Bispecific Antibody Drug Assays

BsAbs are divided into three categories according to their targets: (i) antibodies targeting two different tumor antigens; (ii) antibodies targeting one tumor antigen and one immune-related molecule; (iii) antibodies targeting two immune-related molecules. These three main bispecific antibody fragment formats are: bispecific T-cell engager (BiTE), dual-affinity re-targeting proteins (DARTs) and Tandem diabodies (TandAbs). BiTEs are connected by a linker molecule, which defines the flexibility of the construct and antigen-binding kinetics in conjunction with the specific antigens used. DARTs, on the other hand, have a similar basic structure but include a disulphide linker for additional stability. In the current research scenario. BiTE antibodies are under development with most major pharmaceutical companies for various oncology therapies.

Two other types of bsAbs: bispecific (BiKEs) and trispecific killer cell engagers (TriKEs) consist of either two (BiKE) or three (TriKE) variable antigen regions and activate natural killer cells either by binding to IL16 or containing an IL15 linker. A CD16xCD33 BiKE antibody is currently being studied for treatment of AML.

Since Bispecific antibodies have two different binding sites, their functionality can interfere with multiple surface receptors or ligands. They can also place targets into close proximity, either to support protein complex formation on one cell or trigger contact between cells. Their flexible functionality allows for four main mechanisms of action:

• Blocking immune checkpoints (a hot topic of research!)
• Activating immune cells
• Blocking dual signaling pathways
• Forcing association of protein complexes

Mode of Action of Therapeutic Bispecific Antibodies
Recruitment and Activation of Immune Cells:
Bispecific antibodies bind and activate immune cells at one binding site and recognize specific antigens on tumor cells at the other binding site, which mechanically recruit immune cells (e.g. T cells and NK cells) to the tumor area for killing. Examples include CD3×CD19 BsAbs, CD3×CD20 BsAbs, etc.

Blocking of Dual Signaling Pathways:
The onset and progression of diseases such as tumors usually involve multiple signals, and blocking a single signal may not completely inhibit tumor progression, and sometimes cause activation of other pathways. Bispecific antibodies work by targeting two receptors on the cell surface, inhibiting or activating two signals at the same time. Examples include PD-1 x CTLA-4 BsAbs, HER2 dual epitope BsAbs, etc.

Forcing Association Of Protein Complexes:
Bispecific antibodies can act on factors that are free in the body and promote the formation of protein complexes. A heterodimeric common light chain IgG connects FXIa and FX and thereby overcomes FVIII deficiency.