimulatory effector functions, thereby restricting these chimeric molecules to the minimal functional domains required for activity. Such B7-scFv fusion proteins can be expressed in yeast or E. coli, a potential advantage for their production at higher yields and for further evaluation of their potential for specific cancer immunotherapy in suitable in vivo models.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be
addressed, at Chemotherapeutisches Forschungsinstitut,
Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am
Main, Germany. Phone: 49-69-63395-188; Fax: 49-69-63395-189; E-mail: wels{at}em.uni-frankfurt.de 
2 The abbreviations used are: EGFR, epidermal
growth factor receptor; Mab, monoclonal antibody; sc, single chain;
FBS, fetal bovine serum; AOX1, alcohol oxidase 1; IPTG, isopropyl
ß-D-thiogalactopyranoside; FACS, fluorescence-activated
cell sorting; GST, glutathione S-transferase; PBMC,
peripheral blood mononuclear cell.
Received 2/29/00; revised 8/11/00; accepted 8/14/00.
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  F. Rohrbach, R. Weth, M. Kursar, A. Sloots, H.-W. Mittrucker, and W. S. Wels Targeted Delivery of the ErbB2/HER2 Tumor Antigen to Professional APCs Results in Effective Antitumor Immunity J. Immunol., May 1, 2005; 174(9): 5481 - 5489. [Abstract] [Full Text] [PDF]
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 K. Sandstrom, Z. Xu, G. Forsberg, and P.-A. Nygren Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering Protein Eng. Des. Sel., September 1, 2003; 16(9): 691 - 697. [Abstract] [Full Text] [PDF]
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