Overview of Heavy-Chain Assembly

Bispecific antibodies (BsAbs) are a class of artificially synthesized antibody molecules that can simultaneously recognize two different antigens or epitopes. They have a wide range of biomedical applications, such as cancer immunotherapy, targeted drug delivery, diagnostic reagents, etc. However, the preparation of bispecific antibodies also faces many challenges, such as structural complexity, low stability, and functional loss. To overcome these difficulties, various methods for preparing bispecific antibodies have been developed, such as chemical coupling, fusion protein, and double mutation. Among them, heavy-chain assembly (HCA) is a novel method that uses two different heavy chains to form dimers through disulfide bonds and then binds with corresponding light chains to form bispecific antibody molecules.

The Principle of Heavy-Chain Assembly

The principle of heavy-chain assembly is to use two different heavy chains to form dimers through disulfide bonds and then bind with corresponding light chains to form bispecific antibody molecules. The key to this method is to design suitable heavy chain variants that can spontaneously form stable dimers during the denaturation and renaturation processes. To achieve this goal, two strategies have been adopted—one is to introduce mutations in the heavy chain constant region (CH1) so that it can crosslink with the homologous region (CH3) of another heavy chain through disulfide bonds. The other is to introduce mutations in the heavy chain variable region (VH) so that it can crosslink with the complementary region (VL) of another heavy chain through disulfide bonds. These two strategies are called CH1-CH3 crosslinking and VH-VL crosslinking, respectively. By these two strategies, bispecific antibody molecules with different structures and functions can be prepared. For example, CH1-CH3 crosslinking can prepare bispecific antibodies similar to natural antibodies, that is, each heavy chain binds with a light chain to form a complete antibody structural domain. While VH-VL crosslinking can prepare bispecific antibodies similar to single-domain antibodies, that is, each heavy chain only contains a variable region to form a simplified antibody structural domain.

The Steps of Heavy-Chain Assembly

The specific operation steps of heavy-chain assembly include expression, purification, denaturation, renaturation, assembly, separation and identification. The following table summarizes the conditions and parameters of each step of heavy-chain assembly:

Table 1. Conditions and parameters of each step of heavy-chain assembly

The Characteristics of Heavy-Chain Assembly

The heavy chain assembly method is a new method for preparing bispecific antibodies and has the characteristics of high efficiency, high selectivity, low cost and low toxicity. It does not require complicated chemical conjugation or protein engineering and only needs to use the natural interaction between the heavy chain and the light chain, and through simple steps such as denaturation, refolding, and assembly, the production of bispecific antibodies can be achieved. It does not change the antigen-binding regions on the heavy and light chains and retains the original antibody function. It does not require the use of expensive chemical reagents or enzymes, nor does it require the use of specialized equipment or instruments. It only needs to use conventional biochemical methods and materials to prepare bispecific antibodies. It does not introduce any exogenous or unnatural chemical or biological molecules, nor does it produce any by-products or residues, but only utilizes the interaction between naturally occurring heavy and light chains to form pure and safe bispecific antibody molecules. In this way, potential harm to the environment and human body can be avoided.

The Applications of Heavy-Chain Assembly

The heavy chain assembly method has broad application prospects and practical cases in the development of bispecific antibodies, mainly involving the treatment or diagnosis of cancer, autoimmune diseases and other diseases. It can prepare bispecific antibodies that can simultaneously recognize antigens of different types and structures, such as tumor cells and immune cells, autoantigens and immune regulatory molecules, tumor markers and imaging probes, so as to achieve tumor immunotherapy, autoimmune disease treatment or cancer diagnosis. The bispecific antibodies prepared by the heavy chain assembly method show good safety, efficacy, specificity and sensitivity, and have produced significant clinical improvement or imaging effects on a variety of refractory or active diseases.

Table 2. Application cases and effects of bispecific antibodies prepared by the heavy chain assembly method

References

1. Wu Y, et al. Recent advances and challenges of bispecific antibodies in solid tumors. Exp Hematol Oncol. 2021 Dec 18;10(1):56.
2. Slamon DJ, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001 Mar 15;344(11):783-92.
3. Lynch TJ, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004 May 20;350(21):2129-39.
4. Kontermann RE, et al. Bispecific antibodies: a historical perspective and guide to the future. Nat Rev Drug Discov. 2020 Nov;19(11):797-812.
5. Topp MS, et al. Phase II trial of the anti-CD19 bispecific T cell-engager blinatumomab shows hematologic and molecular remissions in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia. J Clin Oncol. 2014 Dec 1;32(36):4134-40.
6. Martinelli G, et al. Complete hematologic and molecular response in adult patients with relapsed/refractory Philadelphia chromosome-positive B-precursor acute lymphoblastic leukemia following treatment with blinatumomab: results from a phase II, single-arm, multicenter study. J Clin Oncol. 2017 Sep 10;35(26):3011-8.
7. Spiess C, et al. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 2015 Oct;67(2 Pt A):95-106.
8. Zou W, et al. T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol. 2010 Apr;10(4):248-56.
9. Ma J, et al. Bispecific Antibodies: From Research to Clinical Application. Front Immunol. 2021 May 5;12:626616.
10. Nagorsen D, et al. AFM13: a first-in-class tetravalent bispecific anti-CD30/CD16A antibody for NK cell-mediated immunotherapy of CD30-positive malignancies. Oncoimmunology. 2016 Oct 14;5(12):e1253656.
11. Liu L, et al. A novel bispecific antibody targeting interleukin (IL)-4 and IL-13 for the treatment of rheumatoid arthritis (RA). Ann Rheum Dis. 2019 Jun;78(Suppl 2):A101-A102.
12. Wang Q, et al. Design and Production of Bispecific Antibodies. Antibodies (Basel). 2019 Aug 2;8(3):43.