Common Methods for Humanizing Antibodies

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 humanized monoclonal antibody production has three stages: chimeric antibody humanization, humanized antibody, and fully humanized antibody. Commonly Modification method of humanized antibody (Humanized antibody production) are as follows:

Fig 1 Direction of antibody humanization

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. Reopro, Sylant, Rituximab, Infliximab, and other chimeric antibody humanization have been marketed.

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.

CDR Transplantation

CDR transplantation, 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. It retains the specificity of mouse monoclonal antibodies and the affinity of humanized antibodies, but it has potential immunogenicity.

SDR Transplantation

SDR transplantation also known as specific determination of disability transplantation which the SDR region refers to the existence of CDR region contact with the antigen of the amino acid sequence. The SDR region can produce immunogenicity transplanted into the human antibody framework.

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.

Transgenic Mouse Technology

Transgenic mouse technology is the technology of transferring the optimized human antibody DNA sequence into the mouse genome. Because the immune system is the mouse, the antibodies are not the same as those of the human body.

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.

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. However, this technique is limited by the capacity of antibody libraries and host modification of amino acids.

Necitumumab and Adalimumab were screened by phage display technology.

Ribosome Display Technology

This technique works by displaying the target gene on the surface of the ribosome, forming an MRNa-protein-ribosome complex, and then adding antigens to the mixture for screening. Compared with phage display technology, this method has a higher storage capacity.

mRNA Display Technology

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

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. It can also label cells with substances such as fluorescein and perform timed and quantitative screening by flow cytometry.

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 humanized Modification methods of humanized antibodies, humanized antibody production, humanized monoclonal antibody production services, 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.