Format of bispecific antibodies (BsAbs)-Tandem VHH

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Since 1975, monoclonal antibodies have been successfully used for the diagnosis and therapeutics. In 1993, heavy-chain-only antibodies which bind to antigens was discovered in camelids. The variable domain of heavy chain antibody (VHH) was called as nanobodies and it was anticipated as the better therapeutic antibodies in future due to its high stability, refoldability, and manipulable characteristics. VHH has also been effectively used for the diagnosis, bioimaging, immunoassay and affinity purification. The production of nanobodies is simple and cost effective compared to the polyclonal antibodies. Besides, Nbs are stable in a wide range of temperatures and pH levels. Nanobodies are also compatible with genetic engineering methods, which allow scaffolding and alteration of amino acids to improve binding. Relating to structure, the hydrophilic side of nanobodies, that is not present in conventional antibodies, means they do not have issues with solubility and aggregation. The conventional antibodies do not bind well in grooves or cavities on the surface of the antigen. But, Nanobodies bind with the clefts of the active site of antigen.

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