To engineer bispecific antibodies, the early stages of development require a lot of planning in the design phase. ProMab Biotechnologies experience in the antibody development field have allowed us to successfully develop a platform to design, engineer and generate bispecific antibodies.
Alongside our antibody development platform, we have a dedicated team to help you engineer and develop these bispecific antibodies to meet every criteria you need to further your research.
ProMab's Advantage In Designing Bispecific Antibodies
Our extensive antibody development platform gives us a huge advantage over our competitors.
For example, here is a list of antibodies with sequences that can be used for bispecific antibody design.
|Antibody||Sequence Source||Clone #||Humanized?||For Licensing?|
|Mesothelin||Public||P4 / 7E6A6|
Example Bispecific Antibody Designs
There are hundreds of different combinations of bispecific antibodies. A few have been highlighted here:
Triomab / Quadroma
Fusion of two antibody-producing cell lines, e.g., generating a hybrid-hybridoma (quadroma), allows the combination of the heavy and light chains of two different antibodies. An unintended consequence from the combination due to the statistical association of the two heavy and light chains were a "HC heterodimerization problem" as well as "LC mispairing problem." The intended compound was only a minor component of a mixture of a variety of species, and it would be nearly impossible to develop a purification method to provide the compound on a scale necessary for therapeutic purposes. To address this problem, the rat/mouse bispecific antibodies (Triomabs) with species-restricted HC and LC chain pairing was developed and reduced the mismatch variants to <10%.
Symmetric bispecific IgG molecules that are modified so that they are able to recognize two different antigens, have been obtained by either modifying the existing antigen-binding site formed by the VH and VL domains (two-in-1 and four-in-1 antibodies) or by grafting an additional binding site into the bottom region of the Fc fragment (mAb2).
DVD-IgG consists from two parental monoclonal antibodies by fusing two variable domains from one parental antibody onto the heavy chain and the light chain of another parental antibody, instead of one variable domain.
The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. The antibody chains are co-expressed recombinantly with mutations to create knob on one end and hole on the other such that heterodimer is the predominant product.
Using an Fc region, which might further comprise the hinge region for covalent connection, bispecific antibodies can be generated by fusing scFvs, to the N- and C-terminus, generating bispecific, tetravalent molecules.
Bi-specific antibodies without Fc region
Diabodies (Db) are bivalent molecules composed of two chains, each comprising a VH and VL domain, either from the same or from different antibodies. The single chain (sc) diabody (Db) format was used to generate tetravalent bispecific molecules. This can be achieved by shortening the middle linker, resulting in head-to-tail assembly of the scDb polypeptide chains into a dimeric molecule (tandem diabody, TandAb).
Fusing two scFvs or bispecific antibody molecules to albumin combines bispecificity with albumin-like properties. Albumin is a plasma protein that lacks any antibody-like effector functions, but exhibits a plasma half-life similar to IgG molecules due to FcRn recycling. Thus fusion of two different scFvs to human serum albumin (HSA), one at the N-terminus and another on C-terminus increased the half-life of the scFvs.
The bispecific tandem scFv format has been applied in cancer immunotherapy for the retargeting of T cells to tumor cells. This format used for bispecific T-cell engager (BiTE) molecules such as (Blincyto_, blinatumomab) approved for the treatment refractory acute lymphoblastic leukemia (ALL). This approach allows further extension of tandem scFvs to generate a trivalent, bispecific molecules.
The approach applied to diabodies with the covalent linkage of the two chains through C-terminal cysteine generated dual-affinity retargeting (DART) proteins.
The Nanobody technology was originally developed based on the discovery that camelidae (e.g. camels and llamas) possess fully functional antibodies that consist of heavy chains only without light chains. Nanobodies are a novel class of proprietary therapeutic proteins based on single-domain antibody fragments that contain the unique structural and functional properties of naturally-occurring heavy chain only antibodies.
Example Bispecific Antibody Drug Candidates
These bispecific antibodies are in different phases of clinical development. ProMab can help you design, engineer, and generate bispecific antibodies utilizing the many different therapeutics in trial.
|CD19 + CD3||Acute lymphoblastic
|AMG420, BI 836909||BCMA + CD3||Multiple Myeloma||1||Boehringer
Ingelheim, Amgen (Micromet)
|Angiopoietin 2 +
|RG7802 (2+1)||CEA + CD3||CEA pos. solid
|DVD-Ig||ABT165||DLL4 + VEGF||Adv solid tumors||1||Abbvie|
|TNFalpha + IL17||Psoriatic arthritis||2||Abbvie|
|TandAb||AFM111||CD19 + CD3||Non Hodgkins Lymphoma, Acute lymphoblastic leukemia||1||Affimed|
|AFM113||CD30 + CD16A||Hodgkins
|DART||MGD006 S80880||CD123 + CD3||AML||1||Macrogenics,
MGD 011, JNJ64052781
|CD19 + CD3||Solid Tumors||2||Macrogenics,
|Bi-nanobody||BI836880||VEGF + Ang2||Solid tumors||1||Ablynx,
|TNF + albumin||Rheumatoid
|Xmab14045 Fab-scFv-Fc||CD123 + CD3||Acute myeloid
|GBR1302 Fab-scFv-Fc||Her2 + CD3||Her2 pos. cancer||1||Glenmark|
- Ulrich Brinkmann a and Roland E. Kontermann. The making of bispecific antibodies, MABS, 2017, VOL. 9, NO. 2, 182–212
- Klein, C., Schaefer, W., & Regula, J. T. (2016). The use of CrossMAb technology for the generation of bi- and multispecific antibodies. mAbs, 8(6), 1010–1020. doi:10.1080/19420862.2016.1197457