Monoclonal antibodies (abbreviated as MAbs) are not only used in basic science, but also in the fields of drugs, biosensors, etc., and are of great significance in medicine. Among them, mouse MAbs do not contain human gene components and can be recognized by the human immune system. Therefore, when used to treat diseases, they are prone to cause human rejection immune responses. In order to overcome this problem, the emerging antibody humanization technology.

Antibody humanization is the process of modifying non-human antibodies (such as mouse, rabbit or camel antibodies) through genetic engineering technology to make their structure closer to human antibodies. Traditional animal-derived antibodies may cause immune rejection reactions or be cleared by the body's immune system in clinical applications, resulting in reduced efficacy or increased side effects. In order to solve this problem, antibody humanization technology came into being. Its main purpose is to replace the structure of non-human antibodies with human framework regions by modifying the framework region of antibodies, retaining the antigen binding sites and biological activity of antibodies, thereby reducing immunogenicity, extending the half-life of drugs, and improving efficacy.

The core of antibody humanization technology lies in the optimization of framework regions and antigen binding sites. Through methods such as gene mutation, computer-aided design or directed evolution, the affinity and specificity of antibodies are maintained, while making them more adaptable to the human immune system and reducing immune responses. In addition, humanized antibodies often have better stability and lower immunogenicity, can circulate in the human body for a long time, and improve the therapeutic effect.

This technology is widely used in tumor immunotherapy, antibody drug development, autoimmune diseases and other fields, and has become an important part of modern biopharmaceuticals. With the continuous advancement of technology, antibody humanization will provide effective treatments for more clinical diseases. Commonly used antibody humanization transformation methods (antibody humanization production methods) are as follows:

Figure 1 Directions of antibody humanization

1. Chimeric IgG Antibody

Chimeric IgG antibody is a recombination of the variable region (V region) gene of the mouse antibody in the hybridoma cell with the constant region (C region) gene of the human. The recombinant antibody is cloned into a vector and transferred into the recipient cell for expression. The key to chimeric antibody technology is to retain the antigen specificity of the antibody (through the mouse variable region) while reducing immunogenicity through the human constant region. Reopro, Sylant, Rituximab, Infliximab, etc. are human-mouse chimeric antibodies that have been marketed.

2. Chimeric Fab and F(ab')2 Antibodies

For most antibodies of this type, due to their low affinity and small molecular weight, they are easily filtered by the glomerulus and disappear from the blood, so most of them are not suitable for single use in clinical treatment.

3. CDR Transplantation (Antibody Reconstruction)

CDR transplantation technology is a classic antibody humanization method. The process is to retain the complementary determining region (CDR) sequence of the mouse antibody and clone the human framework region (FR) sequence to the corresponding position of the antibody. This method greatly reduces the immune response while retaining the original antibody binding characteristics, thereby optimizing the antibody. Usually, when performing CDR transplantation, it is also necessary to further transform the affinity and stability of the antibody through technical means to meet the needs of clinical applications.

4. SDR Transplantation (Specificity Determining Residue Transplantation)

SDR transplantation (Specificity Determining Residue) is the transplantation of specificity determining residues (located in the CDR region) that can produce immune responses from non-human antibodies to human antibody framework regions. Unlike CDR transplantation, SDR transplantation focuses on adjusting those amino acid residues that directly contact the antigen to avoid immune responses and maintain the specificity of the antibody. This technology helps to reduce immunogenicity while improving the stability and affinity of the antibody.

5. Framework Region Reconstruction

Framework region reconstruction is based on the screening of FRs. The screening methods include surface remodeling and glycosylation modification. The former is to humanize amino acid residues, and the latter is to change glycosylation sites. This technology gives antibodies higher affinity.

6. Transgenic Mouse Technology

Transgenic mouse technology produces human antibodies by transferring the DNA sequence of human antibodies into the mouse genome. Since the immune system is still that of mice, the antibodies generated by transgenic mice are very similar to human antibodies, but there are still minor differences. This technology can be used to develop large-scale antibody drugs, such as Nivolumab (anti-PD-1 antibody), Denosumab (anti-RANKL antibody), and Panitumumab (anti-EGFR antibody). These antibodies are all produced by transgenic mouse technology and have been successfully used in clinical applications.

7. Antibody Library Technology

The immune antibody library is established by single-cell cloning technology.

Non-immune antibody libraries are divided into: ① Semi-synthetic antibodies: composed of randomly synthesized CDR sequences; ② Fully synthetic antibody library: variable regions are artificially synthesized; ③ Natural antibody library: antibody genes are derived from lymphocytes.

8. Phage Display Technology

Phage display technology screens antibodies with specific affinity by displaying antibodies or their fragments on the surface of phages. Phage display technology can construct an antibody library with a large capacity and quickly screen out antibodies with high affinity. Representative drugs such as Necitumumab (anti-EGFR antibody) and Adalimumab (anti-TNF-α antibody) were obtained through screening with this technology. The advantage of this technology is that it can carry out large-scale antibody screening, and the disadvantage is that it is limited by factors such as amino acid modification and library capacity.

9. Ribosome Display Technology

Ribosome display technology is based on mRNA-protein-ribosome complex for antibody screening. By combining antigen with ribosome complex, antibody screening and verification can be completed in a very short time. This technology has higher screening accuracy and is suitable for precise antibody screening and optimization.

10. mRNA Display Technology

mRNA display technology is based on ribosome display technology and has more accurate screening.

11. Yeast Display Technology

Yeast display technology locates exogenous genes on the surface of yeast cells for screening.

12. Single Cell Technology

This technology can screen out single cells from the established antibody library based on cell surface marker molecules, and directly obtain the heavy chain and light chain genes of antibodies by RT-PCR after cell enrichment.

TekBiotech has been committed to the field of antibodies for many years, with rich experience and mature technology, and can provide customers with high-quality antibody humanization transformation methods, antibody humanization production, and humanized monoclonal antibody preparation services. In addition, TekBiotech can also provide customized antibody services, including antibody expression and purification, affinity determination, antibody sequencing, etc., to meet customer needs.