All about Bispecific Antibodies

What are Monoclonal Antibodies?

Monoclonal antibodies (mAbs) are antibodies that are produced by a single clone of cells or cell line and consist of identical antibody molecules. They can recognize and bind to a specific antigen or epitope with high specificity and affinity. MAbs are produced by hybridoma technology, which involves fusing antibody-producing B cells from an immunized animal with immortalized myeloma cells. The resulting hybrid cells can secrete large amounts of mAbs in culture.

MAbs have many advantages in therapeutics, such as targeting specific molecules or cells involved in disease processes, modulating immune responses, delivering drugs or toxins to specific sites, and enhancing imaging and diagnosis. However, mAbs also have some limitations, such as immunogenicity, toxicity, resistance, and cost. Some examples of approved mAbs and their indications are rituximab for non-Hodgkin lymphoma, trastuzumab for breast cancer, and nivolumab for melanoma.

What Are Bispecific Antibodies?

Bispecific antibodies (BsAbs) are artificial proteins that can simultaneously bind to two different antigens or epitopes on the same antigen. They are derived from mAbs by various engineering and manufacturing methods. BsAbs can differ from mAbs in structure, function, and mechanism of action.

BsAbs have potential benefits in therapeutics, such as engaging two targets at once, redirecting immune cells to kill tumor cells, blocking multiple signaling pathways, and enhancing specificity and efficacy. However, BsAbs also face some challenges, such as complexity, stability, immunogenicity, and scalability. Some examples of BsAbs in development and their mechanisms of action are blinatumomab for acute lymphoblastic leukemia, which recruits T cells to kill CD19-positive B cells; emicizumab for hemophilia A, which bridges factor IXa and factor X to restore blood clotting; and faricimab for age-related macular degeneration, which inhibits both vascular endothelial growth factor and angiopoietin-2 to prevent abnormal blood vessel growth.

Formats of Bispecific Antibodies

Bispecific antibodies (BsAbs) can have different formats, depending on their structure and composition. They can be classified into two main categories: IgG-like and non-IgG-like.

IgG-like BsAbs retain the basic structure of a monoclonal antibody (mAb), with two Fab arms and one Fc region, but each Fab arm can bind to a different antigen or epitope. The Fc region can also have a third binding site, resulting in trifunctional BsAbs. IgG-like BsAbs can be produced by various methods, such as quadroma, knobs-into-holes, CrossMab, and dual variable domain technologies. IgG-like BsAbs have the advantages of long half-life, effector functions, and easy purification, but they also have the disadvantages of complexity, immunogenicity, and aggregation.

Non-IgG-like BsAbs have different formats that do not resemble a conventional mAb, such as single-chain variable fragment (scFv), diabody, tandem diabody, bispecific T-cell engager, dual-affinity re-targeting. Non-IgG-like BsAbs can be produced by various methods, such as chemical conjugation, genetic fusion, and hybridoma fusion. Non-IgG-like BsAbs have the advantages of small size, high specificity, and novel mechanisms of action, but they also have the disadvantages of short half-life, low stability, and difficult production.

Methods to Generate Bispecific Antibodies

There are many methods of generating BsAbs, but the three main categories are quadromas, chemical conjugation, and genetic recombination. Each method has its own advantages and disadvantages and can result in different formats of BsAbs.

Quadromas are hybrid cells that are formed by the fusion of two different hybridoma cells, each producing a unique mAb. The quadromas can secrete BsAbs that have two different Fab arms, but the process is random and inefficient, and requires extensive screening and purification. Quadromas can produce IgG-like BsAbs, such as trifunctional antibodies.

Chemical conjugation is a method that involves the covalent linkage of two different antibodies or antibody fragments, using various cross-linking agents or bioorthogonal reactions. The chemical conjugation can be site-specific or random, and can generate different formats of BsAbs, such as scFv, diabody, and bispecific T-cell engager antibodies. Chemical conjugation can offer modularity, reproducibility, and diversity, but it can also introduce heterogeneity, instability, and toxicity.

Genetic recombination is a method that involves the engineering of DNA sequences that encode for BsAbs, using various techniques such as PCR, cloning, and gene synthesis. The recombinant DNA can be expressed in different host cells, such as bacteria, yeast, or mammalian cells, to produce BsAbs. Genetic recombination can generate different formats of BsAbs, such as knobs-into-holes, CrossMab, and tandem diabody antibodies. Genetic recombination can offer flexibility, scalability, and quality, but it can also involve complexity, cost, and regulatory issues.

Selected Technology Platforms for Bispecific Antibodies

There are many technology platforms for bispecific antibody generation, but some of the most widely used or promising ones are bispecific T-cell engager, DuoBody, and CrossMab. These platforms have different characteristics and can overcome some of the challenges of bispecific antibody engineering and production.

Bispecific T cell engager is a platform developed by Micromet (acquired by Amgen in 2012) that generates bispecific antibodies composed of two single-chain variable fragments (scFvs) linked by a short peptide. One scFv binds to CD3 on T cells, while the other scFv binds to a tumor-associated antigen. These antibodies can recruit and activate T cells to kill tumor cells in a major histocompatibility complex (MHC)-independent manner, and have the advantages of small size, high specificity, and potent cytotoxicity, but they also have the disadvantages of short half-life, low stability, and immunogenicity.

Dual-affinity re-targeting is a platform developed by MacroGenics that generates bispecific antibodies composed of two diabodies, each consisting of two scFvs with different specificities. The diabodies are linked by a flexible linker that allows for optimal orientation and avidity. These antibodies can bind to two different targets on the same or different cells, such as T cells and tumor cells, or two signaling molecules on tumor cells. They can modulate the interactions between cells, molecules, and pathways involved in disease processes, and have the advantages of modularity, diversity, and functionality, but they also have the disadvantages of short half-life, low stability, and immunogenicity. 

DuoBody is a platform developed by Genmab that generates bispecific antibodies with an IgG-like structure, but with two different Fab arms. The Fab arms are produced by separate expression of heavy and light chains, followed by controlled Fab arm exchange. The resulting bispecific antibodies have two different antigen-binding sites and one Fc region. DuoBody antibodies can bind to two different targets on the same or different cells, such as T cells and tumor cells, or two signaling molecules on tumor cells. DuoBody antibodies can modulate the interactions between cells, molecules, and pathways involved in disease processes. DuoBody antibodies have the advantages of long half-life, effector functions, and easy purification, but they also have the disadvantages of complexity, immunogenicity, and aggregation. Epcoritamab is the first approved DuoBody antibody for the treatment of B-cell malignancies.

CrossMab is a platform developed by Roche that generates bispecific antibodies with an IgG-like structure, but with two different Fab arms. The Fab arms are engineered by swapping the CH1 and CL domains between the heavy and light chains, creating CH1-CL and CL-CH1 fusions. The resulting bispecific antibodies have two different antigen-binding sites and one Fc region. CrossMab antibodies can bind to two different targets on the same or different cells, such as T cells and tumor cells, or two signaling molecules on tumor cells. CrossMab antibodies can modulate the interactions between cells, molecules, and pathways involved in disease processes. CrossMab antibodies have the advantages of long half-life, effector functions, and easy purification, but they also have the disadvantages of complexity, immunogenicity, and aggregation. Faricimab is the first approved CrossMab antibody for the treatment of age-related macular degeneration.

Mechanisms of Bispecific Antibodies

Bispecific antibodies (BsAbs) can have different mechanisms of action, depending on their format, target, and function. Some of the main mechanisms of action of BsAbs are:

• T cell redirection: BsAbs can bind to CD3 on T cells and a tumor-associated antigen on tumor cells, and bring them into close proximity. This can activate the T cells and induce them to release cytotoxic molecules, such as perforin and granzyme, that can kill the tumor cells. This mechanism is independent of MHC presentation and T cell receptor specificity, and can overcome tumor immune evasion.
• NK cell redirection: BsAbs can bind to CD16 on NK cells and a tumor-associated antigen on tumor cells, and bring them into close proximity. This can activate the NK cells and induce them to release cytotoxic molecules, such as perforin and granzyme, that can kill the tumor cells. This mechanism is independent of MHC presentation and NK cell receptor specificity, and can overcome tumor immune evasion.
• Dual blockade: BsAbs can bind to two different signaling molecules on tumor cells, such as growth factor receptors or immune checkpoints, and block their activation or interaction. This can inhibit the proliferation, survival, invasion, and metastasis of tumor cells, and enhance the anti-tumor immune response.
• Payload delivery: BsAbs can bind to a tumor-associated antigen on tumor cells and a toxic payload, such as a drug, a toxin, or a radionuclide, and deliver it to the tumor site. This can increase the specificity and efficacy of the payload, and reduce its systemic toxicity and side effects.

Application of Bispecific Antibodies

Bispecific antibodies (BsAbs) have a wide range of applications in various therapeutic areas and indications, such as cancer, infectious diseases, autoimmune diseases, hemophilia, and ophthalmology. BsAbs can address the unmet medical needs and improve the outcomes of patients in these areas by exploiting their unique features and mechanisms of action.

Cancer is one of the main therapeutic areas for BsAbs, as many cancers are driven by multiple factors and pathways that cannot be effectively targeted by conventional monoclonal antibodies (mAbs). BsAbs can enhance the anti-tumor activity of mAbs by engaging immune cells to kill tumor cells, blocking two or more signaling molecules or pathways, or delivering toxic payloads to tumor sites. Some examples of BsAbs in cancer therapy are blinatumomab, catumaxomab, epcoritamab, faricimab, and margetuximab.

Infectious diseases are another important therapeutic area for BsAbs, as many pathogens can evade the host immune system or resist the existing drugs. BsAbs can enhance the anti-infective activity of mAbs by neutralizing two or more strains or variants of the same pathogen, blocking the entry or replication of the pathogen, or recruiting immune cells to eliminate the pathogen. Some examples of BsAbs in infectious diseases are REGN-COV2, a combination of two BsAbs against SARS-CoV-2, and epcotamab, a BsAb against HIV-1.

Autoimmune diseases are another promising therapeutic area for BsAbs, as many autoimmune diseases are mediated by multiple cytokines or immune cells that cause inflammation and tissue damage. BsAbs can modulate the immune system by inhibiting two or more cytokines or receptors, depleting or activating specific immune cells, or inducing immune tolerance. Some examples of BsAbs in autoimmune diseases are emicizumab, a BsAb that restores blood clotting in hemophilia A, and ABT-122, a BsAb that inhibits TNF-alpha and IL-17A in rheumatoid arthritis and psoriatic arthritis.

Ophthalmology is another emerging therapeutic area for BsAbs, as many eye diseases are associated with abnormal angiogenesis or inflammation that affect vision and quality of life. BsAbs can target the underlying causes of eye diseases by inhibiting two or more angiogenic or inflammatory factors, reducing vascular leakage or edema, or protecting the retina or optic nerve. Some examples of BsAbs in ophthalmology are faricimab, a BsAb that inhibits VEGF and angiopoietin-2 in age-related macular degeneration and diabetic macular edema, and RO7248824, a BsAb that inhibits VEGF and PDGF in neovascular age-related macular degeneration.

Clinical Status of Bispecific Antibodies

BsAbs are subject to the same regulatory standards and guidelines as mAbs for the development and approval of therapeutic products. However, BsAbs also pose some specific challenges and considerations, such as the selection of optimal targets and formats, the demonstration of added value and safety over mAbs, and the establishment of reliable and scalable manufacturing processes.

Currently, there are three BsAbs that have been approved by the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA) for clinical use: catumaxomab, blinatumomab, and emicizumab. Catumaxomab is a trifunctional BsAb that binds to EpCAM on tumor cells, CD3 on T cells, and Fc gamma receptors on accessory cells. It was approved by the EMA in 2009 for the treatment of malignant ascites in patients with EpCAM-positive tumors, but it was withdrawn from the market in 2017 due to manufacturing issues. Blinatumomab is a bispecific T-cell engager antibody that binds to CD19 on B cells and CD3 on T cells. It was approved by the FDA in 2014 and by the EMA in 2015 for the treatment of acute lymphoblastic leukemia. Emicizumab is a CrossMab BsAb that binds to factor IXa and factor X, mimicking the function of factor VIII. It was approved by the FDA in 2017 and by the EMA in 2018 for the treatment of hemophilia A.

In addition to the approved BsAbs, there are more than 100 BsAbs that are in various stages of clinical trials for different therapeutic areas and indications. Some of the most advanced BsAbs in clinical development are epcoritamab, a DuoBody BsAb that binds to CD20 on B cells and CD3 on T cells, for the treatment of B-cell malignancies; faricimab, a CrossMab BsAb that binds to VEGF and angiopoietin-2, for the treatment of age-related macular degeneration and diabetic macular edema; and margetuximab, a dual-affinity re-targeting antibody that binds to HER2 and CD16A, for the treatment of HER2-positive breast cancer.

The future prospects and challenges of BsAbs in clinical practice depend on several factors, such as the identification of novel targets and mechanisms, the optimization of the design and engineering of BsAbs, the improvement of the efficacy and safety profiles of BsAbs, and the reduction of the cost and complexity of BsAbs production.

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