Format of bispecific antibodies (BsAbs)-Tandem scFv

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SOCAIL MEDIA

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.

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.

Format	Description	Example BsAb	Trademark	Company
tandem VHH	Tandem VHH fragment-based BsAb	N/A
tandem scFv	Tandem ScFv fragment-based BsAb	AMG330	BiTE^TM	Amgen
Dual-affinity re-targeting antibody	Tandem domain-exchanged Fv (can also be used to fuse with Fc domain to create whole Abs)	Flotetuzumab	DART^TM	Macrogenics
Diabody	dimer of single-chain Fv (scFv) fragment	vixtimotamab	ReSTORE^TM	Amphivena Therapeutics
(scFv)2-Fab	a Fab domain and two scFv domains bind	A-337	ITab^TM	Generon/EVIVE Biotech
Rat–mouse hybrid IgG	Full-size IgG-like half antibodies from two different species	Catumaxomab	Triomab^TM	Trion Pharma
Hetero heavy chain, Common light chain	Hetero heavy chain, Common light chain	Emicizumab	ART-Ig^TM	Genentech/ Chugai/Roche
Controlled Fab arm exchange	Recombin the parental half antibodies	JNJ-64007957	Duobody^TM	Genmab/ Janssen
Hetero H, forced HL IgG1	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 paring	MEDI5752	DuetMab^TM	MedImmune/ AstraZeneca
cH IgG1	Identical heavy chains; 2 different light chains: one kappa (κ) and one lambda (λ)	NI-1701	κλ body^TM	Novimmune SA
Hetero H, CrossMab	KIH technology; domain crossover of immunoglobulin domains in the Fab region	Vanucizumab	CrossMab^TM	Roche
scFv-Fab IgG	Fab-Fc; ScFv-Fc	Vibecotamab; M802	Xmab^TM (the engineered Fc to enhance the generation of heterodimeric Fc); YBODY^TM	Xencor/Amgen; YZYBio
VH1-VH2-CH1-Fc1(G1) x VL2-VL1-CL-Fc2(G1)	2 binding motif in one half antibody	SAR440234	CODV-Ig^TM	Sanofi
VL1-CL1-VH2-CH2-Fc x VH1-CH1 x VL2-CL2	2 binding motif in one half antibody	EMB-01	FIT-Ig^TM	EPIMAB BIOTHERAPEUTICS
VH-1-TCR Cα x VL-1-TCR Cβ; VH-2-CH-2-Fc x VL-2-CL-2	KIH technology; TCR Cα/Cβ is used to substitute the CH1 and CL domain in one arm		WuXibody^TM	WuXi Biologics
C-terminal linker of Fc	Link the other molecules at the C-terminal of Fc	APVO442	ADAPTIR-FLEX^TM	Aptevo Therapeutics
Fc antigen binding site	2 natural binding sites; 2 additional binding sites in the Fc loop	FS118	mAb²	F-star Therapeutics