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 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 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
| Step | Condition | Parameter |
|---|---|---|
| Expression | Expression vector: mammalian cell, yeast cell, or E. coli | Transfection or transformation efficiency, expression level, expression time, etc. |
| Purification | Affinity chromatography: protein A, protein G, nickel column, etc. | Type and concentration of affinity ligand, pH and salt concentration of washing and elution buffer, etc. |
| Denaturation | Denaturant: urea, guanidine hydrochloride, ammonium sulfate, etc. | Type and concentration of denaturant time, temperature of denaturation, etc. |
| Renaturation | Buffer solution: Tris-HCl PBS, etc. | pH and salt concentration of buffer solution, method of renaturation (dilution or dialysis) time, temperature of renaturation, etc. |
| Assembly | Buffer solution: Tris-HCl PBS, etc. | Ratio of heavy chain and light chain reaction time, temperature, pH, etc. |
| Separation | Gel filtration chromatography: Sephacryl S-200 HR etc. | Type and pore size of gel flow rate, volume of elution buffer, etc. |
| Identification | Biochemical methods: SDS-PAGE, western blotting, mass spectrometry, circular dichroism spectroscopy, surface plasmon resonance spectroscopy, etc. | Sensitivity and specificity of method, sample processing method and amount, etc. |
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 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
| Application | Disease | Bispecific antibody | Target 1 | Target 2 | Effect |
|---|---|---|---|---|---|
| Cancer treatment | B-cell lymphoma (B-ALL) | Blinatumomab | CD19 (on B-ALL cells) | CD3 (on T cells) | Activate T cells to kill B-ALL cells |
| Autoimmune disease treatment | Rheumatoid arthritis (RA) | MEDI4920 | IL-13 (pro-inflammatory factor) | IL-4 (pro-inflammatory factor) | Neutralize IL-13 and IL-4 signals to reduce inflammation |
| Cancer diagnosis | Colorectal cancer (CRC) | 99mTc-HCAb | CEA (tumor marker on CRC cells) | 99mTc (radioactive nuclide) | Deliver 99mTc to CEA-positive CRC cells for imaging |
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