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USA , 90 , — USA , 92 , — Cancer , 62 , — Oxford University Press is a department of the University of Oxford. A standard curve was calibrated using clinical grade antibodies, C or c-p1C Two different assays were carried out to demonstrate the dual specificity of the BsAb. 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. 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. The cells were then lysed and the cell lysate was used for KDR phosphorylation assays. The plates were washed and developed as described above. The spatial relationship or the geometry of the two epitopes could place some degree of restraint on the effectiveness of BsAb binding. Bs scFv 4 -IgG, however, possesses an approximately 8-fold slower association rate k on and a fold faster dissociation rate k off than DAB p4G7. Phosphorylation of KDR was analyzed followed the protocol described previously Zhu et al. We believe this approach is applicable to scFv antibodies recognizing virtually any pair of antigens. The original V H or V L 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 1. 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. 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. The conditioned supernatant was collected at 48 and h after the transfection. Natl Acad. KDR-AP or FlkAP fusion proteins were immobilized onto a sensor chip and various antibodies were injected at concentrations ranging from 25 to nM. As expected, C, a chimeric antibody directed against human EGFR, did not bind to either of the antigens. These unwanted pairings often greatly compromise the yield of BsAb and impose significant problems with purification. Further, the Bs scFv 4 -IgG also retains the antigen-binding efficacy and biological activity of its component antibodies. Association between the heavy and the light chains forms a covalently linked hetero-tetramer with dual specificity. 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. 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. Sensorgrams were obtained at each concentration and were evaluated using a program, BIA Evaluation 2. The mixtures were transferred to a well plate coated with KDR Ig1—3 untagged and incubated at room temperature for 2 h. 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. Co-expression of two different IgGs in a hybrid hybridoma may produce up to 10 heavy- and light-chain pairs Suresh et al. 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. 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, 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. E-mail: zhenping imclone. 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. All constructs were examined by restriction enzyme digestion and verified by dideoxynucleotide sequencing. Note: the diagram is not to scale. 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. The authors thank their colleagues at ImClone Systems, H. 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. 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. 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. 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. 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. The two polypeptides are co-expressed in mammalian COS cells. The mixtures were then transferred to VEGF -coated well plates and incubated at room temperature for an additional 2 h, after which the plates were washed five times. 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. Also shown are the positions of molecular mass standards. 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. Methods , , — Kluwer, Dordrecht, pp. 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 -C H1 -C H2 -C H3 and two copies of scFv 2 -C L fusion polypeptides Figure 1. De Kruif,J. It is also possible to produce a similar molecule that is tetravalent to the same epitope. Kotanides for providing the KDR-transfected cells, K. Further, bivalent binding may cause receptor cross-linking or dimerization which, in many cases, is required to trigger biological responses. 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. The cells were washed and analyzed with a flow cytometer Zhu et al. 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. 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. Light-chain mispairing was prevented by using an identical light chain for each arm of the resulting BsAb-IgG Merchant et al. KDR phosphorylation assay was carried out following the procedure described previously Zhu et al. To whom correspondence should be addressed. In conclusion, we constructed a novel bispecific IgG-like antibody that is also bivalent to each of its target antigen. Chemical cross-linking is often inefficient and can lead to the loss of antibody activity. De Gast,G. 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 major hurdles in traditional methods of BsAb production include heterogeneity of the products, low yield and difficulty with the purification process. Sign In or Create an Account. Bs scFv 4 -IgG retained the biological functions of both of its components. The cells were switched to serum-free medium 24 h after the transfection. Broad clinical evaluation of BsAb has been hampered, however, by the lack of efficient production methods. A Schematic diagrams showing the expression constructs for the BsAb. Sign In.{/INSERTKEYS}{/PARAGRAPH} It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. 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 3B. 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, 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. The BsAb retains not only antigen binding efficiency but also the biological activity of its component antibodies. The tetravalency of the BsAb, plus the capability of intramolecular cross-linking i. 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. This arrangement replaces the original V H and V L domains of an IgG with two scFv molecules, each constituting an independent antigen-binding unit Figure 1. In Bs scFv 4 -Fab, a stop codon is introduced at the C-terminal end of the heavy-chain C H1 domain, which results in the expression of a bivalent, Fab-like bispecific molecule also see Figure 2A. Antibodies of different specificity that use identical light chain are frequently isolated from phage display libraries with limited light-chain diversity Vaughan et al. 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. Lanes 4—6 were run under reducing conditions. High affinities are desirable and may be necessary for each arm of a BsAb destined for human therapy. 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. 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. Because of the avidity factor of bivalent binding, our format may be of great advantageous over the monovalent BsAb. 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. We describe here a recombinant method for the production of BsAb-IgG that eliminates mispairing between antibody heavy and light chains. {PARAGRAPH}{INSERTKEYS}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. First, bivalency may increase binding avidity, which can compensate for the lower affinity of each individual components of the BsAb. Pereira for performing the FACS analysis. Bivalent binding can be of advantageous in several respects. As previously seen with c-p1C11 Zhu et al. Lanes 1—3 are run under non-reducing conditions.