Since the birth of hybridoma technology in 1975, monoclonal antibodies (MAbs) have been regarded as "magic bullets". Mouse-derived MAbs have many disadvantages, such as strong possibility of producing human anti-mouse antibody (HAMA) response, short half-life in human body, inability to perform Fc biological function in human body, and hypersensitivity in human body caused by repeated use, which limit its large-scale use in clinic. In order to overcome these disadvantages, chimeric antibodies, humanized antibodies and human antibodies were developed successively by recombinant DNA technology. After nearly 40 years of development, the cell productivity and expression level of recombinant antibodies have been significantly improved, which largely depends on the construction of antibody expression vector, the optimization of antibody gene sequence, the construction of engineering cell lines, the establishment of rigorous screening methods and the overcoming of chromosome location effect. Today, let's look at the optimum conditions for efficient expression of recombinant antibodies.



Figure1. Recombinant antibodies

Construction of an efficient expression vector

High expression vector is the main factor to achieve high unit productivity of antibody. Various expression vectors have been constructed according to the structure and arrangement of expression vectors and the differences of related regulatory elements. Reasonable structure arrangement and appropriate expression regulation sequence can not only improve the level of antibody expression, but also increase the stability of engineering cell lines.

Structural Arrangement of Expression Vectors
The classical structural arrangement is to clone the light and heavy chain genes into 2 expression vectors. Each carrier carries a selection marker gene or a co-amplification gene, and then co-transfected the host cell to a certain proportion, and that is called  co-transfection vector. The main disadvantage is that there are differences in the number and location of random integration on chromosomes, resulting in imbalance of light and heavy chain expression, especially when co-amplified genes are used. Another arrangement is to construct independent light and heavy chain expression units on the same expression vector, screen marker genes or co-amplified genes to exist as independent expression units, or use IRES sequence to connect the 3'end of light or heavy chain, i.e. multi-promoter single vector. Its main disadvantage is that closely linked multiple promoters can cause transcriptional interference, especially when the direction of the promoter is between the two. Some scientists constructed light and heavy chains of antibodies into the same expression unit by selective splicing function, and controlled the expression ratio of light and heavy chains by mutation of splicing sites, so as to ensure the correct folding and assembly of antibodies and improve the secretion level of functional antibodies.

Promoter selection
In order to achieve high level of antibody expression, a strong promoter must be used. CMV promoter is commonly used in industrial production, but it is related to cell cycle and has the best activity in G1 phase. Promoters EF-1alpha and beta-actin from housekeeping genes can be used to maintain high transcriptional activity during cell culture. In addition, heterozygous promoters, such as CMV/EF-1alpha heterozygous promoter and heterozygous promoter composed of ubiquitin C promoter sequence and CMV enhancer, may also overcome their disadvantages.

Enhancers and introns
Enhancers and introns are inserted between promoters and target genes in the commonly used antibody expression vectors. Enhancer, mainly derived from some eukaryotic cells or non-coding regions of the virus, can increase the transcription frequency of promoter, improve the stability and translation efficiency of RNA.

Transcription regulatory elements
An important factor affecting the efficient expression of antibody genes is the "location effect" of chromatin. Integration into inactive heterochromatin regions results in no or low expression of target genes, while integration into transcriptional active euchromatin regions does not ensure long-term stable expression of target genes. The adjacent condensed chromatin of the target gene silences its expression, which may be related to low acetylation of histone, methylation of histone H3 9 lysine and increased methylation of vector promoter CpG. Several strategies have been developed to overcome the "location effect" to increase antibody expression, such as the use of protective cis-regulatory elements including nuclear skeleton/matrix attachment regions (S/MARs), Ubiquitous chromatin opening elements (UCOEs), repressor elements and insulators, and Woodchuck hepatitis virus post-transcriptional regulatory elements. Is post-transcriptional regulatory element, WPRE, etc. are inserted into the appropriate position on the carrier.

Polyadenylation tailing signal (poly A)
The 3'terminal region of eukaryotic cells usually contains three functional elements: poly A, splicing sites and transcription termination elements. Strong transcription termination signal and polyadenylate plus tail signal can ensure that the target gene terminates transcription and polyadenylation in time to enhance the stability of the RNA. With the development of transcriptome, the understanding of selective polyadenylation has been deepened, which provides a new method for finding more efficient poly A signal and improving the expression level of antibodies. Strong transcription termination elements can terminate transcription in time and avoid interference from upstream promoters to downstream promoters.

Optimization of antibody gene sequence

More and more studies have shown that splicing signal, secondary structure, GC content and codon preference in the sequence of antibody genes affect the expression level of antibody. Signaling peptides can affect the efficiency of folding, assembly and modification of antibodies, and then affect their secretion level.

Codon optimization
Another strategy to improve the expression of target protein is to replace the codon of the target gene with the preferred codon of the host cell. However, the use of preferred codons does not always improve the expression level, there are also examples of reduction of the expression level, which suggests that the evaluation of optimized codons is necessary.

Gene structure optimization
There may be some unfavorable elements in gene sequence, such as covert splicing sites, TATA frames, gene internal termination signals, negative CpG islands, hairpin structure, poly(A) early signals, potential Chi sequences and ribosome binding sites. Using bioinformatics technology to replace or delete negative elements in antibody gene sequence and increase GC content can increase the stability of RNA, which contributes to the improvement of antibody expression level.

Signal peptide
Antibody is a tetramer consisting of light and heavy chains. Light and heavy chains need to be folded and assembled into endoplasmic reticulum under the action of signal peptide, and then secreted to extracellular. Therefore, the selection of appropriate signal peptide will accelerate the completion of this process, thereby improving the expression level of antibody.

Antibody expression system and its screening strategy

In order to obtain highly expressed and stable cell lines for long-term passage, it is essential to develop and select appropriate antibody expression system. Generally, antibody genes are randomly integrated into the chromosomes of host cells. Only integrated into the transcriptional active region can antibody genes achieve high level expression. Therefore, screening highly expressed cell clones is particularly critical to shorten the development cycle, save labor and cost.

Increased antibody expression system
The copy number of the target gene is an effective way to improve the productivity of antibody unit.

Strict screening methods
In order to improve the screening efficiency and reduce the production of non-specific clones, it is very important to design and construct appropriate rigorous expression vectors, which is usually achieved by modifying the co-amplified genes of vectors and/or antibiotic antibody genes.

With the development of genomics, transcriptome, proteomics and metabonomics, the research on the efficient expression mechanism of exogenous genes in host cells will be further studied, which will greatly promote the development of personalized medium and the optimization of mammalian cell culture process. Therefore, owing to the improvement of vector, the transformation of host cells, the efficient delivery of target genes, the development of personalized culture media, and the optimization of screening methods and process development, the cell productivity and antibody expression level have been significantly improved.