Technical Service

Bispecific Antibody


Bispecific antibodies (hetero-conjugated antibodies) are a class of bifunctional hybrid antibody molecules with two different antigen-specific binding Fab segments, which can bind to different ligands and simultaneously bind to tumor antigens by specific binding. Different effector cells and molecules achieve targeted killing of tumor cells.

2.1Bispecific Antibodies with Fc Region

The biological functions of the Fc domain of antibodies include antibody -dependent cell-mediate cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC). There are several technical means for the preparation of bispecific antibodies:

Triomabs: Triomabs bind to tumor cells and T cells respectively through the Fc functional region, and through Fc functional region, they recruit Fc R-expressing functional cells, such as NK cells, monocytes, macrophages, granulocytes, and dendritic cells to form complex that stimulates T cells to secrete cytokines to eliminate tumor cells. Triomabs are also called trifunctional antibodies. The Triomabs bispecific antibody technology platform was jointly developed by the German company Fresenius and Trion Pharma.

Knobs-into-holes: This technology was developed by Genentech. The specific method is to mutate the smaller threonine (T) at position 366 of the heavy chain CH3 region of one of the antibodies to the larger tyrosine (Y) to form a prominent "Knobs" type structure (T366Y); The larger tyrosine (Y) residue at position. 407 of the CH3 region of another antibody heavy chain was mutated to a smaller threonine (T) to form a recessed “holes" type structure (Y407T); using "Knobs - The steric hindrance effect of the "into-holes" structure enables the correct assembly of two different antibody heavy chains. After mutation, the correct assembly rate of the product is increased from 57% of wild type to 92%, which can meet the requirements of large-scale production. However, this modification of the heavy chain CH3 reduces the stability of the antibody structure. To overcome the shortcoming, the researchers conducted random mutation screening through phage display technology and constructed a more stable "3+1" model, Knobs-holes Structure: T366W mutation forms a prominent "Knobs" type, and 3 amino acid mutations (T366S, L368A and Y407V) form a recessed "holes” Type. Knobs-holes structural design facilitates the assembly of 2 hetero logous antibody heavy chains.

Crossmab: The representative products of Crossmab technology are Roche's RG7221 and RG7716, both of which are anti-Ang- 2/VEG Fbi specific antibodies. Its structure is based on the “knobs -holes”structure, through chain exchange technology, the CL and CH1 in the Fab domain of the Ang-2 antibody are exchanged, while the Fab structure of the VEGF antibody remains unchanged. The engineered Ang- 2 antibody light chain is not prone to mismatch with the heavy chain of vascular endothelial growth factor (VEGF) antibody, and the “knobs-holes “structure promotes heterodimerization of the two heavy chains.

Ortho - Fab: This technique is a design strategy to overcome light chain mismatches reported by Lewis et al. Lewis et al. carried out orthogonal complementary mutation design of VH/VL and CH1/CL through computer modeling combined with X-crystal diffraction technology, thereby reducing the phenomenon of light chain mismatch. This technology combined with heavy chain heterodimerization method can achieve efficient expression of bispecific antibodies in single cells. Recently, electrostatic steering has also been used to construct Orthogonal Fab bispecific antibodies.

IgG-sc Fv & sc Fv2-Fc: IgG-sc Fv bispecific antibody is a single-chain antibody (sc Fv) linked to the C -

terminus of a normal IgG antibody molecule, and the CDR regions at both ends of the molecule are combined with the target molecule to achieve dual functions. The sc Fv2-Fc molecule is similar in structure to IgG -sc Fv, and two sc Fv molecules are connected at both ends of the Fc functional domain to form a bifunctional domain.

2.2 Bispecific Antibodies without Fc Region

Another type of bispecific antibody does not contain an Fc region, which has the advantage of a small relative molecular mass, can be expressed in prokaryotic cells and can more easily pass through tissues and tumor cells to reach the target; the disadvantage is that the absence of the antibody Fc region does not mediate the corresponding biological function and the half-life is usually short. For example, the blood half-life of the marketed blinatumomab is only 2.11 h, and it needs to be continuously administered by a syringe pump for 28 days. At present, such bispecific antibodies mainly include BiTE, DART, TandAbs, and bi-Nanobody, etc.

BiTE Bispecific Antibody: BiTE series products developed by Micromet company in Germany are typical representatives of bispecific antibodies without Fc region structure. BiTE is obtained by linking anti-CD3 single-chain antibody (sc Fv) with different anti-tumor cell surface antigen single-chain antibodies through peptide fragments, which can simultaneously bind CD3-positive T cells and tumor cells. BiTE recruits T cells to the surface of tumor cells by binding to CD3 on the surface of T cells, thereby activating T cells for tumor killing. BiTE technology researchers overcame the production problems of poor sc Fv stability, low expression, and low solubility, and successfully commercialized BiTE products.

Bi-Nanobody Bispecific Antibody: Nanobody is Ablynx's reference camel and llama single-domain antibody structure (without light chain and CH1 region) ,a patented platform technology developed by simplifying the structure and retaining only the VH region. In practical applications, Nanobodies link the VH regions of two or more antibody molecules to achieve multispecific binding. The main advantages of this type of products are small molecules, high stability, easy humanization, easy connection, and can be administered through a variety of routes. In addition, a functional domain that binds to human album in can be optionally added during molecular design to extend the half-life to 2 to 3 weeks, and the drug can be transported to the target site through album in.

AtaGenix has an experienced bispecific antibody development team that can provide the design of the bispecific antibody types above.





Time Frame



Antibody development



Gene synthesis & codon optimization

Vector construction

Expression & purification

QC analysis

   5-6 Weeks

Purified antibody

Technical service report

Case Study