An efficient route to the production of an IgG-like bispecific antibody

Zhuang Zuo, Xenia Jimenez, Larry Witte and Zhenping Zhu1

Department of Molecular and Cell Biology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human {kappa} chain (CL) and the first constant domain of human heavy chain (CH1), to form two polypeptides, (scFv)1-CL and (scFv)2-CH1-CH2-CH3, respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)4-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)4-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)4-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)4-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.

Keywords: antibody engineering/bispecific antibody/KDR/single-chain Fv/VEGF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bispecific antibodies (BsAb) have significant potential for human applications and have shown promise in several small-scale clinical trials as cancer imaging and therapy agents (Fanger et al., 1993Go; Curnow, 1997Go; De Gast et al., 1997Go). Broad clinical evaluation of BsAb has been hampered, however, by the lack of efficient production methods. In recent years, a variety of recombinant methods have been developed for the production of bispecific and/or bivalent and multivalent antibody fragments (for reviews, see Holliger and Winter, 1993; Carter et al., 1995; Plückthun and Pack, 1997). Bispecificity and/or bivalency have been accomplished by fusing two single-chain Fv (scFv) or Fab via flexible linkers (Mallender and Voss,1994Go; Mack et al., 1995Go; Zapata et al., 1995Go), leucine zipper (De Kruif and Logtenberg, 1996Go), CHCL-heterodimerization (Müller et al., 1998aGo) and diabody format (Holliger et al., 1993Go; Zhu et al., 1996Go). On the other hand, multivalency is achieved by the addition of multimerization sequences at the carboxyl or amino terminus of the scFv or Fab fragments, for example, by using p53 (Rheinnecker et al., 1996Go), streptavidin (Dübel et al.,1995Go; Kipriyanov et al., 1996Go) and helix–turn–helix motifs (Müller et al., 1998bGo). Combining multi-specificity and multi-valency engineering has led to the production of multivalent bi- or tri-specific antibodies that possess more than one binding site to each of their target antigens. For example, by dimerizing two scFv fusions via the helix–turn–helix motif, (scFv)1–hinge–helix–turn–helix–(scFv)2, a tetravalent bispecific miniantibody was produced (Müller et al., 1998bGo). The so-called `di-bi-miniantibody' possesses two binding sites to each of it target antigens.

Compared with the advance in producing BsAb fragments, progress in methods to prepare IgG-form BsAb (BsAb-IgG) has been modest. The traditional methods for producing BsAb-IgG include chemical cross-linking of two different IgG molecules (Karpovsky et al., 1984Go; Zhu et al., 1994Go) or co-expressing two IgG in hybrid hybridomas (Milstein and Cuello, 1983Go; Suresh et al., 1986Go). Chemical cross-linking is often inefficient and can lead to the loss of antibody activity. Co-expression of two different IgGs in a hybrid hybridoma may produce up to 10 heavy- and light-chain pairs (Suresh et al., 1986Go), hence compromising the yield of BsAb-IgG. In both methods, purification of the BsAb-IgG from the non-functional species, such as multimeric aggregates resulting from chemical modification and homodimers of heavy or light chains and non-cognate heavy–light chain pairs, is often difficult and the yield is usually low.

We describe here a recombinant method for the production of BsAb-IgG that eliminates mispairing between antibody heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human {kappa} chain (CL) and the first constant domain of human heavy chain (CH1) to form two polypeptides, (scFv)1-CL and (scFv)2-CH1-CH2-CH3, respectively. The two polypeptides are co-expressed in mammalian COS cells. Association between the heavy and the light chains forms a covalently linked hetero-tetramer with dual specificity. This approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens (Figure 1Go). The BsAb retains not only antigen binding efficiency but also the biological activity of its component antibodies.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Schematic diagrams of Bs(scFv)4-IgG and Bs(scFv)2-Fab molecules. In Bs(scFv)4-IgG, the VH and VL domains of a human IgG1 molecule are replaced by two scFv antibodies of different specificity. Co-expression of the scFv–light- and scFv–heavy-chain fusion polypeptides in mammalian cells results in the formation of a tetravalent, IgG-like bispecific molecule. In Bs(scFv)4-Fab, a stop codon is introduced at the C-terminal end of the heavy-chain CH1 domain, which results in the expression of a bivalent, Fab-like bispecific molecule (also see Figure 2AGo).

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proteins and antibodies

Vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor-alkaline phosphatase fusion protein (KDR-AP) and its mouse homolog, fetal liver kinase 1 (Flk-1)-AP, were expressed in baculovirus and NIH 3T3 cells, respectively, and purified following the procedures described (Zhu et al., 1998Go). KDR extracellular domain (ECD) immunoglobulin (Ig) domain deletion mutants were constructed by PCR cloning, expressed in NIH 3T3 cells and purified as described (Lu et al., 2000Go). The definition of KDR ECD Ig domain deletion mutants are as follows: KDR(Ig1–7), the full length KDR ECD containing all seven Ig domains of the receptor (from amino acid Met1 to Val742); KDR(Ig1–3), the mutant containing the three N-terminal ECD Ig domains (from amino acid Met1 to Lys327); and KDR(Ig3–7), the mutant containing KDR ECD Ig domains 3–7 (from amino acid Asp225 to Val742). The isolation of the anti-KDR single-chain Fv (scFv) p1C11 and scFv p4G7 from a phage display library constructed from a mouse immunized with KDR has been reported previously (Zhu et al., 1998Go; Lu et al., 1999Go). Diabody DAB p4G7, a form of bivalent scFv fragment (Holliger et al., 1993Go; Zhu et al., 1996Go) was constructed from scFv p4G7 as described previously (Zhu et al., 1996Go; Lu et al., 1999Go). C-p1C11, a mouse–human chimeric IgG1 antibody constructed from scFv p1C11 and C225, a chimeric IgG1 antibody directed against epidermal growth factor (EGF) receptor, were both produced at ImClone Systems (New York) (Zhu et al., 1999Go).

Construction of expression vectors for BsAb-IgG [Bs(scFv)4-IgG] and BsAb-Fab[Bs(svFv)2-Fab]

The gene encoding scFv p4G7 was amplified from the scFv expression vector by PCR using primers, JZZ-2 and JZZ-3. The leader peptide sequence for protein secretion in mammalian cells was then added to the 5' of the scFv encoding sequence by PCR using primers JZZ-12 and JZZ-3. Similarly, the gene encoding scFv p1C11 was amplified from the scFv expression vector by PCR using primers JZZ-2 and p1C11VL3-2, followed by PCR with primers JZZ-12 and p1C11VL3-2 to add the leader peptide sequence. The same leader peptide consisting of 19 amino acids, MGWSCIILFLVATATGVHS, was used for the secretion of both the light and the heavy chains.

JZZ-2: 5' CTA GTA GCA ACT GCC ACC GGC GTA CAT TCA CAG GTC AAG CTG C 3'

JZZ-3: 5' TCG AAG GAT CCA CTC ACC TTT TAT TTC CAG C 3'

BamHI

p1C11VL3-2: 5' TCG ATC TAG AAG GAT CCA CTC ACG TTT TAT TTC CAG 3'

BamHI

JZZ-12: 5' GGT CAA AAG CTT ATG GGA TGG TCA TGT ATC ATC CTT TTT CTA GTA GCA ACT 3'HindIII

JZZ-18: 5' TCT CGG CCG GCT TAA GCT GCG CAT GTG TGA GT 3'

NaeI

Separate expression vectors for the light and heavy chains of Bs(scFv)4-IgG were constructed (Figure 2AGo). The cloned scFv p4G7 gene was digested with HindIII and BamHI and ligated into the vector pKN100 (a gift from Dr T.Jones, MRC Collaborative Centre, London, UK) containing the human {kappa} light-chain constant region (CL) to create the expression vector for the BsAb-IgG light chain, BsIgG-L. The cloned scFv p1C11 gene was digested with HindIII and BamHI and ligated into the vector pG1D105 (a gift from Dr T.Jones) containing the human IgG1 ({gamma}) heavy-chain constant domain (CH) to create the expression vector for the BsAb-IgG heavy chain, BsIgG-H.



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 2. Expression and purification of Bs(scFv)4-IgG and Bs(scFv)2-Fab antibodies. (A) Schematic diagrams showing the expression constructs for the BsAb. The individual scFv antibodies were precisely fused in their 5' to a leader sequence for secretion in mammalian cells and in their 3' to the CL or CH1 domains of a human IgG1 molecule. Note: the diagram is not to scale. (B) SDS–PAGE analysis of the Protein G purified Bs(scFv)4-IgG and Bs(scFv)2-Fab antibodies. Lanes 1–3 are run under non-reducing conditions. Lane 1, c-p1C11, a chimeric IgG1; lane 2, Bs(scFv)4-IgG; lane 3, Bs(scFv)2-Fab. Lanes 4–6 were run under reducing conditions. Lane 4, c-p1C11; lane 5, Bs(scFv)4-IgG; lane 6, Bs(scFv)2-Fab. Also shown are the positions of molecular mass standards.

 
To prepare the expression vector for Bs(scFv)2-Fab, a stop codon was introduced into vector BsIgG-H immediately after the first constant domain (CH1) to terminate the protein translation, by PCR using primers JZZ-12 and JZZ-18. The gene fragment was digested with HindIII and NaeI and cloned into vector pG1D105 to create vector BsFab-H. All constructs were examined by restriction enzyme digestion and verified by dideoxynucleotide sequencing.

Antibody expression and purification

COS cells were co-transfected with equal amounts of DNA from vector BsIgG-L and BsIgG-H or BsIgG-L and BsFab-H, for transient expression of Bs(scFv)4-IgG and Bs(scFv)2-Fab, respectively, following the procedure described previously (Zhu et al., 1999Go). The cells were switched to serum-free medium 24 h after the transfection. The conditioned supernatant was collected at 48 and 120 h after the transfection. The Bs(scFv)4-IgG and Bs(scFv)2-Fab were purified from the pooled supernatant by affinity chromatography using a Protein G column following the protocol described by the manufacturer (Pharmacia Biotech, Piscataway, NJ). The antibody-containing fractions were pooled, buffer exchanged into PBS and concentrated using Centricon 10 concentrators (Amicon, Beverly, MA). The purity of the antibodies was analyzed by SDS–PAGE. The concentration of purified antibody was determined by ELISA using goat anti-human IgG Fc specific antibody as the capture agent and HRP-conjugated goat anti-human {Phi} chain antibody as the detection agent. A standard curve was calibrated using clinical grade antibodies, C225 or c-p1C11.

Bispecific binding of the BsAb to KDR

Two different assays were carried out to demonstrate the dual specificity of the BsAb. In the direct binding assay, a 96-well plate (Nunc, Roskilde, Denmark) was first coated with KDR(Ig1–7)-AP, KDR(Ig1–3)-AP or KDR(Ig3–7)-AP fusion proteins (1.0 µg/mlx100 µl per well) using a rabbit anti-AP antibody (DAKO-immunogloblins, Denmark) as the capturing agent. The plate was then incubated with the BsAb, c-p1C11 or DAB p4G7 at room temperature for 1 h, followed by incubation with rabbit anti-human IgG Fc specific antibody–HRP conjugate (Cappel, Organon Teknika, West Chester, PA) for the BsAb and c-p1C11 or mouse anti-E tag antibody–HRP conjugate (Pharmacia Biotech) for DAB p4G7. The plates were washed five times, TMB peroxidase substrate (KPL, Gaithersburg, MD) was added and the OD at 450 nm read using a microplate reader (Molecular Device, Sunnyvale, CA) (Zhu et al., 1998Go). In the cross-linking assay, the antibodies were first incubated in solution with KDR(Ig1–7)-AP, KDR(Ig1–3)-AP or KDR(Ig3–7)-AP. The mixtures were transferred to a 96-well plate coated with KDR(Ig1–3) (untagged) and incubated at room temperature for 2 h. The plate was washed and the KDR(Ig1–3) (untagged)-bound AP activity was measured by the addition of AP substrate, p-nitrophenyl phosphate (Sigma), and the OD was read at 405 nm (Zhu et al., 1998Go).

Quantitative binding of Bs(scFv)4-IgG and Bs(scFv)2-Fab to KDR and Flk-1

Various amounts of Bs(scFv)4-IgG, Bs(scFv)2-Fab, c-p1C11 or scFv p4G7 were added to 96-well Maxi-sorp microtiter plates (Nunc) coated with either KDR-AP or Flk-1-AP (100 ng protein per well) and incubated at room temperature for 1 h, followed by incubation at room temperature for 1 h with rabbit anti-human IgG Fc specific antibody-HRP conjugate for bispecific antibodies and c-p1C11 or mouse anti-E tag antibody-HRP conjugate for scFv p4G7. The plates were washed and developed as described above.

FACS analysis

Early-passage HUVEC cells were grown in growth factor-depleted EBM-2 medium overnight to induce the expression of KDR receptor. The cells were harvested and washed three times with PBS, incubated with 5 µg/ml Bs(scFv)4-IgG or c-p1C11 for 1 h at 4°C, followed by incubation with a FITC-labeled rabbit anti-human Fc antibody (Cappel, Organon Teknika) for an additional 1 h. The cells were washed and analyzed with a flow cytometer (Zhu et al., 1999Go).

Binding kinetic analysis

The binding kinetics of the BsAb to both KDR and Flk-1 were measured by surface plasmon resonance, using a BIAcore biosensor (Pharmacia Biosensor). KDR-AP or Flk-1-AP fusion proteins were immobilized onto a sensor chip and various antibodies were injected at concentrations ranging from 25 to 200 nM. Sensorgrams were obtained at each concentration and were evaluated using a program, BIA Evaluation 2.0, to determine the rate constants kon and koff. Kd was calculated as the ratio of rate constants koff/kon.

VEGF blocking and phosphorylation inhibition assay

In the blocking assay, various amounts of BsAb or c-p1C11 were mixed with a fixed amount of KDR-AP or Flk-1-AP and incubated at room temperature for 1 h. The mixtures were then transferred to VEGF165-coated 96-well plates and incubated at room temperature for an additional 2 h, after which the plates were washed five times. The VEGF-bound AP activity was quantified as described (Zhu, et al., 1998Go, 1999Go).

KDR phosphorylation assay was carried out following the procedure described previously (Zhu et al., 1998Go, 1999Go), using a stable 293 cell line transfected with the full length KDR (ImClone Systems). Briefly, the transfected 293 cells (~3x106 cells per plate) were incubated in the presence or absence of antibodies for 15 min, followed by stimulation with 20 ng/ml of VEGF165 at RT for an additional 15 min. The cells were then lysed and the cell lysate was used for KDR phosphorylation assays. The KDR receptor was immunoprecipitated from the cell lysates with Protein A Sepharose beads (Santa Cruz Biotechnology, CA) coupled to an anti-KDR antibody, Mab 4.13 (ImClone Systems). Proteins were resolved with SDS–PAGE and subjected to Western blot analysis. To detect KDR phosphorylation, blots were probed with an anti-phosphotyrosine Mab, PY20 (ICN Biomedicals, Aurora, OH). The signals were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL). The blots were reprobed with a polyclonal anti-KDR antibody (ImClone Systems) to ensure that an equal amount of protein was loaded in each lane of the SDS–polyacrylamide gels.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of Bs(scFv)4-IgG and Bs(scFv)2-Fab

Two anti-KDR scFv antibodies, scFv p1C11 and p4G7, were used for the construction of Bs(scFv)4-IgG and Bs(scFv)2-Fab (Figure 2AGo). ScFv p1C11 binds specifically to KDR and blocks KDR/VEGF interaction, whereas scFv p4G7 binds to both KDR and its mouse homolog, Flk-1, but does not block either KDR/VEGF or Flk-1/VEGF interaction (Zhu et al., 1998Go; Lu et al., 1999Go). Epitope mapping studies revealed that p1C11 binds to epitope(s) located within KDR ECD Ig domain 1–3, whereas the epitope(s) for p4G7 are located within Ig domains 6 and 7 (Lu et al., 2000Go). Gene segments encoding scFv p1C11 and p4G7 were fused to the N-terminus of CH1 and CL of a human IgG1 molecule, respectively, to create expression vectors BsIgG-H and BsIgG-L (Figure 2AGo). This arrangement replaces the original VH and VL domains of an IgG with two scFv molecules, each constituting an independent antigen-binding unit (Figure 1Go). Co-expression of BsIgG-H and BsIgG-L yields an IgG-like tetravalent molecule, Bs(scFv)4-IgG, with dual specificity (Figure 1Go). A bispecific, bivalent Fab-like molecule (Figure 1Go), Bs(scFv)2-Fab, was also produced by co-expression of BsIgG-L and BsFab-H. Vector BsFab-H was constructed from BsIgG-H by introducing a stop codon at the end of CH1 domain (Figure 2AGo).

Expression and purification of Bs(scFv)4-IgG and Bs(scFv)2-Fab

The Bs(scFv)4-IgG and Bs(scFv)2-Fab were transiently expressed in COS cells and purified from the cell culture supernatant by an affinity chromatography using a Protein G column. The purified BsAb were analyzed by SDS–PAGE (Figure 2BGo). Under non-reducing condition, Bs(scFv)4-IgG gave rise to a single band with a molecular mass of ~200 kDa, whereas Bs(scFv)2-Fab gave a major band of ~75 kDa (Figure 2BGo, lanes 2 and 3). Under reducing conditions, Bs(scFv)4-IgG yielded two major bands with the expected mobility for scFv-CH1-CH2-CH3 fusion (~63 kDa) and scFv-CL fusion (~37 kDa), respectively (Figure 2BGo, lane 5). On the other hand, Bs(scFv)2-Fab gave rise to two major bands with molecular mass of ~38 kDa and 37 kDa, representing the scFv-CH1 and scFv-CL fusions, respectively (Figure 2BGo, lane 6). As a control, c-p1C11, a chimeric IgG1 antibody, gave rise to one band of ~150 kDa under non-reducing conditions (Figure 2BGo, lane 1) and two bands of ~50 kDa (the heavy chain, VH-CH1-CH2-CH3 fusion) and ~25 kDa (the light chain, VL-CL fusion) under reducing conditions (Figure 2BGo, lane 5).

Dual specificity of the BsAb

Dual specificity of the BsAb was assayed using the full-length KDR ECD and two of its Ig domain-deletion mutants (Figure 3AGo). As seen previously, p1C11 only binds to KDR mutants containing Ig domains 1–3 (Zhu et al., 1999Go), whereas p4G7 only binds to mutants containing Ig domains 6 and 7 (Lu et al., 1999Go). In contrast, both Bs(scFv)4-IgG and Bs(scFv)2-Fab bind to all three KDR variants, indicating that the BsAb possess two binding sites, one to the epitope on Ig domains 1–3 and the other to the epitope on Ig domains 6 and 7.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. ELISA assays for the bispecificity of Bs(scFv)4-IgG and Bs(scFv)2-Fab antibodies. (A) Binding of Bs(scFv)4-IgG, Bs(scFv)2-Fab and its parent antibodies to KDR ECD Ig domain deletion mutant-AP fusion proteins. (B) Cross-linking ELISA for detection of simultaneous binding by Bs(scFv)4-IgG and Bs(scFv)2-Fab to the two different epitopes that are located on separate KDR ECD Ig domain deletion mutants, KDR(Ig1–3) and KDR(Ig3–7)-AP. The BsAb are incubated in solution with KDR(Ig1–7)-AP, KDR(Ig1–3)-AP or KDR(Ig3–7)-AP and transferred to a plate coated with untagged KDR(Ig1–3). The cross-linking complexes formed between the soluble phase antibody–KDR variant-AP complex and the immobilized KDR(Ig1–3) are detected by measuring the plate-bound AP activity. Data shown are mean ± SD of triplicate determinations.

 
To investigate whether the BsAb are capable of simultaneous binding to both epitopes, a cross-linking assay was carried out using several KDR ECD Ig domain-deletion mutants that are either untagged or tagged with AP. In this assay, the BsAb were first incubated with KDR(Ig1–7)-AP, KDR(Ig1–3)-AP or KDR(Ig3–7)-AP. The mixtures were transferred to a microtiter plate coated with KDR(Ig1–3) (untagged), followed by measuring KDR(Ig1–3) (untagged)-bound AP activity (Figure 3BGo). Both Bs(scFv)4-IgG and Bs(scFv)2-Fab bind effectively to all three KDR-AP variants in solution and form cross-linking complexes with the immobilized KDR(Ig1–3) (untagged), as demonstrated by plate-bound AP activity (Figure 3BGo). In contrast, c-p1C11 only cross-links KDR(Ig1–3) (untagged) with KDR variants containing Ig domains 1–3, i.e. KDR(Ig1–7)-AP and KDR(Ig1–3)-AP, but not KDR(Ig3–7)-AP. As expected, p4G7 fails to cross-link any KDR variants to the immobilized KDR(Ig1–3) (untagged), since p4G7 does not bind KDR(Ig1–3) mutant.

Antigen binding by BsAb

The antigen binding efficiency of the BsAb was determined on immobilized KDR (Figure 4AGo) and Flk-1 (Figure 4BGo). Figure 4AGo shows the dose-dependent binding of Bs(scFv)4-IgG and Bs(scFv)2-Fab to KDR. Both Bs(scFv)4-IgG and Bs(scFv)2-Fab bind KDR as efficiently as one of the parent antibodies, c-p1C11. In addition, Bs(scFv)4-IgG and Bs(scFv)2-Fab, but not c-p1C11, also bind to Flk-1 in a dose-dependent manner similar to scFv p4G7 (Figure 4BGo). As expected, C225, a chimeric antibody directed against human EGFR, did not bind to either of the antigens.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Dose-dependent binding of Bs(scFv)4-IgG, Bs(scFv)2-Fab and its parent antibodies to immobilized full-length KDR-AP (A) and Flk-1-AP (B). Data shown are mean ± SD of triplicate determinations.

 
Binding of the BsAb to cell surface-expressed receptor was assayed by FACS analysis. As previously seen with c-p1C11 (Zhu et al., 1999Go), Bs(scFv)4-IgG binds efficiently to KDR expressed on early passage HUVEC (data not shown).

The binding kinetics of the BsAb to KDR and Flk-1 were determined by surface plasmon resonance using a BIAcore instrument (Table IGo). The overall affinities (Kd) or avidity of Bs(scFv)4-IgG and Bs(scFv)2-Fab to KDR were 1.4 and 1.1 nM, respectively, which are similar to those of the monovalent scFv p1C11 and p4G7, but are 4–10-fold weaker than those of the bivalent c-p1C11 or DAB p4G7. On the other hand, Bs(scFv)4-IgG, which is bivalent to Flk-1, showed an avidity (Kd 0.33 nM) that is similar to that of the bivalent DAB p4G7 (Kd 0.18 nM). Bs(scFv)2-Fab and scFv p4G7, both monovalent to Flk-1, bind to Flk-1 with similar affinity (Kd 1.7 and 4.2 nM, respectively), which are 5–20-fold weaker than those of their bivalent counterparts.


View this table:
[in this window]
[in a new window]
 
Table 1. Binding kinetics of various antibodies to KDR and Flk-1 as determined by BIAcorea
 
VEGF blocking by Bs(scFv)4-IgG

Figure 5Go shows that Bs(scFv)4-IgG effectively block KDR-AP from binding to immobilized VEGF. The IC50, the antibody concentrations required to block 50% of KDR binding, of Bs(scFv)4-IgG and c-p1C11 are 4 and 1 nM, respectively. As seen with scFv p4G7, Bs(scFv)4-IgG did not block Flk-1 binding to VEGF (not shown). C225, an anti-EGFR antibody, showed no effect on KDR binding to VEGF.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of binding of KDR to immobilized VEGF by Bs(scFv)4-IgG and c-p1C11. Data shown are mean ± SD of triplicate determinations.

 
KDR phosphorylation inhibition by the BsAb

The biological effect of Bs(scFv)4-IgG on VEGF-induced receptor phosphorylation was determined using KDR-transfected 293 cells. As shown in Figure 6Go, VEGF treatment induces strong phosphorylation of KDR receptor. Pretreatment with Bs(scFv)4-IgG inhibits VEGF-induced receptor phosphorylation in a dose-dependent manner (Figure 6Go). Further, Bs(scFv)4-IgG is equally potent as c-p1C11 at each antibody concentration assayed.



View larger version (97K):
[in this window]
[in a new window]
 
Fig. 6. Dose-dependent inhibition of VEGF-stimulated phosphorylation of KDR receptor by Bs(scFv)4-IgG and c-p1C11. The KDR-transfected 293 cells were treated with various amounts of antibodies at room temperature for 15 min, followed by incubation with 20 ng/ml of VEGF (except the control group) at room temperature for an additional 15 min. Phosphorylation of KDR was analyzed followed the protocol described previously (Zhu et al., 1998Go, 1999Go).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have developed an efficient method for the production of IgG-like bispecific antibodies using the natural dimerization mechanism between IgG heavy and light chains. The original VH or VL domains of an IgG molecule were replaced by two scFv with different specificity, resulting in a hybrid bispecific antibody that is bivalent to each of its target antigens (Figure 1Go). Bs(scFv)4-IgG is functionally expressed and assembled in mammalian cells and is capable of binding to two different epitopes simultaneously, as demonstrated by the cross-linking assay (Figure 3BGo). We believe this approach is applicable to scFv antibodies recognizing virtually any pair of antigens. It is also possible to produce a similar molecule that is tetravalent to the same epitope.

The small-size BsAb fragments may be preferable to the full-size BsAb-IgG for some clinical applications, for example, to promote rapid clearance for improved tumor imaging and to facilitate efficient tumor penetration. In many other applications, however, BsAb-IgG may offer additional advantages as this format provides an IgG Fc region that can confer long serum half-life and support secondary immune functions, for example, antibody-dependent cellular cytotoxicity (ADCC) and complement mediated cytotoxicity (CMC). The major hurdles in traditional methods of BsAb production include heterogeneity of the products, low yield and difficulty with the purification process. For example, co-expression of two sets of IgG heavy and light chains in a hybrid hybridoma may produce up to 10 pairings (Suresh et al., 1986Go). These unwanted pairings often greatly compromise the yield of BsAb and impose significant problems with purification. Recently, two recombinant methods were developed to improve the production of IgG-like bispecific and/or multivalent molecules (Coloma and Morrison, 1997Go; Merchant et al., 1998Go; for comments and review, see Hoogenboom, 1997; Dall'Acqua and Carter, 1998). Coloma and Morrison fused an scFv fragment to the C-terminus of the heavy chain of the second antibody to create an antibody heavy-chain–scFv fusion. Co-expression of the antibody light chain along with the modified heavy-chain–scFv fusion results in the production of a homogeneous population of IgG-like, tetravalent bispecific molecule that binds to antigen A at one end and to antigen B at the other end (Coloma and Morrison, 1997Go). On the other hand, strategies were developed to overcome the unwanted pairings between two different sets of IgG heavy and light chains co-expressed in transfected cells (Merchant et al., 1998Go). The CH3 domains of heavy chains were first remodeled for heterodimerization using `knobs-into-holes' mutations in conjunction with engineered disulfide bonds, to reduce dramatically the homodimerization between antibody heavy chains of the same specificity (Ridgway et al., 1996Go). Light-chain mispairing was prevented by using an identical light chain for each arm of the resulting BsAb-IgG (Merchant et al., 1998Go). Antibodies of different specificity that use identical light chain are frequently isolated from phage display libraries with limited light-chain diversity (Vaughan et al., 1996Go; Merchant et al., 1998Go). Co-expression of one common set of light chains along with two sets of `knobs-into-holes'-modified heavy chains resulted in the production of ~95% functional BsAb-IgG (Merchant et al., 1998Go). In our approach, the VH and VL domains of IgG are replaced by scFv antibodies of different specificity to form (scFv)1-CH1-CH2-CH3 and (scFv)2-CL fusion polypeptides, respectively. Co-expression of the two fusion polypeptides in mammalian cells results in the formation of an IgG-like hybrid molecule, each comprising of two copies of (scFv)1-CH1-CH2-CH3 and two copies of (scFv)2-CL fusion polypeptides (Figure 1Go). The hybrid molecule, Bs(scFv)4-IgG, is bispecific and tetravalent since each of its two Fab-like arms possesses two different antigen binding sites. Both scFv displayed on each Fab-like arm are properly folded and immunoreactive, as demonstrated by fact that Bs(scFv)2-Fab is bifunctional and capable of cross-linking the two target antigens (Figure 3BGo). It is not clear, however, whether all the four antigen-binding sites within a Bs(scFv)4-IgG molecule are accessible to the target epitopes at the same time.

Our BsAb format, Bs(scFv)4-IgG, combines the features of bispecificity and bivalency. High affinities are desirable and may be necessary for each arm of a BsAb destined for human therapy. For example, a bispecific anti-p185HER2/anti-CD3 F(ab')2 constructed with a higher affinity variant of an humanized anti-CD3 antibody was much more potent in mediating tumor cell-killing than one constructed with an anti-CD3 variant of lower affinity (Zhu et al., 1995Go). Because of the avidity factor of bivalent binding, our format may be of great advantageous over the monovalent BsAb. This is particularly relevant when constructing BsAb from antibodies of low affinity; such antibodies may require laborious and time-consuming affinity maturation processes to achieve a `minimal' functional affinity. For example, our studies showed that Bs(scFv)4-IgG, which is bivalent to Flk-1, had an avidity similar to DAB p4G7, a bivalent diabody to Flk-1. The avidity of Bs(scFv)4-IgG and DAB p4G7 is ~10–23-fold higher than that of their respective monovalent counterparts, the divalent Bs(scFv)2-Fab and the scFv p4G7 (Table IGo), demonstrating clearly the avidity enhancement from bivalent binding. Bs(scFv)4-IgG, however, possesses an approximately 8-fold slower association rate (kon) and a 10-fold faster dissociation rate (koff) than DAB p4G7. This may reflect decreased accessibility of the antigen epitope to the binding sites of scFv p4G7 because of the presence in the close vicinity of the second (and non-functional) molecule, the KDR-specific scFv p1C11 (see Figure 1Go). It is of interest that the tetravalent Bs(scFv)4-IgG showed the same avidity for KDR as the divalent Bs(scFv)2-Fab. Further, the avidity of Bs(scFv)4-IgG to KDR is 5–7-fold lower than that of both the bivalent c-p1C11 and DAB p4G7 (Table IGo). Unlike in the case of Flk-1 binding, the four binding sites of Bs(scFv)4-IgG are directed to two distinct epitopes within the same KDR molecule. The spatial relationship or the geometry of the two epitopes could place some degree of restraint on the effectiveness of BsAb binding. For example, the distance between the two epitopes, the steric distribution and accessibility of the epitopes and the density of the antigen on the surface can affect significantly the binding kinetics of the BsAb to the immobilized KDR molecules. Further, the `crowd' resulting from displaying four individual scFv units on the antigen-binding surface of a single IgG molecule may have an effect on antigen binding. The spatial arrangement of the four scFv may not allow each binding unit to rotate freely and fully adapt to the steric distribution of individual antigen epitopes, thus affecting the binding of the BsAb to the antigen. An early study on the functional properties of a Waldenstrom macroglobulin antibody (IgM) demonstrated that although the pentameric IgM has 10 possible binding sites, the functional valence of the antibody is five or even lower (Stone and Metzger, 1968Go). Taken together, we speculate that although all the four component scFv are immunoreactive, the functional valence of our Bs(scFv)4-IgG for binding KDR is most likely lower than that which is expected.

Bs(scFv)4-IgG retained the biological functions of both of its components. First, the BsAb binds as efficiently as the individual parent antibodies to both of its targets, KDR and Flk-1 (Figure 4Go). The BsAb also binds to the surface-expressed KDR molecule on human endothelial cells. Further, the BsAb blocks KDR–VEGF interaction and efficiently neutralizes VEGF-induced KDR receptor phosphorylation in a dose-dependent manner (Figures 5 and 6GoGo). It is noteworthy that the BsAb is equally potent as c-p1C11 in neutralizing VEGF-induced receptor phosphorylation despite the fact that the BsAb binds to KDR with a 5-fold lower affinity than c-p1C11 and is 4-fold less effective in blocking KDR–VEGF interaction in an ELISA assay. The tetravalency of the BsAb, plus the capability of intramolecular cross-linking (i.e. cross-linking two epitopes within the same KDR molecule) and/or intermolecular cross-linking to form a multimolecular complexes on the cell surface, may account for the enhanced biological activity of the BsAb. Our Bs(scFv)4-IgG antibody, as well as the bivalent c-p1C11, failed to induce significant ADCC and CMC on the KDR-transfected 293 cells (data not shown). This is probably due to the low receptor density of the transfected cells: there are only several thousand molecules of KDR expressed on each transfected cell (our unpublished observations), a number that might be below the threshold required to trigger an efficient ADCC and CMC. A number of reports, however, have demonstrated that Fc-containing BsAb and other fusion proteins retained full effector mechanisms of the Fc component (Ridgway et al., 1996Go; Merchant et al., 1998Go).

In conclusion, we constructed a novel bispecific IgG-like antibody that is also bivalent to each of its target antigen. Bivalent binding can be of advantageous in several respects. First, bivalency may increase binding avidity, which can compensate for the lower affinity of each individual components of the BsAb. Further, bivalent binding may cause receptor cross-linking or dimerization which, in many cases, is required to trigger biological responses. Finally, the IgG-like BsAb provides an IgG Fc region that can confer a long serum half-life and support secondary immune functions, such as ADCC and CMC (Coloma and Morrison, 1997Go; Merchant et al., 1998Go).


    Notes
 
1 To whom correspondence should be addressed.E-mail: zhenping{at}imclone.com Back


    Acknowledgments
 
The authors thank their colleagues at ImClone Systems, H.Kotanides for providing the KDR-transfected 293 cells, K.Persaud for providing the KDR Ig domain-deletion mutant proteins and D.Pereira for performing the FACS analysis.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carter,P., Ridgway,J. and Zhu,Z. (1995) J. Hematother., 4, 463–470.[Medline]

Coloma,M.J. and Morrison,S.L. (1997) Nature Biotechnol., 15, 159–163.[ISI][Medline]

Curnow,R.T. (1997) Cancer Immunol. Immunother., 45, 210–215.[ISI][Medline]

Dall'Acqua,W. and Carter,P. (1998) Curr. Opin. Struct. Biol., 8, 443–450.[ISI][Medline]

De Gast,G.C., van de Winkel,J.G.J. and Bast,B.E.J.E. J. (1997) Cancer Immunol. Immunother., 45, 121–123.[ISI][Medline]

De Kruif,J. and Logtenberg,T. (1996) J. Biol. Chem., 271, 7630–7634.[Abstract/Free Full Text]

Dübel,S., Breitling,F., Kontermann,R., Schmidt,T., Skerra,A. and Little,M. (1995) J. Immunol. Methods, 178, 201–209.[ISI][Medline]

Fanger,M.W., Morganelli,P.M. and Guyre P.M. (1993) In Rosen, Steven,T. and Kuzel,T.M. (eds), Immunoconjugate Therapy of Hematologic Malignancies. Kluwer, Dordrecht, pp. 181–194.

Holliger,P. and Winter,G. (1993) Curr Opin. Biotechnol., 4, 446–449.[Medline]

Holliger,P., Prospero,T. and Winter,G. (1993) Proc. Natl Acad. Sci. USA, 90, 6444–6448.[Abstract]

Hoogenboom,H.R. (1997) Nature Biotechnol., 15, 125–126.[ISI][Medline]

Karpovsky,B., Titus,J.A., Stephany,D.A., Segal,D.M. (1984) J. Exp. Med., 160, 1686–1701.[Abstract]

Kipriyanov,S.M., Little,M., Kropshofer,H., Breitling,F., Gotter,S. and Dübel,S. (1996) Protein Engng, 9, 203–211.[Abstract]

Lu,D., Kotanides,K., Jimenez,X., Zhou,Q., Persaud,K., Bohlen,P., Witte,L. and Zhu,Z. (1999) J. Immunol. Methods, 230, 159–171.[ISI][Medline]

Lu,D., Kussie,P., Pytowski,B., Persuad,K., Bohlen,P., Witte,L. and Zhu,Z. (2000) J. Biol. Chem., in press.

Mack,M., Riethmüller,G. and Kufer,P. (1995) Proc. Natl Acad. Sci. USA, 92, 7021–7025.[Abstract]

Mallender,W.D. and Voss,E.W.,Jr (1994) J. Biol. Chem., 269, 199–206.[Abstract/Free Full Text]

Merchant,A.M., Zhu,Z., Yuan,J., Goddard,A., Adams,C.W., Presta,L.G. and Carter,P. (1998) Nature Biotechnol., 16, 677–681.[ISI][Medline]

Milstein,C. and Cuello,A.C. (1983) Nature, 305, 537–540.[ISI][Medline]

Müller,K., Arndt,K.M., Strittmatter,W. and Plückthun,A. (1998a) FEBS Lett., 422, 259–264.[ISI][Medline]

Müller,K., Arndt,K.M. and Plückthun,A. (1998b) FEBS Lett., 432, 45–49.[ISI][Medline]

Plückthun,A. and Pack,P. (1997) Immunotechology, 3, 83–105.

Rheinnecker,M., Hardt,C., Ilag,L.L., Kufer,P., Gruber,R., Hoess,A., Lupas,A., Rottenberger,C., Plückthun,A. and Pack,P. (1996) J. Immunol., 157, 2989–2997.[Abstract]

Ridgway,J.B., Presta,L.G. and Carter. P. (1996) Protein Engng, 9, 617–621.[Abstract]

Stone,M.J. and Metzger,H. (1968) J. Biol. Chem., 243, 5977–5984.[Abstract/Free Full Text]

Suresh,M.R., Cuello,A.C. and Milstein,C. (1986) Methods Enzymol., 121, 210–228.[ISI][Medline]

Vaughan,T.J., Williams,A.J., Pritchard,K., Osbourn,J.K., Pope,A.R., Earnshaw,J.C., McCafferty,J., Hodits,R.A., Wilton,J. and Johnson,K.S. (1996) Nature Biotechnol., 14, 309–314.[ISI][Medline]

Zapata,G., Ridgway,J.B.B., Mordenti,J., Osaka,G., Wong,W.L.T., Bennet,G.L. and Carter,P. (1995) Protein Engng, 8, 1057–1062.[Abstract]

Zhu,Z., Ghose,T., Lee,S.H.S., Fernandez,L.A., Kerr,L.A., Donohue,J.H. and McKean,D.J. (1994) Cancer Lett., 86, 127–134.[ISI][Medline]

Zhu,Z., Lewis,G.D. and Carter,P. (1995) Int. J. Cancer, 62, 319–324.[ISI][Medline]

Zhu,Z., Zapata, G, Shalaby,M.R., Snedecor,B., Chen,H. and Carter,P. (1996) Bio/Technology, 14, 192–196.[ISI][Medline]

Zhu,Z., Rockwell,P., Lu,D., Kotanides,H., Pytowski,B., Hicklin,D.J., Bohlen,P. and Witte,L. (1998) Cancer Res., 58, 3209–3214.[Abstract]

Zhu,Z., Lu,D., Kotanides,H., Santiago,A., Jimenez,X., Simcox,T., Hicklin,D.J., Bohlen,P. and Witte,L. (1999) Cancer Lett., 136, 203–213.[ISI][Medline]

Received September 30, 1999; revised January 21, 2000; accepted February 20, 2000.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (9)
Request Permissions
Google Scholar
Articles by Zuo, Z.
Articles by Zhu, Z.
PubMed
PubMed Citation
Articles by Zuo, Z.
Articles by Zhu, Z.