Formats of Bispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. Compared to monoclonal antibodies (mAbs), BsAbs have several advantages, such as enhanced clinical efficacy, improved specificity, and expanded functionality. For example, BsAbs can simultaneously target two tumor-associated antigens, block two signaling pathways, recruit immune cells, or deliver cytotoxic agents to tumor cells. BsAbs have been developed for various therapeutic applications, such as cancer, autoimmune diseases, infectious diseases, and hemophilia. The first BsAb, catumaxomab, was approved by the European Medicines Agency in 2009 for the treatment of malignant ascites. Since then, more than 100 BsAbs have entered clinical trials, and several BsAbs have reached late-stage development or market approval. However, the development of BsAbs is not without challenges, as BsAbs have to meet multiple criteria, such as high affinity, specificity, stability, solubility, expression, purification, and safety. To address these challenges, various formats of BsAbs have been designed and optimized, each with its own advantages and disadvantages.

Overview of Bispecific IgG (BsIgG)

Bispecific IgG (BsIgG) is a format of BsAbs that is based on the natural IgG scaffold with two heavy chains and two light chains. BsIgG has the same structure and size as conventional IgG, except that each heavy chain is paired with a different light chain, resulting in two different antigen-binding sites. BsIgG can be generated by several strategies, such as knobs-into-holes, CrossMab, dual variable domain, and asymmetric Fc. These strategies aim to ensure the correct assembly and expression of BsIgG, as well as to avoid the formation of unwanted homodimers. BsIgG has several advantages over other formats of BsAbs, such as high stability, long half-life, Fc-mediated effector functions, and compatibility with existing IgG production and purification platforms. However, BsIgG also has some disadvantages, such as potential immunogenicity due to the introduction of unnatural amino acids or domains, and limited diversity due to the constraints of the IgG scaffold. Some examples of BsIgG in development or in clinical trials are emicizumab, faricimab, and epcoritamab. Emicizumab is a BsIgG that binds to factor IXa and factor X, mimicking the function of factor VIII, and is approved for the treatment of hemophilia A. Faricimab is a BsIgG that binds to vascular endothelial growth factor (VEGF) and angiopoietin-2, and is in phase III trials for the treatment of diabetic macular edema and age-related macular degeneration. Epcoritamab is a BsIgG that binds to CD3 and CD20, and is in phase I/II trials for the treatment of B-cell malignancies.

Overview of Appended IgG

Appended IgG is a format of BsAbs that is a fusion of a conventional IgG with an additional antigen-binding moiety. Appended IgG has a similar structure and size as conventional IgG, except that it has an extra domain attached to the N-terminus or the C-terminus of the heavy chain or the light chain, resulting in three or four different antigen-binding sites. Appended IgG can be generated by several strategies, such as scFv-Fc and diabody-Fc. These strategies aim to increase the valency, flexibility, and diversity of BsAbs, as well as exploit the benefits of the IgG scaffold. Appended IgG has several advantages over other formats of BsAbs, such as increased avidity, enhanced functionality, and modularity. However, appended IgG also has some disadvantages, such as potential steric hindrance, aggregation, and immunogenicity due to the presence of the extra domain. One example of appended IgG in development or in clinical trials is blinatumomab, an appended IgG that consists of two scFv domains fused to the C-terminus of the heavy chain, and binds to CD3 and CD19, and is approved for the treatment of acute lymphoblastic leukemia.

Overview of Bispecific Antibody Fragments

Bispecific antibody fragments are small molecules composed of two antigen-binding domains without an Fc region. Bispecific antibody fragments have a lower molecular weight and a simpler structure than conventional IgG or appended IgG, resulting in two different antigen-binding sites. Bispecific antibody fragments can be generated by several strategies, such as scFv, diabody, and tandem scFv. These strategies aim to reduce the size, increase the penetration, and enhance the versatility of BsAbs, as well as to avoid the limitations of the IgG scaffold. Bispecific antibody fragments have several advantages over other formats of BsAbs, such as low immunogenicity, high expression, and easy modification. However, bispecific antibody fragments also have some disadvantages, such as a short half-life, low stability, and a lack of Fc-mediated effector functions. Some examples of bispecific antibody fragments in development or in clinical trials are SAR441236, ABT-981, and ALX-1141. SAR441236 is a bispecific antibody fragment that consists of two scFv domains linked by a flexible peptide, binds to CD28 and CD3, and is in phase I trials for the treatment of solid tumors. ABT-981 is a bispecific antibody fragment that consists of two tandem scFv domains, binds to interleukin-1α (IL-1α), and interleukin-1β (IL-1β), and is in phase II trials for the treatment of osteoarthritis. ALX-1141 is a bispecific antibody fragment that binds to lipopolysaccharide (LPS) and CD14 and is in phase I trials for the treatment of sepsis.

Overview of Bispecific Antibody Conjugates

Bispecific antibody conjugates are molecules that combine two antigen-binding domains with a payload, such as a toxin, a cytokine, or a radionuclide. Bispecific antibody conjugates have a complex structure and a high molecular weight, resulting in two different antigen-binding sites and a cytotoxic or immunomodulatory effect. Bispecific antibody conjugates can be generated by several strategies, such as chemical conjugation, genetic fusion, and click chemistry. These strategies aim to enhance the cytotoxicity, specificity, and functionality of BsAbs, as well as to overcome the resistance and toxicity of conventional antibody-drug conjugates. Bispecific antibody conjugates have several advantages over other formats of BsAbs, such as improved efficacy, reduced dosage, and broadened applicability. However, bispecific antibody conjugates also have some disadvantages, such as potential toxicity, immunogenicity, and complexity due to the presence of the payload. Some examples of bispecific antibody conjugates in development or in clinical trials are catumaxomab, RG7828, and 177Lu-IPN01087. Catumaxomab is a bispecific antibody conjugate that consists of a rat-mouse hybrid antibody chemically linked to a human anti-EpCAM antibody, binds to CD3 and EpCAM, and is approved for the treatment of malignant ascites. RG7828 is a bispecific antibody conjugate that consists of a humanized anti-CD20 antibody genetically fused to a humanized anti-CD3 antibody, binds to CD3 and CD20, and is in phase I trials for the treatment of B-cell malignancies. 177Lu-IPN01087 is a bispecific antibody conjugate that consists of a humanized anti-CEA antibody chemically conjugated to a humanized anti-CD3 antibody, binds to CD3 and CEA, and is in phase I trials for the treatment of solid tumors.

Overview of Bispecific Fusion Proteins

Bispecific fusion proteins are molecules that combine two antigen-binding domains with a non-antibody protein, such as a receptor, a ligand, or an enzyme. Bispecific fusion proteins have a hybrid structure and a variable molecular weight, resulting in two different antigen-binding sites and novel functionality. Bispecific fusion proteins can be generated by several strategies, such as genetic fusion, peptide linkers, and glycosylation. These strategies aim to create novel functionality, modularity, and diversity of BsAbs, as well as exploit the benefits of the non-antibody protein. Bispecific fusion proteins have several advantages over other formats of BsAbs, such as novel functionality, modularity, and diversity. However, bispecific fusion proteins also have some disadvantages, such as potential immunogenicity, instability, and complexity due to the presence of the non-antibody protein. Some examples of bispecific fusion proteins in development or in clinical trials are MEDI-565, AMG 110, and ALX-0171. MEDI-565 is a bispecific fusion protein that consists of a humanized anti-CD3 antibody genetically fused to a humanized anti-CEA antibody, binds to CD3 and CEA, and is in phase I trials for the treatment of solid tumors. AMG 110 is a bispecific fusion protein that consists of a humanized anti-CD3 antibody genetically fused to a human epidermal growth factor receptor 2 (HER2) ligand, binds to CD3 and HER2, and is in phase I trials for the treatment of HER2-positive cancers. 

Conclusion

BsAbs are a promising class of therapeutics that can overcome the limitations of MoAbs and provide novel functionality, specificity, and efficacy. BsAbs have been developed for various therapeutic applications, such as cancer, autoimmune diseases, infectious diseases, and hemophilia. However, BsAbs are not without challenges, as they have to meet multiple criteria, such as high affinity, specificity, stability, solubility, expression, purification, and safety. Moreover, BsAbs have to face the regulatory hurdles, manufacturing issues, and market competition that are common to all biologics. To address these challenges, various formats of BsAbs have been designed and optimized, each with its own advantages and disadvantages. The choice of the optimal format depends on the desired target, mechanism of action, pharmacokinetics, and pharmacodynamics of the BsAb. Future directions of BsAbs research and development include the discovery of new targets and mechanisms of action, the optimization of the existing formats and the development of new formats, the improvement of the production and purification processes, and the evaluation of the safety and efficacy of BsAbs in clinical trials and real-world settings.

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