Bispecific antibodies (BsAb) are promising biotherapeutics for an increasing range of diseases. There are currently seven BsAb approved by the U.S. Food and Drug Administration (FDA) and/or the European Medicines Agency (EMA) for a range of indications:

• Relapsed or refractory precursor B-cell acute lymphoblastic leukemia
• Bleeding due to hemophilia A
• Non-small cell lung cancer
• Unresectable or metastatic uveal melanoma
• Wet (neovascular) age-related macular degeneration and diabetic macular edema
• Relapsed or refractory follicular lymphoma
• Relapsed and refractory multiple myeloma

Other than these approved bispecific antibodies, there are more than 50 BsAb currently in clinical trials, with the majority of late-stage candidates targeting hematological cancers and solid tumors. The number in preclinical development continues to grow as well.

BsAb structure and mechanisms

BsAb are complex both structurally and functionally. As the name denotes, BsAb have two active regions, one on each arm of the molecule, that simultaneously bind to two epitopes of a single target or two different targets on one cell or two different cells. They have a traditional Y shape composed of two heavy and two light chains. What is unique about BsAb is the heterologous nature of the two arms of the molecule – each arm has a unique active site that binds to a different target antigen.

The dual binding sites of BsAb make their functional pathways complex. BsAb have four primary modes of action.

• Immune Cell Activation: One of the antigen-binding arms of BsAb binds to a target cell antigen while the second binds to an antigen on an immune effector cell. This activates the effector cell to kill the tumor cell.
• Signaling Pathway Blocking: BsAb simultaneously interfere with or completely block multiple signaling pathways. This reduces tumor cell escape and improves therapeutic efficacy.
• Immune Checkpoint Blocking: BsAb target two immune checkpoints on the immune cell surface, creating a synergistic blocking effect. This mechanism is proving to be more effective than single or combinational monoclonal antibody approaches.
• Functional Protein Complex Formation: Each BsAb arm binds to a different protein molecule, forming a functional complex. This mechanism has been used to facilitate blood clotting by forming a complex that helps overcome factor VIII deficiencies in persons with Hemophilia A.

Advantages of bispecific antibodies

The dual binding ability of BsAb has advantages over conventional therapies that target a single binding site. A few of the key advantages are:

• BsAb binding of two targets on the same cell increases drug specificity and reduces off-target side effects.
• Targeting multiple signaling receptors or immune checkpoints on the same cell reduces the development of drug resistance.
• The simultaneous bonding of a BsAb to a cancer cell and an immune cell brings them into proximity of each other which promotes their interaction and improves treatment efficacy.

These advantages increase BsAb drug efficacy and dosing efficiency, which in turn lead to lower treatment costs compared to single bonding drugs and combinational drug approaches.

Challenges in BsAb development

Much progress has been made in bispecific antibody development over the last decade. There are, however, remaining challenges that researchers are endeavoring to address, such as:

• Optimizing target antigen selection to achieve the desired synergistically therapeutic effect
• Developing improved methods that ensure correct matching of heavy and light chains in the antigen-binding arms of the BsAb
• Optimizing dosing protocols to minimize the potential for adverse inflammatory responses, some of which can be life threatening
• Designing BsAb oncotherapies that address perennial barriers such as tumor heterogeneity, mutational burden, and intractable tumor microenvironment

Researchers are using biophysical data in tandem with functional assays to tackle these challenges.

Overcoming the challenges

Scientists are continuously seeking advanced methods to address the challenges they face in their BsAb research. Two researchers who are doing just that are Dr. Elizabeth Christian and Dr. Devon Stork.

Dr. Christian is a senior scientist at AstraZeneca. She measures antibody degradation as part of her research and was in need of a bispecific binding assay that could generate more data without increasing the bench workload or sacrificing sensitivity and accuracy. She started using AlphaPlex™ and found it highly customizable to different aspects of her work. It also increased her throughput while maintaining the high levels of sensitivity and accuracy she required.

Dr. Devon Stork is a principal scientist at Tenza where he is developing probiotics that can be used as a safer and more effective alternative to conventional intravenous drug delivery systems. He found he needed a customized assay to better determine the level of binding activity between a payload protein and a therapeutic target. Dr. Stork chose AlphaLISA™ to screen a small library of hits for improved activity and optimized drug delivery. He found the assay to be highly sensitive, streamlined, and cost-effective.

These and other BsAb researchers are finding ways to overcome their challenges and lead in the design of new biotherapeutics.