Format of bispecific antibodies (BsAbs)-Tandem scFv


A tandem scFv links two or more scFvs with the helical peptide linkers in the orientation NH2–VL1–VH1–(linker–VL2–VH2)n–COOH, resulting in a single chain bivalent and bi-specific molecule encoded by a single gene (Figure 2). Tandem scFv can be used to target two different antigens on two different cells, two different antigens on the same cell, or two different epitopes on the same antigen with increased avidity.

Figure 2. The tandem scFv platform. (A) A monospecific bivalent tandem scFv composed of two identical scFvs joined by a helical linker. (B) A bispecific bivalent scFv composed of two different scFvs joined by a helical linker (adopted from: Bates A, Power CA. (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies, 8:28).

Bispecific T-cell engagers (BiTE) are a type of tandem scFv used to redirect cytotoxic T-cells to tumors. The induced response is highly selective against the target tumor cells (Figure 3). It contains two antigen binding sites. The first is focused towards a tumor antigen, whereas the second is focused towards the T-cell receptor (TCR) signaling complex CD3. Simultaneous binding of the BiTE to CD3 and the tumor antigen evades pMHC restriction and encourage T-cell activation and cytokine production towards the tumor-directed cytotoxic response, and the activation of other host immune responses. Bispecific T-cell engagers has been used to treat the non-Hodgkin’s lymphoma and Philadelphia chromosome negative acute lymphoblastic. In addition, Bispecific killer cell engagers (BiKEs) and trispecific killer cell engagers (TriKEs) are bi- or trispecific tandem scFvs has been developed to redirect natural killer (NK) cells via an anti-CD16 scFv.

Figure 3. The structure and mechanism of action of MT103, an α-CD3/α-CD19 bispecific T-cell engager (BiTE). The antigen binding site of each parental antibody is isolated and converted into an scFv format. The two scFvs are then joined by a flexible peptide linker to produce a bispecific moiety. The anti-CD19 scFv binds to tumour cells whilst the anti-CD3 scFv will bind passing T-cells, re-directing them to attack the tumour cell (adopted from Bates A, Power CA. (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies, 8:28).

Formats of bispecific antibodies (BsAbs)

Many formats have been developed for BsAb generation as listed in the following table.

FormatSchematic structureDescriptionExample BsAbTrademark Company
tandem VHHTandem VHH fragment-based BsAbN/A
tandem scFvPicture loading failed.Tandem ScFv fragment-based BsAbAMG330BiTETMAmgen
Dual-affinity re-targeting antibodyPicture loading failed.Tandem domain-exchanged Fv (can also be used to fuse with Fc domain to create whole Abs)FlotetuzumabDARTTMMacrogenics
DiabodyPicture loading failed.dimer of single-chain Fv (scFv) fragmentvixtimotamabReSTORETMAmphivena Therapeutics
(scFv)2-FabPicture loading failed.a Fab domain and two scFv domains bindA-337ITabTMGeneron/EVIVE Biotech
Rat–mouse hybrid IgGPicture loading failed.Full-size IgG-like half antibodies from two different speciesCatumaxomabTriomabTMTrion Pharma
Hetero heavy chain, Common light chainPicture loading failed.Hetero heavy chain, Common light chainEmicizumabART-IgTMGenentech/ Chugai/Roche
Controlled Fab arm exchangePicture loading failed.Recombin the parental half antibodies JNJ-64007957DuobodyTMGenmab/ Janssen
Hetero H, forced HL IgG1Picture loading failed.KIH technology for heterodimerization of 2 distinct H chains, replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond to enhance the cognate of H and L paringMEDI5752DuetMabTMMedImmune/ AstraZeneca
cH IgG1Picture loading failed.Identical heavy chains; 2 different light chains: one kappa (κ) and one lambda (λ)NI-1701κλ bodyTMNovimmune SA
Hetero H, CrossMabPicture loading failed.KIH technology; domain crossover of immunoglobulin domains in the Fab regionVanucizumabCrossMabTMRoche
scFv-Fab IgGPicture loading failed.Fab-Fc; ScFv-FcVibecotamab;
XmabTM (the engineered Fc to enhance the generation of heterodimeric Fc);
Xencor/Amgen; YZYBio
VH1-VH2-CH1-Fc1(G1) x VL2-VL1-CL-Fc2(G1)Picture loading failed.2 binding motif in one half antibodySAR440234CODV-IgTMSanofi
VL1-CL1-VH2-CH2-Fc x VH1-CH1 x VL2-CL2Picture loading failed.2 binding motif in one half antibodyEMB-01FIT-IgTMEPIMAB BIOTHERAPEUTICS
VH-1-TCR Cα x VL-1-TCR Cβ; VH-2-CH-2-Fc x VL-2-CL-2Picture loading failed.KIH technology; TCR Cα/Cβ is used to substitute the CH1 and CL domain in one armWuXibodyTMWuXi Biologics
C-terminal linker of FcPicture loading failed.Link the other molecules at the C-terminal of FcAPVO442ADAPTIR-FLEXTMAptevo Therapeutics
Fc antigen binding sitePicture loading failed.2 natural binding sites; 2 additional binding sites in the Fc loopFS118mAb2F-star Therapeutics