Bispecific antibodies (BsAbs) are a class of antibody molecules that can simultaneously recognize two different antigens or antigen epitopes. BsAbs have various structural forms, such as bivalent, trivalent, tetravalent, etc., and can be composed of monoclonal antibodies (mAbs) from different sources or subclasses. BsAbs play an important role and have application prospects in drug development, such as targeted therapy, immunomodulation, diagnosis, and prevention. However, the generation methods of BsAbs have always been a challenge and a bottleneck. Traditional methods such as chemical cross-linking, hybridoma cells, and genetic engineering have many limitations, such as low efficiency, poor stability, poor reproducibility, etc. Therefore, developing novel BsAbs generation methods is an urgent need and a research hotspot.
In recent years, a novel method called the co-culture method has attracted people's attention and interest. This method is a way to simulate the complex ecological environment of microorganisms. It uses an ecological-driven approach to induce the expression of silent gene clusters by the interaction between different species or sources of microorganisms, thereby producing diverse and novel BsAbs. This method can not only activate the hidden antibody molecules in microorganisms but also produce antibody molecules with bispecificity or multispecificity, providing a new strategy and approach for the discovery and development of BsAbs.
Using the interaction between different species or sources of microorganisms, the co-culture method induces the expression of silent gene clusters, thereby producing diverse and novel BsAbs. Silent gene clusters are a group of genes that encode specific metabolic products but do not express or express very low under conventional culture conditions. There are a large number of silent gene clusters in microorganisms, which may be the result of microorganisms adapting to complex ecological pressures in the natural environment. However, in the laboratory, these silent gene clusters are often not activated, resulting in many potential antibody molecules that cannot be discovered and utilized. The co-culture method is a way to simulate the microbial interactions in the natural environment by changing the ecological balance between microorganisms, stimulating the expression of silent gene clusters, and obtaining antibody molecules with bispecificity or multispecificity. The main mechanisms of the co-culture method include metabolic complementation, signal molecule induction, competition inhibition, and symbiosis cooperation. Metabolic complementation refers to the promotion of each other's growth and differentiation by sharing or exchanging metabolic products between different microorganisms. Signal molecule induction refers to the influence of one microorganism's gene expression and metabolic activity by the signal molecule secreted by another microorganism. Competition inhibition refers to the inhibition of another microorganism's growth and activity by one microorganism producing inhibitory substances or occupying dominant resources. Symbiosis cooperation refers to the mutual dependence and coordination between different microorganisms by forming tight cell structures or signal networks.
The steps of the co-culture method are the experimental procedures, conditions, and techniques required to implement this method. The steps of the co-culture method can be divided into the following stages: (1) Microorganism screening; (2) Microorganism culture; (3) Microorganism separation; (4) Microorganism purification; (5) Microorganism structure identification.
Table 1. Co-culture method experimental procedures and precautions
| Stage | Description | Precautions |
|---|---|---|
| Microorganism screening | Select microorganisms with potential silent gene clusters from different sources or environments, such as fungi, bacteria, actinomycetes, etc | Choose microorganisms that are compatible and non-pathogenic; avoid contamination and cross-infection. |
| Microorganism culture | Mix and culture different species or sources of microorganisms in the same culture medium, or use one microorganism as an additive for another microorganism, adjust the culture conditions, such as temperature, pH, time, etc., to promote the interaction between microorganisms | Optimize the ratio and concentration of microorganisms; monitor the growth and activity of microorganisms; avoid overgrowth or death of microorganisms. |
| Microorganism separation | Use different methods, such as solid-phase extraction, liquid chromatography, mass spectrometry, etc., to separate the microorganisms containing BsAbs molecules from the culture liquid | Choose appropriate separation methods according to the properties of microorganisms and BsAbs molecules; avoid loss or damage of BsAbs molecules. |
| Microorganism purification | Use different methods, such as gel filtration, affinity chromatography, ion exchange, etc., to purify the BsAbs molecules from the separated microorganisms | Choose appropriate purification methods according to the properties of BsAbs molecules; avoid contamination or degradation of BsAbs molecules. |
| Microorganism structure identification | Use different methods, such as nuclear magnetic resonance, X-ray diffraction, mass spectrometry, etc., to identify the structure and function of the purified BsAbs molecules | Choose appropriate identification methods according to the properties of BsAbs molecules; verify the specificity and activity of BsAbs molecules. |
Co-culture method has some advantages and disadvantages compared with other generation methods.
Table 2. Comparison of the advantages and disadvantages of co-culture method and other generation methods
| Method | Advantages | Disadvantages |
|---|---|---|
| Co-Culture Method | It can activate silent gene clusters and produce diverse and novel BsAbs; it can produce BsAbs with bispecificity or multispecificity; it can simulate the microbial interactions in the natural environment. | The compatibility between microorganisms is difficult to control; the culture conditions are difficult to optimize; the BsAbs molecules are difficult to identify and verify. |
| Chemical cross-linking | It is simple to operate and low-cost; it can use existing mAbs. | It has low efficiency and poor stability; it may produce toxic or immunogenic by-products. |
| Hybridoma cells | It can produce high-efficiency and high-stability BsAbs; it can use existing mAb hybridoma cells. | It is complex to operate and high-cost; it may produce heterogeneous or incomplete BsAbs. |
| Genetic engineering | It can produce various structures and functions of BsAbs; it can precisely control the composition and ratio of BsAbs. | It is complex to operate and high-cost; it may produce non-foldable or non-expressible BsAbs. |
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