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The Advantages, Manufacture, and Applications of Bispecific Antibodies
By: Rachel Kaplan


Bispecific antibodies (BsAbs) are antibodies that have two binding sites with different specificities. BsAbs provide advantages over monospecific antibodies, such as increased binding site specificity, better recognition of single antigens with multiple epitopes, and the ability to block two different antigens simultaneously. They address the issue of patients developing resistance to antibody treatment and can reduce expenses associated with multiple monospecific antigens. BsAbs can also recruit one cell type to another, creating a powerful tool for immunotherapy. Bispecific antibodies were first created in the 1960s by Nisonoff and his collaborators, and over time, a multitude of methods were developed to engineer BsAbs. The manufacture of BsAbs is particularly difficult due to the variabilities in recombination of their four polypeptide chains. The knob-in-hole technique is one of the most well-known methods for BsAb engineering, but it has limitations. The SEED method, along with other techniques, address some of these limitations. Three types of BsAbs are commercially available today to treat cancers, yet research in this field continues to advance.


Antibodies are crucial components of the immune system. These specialized, Y-shaped proteins are excellent “scouts” for potential pathogens. They search for and either destroy antigens directly or tag the antigens through the use of unique binding sites. While naturally occurring antibodies have an extensive range of antigen-binding center variants, each individual antibody is monospecific. In other words, the two binding sites located on the tips of antibodies are identical. 1 Bispecific antibodies, or BsAbs, are artificially manufactured antibodies that contain two binding sites with different specificities, whether that be to different antigens or to different epitopes on the same antigen.

Advantages of Bispecific Antibodies:

Bispecific antibodies provide a number of advantages over monospecific antibodies, as they provide more binding site specificity due to their ability to recognize and bind to two different surface antigens. It has been found that many tumors coexpress more than one surface antigen, requiring more than one monospecific antibody. Oftentimes, to approach these types of pathogens more efficiently, one bispecific antibody can be used. 2  Further, if a single antigen has more than one epitope, bispecific antibodies can allow for more effective recognition of that antigen. It is known that single-target antibodies do not sufficiently destroy tumor cells due to the complex signaling pathways and mechanisms associated with many cancers. BsAbs can pose a solution to this issue because of their ability to block two different antigens involved in pathogenesis simultaneously. 3 Bispecific antibodies are especially compelling because of their ability to recruit one cell type to another, such as their ability to target proteins on cancerous B-cells while the other recognition site can recruit immune cells, such as killer T-cells in order to detect and destroy the cancer. BsAbs also address the issue of patients developing resistance to antibody treatment. Due to their increased specificity, BsAbs can help to avoid the development of immunity to treatment. 4 The use of BsAbs can potentially reduce expenses associated with and enhance the development of clinical trials that might otherwise use multiple monospecific antigens. 2 BsAbs can also be useful in creating bridges to combine two protein complexes, which expands their relevance beyond immunotherapy. 5


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Figure 1

Early Development of Antibody Engineering

Bispecific antibodies were first created in the 1960s by Nisonoff and his collaborators. They combined antigen-binding fragments from two different immune cell mixtures in solution and found that they reassociated into “hybrid” antigen-binding fragment complexes. 6

In 1975, Milstein and Köhler developed a technique for the production of large amounts of monoclonal antibodies through the use of hybridomas, which was later applied to the synthesis of bispecific antibodies. By fusing myelomas, or cancerous forms of plasma cells that do not produce antibodies, with wild-type plasma cells that can produce antibodies, hybridomas are formed. These ‘fusion cells’ combine the longevity of myelomas with the antibody-producing properties of plasma cells to allow for extensive production of antibodies. 7 Through the fusion of two separate hybridomas to form a quadroma, identical, bispecific antibodies can be formed in extensive amounts. 8 It was also discovered that these hybrid antibodies can be artificially synthesized by directly chemically conjugating two monospecific antibodies. Over time, a multitude of methods were developed to engineer bispecific antibodies with unique biochemical properties, which allowed for a platform to create potential immunotherapies that be curated to fit specific pharmacological needs. 6

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Figure 2

Limitations of Bispecific Antibody Development:

The development of bispecific antibodies poses a certain challenge due to their molecular makeup and the variabilities in the recombination of their four polypeptide chains. Standard, naturally occurring antibodies are made up of two identical heavy polypeptide chains and two identical light polypeptide chains. These chains are held together with disulfide bonds. At the ends of the Y-shaped structure, there are two identical antigen binding fragments, referred to as the Fab region. 9 Naturally occurring antibodies are monospecific, bivalent, and symmetrical, due to their makeup of two identical heavy chains and two identical light chains. In order to create bispecific antibodies, one heavy and one light polypeptide chain from one type of antibody must be chemically combined with a heavy and light chain from another antibody. Unfortunately, this recombination process results in the formation of undesirable protein complexes. Theoretically, the recombination of heavy and light chains from two different antibodies can result in 16 different complexes, only one of which is bispecific with the others either monospecific or nonfunctional. 10 Even the hybridoma technique results in numerous, non-functional side products, as the hybridoma cells produce two different heavy and two different light chains, allowing for the polypeptide chains to associate in a variety of ways. 6  Purification of the bispecific antibodies from these recombinants is not readily available as is difficult and expensive. 11 To combat this issue, many biochemical techniques have been engineered to allow for preferential formation of functional, bispecific antibodies.  

Methods of Bispecific Antibody Development:

One of the most well-known methods for bispecific antibody engineering is the knob-in-hole technique. This technique was originally developed by Crick in 1952 in order to pack amino acid side chains within alpha helix structures. In 1996, John B. Ridgway and his colleagues applied the knob-in-hole method for the adjustment of protein structure to the manufacture of heterodimer antibody proteins. Essentially, this technique replaces a small amino acid in the CH3 region of the heavy chain with a larger amino acid, creating a protrusion in the protein dimer, or a “knob”. In the other heavy chain (with different antibody specificity), a large amino acid in the CH3 region is replaced with a small amino acid, creating a “hole”. This method structurally facilitates the polypeptide chains with proper recombination. This technique allows for a recombination efficiency of 57%. The knob-in-hole technique is an incredibly novel method to enhance the formation of BsAbs. Still, it has its limitations. Due to the change in amino acids, the stability of the protein structure is negatively affected. Therefore, other methods to create these bispecific antibodies were investigated. 12 

In 2009, Jonathan H. Davis and his colleagues created fusion antibody proteins using a method called strand-exchange engineered domain (SEED). This team aimed to improve upon the classic knob-in-hole approach, as it was found that to optimize the stability of BsAbs made with this technique, a non-native disulfide bridge between the heterodimers was required. 13 To avoid the introduction of an external disulfide bond, the SEED technique to create a more stable heterodimer product was investigated. Two protein derivatives were manufactured from Immunoglobin G and Immunoglobin A CH3 domains. The two derivatives of these parent polypeptides were called AG and GA SEED CH3 domains. This pair of SEED CH3 domains preferentially combine to produce heterodimer antibody proteins. Davis and his team created two different bispecific antibodies using the SEED method as models. 11 This method is particularly significant as it maintains the binding affinity of bispecific antibodies in comparison to their parent, homodimer proteins and allows for the manufacturing of biochemically stable products. 14

Applications in Medicine and Immunotherapy:

Today, three types of bispecific antibodies are commercially available for treatment15 and over 90 BsAbs are currently being investigated in pre-clinical and clinical trials. 2 Catumaxomab, one of these commercially available BsAbs, treats tumor cells by recognizing the EpCAM antigen. It also has specificity for the CD3 antigen on T cells, which, upon binding, stimulates T cells to secrete cytokines and promotes the elimination of tumor cells. Essentially, this BsAb recruits pathogen-fighting T cells directly to the site of the cancerous cell. Blinatumomab, another commercially available BsAb, helps to treat precursor B ALL leukemia due to the Philadelphia chromosome, which is notorious for its complications and difficulties in treatment. Patients with this type of leukemia always have some measurable residual disease even after extensive treatment, which often causes relapse. The Blinatumomab hybrid, by recruiting T cells to the antigen, has been shown to cause some degree of complete remission in patients. This is especially significant as leukemia is referred to as a liquid tumor, which means that the cancerous growth can not be surgically removed or easily identified. The Blinatumomab is helpful in combating this disease as it is able to locate and tag cancerous cells to promote antibody-directed cytotoxicity. The third type of BsAb commercially available is called Emicizumab, which is used to treat patients with congenital factor VIII deficiency, or hemophilia. People without this disease are able to produce three clotting factors: factor FIXa, factor FX and factor VIII. Factor VIII, which hemophilic patients lack the ability to produce, will gather factors FIXa and FX to form a coagulation complex protein and stop blood flow. Emicizumab has specificity for both factors FIXa and FX and links these two proteins together, which is effective at creating a protein complex that causes blood clotting. Emicizumab is especially interesting as it acts as a protein linker to provide treatment instead of interacting with immune system cells. 15



Bispecific antibodies have emerged as promising therapeutic tools for the treatment of various diseases, including cancer, autoimmune disorders, and infectious diseases. Their ability to be manufactured to meet the needs of a particular disease or match the specificity of a multitude of antigens and other proteins allows them to be incredibly versatile. They offer several advantages over traditional monospecific antibodies, such as the ability to simultaneously target two different antigens, their enhanced resistance to immunity, and the ability to treat complex diseases with multiple pathogenic pathways. As research in this field continues to advance, it is likely that bispecific antibodies will play an increasingly important role in the development of new and effective treatments for a wide range of diseases.


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  2. Sedykh SE, Prinz VV, Buneva VN, Nevinsky GA. Bispecific antibodies: design, therapy, perspectives. Drug Des Devel Ther. 2018;12:195-208


  4. Fan G, Wang Z, Hao M, Li J. Bispecific antibodies and their applications. J Hematol Oncol. 2015;8:130. doi:10.1186/s13045-015-0227-0

  5. Kontermann R. Dual targeting strategies with bispecific antibodies. mAbs. 2012;4(2):182-197. doi:10.4161/mabs.4.2.19000 

  6. Carter P. A bispecific revolution. Genentech: Breakthrough science. One moment, one day, one person at a time. Published December 8, 2017. Accessed March 29, 2023. 

  7. Brinkmann U,  Kontermann RE. The making of bispecific antibodies. mAbs. 2017;9:2, 182-212, doi: 10.1080/19420862.2016.1268307

  8. César Milstein. Encyclopædia Britannica. Published March 20, 2023. Accessed March 29, 2023. 

  9. Milstein C, Cuello AC. Hybrid hybridomas and their use in immunohistochemistry. Nature. 1983;305(5934):537-540. doi:10.1038/305537a0 

  10. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. B Cells and Antibodies. Available from:

  11. Schaefer W,  et al. Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry. MAbs 2016; 8:49-55; PMID:26496506;

  12. Davis JH, Aperlo C, Li Y, et al. Seedbodies: Fusion proteins based on Strand-exchange engineered domain (SEED) CH3 heterodimers in an FC analogue platform for asymmetric binders or immunofusions and bispecific antibodies†. Protein Engineering, Design and Selection. 2010;23(4):195-202. doi:10.1093/protein/gzp094 

  13. Ridgway JBB, Presta LG, Carter P. ‘Knobs-into-holes’ provides a rational design strategy for engineering antibody CH3 domains for heavy chain heterodimerization. Immunotechnology. 1996;2(1):73. doi:10.1016/1380-2933(96)80685-3 

  14. Merchant AM, Zhu Z, Yuan JQ, et al. An efficient route to human bispecific IGG. Nature Biotechnology. 1998;16(7):677-681. doi:10.1038/nbt0798-677 

  15. Muda M, Gross AW, Dawson JP, et al. Therapeutic assessment of seed: A new engineered antibody platform designed to generate mono- and bispecific antibodies. Protein Engineering Design and Selection. 2011;24(5):447-454. doi:10.1093/protein/gzq123 

  16. Ma J, Mo Y, Tang M, et al. Bispecific antibodies: From research to clinical application. Frontiers in Immunology. 2021;12. doi:10.3389/fimmu.2021.626616

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