Definition of antibody affinity maturation
Antibody affinity maturation refers to a state of immune function that normally exists in the body. In humoral immunity, the average affinity of the antibody produced by the second response is higher than the initial immune response. This phenomenon is called antibody affinity maturation. It is due to the genetic mutation of the antibody forming cell itself and the selective activation of the B cell clone by the antigen. This functional state of the body is the result of long-term evolution and continuous adaptation to the external environment, and is of great significance for the body's defense and maintenance of its own immune monitoring.
Significance of antibody affinity maturation
Currently, the development of pharmaceutical antibodies is mainly based on hybridoma cells or in vitro antibody libraries. Through antigen design and antibody screening, people initially obtain positive hits, and then further pass a series of biological property detection and functional verification, and finally obtain antibodies with medicinal potential. In the actual research and development process, the antibodies obtained after routine screening have many aspects that need more detailed improvement, including affinity, immunogenicity, half-life, etc. In the field of antibodies, many related researches and practices have been done over the years. Among them, antibody affinity maturation is one of the important research directions.
Theoretically, the increased affinity of antibodies will help to improve the specificity and efficacy of antibodies, help to reduce the dosage of drugs, and reduce toxic and side effects. Although the actual research work proves that the increase in affinity and the increase in antibody titer are not always linear, especially in the treatment of solid tumors (refer to Weinstein's "binding site barrier" hypothesis), in many cases, this linear relationship is obvious. In addition, the development of mature technology of antibody affinity not only helps the development and quality improvement of antibody drugs, but also helps people better understand the mechanism of antibody interaction with the target and better understand the function of the target.
Commonly strategies used for in vitro antibody affinity maturation
According to the principle of in vivo antibody affinity maturation, in the process of in vitro antibody affinity maturation, the selection of mutation regions and how to introduce mutations are a key issue. The current mutation strategies are mainly divided into three categories, random mutation, substitution and directed mutation. One of the strategies for antibody affinity maturation is to simulate high-frequency mutations in somatic cells, and screen for antibodies with high affinity for antigens through cell mutation and display of antibody proteins.
- Chain replacement
Chain replacement is to retain the heavy chain or light chain of a specific antibody, and the other chain is combined with a randomized complementary chain to select higher activity mutant strains. By fixing one of the two chains of the antibody and constructing a replacement library with sufficient diversity for the other chain, random combinations are likely to produce the best chain combination, and new antibodies with high affinity can be obtained through phage display antibody screening. Light chain replacement is often used in chain replacement.
- Site-directed mutation
Due to the affinity maturation of natural antibodies, the regions where high-frequency mutations of somatic cells occur are not evenly distributed, but are mainly concentrated in the CDR regions that directly contact the antigen. During the in vitro maturation of the antibody's affinity, the CDR region is the most commonly selected site-directed mutation region, so that it can obtain sufficient sequence diversity without destroying the protein structure. When performing site-directed mutation on CDRs, multiple CDRs can be changed in parallel or optimized step by step.
- DNA shuffling
DNA shuffling is a technology that uses homologous antibody genes to cut them into fragments of no more than 50 bp using deoxyribonuclease I, and then randomly combines them to perform PCR amplification to complete anti-rest genes. It contains the process of randomized cutting, recombination and screening of antibody fragments, which mimics the natural process of affinity maturation of antibodies and accelerates the speed of directed evolution in vitro.
Antibody affinity maturation based on antibody libraries is not essentially different from antibody library-based antibody screening. Both are in vitro high-affinity antibody screening. The focus is still on two aspects, namely, library construction and selection of screening systems. The difference is that the library used in the latter is not biased in construction or synthesis or only has a limited bias against an antigen; while the library used in the former is constructed based on the determined antibody sequence template. The strategy of building a library can be divided into two large categories: one is to build a larger library, random mutation of antibody CDR regions or even the entire V region; the other is to build a smaller library, focusing mutations on antibody sequences on a specific area. As for the method of introducing mutations, the simpler is error-prone PCR, but it is difficult to achieve good mutation effects. Another popular method is to design mutations on primers, that is, artificially synthesize primer libraries with mutation regions. There are many specific design methods, including controlling the number of mutated bases, controlling the probability of occurrence of various bases at each site, etc., in order to meet the needs of library capacity and bias.
- Error-prone PCR
Error-prone PCR is currently the most commonly used antibody mutation technique, which can randomly introduce mutations in the entire length or part of the region of the antibody gene. When the polymerase amplifies the target sub-cause, by applying a polymerase with a high mismatch rate or adjusting the reaction conditions, a mutation is randomly introduced into the target gene at a certain frequency, and random mutagenesis is repeated through multiple rounds of PCR, Cumulative mutation effect, and finally obtain a random mutant of the target protein.