The IgG CH1 and CL domains were also utilized to form heterodimeric, bispecific scFv fusion proteins (Fig. 2, box 4) shown for a bispecific scFv-CH1/scFv-CL fusion protein directed against EGFR and CD2 and using the human γ1 CH1 and Cκ domains as heterodimerization module.121 The CH1 and CL domains are covalently linked through a naturally occurring disulfide bond, which results in stable heterodimers. The option of varying the length of the flexible linkers connecting the N-terminal scFvs to the constant domains to allow molecules to span distant antigens was discussed. The CH1-CL heterodimerization module was further applied to generated bispecific single-domain antibody fusion proteins (Fig. 2, box 4), e.g., for the retargeting of NK cells to tumor cells through binding to CD16 and carcinoembryonic antigen (CEA).122 Extending the CH1 with the hinge region futher allows homodimerization of two scFv-CH1-hinge/scFv-CL fusion proteins, generating tetravalent bispecific molecules (Fig. 2, box 17). This was applied to bispecific antibodies targeting HIV gp41 and the IgA receptor (CD89) on neutrophils.123 Significant antibody-dependent cell-mediated viral inhibition (ADCVI) was observed for the bivalent scFv-CH1/scFv-CL fusion protein and the tetravalent scFv-CH1-hinge/scFv-CL fusion protein, while a tandem scFv against the same targets was described to be inactive.
X-force Fusion Connect 2011 Key
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Fusion of a VH domain to the C-terminus of one Fc(kih) chain and the VL domain either expressed separately or fused to the C-terminus of the other resulted in a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein, with the Fv stabilized by a interdomain disulfide bond (Fig. 2, box 8).158 Flexibility of the Fv in the IgG-Fv fusion could be further increased by introducing a proteolytic cleavage site, e.g., for furin or MMP, in the linker connecting the VL domain with the Fc chain (Fig. 2, box 8). After cleavage, this resulted in a bispecific molecule with the C-terminal Fv connected only through the VH domain to the IgG. Similarly, a scFv-Fc-Fv fusion protein was generated that exhibited two binding sites for EGFR (scFv fused to the N-terminus of the Fc chain) and one for LPS (VH fused to the C-terminus of the Fc(knob) and VL fused to the C-terminus of Fc(hole)) (Fig. 2, box 9).162 Further derivatives of bispecific IgG(kih) antibodies include TriMAbs.157 Here, one or two disulfide-stabilized scFvs are fused to one or both Fc(kih) chains resulting in trispecific, trivalent or tetravalent antibodies, respectively (Fig, 2, box 8). This was shown for TriMAbs targeting EGFR, IGF-1R and either cMet or HER3. In this approach, the Fab was composed of a single-chain Fab (see section 3.3) with disulfide-stabilized Fv domains (scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv).
Several of the Fc modifications described above utilized scFvs fused to the Fc chains to generate bispecific antibodies in order to circumvent the light chain problem.155 Based on the finding that Fabs can be expressed as single-chain derivatives (scFab to connect the C-terminus of the light chain with the N-terminus of the VH domain), full IgG molecules were generated by the expression of a single polypeptide comprising a light chain connected to a heavy chain.192-194 Linkers with a lengths of 30 residues, e.g., (G4S)6, to 38 residues have been utilized, including deletions of the connecting disulfide bond between CH1 and CL. An improved scFab platform was described for disulfide-linked scFab molecules using a linker of 60 flexible residues.195 A (G4S)6 linker was applied to generate a bispecific Fab-Fc fusion protein combined with knobs-into-holes mutations in the Fc region, which was further modified through C-terminal fusion of scFvs to obtain trispecific, trivalent and tetravalent molcules (Fig. 2, box 7, 9), e.g., for targeting EGFR, IGF-1R, and either cMet or HER3.157 This was recently extended to generate tetravalent, tetraspecific IgG-scFv fusion proteins directed against EGFR, IGF-1R, cMet, and HER3 by utilizing either one Fab and one scFab arm, or two scFab with different specificity (Fig. 2, box 8).196 The scFab format was also combined with the LUZ-Y Fc heterodmerization strategy (Fig. 2, box 7). Here, proteolytic cleavage sites were introduced into the Fab linker to allow removal of the linkers from the correctly assembled bispecific IgG molecules.185 Furthermore, scFab were combined with unmodified Fabs to generate bispecific Fab-scFab-Fc fusion proteins in combination with Fc(kih) (OAscFab-IgG format), as shown for a bispecific IgG targeting EGFR and IGF-1R, which could be expressed at high yields.160
This approach was first described in 1997 by Coloma and Morrison107 who fused an anti-dansyl scFv to either the C-terminus of the heavy chain CH3 domain of an anti-dextran antibody or to the C-terminal end of the hinge region. A short G3S linker was used to connect the C-terminus of the heavy chain fragment to the N-terminus of the VH domain of the scFv. Coexpression of the corresponding anti-dextran light chain resulted in an IgG-HC-scFv (CH3-scFv) or F(ab')2-scFv2 (Hinge-scFv) fusion protein comprising four binding sites, two for each antigen (Fig. 2, box 10, 16). The molecular mass of these bispecific fusion proteins is 200 kDa for the IgG-scFv and 150 kDa for the F(ab')2-scFv2. These bispecific antibodies were able to bind to both antigens, although a somewhat reduced affinity for the scFv-encoded specificity was found.
A critical issue to be considered for IgG scFv fusion proteins is the linker connecting the scFv moiety to the IgG. The linker has to be stable and ideally flexible, and fusion should not interfere with antigen binding activity of the scFv and the IgG binding site. Fusing the scFv to the C-terminus of either the heavy or light chain keeps the IgG binding site unaffected. However, in this case the connection will be to the N-terminus of the scFv, i.e., close to its antigen-binding site. Typical linkers used for IgG-scFv fusion proteins are composed of 2 or 3 repeats of G4S.216,217 Also other linkers such as a hydrophilic helical linker, e.g., with the sequence SNS(EEAKK)3SNS, have been used to fuse an scFv to the heavy chain C-terminus.104 Short linkers might restrain antigen binding, although recently a three residue linker (GSS) was successfully used to generated an IgG-HC-scFv targeting HER2 and HER3.218
Furthermore, the intrinsic stability of the scFv might affect manufacturability. Therefore, scFvs were optimized for stability by using a longer (20 amino acid residues) linker to connect the VH and VL domain and screening of appropriate positions to introduce an additional disulfide bond between the two domains.110,111,216 Combining the longer linker with a disulfide bond between positions VH44 and VL100 increased the thermal stability of an anti-LTβR scFv by 13 C, resulting in bispecific anti-TRAILR2 x anti-LTβR IgG-scFv and scFv-IgG fusion proteins with properties desirable for pharmaceutical development.
Disulfide linkage was also applied to generate improved versions of bispecific IgG-scFv of the Morrison format (IgG-HC-scFv) stabilizing an anti-digoxigenin-specific scFv moiety through VH-VL interdomain disulfide bonds (VHCys44 to VLCys100) fused to the C-terminus of the heavy chain or hinge region of antibodies of different specificity, including HER2, IG1-R, CD22, and LeY.217,219 Here, the scFv was connected to the heavy chain by two repeats of G4S. All antibodies retained binding specificity and affinity. The stability of these bispecific antibodies was demonstrated by a lack of aggregation propensity. This approach was subsequently applied to other antibodies, e.g., targeting cMet, HER1, HER2, or HER3.106 The influence of linker length was also studied for bispecific IgG scFv fusion proteins targeting two different epitopes of HIV receptor CCR5.209 Linkers of 3 to 6 G4S repeats were analyzed in non-disulfide-stabilized scFvs. Stable molecules with yields similar to that of the parental IgG could be obtained using scFv with a 30 residue long linker, but also for disulfide-stabilized scFv with a 15 residue linker.
Brünker and coworkers243 generated a bispecific IgG-Fab molecule by applying the CrossMab technology to the second Fab fused through a (G4S)4 connector to the C-terminus of an IgG molecule (Fig. 2, box 7). This approach, in which the VH of the fused Fab is linked to a CL domain, and the VL domain to a CH1 domain, was applied to generate a tetravalent, bispecific antibody directed against fibroblast activation protein (FAP), for targeting of activated tumor stroma fibroblasts, and DR5 (TRAIL receptor 2), to induce apoptosis by activation of this death receptor (Fig. 2, box 10). Differences were observed for a VHCL and VLCH1 configuration. The VLCH1 format, with the VL domain fused to the C-terminus of the HC, showed, compared to the parental antibody, reduced binding to FAP, while binding activity was retained in the VHCL format (VH domain fused the HC). In combination with a knobs-into-holes Fc region, this approach can also be applied to generate trivalent, bispecific IgG-Fab fusion proteins.243
Fab-IgG fusion proteins were also generated using the charged residue mutations or hydrophobicity-polarity-swap mutations introduced into the CH1-CL interface (see above).204 In this study, the Fd fragment of a first antibody was fused to a hinge sequence (either wild-type or with the two cysteine mutated to serines), then connected by a STPPTPSPSGG linker to the N-terminus of the heavy chain carrying the CR3 or MUT4 mutations in the CH1 domain. The cognate light chain of the inner Fab binding site carried the corresponding mutations. Thus, molecules with heavy chains containing the wild-type hinge sequence were covalently linked by disulfide bonds in the additional hinge between the two Fd sequences (Fig. 2, box 10). Functionality was demonstrated for a tetravalent, bispecific Fab-IgG fusion protein directed against HLA-DR (outer Fab arm) and CD5 (inner Fab arm). Good stability, limited aggregation and in vivo activity was observed for the CD5 x HLA-DR(CR3) molecule. Molecules with a cysteine-free hinge in the inter-Fab region showed a somewhat reduced binding and where slightly less effective, e.g., in complement activation. 2ff7e9595c
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