A dimeric bispeci¢c miniantibody combines two speci¢cities with avidity

Abstract Bispecific antibodies extend the capabilities of natureand might be applied in immunotherapy and biotechnology. Byfusing the gene of a single-chain Fv (scFv) fragment to a helicaldimerization domain, followed by a second scFv fragment ofdifferent specificity, we were able to express a functional proteinin E. coli, which is bispecific and has two valencies for eachspecificity. The dimeric bispecific (DiBi) miniantibody preservesthe natural avidity of antibodies in a very small-sized molecule ofonly 120 kDa. The generality of the principle was shown with ascFv fragment binding the EGF-receptor (named scFv 425) inthree combinations with scFv fragments either directed againstCD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein(FITC-E2). Binding was analyzed by sandwich surface plasmonresonance biosensor (BIAcore) measurements.z 1998 Federation of European Biochemical Societies.Key words: Protein design; Dimerization domain; Bispeci¢cantibody; scFv fragment1. IntroductionIn natural antibodies the Fc part is utilized as the secondeiector site and also serves to dimerize the antigen bindingsites. A potentially much wider scope of applications becomesaccessible by engineered antibody constructs with two diier-ent speci¢cities. So far, most of the bispeci¢c antibodies havebeen produced as hybrid hybridoma IgG [1] or chemicallycrosslinked [2] molecules, which assemble as heterodimericmolecules with one binding site for each speci¢city. However,this approach results in the loss of avidity, also named func-tional a⁄nity, which arises from the simultaneous binding totwo antigens. Depending on the spatial arrangement of theantigen binding sites as well as the antigen surface distribu-tion, the avidity can be signi¢cantly higher than the intrinsica⁄nity [3,4]. In addition, divalency is required for several bio-logical functions such as receptor crosslinking on cell surfaces.For some antibody action divalency is therefore essential [5]. Furthermore, for therapeutic approaches the presence of anFc domain might not always be desired. We therefore con-structed a miniantibody, consisting of four single-chain (scFv)fragments and a dimerization domain, which is at the sametime bispeci¢c and bivalent for each speci¢city.Bispeci¢c antibodies have been considered as useful agentsfor example in the treatment of cancer. They can bind withone side to a tumor cell and with the second speci¢city toimmune eiector cells, which are thereby redirected againstthe tumor, or further molecules of pharmacological interest.Several markers on tumor cells (e.g. EGF-R, HER-2/neu, Ep-CAM/EGP-2, CEA, CD19, CD30) and on immune cells (e.g.CD2, CD3, CD16, CD64) have been investigated for thispurpose [6]. The immune therapy approach with bispeci¢cantibodies has come of age and is already evaluated in clinicaltrials [7]. For targeting of tumors overexpressing the epidermalgrowth factor receptor (EGF-receptor), such as the headand neck tumor, the well characterized murine IgG2a anti-body 425 is a promising molecule. It binds to the EGF-recep-tor while preventing growth stimulation [8] and has alreadybeen used in clinical trials [9,10]. We utilize a scFv fragment[11^13] derived from the monoclonal antibody 425 [14] fordesigning bispeci¢c molecules. For the potential redirectionof cytotoxic T-cells and natural killer cells, a scFv fragmenthas been used which binds CD2 and has been derived from amonoclonal antibody named ACID2.M1 [15^17]. To test thegenerality of this concept of combining various speci¢citiestwo additional model scFv fragments have been studied in afusion protein together with the 425 scFv fragment. One isderived from the monoclonal antibody McPC603 [18,19],which binds the hapten phosphorylcholine, and has been en-gineered for improved in vivo expression [20]. The other scFvfragment is named FITC-E2, binds FITC, and has been de-rived from a phage display library [21]. As a dimerizationdomain we used a helix-loop-helix motif, based on designedpeptides [22], which has been shown previously to work wellfor the functional dimerization of miniantibodies [23,24]. Here we demonstrate that a protein resulting from a genefusion of a scFv fragment to a helix-loop-helix motif andfurther to a second scFv fragment can be functionally ex-pressed in E. coli. We show that the molecule is both bispeci¢cand shows avidity resulting from divalency for each speci¢c-ity. As expression and puri¢cation worked with all three com-binations of speci¢cities tested so far, the fusion strategy pre-sented might be applicable to a wide range of scFv fragments.2. Materials and methods