Department of Molecular and Cell Biology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA
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Abstract |
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Keywords: antibody engineering/bispecific antibody/KDR/single-chain Fv/VEGF
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Introduction |
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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., 1984; Zhu et al., 1994
) or co-expressing two IgG in hybrid hybridomas (Milstein and Cuello, 1983
; Suresh et al., 1986
). 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., 1986
), 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 heavylight 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 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 1
). The BsAb retains not only antigen binding efficiency but also the biological activity of its component antibodies.
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Materials and methods |
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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., 1998). 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., 2000
). The definition of KDR ECD Ig domain deletion mutants are as follows: KDR(Ig17), the full length KDR ECD containing all seven Ig domains of the receptor (from amino acid Met1 to Val742); KDR(Ig13), the mutant containing the three N-terminal ECD Ig domains (from amino acid Met1 to Lys327); and KDR(Ig37), the mutant containing KDR ECD Ig domains 37 (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., 1998
; Lu et al., 1999
). Diabody DAB p4G7, a form of bivalent scFv fragment (Holliger et al., 1993
; Zhu et al., 1996
) was constructed from scFv p4G7 as described previously (Zhu et al., 1996
; Lu et al., 1999
). C-p1C11, a mousehuman 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., 1999
).
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 2A). 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
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 (
) heavy-chain constant domain (CH) to create the expression vector for the BsAb-IgG heavy chain, BsIgG-H.
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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., 1999). 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 SDSPAGE. 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
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(Ig17)-AP, KDR(Ig13)-AP or KDR(Ig37)-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 antibodyHRP conjugate (Cappel, Organon Teknika, West Chester, PA) for the BsAb and c-p1C11 or mouse anti-E tag antibodyHRP 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., 1998). In the cross-linking assay, the antibodies were first incubated in solution with KDR(Ig17)-AP, KDR(Ig13)-AP or KDR(Ig37)-AP. The mixtures were transferred to a 96-well plate coated with KDR(Ig13) (untagged) and incubated at room temperature for 2 h. The plate was washed and the KDR(Ig13) (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., 1998
).
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., 1999).
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., 1998, 1999
).
KDR phosphorylation assay was carried out following the procedure described previously (Zhu et al., 1998, 1999
), 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 SDSPAGE 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 SDSpolyacrylamide gels.
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Results |
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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 2A). 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., 1998
; Lu et al., 1999
). Epitope mapping studies revealed that p1C11 binds to epitope(s) located within KDR ECD Ig domain 13, whereas the epitope(s) for p4G7 are located within Ig domains 6 and 7 (Lu et al., 2000
). 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 2A
). This arrangement replaces the original VH and VL domains of an IgG with two scFv molecules, each constituting an independent antigen-binding unit (Figure 1
). Co-expression of BsIgG-H and BsIgG-L yields an IgG-like tetravalent molecule, Bs(scFv)4-IgG, with dual specificity (Figure 1
). A bispecific, bivalent Fab-like molecule (Figure 1
), 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 2A
).
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 SDSPAGE (Figure 2B). 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 2B
, 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 2B
, 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 2B
, lane 6). As a control, c-p1C11, a chimeric IgG1 antibody, gave rise to one band of ~150 kDa under non-reducing conditions (Figure 2B
, 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 2B
, 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 3A). As seen previously, p1C11 only binds to KDR mutants containing Ig domains 13 (Zhu et al., 1999
), whereas p4G7 only binds to mutants containing Ig domains 6 and 7 (Lu et al., 1999
). 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 13 and the other to the epitope on Ig domains 6 and 7.
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Antigen binding by BsAb
The antigen binding efficiency of the BsAb was determined on immobilized KDR (Figure 4A) and Flk-1 (Figure 4B
). Figure 4A
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 4B
). As expected, C225, a chimeric antibody directed against human EGFR, did not bind to either of the antigens.
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The binding kinetics of the BsAb to KDR and Flk-1 were determined by surface plasmon resonance using a BIAcore instrument (Table I). 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 410-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 520-fold weaker than those of their bivalent counterparts.
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Figure 5 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.
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The biological effect of Bs(scFv)4-IgG on VEGF-induced receptor phosphorylation was determined using KDR-transfected 293 cells. As shown in Figure 6, 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 6
). Further, Bs(scFv)4-IgG is equally potent as c-p1C11 at each antibody concentration assayed.
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Discussion |
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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., 1986). 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, 1997
; Merchant et al., 1998
; 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-chainscFv fusion. Co-expression of the antibody light chain along with the modified heavy-chainscFv 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, 1997
). 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., 1998
). 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., 1996
). Light-chain mispairing was prevented by using an identical light chain for each arm of the resulting BsAb-IgG (Merchant et al., 1998
). Antibodies of different specificity that use identical light chain are frequently isolated from phage display libraries with limited light-chain diversity (Vaughan et al., 1996
; Merchant et al., 1998
). 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., 1998
). 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 1
). 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 3B
). 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., 1995). 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 ~1023-fold higher than that of their respective monovalent counterparts, the divalent Bs(scFv)2-Fab and the scFv p4G7 (Table I
), 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 1
). 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 57-fold lower than that of both the bivalent c-p1C11 and DAB p4G7 (Table I
). 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, 1968
). 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 4). The BsAb also binds to the surface-expressed KDR molecule on human endothelial cells. Further, the BsAb blocks KDRVEGF interaction and efficiently neutralizes VEGF-induced KDR receptor phosphorylation in a dose-dependent manner (Figures 5 and 6
). 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 KDRVEGF 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., 1996
; Merchant et al., 1998
).
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, 1997; Merchant et al., 1998
).
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Notes |
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Acknowledgments |
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Received September 30, 1999; revised January 21, 2000; accepted February 20, 2000.