Three Technologies for Bispecific Antibody Construction

Bispecific antibodies (BsAbs) are a class of bifunctional antibody hybrid molecules. The Fab segments in bivalent antibodies have different specificities and can bind to different ligands. Anti-tumor and anti-immunologically active cells CDl6 or CD3 bispecific antibodies not only have the effect of activating NK cells or T cells, but also can specifically act on tumor cells through anti-tumor Fab segments to increase local NK cell or T cell concentration and enhance the ability of effector molecules to kill tumors.

Immunocytokine can also be prepared by fusion of antibodies with certain cytokines, to enhance the concentration of cytokines near tumor cells, stimulate immune function, effectively kill tumors, and reduce side effects. In addition, human anti-tumor monoclonal antibody or human-derived Fc-segment and murine-derived Fab fragment can be used to overcome the immunogenicity of mouse-derived antibodies, enhance antibody-mediated cytotoxicity, and kill tumors. The BsAb contains two different Fab segments that recognize specific antigens, and can effectively kill tumors by specifically binding tumor antigens while binding different effector cells and molecules.

1 Triomab:

Triomab binds tumor cells and T cells through the Fv function, and recruits FcR-expressing functional cells such as NK cells, monocytes, macrophages, granulocytes and dendritic cells through the Fc domain to form complexes. It stimulates T cells to secrete cytokines to clear tumor cells, so Triomabs is also known as a trifunctional antibody. The Triomabs bispecific antibody technology platform was developed in collaboration with Fresenius, Germany and Trion Pharma.

2 Knobs-into-holes:

This technology was developed by Genentech. The specific method is to mutate the 366-volume threonine (T) of the heavy chain CH3 region of one of the antibodies into a larger tyrosine (Y) to form a prominent "Knobs" type structure (T366Y); The tyrosine (Y) residue of the 407-position larger heavy chain CH3 region of another antibody was mutated to a smaller threonine (T) to form a recessed "holes" type structure (Y407T); Using the steric hindrance effect of the Knobs-into-holes structure allows for the correct assembly of the two different antibody heavy chains. After the mutation, the correct assembly rate of the product is increased from 57% of the wild type to 92%, which can meet the requirements of large-scale production. However, this modification of heavy chain CH3 reduced the stability of antibody structure. In order to overcome this shortcoming, the researchers used phage display technology to perform random mutation screening to construct a more stable "3 + 1" model Knobs-holes. The Knobs-holes structure is designed to facilitate the assembly of two heterologous antibody heavy chains.

3 Crossmab:

RG7221 and RG7716 are representative products of CrossMab, both of which are anti-Ang-2 / VEGF bispecific antibodies. Its structure is based on the "knobs-holes" structure through the chain exchange technology, and the CL in the Fab domain of Ang-2 antibody is interchanged with CH1, while the Fab structure of VEGF antibody remains unchanged. The engineered Ang-2 antibody light chain is not easily mismatched with the heavy chain of vascular endothelial growth factor (VEGF) antibodies, and the "knobs-into-holes" structure promotes heterodimerization of the two heavy chains.

4 Ortho-Fab:

Ortho-Fab is a design strategy that overcomes light chain mismatches reported by Lewis et al. who computerized and combined with X-ray diffraction technology to orthogonally mutate VH/VL and CH1 /CL to reduce light chain mismatch. It can be combined with heavy chain heterodimerization, achieving efficient expression of bispecific antibodies in a single cell. Recently, electrostatic steering has also been applied to the construction of Orthogonal Fab bispecific antibodies. DVD-Ig is another design that retains the Fc domain of an antibody. Its structure is to re-access the VL and VH of another antibody at the N-terminus of the light and heavy chains of normal antibodies, and bind the double by two antibody variable regions. The target achieves dual function. Such molecules have the same Fc region as existing antibodies and can therefore be produced using existing universal antibody technology.

Author Bio

Creative Biolabs has been dedicated to the development of our techniques. Now, with the cutting-edge platforms and methods (quadroma development, chemical conjugation, and genetic engineering), a comprehensive list of bispecific antibody products is available to customers in academia and industry fields. According to modular architecture, Creative Biolabs is experienced in producing five major groups of bispecific molecules: bispecific IgGs, appended IgGs, BsAb fragments, bispecific fusion proteins and BsAb conjugates. In addition, our platforms make us the best choice for production of innovative customized designs. With novel strategies and proprietary Creative Discovery Platform™, Creative Biolabs can always adjust the design of the product to meet client’s requirements on format, valency, flexibility and half-life of the BsAb.