A main discrimination of recombinant bispecific antibodies is format and in particular the presence or absecne of an Fc region. Bispecific antibodies (biSpAbs) with no Fc will lack Fc mediated efector functions. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Fc-less bispecific antibody formats:

Diabodies (scFv dimers): Diabodies are scFv dimers in which each chain consists of a variable heavy (VH) domain connected to a variable light (VL) domain using a peptide linker that is too short to permit paring betwen domains on the same chain. Consequently, pairing occurs between complementary domains of two different chains, creating a stable non-covalently bound dimer with two binding sites. 

In the diabody format, the two variable domains are connected by a short linker that is usually 5 resiues, e.g., GGGS. Because the linker lenght is substantially shorter than that required to allow intrachsin assemble of the antigen-bnidng site, which would result in a ScFV, two chains dimerize in a head-to-tail orientation resulting in a compact molecule with a molecular mass similar to tandem scFv. (aobut 5 kDA). Expressing tow chains within the same cell, with either configuration vHA-VLB and VHB-VLA (A and B representing two different specificites) or VLA-VHB and VLB-VHA results in bispecific heterodimers with correct paring of the corresponding variable domains. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Single-chain Fv (scFv)/Single domains specific to different targets genetically fused by peptide linkers: (e.g., WO2008/096158; WO/2007/112940). For reviews, see Enevery Curr Opin Biotechnol, 2009, 29)4), 405-11). 

An scFv is an antibody fragment in which VH and VL domains are joined by a flxible linker that forces them to assemble in a stable manner despite the limited interaction surface. (Fischer “Bispecific antibodies: molecules that enable novel therapeutic strategies” Pathobiology, 2007; 74, 3-14). 

Sgine-chain variable fragments (scFvs) are minimalist forms of a functional antibody, generated by fusing variable domains of the IgG heavy chain (VH) and light chain (VL) through a flexible polypeptide linker. ScFv molecuels have a MW in the range of 25 kDa, with a single antigen binding site that is cimprised of components from each arm of the antibody. (Wang, Design and Production of Bispecific Antibodies” Antibodies, 2019).

Single-domain fusions: Rather than connecting antigen binding sites in a tandem arrangement, single-domain antibodies such as VH or VL domains, VHH and nanobodes can be used to make bispecific molecules. Inclusion of two or more single domain antibodies will result in bivalent, trivalent or even multivalent molecules with one or more specificites. For example, two VHH domains were fused thorugh a long hinge sequence dervied form teh upper hinge of the llama IgG2a, selected for its protease resistance and flexibility. (Kontermann “The making of bispecific antibodies, 9(2), 2017, 182-212)

Bispecific T cell engager  antibodies (BITE®): are recombinant protein constructs made from two flexibly linked antibody derived binding domains. One binding domain is specific for a selected tumor-assocaited surface antigen on target cells; the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. By the design, these antibodies are uniquely suited to transiently connect T cells with target cells and, at the same time, potently activate the inherent cytolytic potential of T cells against target cells. (Battel, WO @016/016859). ‘s binding activity wen assembled. The short flexible linker connecting the two scFvs enables free rotation of the two arms, which is vital for flexible interaction with targeted receptors on the two opposing cells membranes (cytotoxic T cell and Tumor cell) and the subsequent induction of T cell activation. (Wang, Design and Production of Bispecific Antibodies” Antibodies, 2019).

BiTE molecuels ahve been extensively applied in cancer immunotherapy for re-targeting of T cells to tumor cells. They employ scFv fragments form two different mAbs connected by a peptide linker, enabling them to retain each antibody.

–approved BITEs:

Blinatumomab, a bispecific T-cell engager (BiTE) is an example of a formal lacking an Fc region. It is based on the two single-chain variable fragments joined via flexible linker. Blinatumomomab was approved in 2014 for the treatment of Philadelphia chromosome-negative precursor B cell ALL. (Tustian, Biotechnol. Prog., 2018, 34(3)) The FDA and EMA ahve granted approval for blinatumomab, a CD19-CD3-bispecific T cell engager (BiTe)(Dietz “Programmable-multispecific DNA-origami-based T cell engaers” Natura nanotechnology, 2023) 

Dual-affinity re-targeting proteins DARTS): A DART is composed to two Fv fragmetns, with two unique antigne-bnidng sites fomred when two Fc fragmetns hererodimerize. Specifically, Fv1 consists of a VH form antibody A and a VL from antibody B, while Fv2 is made form a VH from antibody B and VL from antibody A. Unlike BiTE antiboides which are connected by a polypetpide linekr, this combination allows dART to mimic natural interation within an IgG molecule. Compared to a BiTE, DART molecules are able to retin potency for both in vitro and in vivo adminsitraiton as well but can be produced at scale with lower aggregation rates. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019).