At present, 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 further followed by a series of biological property testing and functional verification, antibodies with medicinal potential are received. In the actual development process, the antibodies obtained through routine screening require more detailed improvements, including affinity, immunogenicity, half-life, etc. Abundant researches and practicees have been conducted over the years, among which antibody affinity maturation is one of the important directions. In theory, the increase in antibody affinity helps to improve the specificity and potency of the antibody, and helps to cut drug amount, reduce the side effects, and the like. Although the actual research work proves that the improvement of affinity and the increase of antibody titer are not always linear, especially in the treatment of solid tumors (refer to Weinstein's "binding site barrier" hypothesis), but in many cases, this linearity relationship is obvious. In addition, the progress of antibody affinity maturation technology not only contributes to the development and quality improvement of antibody drugs, but also helps people better understand the mechanism of interaction between antibodies and targets, and the function of targets as well.

 

Antibody affinity maturation strategy for mimicking high frequency mutations in somatic cells
After the BCR gene rearranged mature B cells are stimulated by the antigen, high-frequency mutations (mainly point mutations) occur in the heavy and light chain V regions at the germinal center, and then the antigen is captured by the FDC to express the high-affinity BCR. The cells are protected from apoptosis. This process results in an increase in the average affinity of the BCR of the progeny B cells for the antigen. Such somatic high-frequency mutations belong to a secondary immune response, which helps the antibodies effectively recognize the antigen to further mature, and hence the body to effectively resist the re-invasion of foreign antigens. One of the strategies for antibody affinity maturation is to mimic somatic high-frequency mutations, screening for high-affinity antibodies to antigens by cell mutation and display of antibody proteins.



1.1 Screening strategy based on high frequency mutations in B cell lines (human, chicken, etc.)

Ramos is a cell line derived from human Burkitt's lymphoma. During the culture, the rearranged immunoglobulin V region gene continues to undergo constitutive mutation and is expressed on the membrane surface. As early as 2002, Sarah et al. reported the use of Ramos cell lines to screen for IgM with high affinity for streptavidin. They bound streptavidin to the magnetic beads, screened out a population of cells with low affinity for streptavidin from a group of Ramos cells, and then reduced the density of streptavidin on the magnetic beads to improve screening. The harshness further sifted out mutations with higher affinity, which was further increased by flow-wise sorting with streptavidin-labeled FITC-fluorescent. Subsequently, they sequenced the V region genes of each batch of subcloned cells, identified mutation sites, and analyzed the structure-activity relationship between the antibody and the target.

In this article, they also attempted to screen high-affinity IgM against multiple antigens using the XRCC2-deficient chicken-derived B cell line (DT40), which has a high frequency V region gene constitutive mutation and is shorter than the Ramos cell line. The screening results showed that the cell line also achieved affinity maturation of IgM.

The above two examples demonstrate that a suitable B cell line can be used as a tool cell for antibody affinity maturation. In a specific application, a specific antibody template gene can be inserted into the immunoglobulin gene site of the cell, followed by cell culture and screening of high affinity cell populations. However, antibodies affinity maturation technology based on somatic high frequency mutation has certain defects. First, there is usually only one base mutation in each codon during somatic mutation, which seriously restricts the mutation. The diversity of the library; secondly, due to the rapid internalization effect, the affinity of the BCR often encounters the ceiling (the KD value is difficult to reach below 0.1 nM).



1.2 Strategy based on high frequency mutations in other cells

As for the development of antibody affinity maturation based on somatic high frequency mutations, the mainstream methods are around B cell lines. B cell lines have shortcomings, like the difficulty of genetic manipulation, the incomprehensive post-translational modification of proteins. A laboratory has therefore attempted to achieve affinity screening on non-B cell systems to extend the cell line selection surface of this technology.



In addition, there are screening methods based on non-eukaryotic systems, such as the use of E. Coli gene mutator strain to achieve high-frequency mutations in antibody genes, whose principle is similar.

 

Affinity maturation strategy based on antibody library
There is no significant difference between antibody affinity-based antibody affinity maturation and antibody library-based antibody screening. Both are high-affinity antibody screening in vitro, and the focus remains on two aspects, namely, the construction of the library and the selection of screening systems. The difference is that the library used in the latter is unbiased at construction or synthesis or has only a limited bias towards an antigen; the library used in the former is constructed based on a defined antibody sequence template.



2.1 Database Construction Strategy

The strategy of building a database can be divided into two broad categories. One is to build a larger library, and to make random mutations in the CDR regions of the antibody or even the entire V region. The other is to build a smaller one, and focus the mutations on the antibody sequence.

The first kind of methods mainly include CDR walking, chain shuffling, and DNA shuffling. CDR walking refers to a piecewise random mutation and ensures that the two adjacent mutation regions overlap, so that the mutation covers the entire CDR region.

Shuffling reshuffles the pairing of VH and VL of the antibody, while DNA shuffling reshuffles the sequence of the V region of the antibody. In DNA shuffling, mutations are not limited to CDR regions, but also include FR regions, as the FR regions may also contribute to the binding of antibodies to antigens. The libraries built by these methods have a large capacity and a large workload.

Compared to constructing large libraries, small library construction is more sequence-oriented. In the absence of other information, it is customary to prioritize random mutations in the CDR3 region of the heavy chain, since the heavy chain CDR3 region plays a key role in antigen-antibody binding. However, to truly improve the efficiency of affinity optimization, exhausted information is required for finding more precise mutation areas. Selecting current mutation region mainly bases on the structural information of the antigen-antibody complex or the prediction of the germline gene hotspot.

 

1) Mutation region selection based on structural information of antigen-antibody complexes

After resolving the antibody antigen complex, the site information of the interaction between the two is therefore obtained, and then more precise mutation and database construction can be carried out. An article published in MorphoSys AG in 2016 describes their work to increase the affinity of their own anti-human GM-CSF antibody MOR103. After knowing how the antibody binds to the antigen, they randomly mutated the CDR-H2 and CDR-L3 of the antibody, and screened the high-affinity sequence by phage display, and then filtered the heavy and light chains. After pairing, a new "cross-cloned" antibody was obtained: the in vitro inhibition of GM-CSF and GM-CSFR binding compared the potency of these four antibodies. In addition, they also analyzed the structures of the complexes of these four antibodies combined with GM-CSF and compared them to explore the structure-activity relationship.



2) Selection of mutation regions based on germline gene hotspots

Germline hotspots are the preferential mutation regions in the process of high frequency mutations in somatic cells, almost all within the CDR region. It plays a key role in increasing the affinity of the antibody, so the germline hotspots of the antibody can be prioritized when the affinity is matured in vitro. The specific method is to use the database to compare the sequence of the antibody and the antibody germline gene, and to combine the sequence similarity and the coordinate sequence of the RGYW motifs to predict the germline hotspots, then mutate at these hot spots, and construct a small library for screening. As early as 2009, Ira Pastan's group published a protocol, taking anti-CD22 monoclonal antibody as an example, using germline hotspots prediction and phage display technology to improve antibody affinity.

As for the method of introducing mutation, the simpler is error-prone PCR, but it is difficult to achieve a good mutation effect. Another popular one is to design a mutation on the primer, that is, to artificially synthesize a primer library with a mutation region. There are many specific design methods, including controlling the number of mutated bases, controlling the probability of occurrence of various bases at each locus, in order to meet the demand for library capacity and bias.



2.2 Screening methods

Library-based antibody affinity maturation is basically the same as library-based antibody preliminary screening, mainly utilizing phage display, yeast display, ribosome display and other systems. The advantages are: phage display has the relatively simple process; yeast display is combined with flow sorting, detection and screening simultaneously; ribosome display has large capacity system that can be used for screening large libraries. The in vitro evolution of the library can be achieved. These technologies have their own advantages and disadvantages, and they are selected according to actual needs.