Development of Antibody Humanization Production Technology

Monoclonal antibody (mAb) is produced by a single B cell clone only for a specific antigen epitope antibody, that technology was invented in 1975 by Kohler and Milstein, and can be used in basic science, drugs, biosensors, and other fields, especially in medicine has great significance.

In the production of monoclonal antibodies, mouse monoclonal antibodies don’t contain human gene components and can be recognized by the human immune system, thus it’s easy to cause rejection immune response when used to treat diseases. To overcome this problem, humanized antibody technology has been developed to eliminate or reduce antibody immunogenicity. The application of therapeutic antibody discovery is to treat disease, and the first therapeutic antibody drug was introduced in 1986. Since then, many antibody drugs have been discovered that can be used to treat diseases, such as asthma.

The humanization process of antibodies has experienced three stages: human and mouse chimeric antibody, humanized antibody, and fully humanized monoclonal antibody. The humanized antibody platform can be used for therapeutic antibody discovery and antibody drug discovery. KMD Bioscience can provide humanized antibody production services for your research.

Figure 1 Development of antibody humanization

Antibody Chimerization

70% of the genes of mouse and human antibody chimerization are derived from humans. Compared with mouse antibodies, it caused fewer adverse reactions and had a therapeutic effect. The production technology of antibody chimerization is as follows:

① Chimeric IgG antibody

The principle is to obtain the V region gene of the target antibody from hybridoma cells producing a certain mouse monoclonal antibody, recombine with the human C region gene clone it into a suitable vector, and then transfer it to the recipient cell for expression.

②  Chimeric Fab and F (ab ') 2 antibody

The principle is to recombine the V region genes of the functional antibody L and H chains with the human light chain K and H chain CH1, clone them into the expression vector, and transfer them to the host cell for expression. However, due to the low affinity and small molecular weight of the antibody, it is easy to be filtered by the glomeruli and disappear from the blood, which isn’t suitable for clinical treatment alone.

Humanized Antibodies

Based on human and mouse chimeric antibodies, 90% of the genes are derived from human-humanized antibodies. Compared with mouse and human chimeric antibodies, the therapeutic effect is better and the side effects are less. The preparation of humanized antibodies is as follows:

①  CDR transplantation

Antibody reconstruction, also known as antibody reconstruction, is the most commonly used method in the humanization process of monoclonal antibodies, and the human FR sequence is cloned to the corresponding part of the antibody while retaining the mouse CDR sequence.

②  SDR transplantation

SDR region refers to the presence of CDR region that can contact the antigen of the amino acid sequence, SDR cloning into the framework of the antibody.

③  Frame area reconstruction

Frame region remodeling is based on FR screening that includes surface remodeling, which humanizes amino acid residues, and glycosylation modification, which changes the glycosylation site. The technology can improve antibody affinity.

Fully Humanized Monoclonal Antibody

Fully humanized monoclonal antibody has higher safety and efficacy in treatment that could avoid the body's immune response, so they are currently the drugs of choice for diseases, especially cancer and autoimmune diseases. The production method of the technology is as follows:

①  Transgenic mouse technology

Transgenic mouse technology is the technology of transferring the optimized human antibody DNA sequence into the mouse genome.

② Antibody library technology

It is divided into immune antibody library and non-immune antibody library:

An immune antibody library is established by single-cell cloning technology after immunizing the body.

The non-immune antibody library consists of a semi-synthetic antibody library, a fully synthetic antibody library, and a natural antibody library. The semi-synthetic antibody library consists of randomly synthesized CDR sequences. The variable region of the whole synthetic antibody library was synthesized artificially. The antibody genes in the natural antibody library are derived from lymphocytes of non-immune individuals.

③ Phage display technology

The variable region gene in the antibody library is expressed on the phage surface, and the antibody with the highest affinity is screened after binding with the antigen and antibody.

④  Ribosome display technology

The technique is to display the translation products of target genes on the surface of ribosomes to form an MRNA-protein-ribosome ternary complex, and then add antigens to the translation mixture for screening.

⑤ mRNA display technology

mRNA display technology is an improvement and extension of ribosome display technology, which is more conducive to screening.

⑥ Yeast display technology

Yeast display technology uses yeast as a carrier to locate foreign genes on the surface of yeast cells through fusion expression, to screen them.

⑦ Single cell technology

Single cells can be screened from the established antibody library according to cell surface marker molecules, then the heavy chain and light chain genes of antibodies can be obtained by RT-PCR after cell enrichment.

KMD Bioscience has rich experience in antibody engineering construction and mature antibody humanization technology that can provide customers with high-quality antibody humanization production and modification, humanized antibody production, mouse antibody humanization, and other services. In addition, KMD Bioscience can also provide monoclonal antibody production, antibody expression and purification, affinity determination, antibody affinity maturation, antibody sequencing, and other services to meet the needs of different customers.

References

[1] Zinn S, Vazquez-Lombardi R, Zimmermann C, et al. Advances in antibody-based therapy in oncology [J]. Nat Cancer. 2023, 4(2): 165-180.

[2] Nagano K, Tsutsumi Y. Phage Display Technology as a Powerful Platform for Antibody Drug Discovery [J]. Viruses. 2021, 13(2): 178.

[3] Waldmann H. Human Monoclonal Antibodies: The Benefits of Humanization [J]. Methods Mol Biol. 2019, 1904: 1-10.