Recombinant Human Single Chain Fv Antibodies Recognizing Human Interleukin-6
SPECIFIC TARGETING OF CYTOKINE-SECRETING CELLS*

Barbara Krebs, Heather GriffinDagger , Greg WinterDagger , and Stefan Rose-John§

From the Department of Medicine, Section of Pathophysiology, Johannes Gutenberg-University of Mainz, Obere Zahlbacher Straße 63, D-55101 Mainz, Germany and the Dagger  Medical Research Council Centre for Protein Engineering, Cambridge University, Hills Road, Cambridge CB2 2QH, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A human antibody library was displayed on the surface of filamentous bacteriophage and screened for binding to human interleukin-6 (IL-6). Two antibody-bearing phages were selected that bound IL-6. The complementary-determining region 3 loops of the variable heavy chains of these two antibodies differed in length and sequence and recognized two distinct epitopes. One of the single chain Fv fragments isolated (H1) was found to bind human (but not murine) IL-6 with an affinity comparable to that of the human IL-6 receptor. H1 also recognized newly synthesized human IL-6 intracellularly, as shown by indirect immunofluorescence. H1 did not neutralize human IL-6, and the H1 epitope was mapped to a region of IL-6 not involved in interactions with IL-6, IL-6 receptor, or the signal-transducing protein gp130. To target IL-6-secreting cells, we then constructed a bispecific antibody fragment (a diabody) comprising H1 and the antigen binding site of the T-cell activating monoclonal antibody OKT3. The diabody led to T-cell-mediated killing of cells secreting IL-6.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interleukin-6 (IL-6),1 a member of the family of four-helical cytokines (1) is a mediator of hematopoietic cell growth and differentiation acting on B-cells, T-cells, keratinocytes, neuronal cells, osteoclasts, and endothelial cells (2, 3). In the liver, IL-6 modulates the transcription of acute phase response genes during acute and chronic inflammatory states (4). An increased expression of IL-6 has been reported for several diseases, such as plasmacytoma/myeloma, Castleman's disease, mesanglial proliferative glomerulonephritis, osteoporosis, autoimmune diseases, and AIDS (3, 5).

Several strategies have been exploited to neutralize the activity of IL-6. The administration of IL-6-neutralizing monoclonal antibodies to patients with rheumatoid arthritis and multiple myeloma has greatly improved the conditions of the patients for several weeks (6, 7). But the symptoms have returned, because the high stability of antibodies in plasma increased the level of IL-6 that is normally cleared from the circulation within several minutes (8). The identification of the interaction sites between human IL-6, the IL-6 receptor (IL-6R), and the signal-transducing protein gp130 has also made it possible to construct IL-6 receptor antagonists, which have been shown to inhibit IL-6 responses in vitro (9-16).

Here we report on the isolation of the first human recombinant antibodies recognizing a member of the large four-helical cytokine family that comprises most interleukins and colony-stimulating factors. We decided to explore a strategy of using these antibodies to target and destroy cells secreting IL-6. The approach combined the use of phage display technology (17, 18) to make antibody fragments directed against IL-6, and the incorporation of anti-IL-6 antibodies into diabodies (19) that mediate T-cell killing. Thus, human anti-IL-6 antibody fragments were isolated by selection of large repertoires of antibody fragments displayed on filamentous bacteriophage by binding to IL-6. A fragment directed against portions of IL-6 not involved in binding to the IL-6 receptor was then combined with a fragment directed against the T-cell receptor and evaluated for killing by T-cells of cells that secrete IL-6.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial Strains and Plasmids-- The pUC19-derived vector pHEN1 harbors the "single pot library" (20) that encodes the scFv antibodies with NH2-terminal leader sequence, variable domains of heavy and light chain (VH and VL) with a 15-amino acid flexible linker in between and an COOH-terminal peptide tag (myc tag). An amber stop codon between the myc tag and phage minor coat protein encoding gene III makes it possible to produce phage-displayed scFv antibodies in helper phage-infected Escherichia coli TG1 cells, which suppress UAG and expression of soluble scFv in nonsuppressor strain E. coli HB2151. A bispecific scFv recognizing human IL-6 and human CD3 was cloned from the respective scFv parts of anti-IL-6 scFv H1 and anti-CD3 mAb OKT3 as a diabody (19) with the VH H1/VL OKT3 and the VH OKT3/VL H1 fused by a 5-amino acid linker. For high yield expression of the soluble anti-IL-6 antibody H1 and the anti-IL-6/anti-CD3 diabody, the respective scFv parts were subcloned in pUC119SfiI/NotImycHis (pUC119SfiI/NotImyc (21) with hexahistidine tag), a pUC119-derived vector that lacks the gene III and introduces a COOH-terminal hexahistidine tag.

Selection of Phage Library against Human IL-6-- Recombinant phage with scFv expressed as fusion proteins on their tip were rescued from the culture supernatant of VCS-M13 helper phage (Stratagene, Heidelberg, Germany)-infected E. coli TG1 as described previously (20). 1012-1013 phages were panned for three subsequent rounds of binding on immunotubes (Maxisorb, Nunc, Roskilde, Denmark) coated with recombinant human IL-6 at 90 µg/ml. Blocking, panning, washing, elution, and reamplification of eluted phage were carried out as described previously (20). Log phase E. coli TG1 were transduced by eluted phage and plated on ampicillin-containing agar (22). Ampicillin-resistant colonies were scraped and grown to log phase for rescue with helper phage and the next round of panning, or single colonies were picked for screening of selected clones.

Screening and Sequencing of Clones-- As described (17, 23, 24), recombinant phages were rescued after each round from single ampicillin-resistant colonies of infected E. coli TG-1 (UAG suppressor strain) and screened for binding to antigen on ELISA. For each ELISA on IL-6, a parallel plate (Micro Test III, Falcon, Oxnard, CA) was coated with PBS containing 2% skimmed milk powder (ICN, Meckenheim, Germany) as control for unspecifity. After blocking, plates were incubated with bacterial supernatants containing selected phages. Bound phages were detected with sheep antibody to M13 phage 1:1000 (5-Prime-3-Prime, Boulder, CO), following anti-sheep IgG POD conjugate 1:2000 (Sigma). Positive clones were expressed as soluble scFv in isopropyl-beta -D-thiogalactopyranoside-induced E. coli HB2151 (nonsuppressor strain), and bacterial supernatants were again checked for antigen binding in ELISA as described above, utilizing mouse monoclonal anti-myc antibody 1:10 from hybridoma supernatants (mAb 9E10, European Collection of Animal Cell Cultures no. 85102202) and anti-mouse IgG POD conjugate 1:2000 (Sigma). Peroxidase activity was detected with soluble BM blue POD substrate (Boehringer Mannheim). Selected clones were subjected to polymerase chain reaction with primers LMB3 (5-CAGGAAACAGCTATGAC-3) and fdSEQ1 (5-GAATTTTCTGTATGAGG-3). Polymerase chain reaction products were digested with BstNI according to standard protocols (22), and fragments were separated on 4% Nusive agarose gels (FMC, Rockland, ME) yielding typical fingerprint patterns. Polymerase chain reaction products were sequenced with LMB3 and fdSEQ1 using DyeDeoxy chain termination (Applied Biosystems, Weiterstadt, Germany) and 373A DNA sequencer.

Western Blotting-- For detection of human IL-6 or human-mouse IL-6 chimeras (25), purified antigen (hIL-6) or crude bacterial lysates of induced clones (truncated IL-6 or chimeras) were separated on 12,5% SDS-polyacrylamide gels and electroblotted onto nylon filters (GeneScreen plus, NEN Life Science Products). Filters were blocked and incubated with 1011-1012 polyethylene glycol-precipitated phages/ml or bacterial supernatants containing soluble scFv diluted 1:10. After washing, detection of bound phages or soluble scFv was as described above. Peroxidase activity was detected using precipitating BM blue POD substrate (Boehringer Mannheim).

IL-6 Bioassay-- For IL-6 proliferation assay, the IL-6-dependent murine cell line B9 was seeded onto 96-well plates (Falcon, Micro Test III, Becton Dickinson Labware, Oxnard, CA) at 5000 cells/well as described (26) and was incubated with either 0.1 or 0.5 pM IL-6 and increasing amounts of H1 or the neutralizing mAb 8 (27), ranging from 1 nM to 1 µM. For the IL-6 standard curve, B9 cells were stimulated with serially diluted IL-6. The whole assay was performed in triplicate. After 68 h, proliferation was measured with 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as described previously (10).

Expression and Purification of Soluble scFv H1 or Diabody-- Culture supernatants containing hexahistidine-tagged scFv H1 or diabody were purified via Ni-NTA agarose (Qiagen, Hilden, Germany). Column material was equilibrated with 50 mM phosphate buffer, pH 7.5, 500 mM NaCl, 20 mM imidazole after loading with bacterial supernatant washed with 50 mM phosphate buffer, pH 7.5, 500 mM NaCl, 20 mM imidazole and 50 mM phosphate buffer, pH 7.5, 500 mM NaCl, 35 mM imidazole and eluted with 50 mM phosphate buffer, pH 7.5, 500 mM NaCl, 100 mM imidazole. Eluted proteins were pooled and dialyzed against PBS at 4 °C.

H1-D2 Sandwich and IL-6R ELISA-- For both types of ELISA, purified soluble scFv H1 was coated on ELISA plate wells at 10 µg/ml. After blocking of unspecific binding, IL-6 was applied at 10 µg/ml. For the sandwich ELISA, phage-displayed scFv D2 was then added at 108 plaque-forming units/ml and tested for binding to IL-6. After washing, bound phages were detected as described above. For negative controls, either wells were coated with PBS containing 5% skim milk powder and 0.02% Tween 20 instead of H1, no IL-6 was bound to H1, or VCS-M13 helper phage was applied instead of recombinant D2 phage. For the IL-6R ELISA, sIL-6R expressed in Pichia pastoris and purified as described previously (28) was added at 20 µg/ml and was tested for binding to IL-6, which is immobilized via binding to H1. Bound sIL-6R was detected with polyclonal rabbit anti-IL-6R antibody (29) followed by anti-rabbit IgG POD conjugate (Sigma). For negative control, the equivalent experiment was performed without IL-6.

Immunoprecipitation-- Human recombinant IL-6 was expressed, purified, and radiolabeled as described earlier (10). IL-6 was serially diluted in PBS, 0.2% bovine serum albumin, 0.02% Tween 20 and was added to 1.5 ng 125I-labeled IL-6 (10,000 cpm/ng) and 1 µl of bacterial culture supernatant containing soluble scFv H1 in a final volume of 200 µl. IL-6·H1 complexes were immunoprecipitated with 40 µl of mAb 9E10 (from hybridoma supernatants), 0.5 µl of rabbit anti-mouse IgG (DAKO, Hamburg, Germany), and 2 mg of protein A-Sepharose (Pharmacia Biotech Inc.) in a final volume of 400 µl. After washing, precipitated radioactivity was determined by gamma  counting.

Kinetic Assay-- IL-6 was covalently immobilized to a carboxymethyl dextran matrix (FISONS, Cambridge, UK) at 2.25 µg/ml for 2 min in 10 mM sodium acetate buffer, pH 5.0, as recommended by the manufacturer. Binding experiments were performed in triplicate with at least seven different concentrations of purified scFv H1 using the IAsysTM (FISONS) optical biosensor. Association was monitored for 1-2 min, the sample was replaced by PBS containing 0.05% Tween 20, and dissociation of the scFv was monitored for 1-2 min before the cuvette was regenerated with 5 mM HCl and equilibrated again in PBS containing 0.05% Tween 20. Association and dissociation sensorgrams were analyzed by nonlinear regression using the FASTfit (FISONS) software, which uses the Marquardt-Levenburg algorithm for iterative data fitting.

Immunofluorescence-- Murine melanoma cells (B78) and B78 cells stably transfected with H-IL-6 (30) were incubated overnight on tissue culture-treated glass chamber slides (Falcon) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal calf serum (Seromed) and 1% penicillin-streptomycin (Life Technologies, Inc.) solution in a 5% CO2 humidified atmosphere at 37 °C. Cells were fixed in methanol/acetone and air-dried. Nonspecific binding was blocked, and scFv H1 was applied at a concentration of 20 µg/ml. After washing, anti-myc mAb 9E10 hybridoma supernatant diluted 1:1 with PBS was added. After 1 h, fixed cells were washed and incubated with rabbit anti-mouse immunoglobulins-TRITC diluted 1:50 in the dark. After washing, coverslips were mounted with aqua-poly mount (Polysciences Incorporation, Warrington, PA). A magnification of × 400 was used to photograph the cells.

Diabody-directed Cell Killing-- Primary alloreactive cytotoxic T-cells (effector cells or cytotoxic T lymphocytes (CTLs)) were generated from human peripheral blood mononuclear leukocytes of healthy donors cocultured with irradiated (4000 rad) allogene stimulator peripheral blood mononuclear leukocytes in the presence of IL-2 (20 units/ml, Boehringer Mannheim). Target cells (a human hepatoma cell line stably transfected with human IL-6 (31)) were loaded with 51Cr for 1 h (100 µCi of Na251CrO4, Amersham Corp.), washed, and incubated with CTLs at a effector:target ratio of 50:1 in 96-well round-bottomed plates (Falcon). The 51Cr release from the target cells was measured in triplicate in a standard 4-h assay (32) in the presence or absence of the bispecific diabody or the monospecific control antibodies (H1 and OKT3). The percentage of specific lysis was calculated as (E - S)/(M - S), where E is the experimental 51Cr release, S is the spontaneous release from target cells in absence of T-cells, and M is the maximum release from target cells in the presence of detergent (0.25% Nonidet P-40, Sigma).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Screening for Human Recombinant Antibodies Binding to Human IL-6-- The library containing >108 recombinant phages was screened for adsorption to human recombinant IL-6 immobilized to immunotubes (Fig. 1A). After the first round of screening, 4.9 × 103 phages were selected. Among 96 analyzed clones, no phage recognizing human IL-6 was identified. As shown in Fig. 1B, after the second round of screening, 55% of the phages analyzed recognized human IL-6 when absorbed to the ELISA wells. After the third round, 96% of the phages reacted positively in the IL-6 ELISA. We subsequently prepared soluble scFv proteins and reprobed the antibodies in an IL-6 ELISA. It turned out that at this stage only 23% of the soluble scFv antibodies (recruited from clones selected in the second round) recognized human IL-6, indicating that some of the scFv antibodies only recognized human IL-6 when they were exposed on the surface of the filamentous phage (see below). When the DNAs coding for 44 scFv antibodies recognizing IL-6 as soluble scFv were analyzed by fingerprinting (see "Materials and Methods"), they all showed an identical pattern (data not shown). Upon sequencing of 15 scFv DNAs, it was confirmed that all clones were identical and that all belonged to the VH 4 family (germline DP-69). It was confirmed that the germline sequence of these scFv antibodies contained no point mutation (data not shown). The CDR3 loop of all these scFv antibodies consisted of 6 amino acids and had the sequence GLRMDG. We chose clone H1 for further analysis (see Fig. 1B).


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Fig. 1.   Screening for human recombinant antibodies directed against human IL-6. A, scheme illustrating the panning procedure. One round of screening is shown. B, scheme illustrating the detection of phages recognizing human IL-6 immobilized to a 96-well ELISA dish. Well H-12 shows the negative control. Bound phages were detected by sheep anti-phage antibody following POD-conjugated anti-sheep antibody. POD activity was determined with POD substrate BM blue (Boehringer Mannheim). Absorbance was measured at 450 nm, and an extinction of 2.0 at 450 nm was defined as 100% and of 0.0 as 0% black; extinction higher than 0.5 was considered positive when the control equivalent measured on a plastic dish coated with PBS containing 2% skim milk powder, 0.02% Tween 20 (data not shown) was at least 3-fold lower.

Detection of Human IL-6 by ELISA and Western Blot by Antibody H1-- To facilitate expression and purification of the protein, the DNA coding for the scFv antibody H1 was subcloned into the plasmid pUC119SfiI/NotImycHis, which added a sequence of 6 His to the COOH terminus of the protein. The protein was expressed in E. coli, and the bacterial supernatant was analyzed. As shown in Fig. 2, the bacterial supernatant (lane 1) contained a dominant protein with an apparent molecular mass of 33 kDa that was retained on a nickel-chelate column and could be eluted in essentially homogeneous form with imidazole (Fig. 2A). The eluate (Fig. 2, lane 3) but not the flow through (lane 2) could be detected with mAb 9E10, which recognized the myc tagging sequence that is contained NH2-terminal of the His sequence at the COOH terminus of the recombinant antibody (Fig. 2B). The phage carrying the scFv antibody H1 and the purified antibody were used to detect human IL-6 on a Western blot. As shown in Fig. 3B, the scFv antibody H1 recognized human IL-6 in both the phage-associated (lane 1) and soluble (lane 2) forms.


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Fig. 2.   Expression and purification of scFv antibody H1 in bacteria. A, SDS-polyacrylamide gel electrophoresis of scFv antibody H1 expressed in bacteria. B, Western blot with anti-myc mAb 9E10. A and B: lane 1, bacterial supernatant as loaded on Ni-NTA column; lane 2, flow-through; lane 3, eluate. The molecular mass of marker proteins is indicated at the left in kDa.


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Fig. 3.   Detection of human IL-6 by the scFv antibody H1. A, SDS-polyacrylamide gel electrophoresis of 1.5 µg of human recombinant IL-6 (lane 1). The molecular mass of prestained marker proteins (lane M) is indicated on the left in kDa. B, Western blot detection of human IL-6 by phage-exposed scFv antibody (lane 1) and by soluble scFv antibody H1 (lane 2). Note that the prestained marker proteins are not all visible in SDS-polyacrylamide gel electrophoresis and Western blotting due to different amounts of the single proteins (lane M).

Mapping of the Binding Epitope of Antibody H1 on Human IL-6-- We further asked whether recognition of human IL-6 was specific and which region of IL-6 was recognized by the scFv antibody. To answer these questions, we used chimeric human/mouse IL-6 proteins in which different parts of human IL-6 were exchanged for the corresponding murine regions (25). IL-6 is a four-helical protein of 184 amino acids. We used chimeras in which one of the four helices, together with its NH2-terminal loop region, between human IL-6 and murine IL-6 was exchanged (regions 1-4). The regions are shown schematically in Fig. 4, in which the white bars represent human IL-6, and the black bars represent murine IL-6.


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Fig. 4.   Mapping of the IL-6 epitope recognized by the scFv antibody H1. 300 ng of purified human IL-6 (lane 1) and crude lysates of bacterial clones expressing recombinant IL-6 or variants thereof (lane 2, NH2-terminally truncated human IL-6; lanes 3-10, human-mouse IL-6 chimeras) were separated by SDS-polyacrylamide gel electrophoresis, electroblotted, incubated with soluble scFv H1, and detected with anti-myc mAb 9E10. In the chimeric IL-6 molecules, helices with their NH2-terminal loop regions are exchanged between murine and human IL-6 proteins (regions 1-4 (lanes 3-10)) (25). The border between regions 1 and 2 is at amino acid 39; the border between regions 2 and 3 is at amino acid 96; and the border between regions 3 and 4 is at amino acid 134. Human sequences are symbolized by white bars and murine sequences by black bars. The molecular mass of prestained marker proteins (lane M) is indicated at the left in kDa. Note that the higher molecular mass protein band in lane 2, which is recognized by the scFv antibody H1, might correspond to dimeric IL-6, which for the truncated IL-6 has been observed to be stable under denaturing conditions (33).

In addition, we used a truncation mutant of human IL-6 that lacked the NH2-terminal 27 amino acid residues (33). As shown in Fig. 4, the scFv antibody H1 recognized both full-length and NH2-terminally truncated human IL-6 (lanes 1 and 2). None of the mutants in which one murine alpha -helical region together with the NH2-terminal loop region (regions 1-4, corresponding to Fig. 4, lanes 3-6, respectively) was exchanged for the cognate human part was recognized (lanes 3-6). This was somewhat surprising because, taken together, the exchanged human regions in these chimeric proteins covered the total amino acid sequence of human IL-6 (Fig. 4). We reasoned that the recognition epitope for the scFv antibody H1 might be located at the border between two regions, so we used chimeric IL-6 proteins in which the borders between regions 1 and 2, 2 and 3, or 3 and 4 consisted of human amino acid sequences (Fig. 4). As shown in Fig. 4, lanes 7 and 8, chimeric proteins in which the border between regions 2 and 3 is entirely human were recognized by the scFv antibody H1. Chimeric proteins with the border between regions 1 and 2 or regions 3 and 4 consisted of human sequences were not detected by the recombinant antibody (Fig. 4, lanes 9 and 10). From these results, we concluded that the scFv antibody H1 recognized the central portion of the human IL-6 protein. Assuming that a linear epitope is recognized by the scFv antibody H1, we predict that the recognized region is centered around Glu-96 at the border between regions 2 and 3 of human IL-6 (Fig. 4).

Properties of the scFv Antibody H1: Binding and Neutralization-- We next asked whether the scFv antibody H1 also recognized human IL-6 in solution. The scFv antibody was incubated with 125I-labeled IL-6 in the presence of increasing concentrations of nonradioactively labeled IL-6. The complex of scFv antibody with IL-6 was immunoprecipitated with mAb 9E10, which recognized the COOH-terminal myc-tagging sequence of the scFv protein. As shown in Fig. 5A, the H1 antibody bound to 125I-labeled IL-6, and binding could be prevented by an excess of unlabeled human IL-6. These experiments suggest that the scFv antibody H1 recognized human IL-6 in solution, as well as immobilized to a solid support. Using a sandwich ELISA, we analyzed whether binding of H1 to IL-6 inhibits binding of IL-6R. As shown in Fig. 5B, sIL-6R can still be bound by IL-6, which is captured on a ELISA dish by binding to H1. The IL-6-dependent cell line B9 has been widely used to measure the biological activity of human IL-6 (26). As shown in Fig. 5C, addition of up to micromolar concentrations of the scFv antibody H1 to B9 cells stimulated with 0.1 or 0.5 pM human IL-6 did not lead to neutralization of the IL-6 response, whereas even nanomolar concentrations of the neutralizing mAb 8 led to complete inhibition of proliferation. We conclude from these experiments that scFv antibody H1 does not prevent the binding of IL-6 to the cell surface IL-6 receptor complex and does not neutralize human IL-6.


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Fig. 5.   Binding of scFv antibody H1 to IL-6. A, 125I-labeled IL-6 and soluble scFv antibody H1 were incubated in the presence of increasing amounts of nonradioactively labeled human recombinant IL-6. The complexes were precipitated with mAb 9E10 directed against the myc tag of H1, and radioactivity in the pellet was determined by gamma  counting. One representative of three experiments is shown. B, IL-6 was captured by scFv H1 immobilized on an ELISA plate and incubated with sIL-6R. Bound sIL-6R was detected with anti-IL-6R antibody (left column). The average values of triplicate experiments are shown. As a negative control, in the right column, the equivalent experiments were performed without adding IL-6. C, B9 cells were incubated with increasing amounts of human IL-6 (left panel). B9 cells were stimulated with 0.1 pM (middle panel) or 0.5 pM (right panel) of human IL-6 in the presence of increasing amounts of scFv antibody H1 or mAb 8 as indicated in the figure. After 3 days, viable cell numbers were determined using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay (10, 26).

Kinetics of Binding of scFv H1 to Human IL-6-- When human IL-6 was bound to the solid phase of an IAsysTM cuvette (see "Materials and Methods"), real time interaction between immobilized IL-6 and scFv H1 antibody could be analyzed. Fig. 6A shows association and dissociation curves recorded for seven concentrations of scFv H1 protein. The data were used to calculate an association rate, kass = 3.4 × 105 M-1s-1, and a dissociation rate, kdiss = 3.46 × 10-2 (s-1), between IL-6 and scFv H1. From these values, an affinity of KD = 102 nM can be derived (Fig. 6). A similar affinity has been reported for the interaction between immobilized IL-6 or IL-6R and their cognate binding partner (Table I). The measured affinities between immobilized IL-6 and its receptors (34) or between immobilized receptor and IL-6 (35) are about a factor of 50-100 below the values determined in solution (Table I), where affinities of little less than 1 nM have been determined (36). We conclude that in solution, the scFv antibody H1 binds IL-6 with an affinity comparable with the soluble human IL-6R.


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Fig. 6.   Kinetics of binding of scFv H1 to IL-6 bound to a solid phase. A, association of scFv H1 to immobilized IL-6 led to the increase of the resonance angle alpha  as a function of time. The association phase was recorded for different nanomolar scFv concentrations, as indicated. The decrease of the resonance angle indicates the dissociation phase resulting from replacement of the scFv solution from the solid phase with triplicate washing with PBS containing 0.05% Tween 20. B, calculation of the data from A allows the determination of the dissociation rate constant kdiss from the ordinate intercept and the association rate constant kass from the slope of the graph.

                              
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Table I
Comparison of binding affinities between IL-6, soluble IL-6R, and scFv antibody

Isolation of a Second scFv Antibody That Recognizes an IL-6 Epitope Distinct from the H1 Recognition Site-- As mentioned above, some scFv antibodies recognized human IL-6 only when exposed at the surface of the filamentous phage but not in soluble form. A similar phenomenon has been observed before (20) and has been explained by lower avidity of the respective scFv antibodies. We decided to analyze one of these scFv antibodies (antibody D2; compare Fig. 1B) in more detail. It turned out that this antibody belonged to the VH 3 family (germline DP-32) of V genes. The germline sequence of D2 contained no mutations. The CDR3 loop of this antibody consisted of 10 amino acids, with the sequence TRRAGAHSPI. Using a sandwich ELISA, we investigated whether the epitope recognized by the scFv antibody D2 was distinct from that recognized by the scFv antibody H1. As shown in Fig. 7, the scFv antibody D2 recognized human IL-6 bound to scFv antibody H1 that had been immobilized to the ELISA dish. We conclude that the human IL-6 epitopes recognized by the scFv antibodies H1 and D2 are distinct and nonoverlapping. This is consistent with the facts that the scFv antibody D2 belonged to a separate V gene family and that the CDR3 was completely different in length and sequence.


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Fig. 7.   The two scFv antibodies H1 and D2 recognize distinct epitopes of human IL-6. IL-6 was bound by soluble scFv H1 coated on a plastic dish and recognized by phage-exposed scFv D2 as detected with sheep antibody to M13 phage, following anti-sheep IgG POD conjugate. The average values of triple experiments are shown in column A, and equivalent negative controls are shown in columns B-D.

Binding of scFv Antibody H1 to Newly Synthesized Human IL-6-- A potential application of scFv antibodies recognizing human cytokines is the exploitation of specific intracellular or extracellular recognition between scFv and antigen. To test whether scFv antibody H1 recognized newly synthesized human IL-6, we used murine melanoma cells (B78) transfected with the human IL-6-sIL-6R fusion construct H-IL-6 (30). As shown in Fig. 8, the scFv antibody H1 recognized intracellular human IL-6, whereas no staining was seen in untransfected control cells. From these experiments and from the fact that scFv antibody H1 recognizes human natural IL-6 on a Western blot (data not shown), we conclude that posttranslational processing such as glycosylation does not interfere with recognition of human IL-6 by the scFv antibody H1.


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Fig. 8.   Indirect immunofluorescence detection of IL-6. A, B78 cells transfected with the fusion protein of IL-6 and sIL-6R were permeabilized and stained with scFv H1 followed by anti-myc mAb 9E10 and anti-mouse Ig-TRITC (magnification, × 400). B, untransfected B78 cells. Right panels (A' and B') represent the corresponding phase-contrast micrographs.

Killing of IL-6-secreting Cells-- A bispecific anti-human IL-6/anti-human CD3 diabody was tested for its ability to "cross-link" cells secreting human IL-6 with human CTLs and thereby mediating killing of these cells. Human hepatoma cells stably transfected with human IL-6 (HepG2-IL-6) were subjected to a standard cytotoxicity assay (32) in the absence or presence of the diabody. At a concentration of 50 ng/ml, the bispecific diabody specifically activated human CTLs to kill the HepG2IL-6 cells as shown by 51Cr release assay (Fig. 9, column 4). Cell lysis was significantly higher than spontaneous lysis in absence of diabody (Fig. 9, column 1). Cell lysis increased with increasing diabody concentration, whereas the parental HepG2 cells, which do not secrete IL-6, or other human hepatoma cells (Hep3B), which secrete leukemia inhibitory factor but no IL-6, showed no dose-dependent lysis (data not shown). The monospecifc parental antibodies of the diabody (anti-CD3 mAb OKT3 and anti-IL-6 scFv H1) did not induce killing (Fig. 9, columns 2 and 3). The monospecific antibodies OKT3 and H1 competed with the effect of the diabody and thereby reduced killing to control levels (Fig. 9, columns 5 and 6).


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Fig. 9.   Diabody-directed cell killing. Redirected T-cell cytotoxicity as measured by 51Cr release with 50 ng/ml (column 4) or without (column 1) anti-IL-6/anti-CD3 bispecifc diabody at a ratio of 50/1 between T-cells (effector) and 51Cr-loaded hepatoma cells (target), which synthesize and secrete IL-6. Monospecific antibodies OKT3 (column 2) and H1 (column 3) are presented as controls (50 ng/ml). The effect of the diabody could be competed by an 500-fold excess of the monospecifc antibodies (column 5, anti-CD3; column 6, anti-IL-6).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recombinant Human Anti-human Antibodies-- In the present study, we describe the isolation and characterization of two recombinant human scFv antibodies directed against the human four-helical cytokine IL-6 from a semisynthetic phage antibody library (20). The two antibodies that we isolated belong to different VH families and have CDR3 loops that are unrelated in length and sequence. Accordingly, the epitopes of the human IL-6 protein recognized by each of the two antibodies were distinct.

One of the antibodies (H1) could be used as soluble scFv antibody for detection of the antigen by ELISA, Western blotting, epitope mapping, immunoprecipitation, and immunofluorescence. The other antibody (D2) only detected the antigen IL-6 when exposed on the surface of the filamentous phage. This might reflect the greater binding avidity created by the display of multiple copies of the scFv antibody and the moderate binding affinity of the monomeric soluble scFv fragment.

No Neutralizing Properties of the scFv Antibody H1-- Three regions have been mapped on human IL-6 that are responsible for interaction with the IL-6R (site 1) and with two molecules of the signal-transducing protein gp130 (sites 2 and 3). Site 1 is formed by the COOH terminus and the region around Phe-78 of human IL-6 (10, 25, 37). Site 2 comprises amino acids around Lys-54 and around Asp-160 (12, 38), whereas site 3 is formed by the side chains of Tyr-31, Gly-35, Val-121, and Phe-125 (13, 14). We have mapped the epitope recognized by the scFv antibody H1 to a region centered around Glu-96, which is far from the defined interaction sites of IL-6 and the two receptor subunits. Therefore, it is not surprising that the scFv antibody H1 does not exhibit neutralizing properties.

Improving the Properties of Recombinant Antibodies-- It has been observed before that the isolation of scFv antibodies from relatively small phage repertoires of 1 × 107 to 2 × 108 clones yielded antibodies with moderate binding affinities (20, 23, 24, 39). Several strategies have been suggested to affinity maturate such low affinity antibodies (40). The library that we used for the isolation of the IL-6 antibodies consisted of 50 different VH chains with a CDR3 loop randomized in sequence and length combined with a single species of VL chain (20). The IL-6-recognizing scFv antibody H1 had an apparent affinity of 100 nM (Table I), which is much as expected given the library size of about 108 independent clones (41). Randomizing of the CDR3 loop of the VL chain in sequence and/or length might be appropriate to further increase the binding affinity of the scFv antibody. It can, however, be anticipated that the construction of more complex antibody libraries will facilitate the isolation of antibodies with higher affinities that will not have to be affinity maturated (41, 42).

Potential Therapeutic Use of Human Recombinant scFv Antibodies Directed against Human Cytokines-- IL-6 has been implicated to be connected to various diseases, including multiple myeloma, rheumatoid arthritis, Castleman's disease, and proliferative mesanglioglomerulonephritis (3, 5). For the therapy of these diseases, it might be desirable to neutralize in vivo the activity of human IL-6 or to specifically target cells that synthesize IL-6. Several approaches have been exploited, including the administration of humanized mAbs directed against IL-6. Although it was clear that neutralization of IL-6 activity was beneficial for the patients, it soon turned out that high mAb levels resulted in the build-up of high cytokine levels in the plasma of treated patients (6, 7). The in vivo usefulness of recombinant cytokine receptor antagonists has not yet been demonstrated, although the results obtained in vitro are promising. It could be shown that the IL-6-dependent proliferation of human myeloma cells could be completely abrogated by the administration of nanomolar amounts of recombinant IL-6 receptor antagonists (9-16).

With the availability of recombinant human anti-cytokine antibodies, completely different strategies have become possible. Possible targets are cells that secrete IL-6. We have shown here that in the presence of human T-cells, cells that secrete human IL-6 can be specifically killed when treated with a bispecific antibody binding to human IL-6 and the T-cell receptor complex. Cells that do not express IL-6 are unaffected. Because it has been shown before that HepG2 cells transfected with IL-6 cDNA express cell bound IL-6 (43), we hypothesize that the secreted IL-6 for some time remains cell-associated and that this time is long enough to lead to an attachment of T-cells, which in turn perform killing of the HepG2 cells. A similar strategy has recently been employed to specifically kill lymphoma cells with the help of a bispecific antibody directed against an idiotypic marker on mouse B-lymphoma cells and against the murine T-cell receptor complex (44).

A completely different strategy would exploit the intracellular recognition of human IL-6 by the scFv antibody H1. Using this strategy, cells that synthesize human IL-6 could be phenotypically changed not to secrete the cytokine. Such an approach would, however, rely on the specific introduction of DNA into the cells that are to be targeted. By using a KDEL protein (45) sequence that could be added to an scFv on the DNA level, it should be possible to anchor such an scFv protein in the endoplasmic reticulum, which would lead to the retention of IL-6 by such a cell (46). By such a strategy, it would be possible to suppress cellular secretion of IL-6.

We have isolated the first recombinant human scFv antibodies directed against human four-helical cytokines. The availability of such recombinant human antibodies to more cytokines or cytokine receptor subunits will allow for the design of new concepts of therapeutic approaches for many diseases related to dysregulation of the cytokine network.

    ACKNOWLEDGEMENTS

We thank Martina Fischer for the preparation of recombinant human IL-6, Marc Ehlers for the help with the 125I-labeled IL-6 binding assay, and Birgit Ackermann for aid with the CTL assay (Mainz, Germany). The generous support of Dr. A. Skerra (Darmstadt, Germany) is gratefully acknowledged. The truncated IL-6 mutant was a kind gift from Dr. J. Brakenhoff (Amsterdam, the Netherlands). The B78 cell line transfected with the IL-6/sIL-6 fusion protein was provided by Suat Özbek (Mainz, Germany), and the plasmid coding for the scFv of OKT3 was a gift from Philipp Holliger (Cambridge, United Kingdom).

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany and by the Naturwissenschattlich-Medizinisches Forschungszentrum, Mainz, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Department of Medicine, Section of Pathophysiology, Johannes Gutenberg-University of Mainz, Obere Zahlbacher Straße 63, D-55101 Mainz, Germany. Tel.: 49-6131-173363; Fax: 49-6131-173364; E-mail: rose{at}mzdmza.zdv.uni-mainz.de.

1 The abbreviations used are: IL-6, interleukin-6; IL-6R, IL-6 receptor; CDR, complementary-determining region; mAb, monoclonal antibody; PBS, phosphate-buffered saline; POD, peroxidase; scFv, single chain Fv; VH, variable heavy domain; VL, variable light domain; ELISA, enzyme-linked immunosorbent assay; CTL, cytotoxic T lymphocyte.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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