Introduction to Bispecific Antibody

Introduction of Bispecific Antibody

Bispecific antibodies (BsAbs) represent a class of artificially synthesized antibody molecules that possess the unique ability to simultaneously recognize and bind to two distinct antigens or antigen epitopes. The concept of bispecific antibodies was initially proposed by Nisonoff et al. in 1960, who achieved this by cutting and reassembling two different immunoglobulin molecules. Over time, advancements in molecular biology and protein engineering have led to the development of various methods for preparing bispecific antibodies, such as chemical coupling, hybridoma technology, recombinant DNA techniques, and others. To date, researchers have developed nearly 70 different structures of bispecific antibodies, primarily falling into three categories: fragment-based formats, symmetric formats and asymmetric formats

Fig.1 Formats of Bispecific Antibodies and Scaffolds (Liu, 2017)

Notably, bispecific antibodies offer novel functionalities that monoclonal antibodies cannot achieve. These include the ability to recruit and activate immune cells, interfere with receptor signaling, and induce the association of protein complexes. Bispecific antibodies are derived from monoclonal antibodies and can be considered a subclass of monoclonal antibodies with enhanced functionality. They possess various advantages, such as achieving multi-target synergistic effects, enhancing tumor cell killing efficiency, expanding the range of therapeutic indications, reducing drug dosage and toxic side effects, and more. As a result, bispecific antibodies hold significant promise in various fields, including tumor immunotherapy, infectious disease prevention and treatment, autoimmune disease regulation, and beyond. Presently, three bispecific antibody drugs have received worldwide marketing approval: catumaxomab (Removab), blinatumomab (Blincyto), and emicizumab (Hemlibra), used for treating malignant ascites, B-cell acute lymphocytic leukemia and type A hemophilia, respectively. Additionally, there are hundreds of bispecific antibody drugs are currently in clinical or preclinical development stages, targeting multiple diseases and indications.

Design Principles and Methods of Bispecific Antibodies

The design principles behind bispecific antibodies involve leveraging molecular biology and protein engineering techniques to combine two binding elements targeting different antigens or antigen epitopes within a single molecule. This fusion grants the bispecific antibody the ability to simultaneously recognize and bind to two different targets. The design methods of bispecific antibodies primarily fall into two categories: methods based on chemical coupling or cell fusion and methods based on recombinant DNA technology.

Chemical coupling or cell fusion methods involve linking two different specificity antibody molecules or fragments using specific chemical cross-linkers or cell fusion techniques, thereby producing bispecific antibodies. While these methods offer simplicity and rapidity, they come with the drawbacks such as lower purity and stability of the products, potential mismatched antibodies, and issues related to immunogenicity and toxicity. Catumaxomab (Removab), among the three marketed bispecific antibody drugs, is produced using this approach. On the other hand, methods based on recombinant DNA technology employ gene engineering techniques to connect gene fragments encoding the binding elements of two different antigens or antigen epitopes. This results in the construction of an expression vector for bispecific antibodies, which are then expressed and purified in host cells. The advantages of theis approach include higher purity and stability of the products, as well as the ability to design various structures and functions of bispecific antibodies. However, this method comes with technical challenges such as lower expression levels, higher aggregation tendencies, and chain-related issues. Both blinatumomab (Blincyto) and emicizumab (Hemlibra) among the three marketed bispecific antibody drugs, are produced using this method.

According to whether they contain Fc region (fragment crystallizable), bispecific antibodies can be divided into two main formats: scFv-based (single-chain variable fragment-based) and IgG-like (immunoglobulin G-like). The scFv-based format lacks the Fc region, consisting of only two variable regions. This format offers advantages such as a small molecular weight (approximately 25-30 kDa), facilitating tissue and tumor penetration, while avoiding Fc-mediated non-specific binding and effector functions. However, it has drawbacks, including a short half-life (about 1-2 hours), poor stability, and potential aggregation issues. The scFv-based format further divides into single-chain and multi-chain types, connected either on a single polypeptide chain with a peptide linker (e.g., bispecific T cell engager) or on different polypeptide chains, such as diabodies (dimers), triabodies (trimers), tetrabodies (tetramers). On the other hand, the IgG-like format includes the Fc region, resembling natural IgG structure. This format offers advantages such as a longer half-life (about 10-20 days), better stability, and Fc-mediated effector functions. Nonetheless, it also comes with disadvantages, such as a large molecular weight (around 150 kDa), hindering tissue and tumor penetration, and potential Fc-mediated non-specific binding and immunogenicity. The IgG-like format further divides into symmetric and asymmetric types, preserving the natural IgG symmetric structure with variations in size and structure, such as Knobs-into-Holes (concave-convex matching method), CrossMab (crossing method), dual variable domain-Ig, etc., or breaking the natural IgG symmetric structure and combining different variable or constant regions in one molecule, such as Quadroma (quadroma hybridoma method), COCOMA (covalent connection method), dual-affinity retargeting.

Fig.2 The Construction of Three Main Bispecific Antibody Fragment Molecules (Wang, 2019)

Bispecific Antibody Production Process and Challenges

Bispecific antibodies have great potential for achieving improved therapeutic effects compared to conventional monoclonal antibodies due to their ability to target multiple pathways, recruit immune cells, or deliver toxins. However, the production process of bispecific antibodies is challenging due to their complex structure and diverse formats.

The production process involves connecting gene fragments encoding the binding elements of two different antigens or antigen epitopes, constructing the expression vector of bispecific antibodies, and then expressing and purifying them in host cells. The choice of suitable connection mode, linker, expression system, culture conditions, purification methods, and quality control methods largely depends on the specific structure and function of the bispecific antibodies.

Challenges in the production process of bispecific antibodies includes:

• Structure Design: Different formats of BsAbs, such as scFv-based or IgG-like, offer distinct advantages and disadvantages, including pharmacokinetics, half-life, Fc-mediated effector functions and mechanisms of action. Selecting the optimal format for different target combinations requires thoughtful consideration of these factors.
• Expression Level: The complex structure of BsAbs poses difficulties in their efficient expression in host cells, leading to chain-related problems. Enhancing the expression level can be achieved through optimizing gene sequences, connection modes, linkers, promoters and suitable expression systems based on molecular weight, stability, and post-translational modifications. Mammalian cells are usually preferred for IgG-like bispecific antibodies, while microbial cells may be suitable for smaller and simpler bispecific antibody fragments.
• Purification Difficulty: BsAbs may exhibit multiple pairing forms and mismatched molecules due to their two different antigen binding sites. Purification methods, including affinity chromatography, ion exchange chromatography, and gel filtration chromatography, must be employed to eliminate impurities and reduce aggregation tendency and instability.
• Quality Evaluation: Due to their dual antigen binding sites, BsAbs require evaluation of their affinity, specificity, and cross-reactivity for both targets. Various quality testing methods such as ELISA, SPR, and FACS, are employed to be used to determine the function and activity of BsAbs. Safety, immunogenicity, and resistance of BsAbs also necessitate comprehensive evaluation.

Clinical Applications and Prospects of Bispecific Antibodies

BsAbs have demonstrated significant potential in clinical trials across various indications, particularly in cancer immunotherapy. Their applications encompass redirecting immune cells to tumor cells, blocking two different signaling pathways simultaneously, targeting two different disease mediators, and delivering payloads to specific sites. Over 30 mature commercial technology platforms have been developed, yielding BsAbs with diverse formats and properties. Presently, three types of BsAbs have obtained market approval, and more than 110 types are in various stages of clinical development. Beyond cancer immunotherapy, BsAbs hold promise in treating autoimmune and infectious diseases. They modulate the immune balance by targeting two different cytokines, chemokines, growth factors or immune checkpoints involved in inflammation or immunity. Additionally, BsAbs target multiple  antigens or epitopes on pathogens, enhancing neutralization and pathogen clearance. Several BsAbs have entered clinical trials for indications such as rheumatoid arthritis, systemic lupus erythematosus, psoriasis, osteoarthritis, idiopathic pulmonary fibrosis, COVID-19, HIV, etc. Some of these BsAbs are obexelimab (targeting CD40L and CD40), REGN5678 (targeting IL-17A and IL-17F), REGN2477 (targeting IL-23R and IL-12Rβ1), MCLA-145 (targeting CD137 and PD-L1), AZD7442 (targeting two different epitopes on SARS-CoV-2 spike protein), and 10E8v4.0/PGT121 (targeting two different epitopes on HIV-1 envelope glycoprotein).

BsAbs still face challenges related to production, stability, pharmacokinetics, safety, and efficacy. Optimal formats and dosing regimens must be tailored to the specific mechanisms of action and clinical scenarios. Immunogenicity and toxicity of BsAbs require diligent evaluation and monitoring. Combinations with other therapies, such as checkpoint inhibitors or chemotherapy, hold potential to enhance therapeutic outcomes. BsAbs are anticipated to become a valuable therapeutic arsenal in the treatment of diverse diseases in the near future.

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