Construction and Characterization of Bispecific Costimulatory Molecules Containing a Minimized CD86 (B7-2) Domain and Single-Chain Antibody Fragments for Tumor Targeting

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.

CHO and CHO-CTLA-4 cells expressing glycosylphosphatidyl inositol-anchored human CTLA-4 on the cell surface (17) were maintained in MEMα with deoxyribonucleosides(Life Technologies, Inc., Karlsruhe, Germany), containing 2 mm glutamine, 50 μm β-mercaptoethanol, 10%heat-inactivated FBS, and 1 mg/ml G418 (CHO-CTLA-4). Murine renal carcinoma (Renca) cells stably expressing Escherichia coliβ-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, 100μg/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.

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 CD86₁₁₁fragment 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.

The pPIC9-derived expression plasmid encodes, under the control of the methanol-inducible AOX1 promoter, the CD86₁₁₁fragment fused to an N-terminal α-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 × 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 mmimidazole. 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.

Plasmids pSW5-CD86₁₁₁,pSW5-CD86₁₁₁-5 and pSW5-CD86₁₁₁-14E1 were transformed into E. coli BL21 (λDE3)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 mmIPTG for 1 h at room temperature. Cells were harvested by centrifugation at 5000 × 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 × 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.

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 ×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 mmTris-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 50μg/ml propidium iodide (Roth, Karlsruhe, Germany). Coverslips were mounted on glass slides and analyzed under a fluorescence microscope(Zeiss Axiophot; Carl Zeiss, Jena, Germany).

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, 100μg/ml streptomycin, 50 μm β-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 × 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 × 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.

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 α-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 CD86₁₁₁protein, a bacterial expression system was used. The CD86₁₁₁ cDNA fragment and Myc and polyhistidine sequences were assembled in a pFLAG-1-derived E. coliexpression 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 CD86₁₁₁protein 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).

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. colito 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.

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).

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).

To confirm that the chimeric CD86₁₁₁-scFv proteins are bispecific and in addition to tumor cell recognition facilitate interaction with B7 counter receptors, the binding of CD86₁₁₁-scFv(FRP5) and CD86₁₁₁-scFv(14E1) to CTLA-4 was investigated by FACS analysis. CHO-CTLA-4 cells were incubated with purified CD86₁₁₁-scFv(FRP5) and CD86₁₁₁-scFv(14E1) proteins. Specifically bound protein was detected with Mab 9E10 and FITC-labeled goat antimouse IgG. As shown in Fig. 5, 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.

We have previously shown that recombinant human CD86 comprising the complete extracellular domain of the protein(CD86₂₂₅) provides a costimulatory signal to murine T cells when immobilized on a plastic surface but not in solution (9, 17). Similarly, truncated yeast or bacterially expressed CD86₁₁₁ proteins characterized in this study enhanced the proliferation of murine T cells when coated on a plastic surface together with an anti-CD3 antibody (data not shown). In the case of chimeric CD86₁₁₁-scFv proteins, binding to their target antigens is intended to immobilize the B7 domain in high density on the surface of tumor cells, thereby facilitating multiple contacts with neighboring T cells and enabling the CD28 clustering required for efficient costimulation (30).

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.

The costimulatory activity of the EGFR-specific CD86₁₁₁-scFv(14E1) protein was tested in a similar assay using human PBMCs. The cells were grown for 3, 6, or 8 days in the presence of purified CD86₁₁₁-scFv(14E1) on tissue culture plates precoated with a recombinant protein comprising the extracellular domain of human EGFR (19). Control cells were grown in the absence of the CD86 fusion protein. T-cell proliferation was then measured by [³H]thymidine incorporation. As shown in Fig. 6 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.

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 CD86₁₁₁fragment 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.