Effects of Glycosylation on the Stability and Half-life of Antibody Drugs


Read Effects of Glycosylation on the Stability and Half-life of Antibody Drugs

Effects of Glycosylation on the Safety of Antibody Drugs

The Critical Quality Attributes (CQA) of antibody is the standard to judge whether it is qualified. CQA must be in a proper range to ensure the effectiveness of drugs, and more importantly, the safety of drugs. By affecting the effect function of Fc end, glycosylation modification of Fc end will affect the safety of antibody, which is manifested in immunogenicity, PK/Pd (pharmacokinetics/pharmacodynamics), etc.

The development of antibody has experienced mouse antibody, chimeric antibody, humanized antibody, and then to the last all human antibody, throughout which the immunogenicity of antibody has been constantly reduced. For glycosylation, human cells specifically synthesize Neu5Ac sialic acid, while other mammalian cells can not only synthesize Neu5Ac sialic acid, but also synthesize Neu5Gc sialic acid with immunogenicity to human as well. In antibody production, CHO cells are often used as host cells, while CHO cells usually express sialic acid highly, and the proportion of Neu5Gc sialic acid which can cause human immune response is up to 3%. Therefore, when CHO cells produce antibodies, there may be a risk of human immune response if Neu5Gc sialic acid cannot be removed safely.

Similar to sialic acid, human cells only express β - 1,4-galactose, while other mammalian cells also expressα-1,3-galactose, which has immunogenicity to human. This modification of α - 1,3-galactose has brought obstacles to human xenotransplantation of pig organs. At the same time, the antibody produced by NS0 and SP2/0 cells of mouse family may also be modified by galactose, which should be strictly controlled when used, so as to reduce its immunogenicity within the acceptable range.

Antibodies have been developed to all human antibodies. Immunogenicity caused by different glycosylation modifications should not be considered. However, in order to increase the efficacy of the antibody or extend its half-life, some amino acid mutations and glycosylation changes are often introduced artificially, and its potential immunogenicity needs to be considered when making such changes. Moreover, immunogenicity is not only caused by internal factors, but also by external factors such as the formation of materials, polymers and the introduction of surfactants.

PK/PD mainly considers the minimum dose and time of existence of the drug in vivo. It is very important to control the immunogenicity of the antibody which can not completely eliminate the immunogenicity. Glycosylation affects PK mainly by affecting the stability and half-life of antibody. In addition, glycosylation also affects the binding of FcRn to antibody and then affects PK of drug. The effect of glycosylation on antibody activity acts on PD, which will be discussed in the next section. In the study of PK/PD of antibody, the clearance rate is sometimes introduced, and the clearance rate of antibody with strong binding to Fc receptor is low, while glycosylation will affect the binding strength of antibody to Fc receptor or C1q complement. If the clearance rate of antibody is low, the antibody combined with Fc receptor or C1q complement may cause the immune system to attack normal cells and produce serious Miss target effect.

Effects of Glycosylation on the Bioactivity of Antibody Drugs

In the treatment of tumor, the two important ways for antibody to play a role are Antibody-dependent cell-mediated cytotoxicity (ADCC) and Complement-dependent cytotoxicity (CDC), the mechanism of which is shown in Figure 2.




Fig.2 Mechanism of ADCC and CDC

The glycosylation modification site of FC end of antibody is usually the binding site of Fc receptor (ADCC mechanism) and C1q (CDC mechanism). Through the adjustment of glycosylation, the binding of Fc receptor and C1q with antibody can be increased, thus the activity of ADCC and CDC of antibody can be increased.

It has been reported in many studies that sialic acid glycosylation can affect the binding of antibody to FC γ RIIA, thus reducing the activity of antibody ADCC.

Fucose is considered to be the most important monosaccharide in the glycosylation of antibody. The affinity of antibody to FC γ riiia and ADCC activity (up to 100 times) can be significantly improved by de fucose. Currently, no fucose has been found to affect the activity of CDC. As shown in Figure 3, Tsuguo Kubota knocked out fut8 gene (encoding fucose) of CHO cells by gene means to produce antibody without fucose residue, and found that its ADCC activity was significantly enhanced.


Fig.3 Fucosylation of antibody and its effect on ADCC activity

Related in vitro studies show that galactosylation can increase the binding of antibody and complement, thus increasing the activity of antibody CDC. In addition, the activity of ADCC can be increased by increasing galactosylation, but it is only an auxiliary function compared with fucosylation. Galactosylation itself has little effect on ADCC activity, and fucosylation is the main factor affecting ADCC activity.

In addition, the effect of Mannose on antibody activity is not clear. Although research shows that high mannose type can significantly increase the combination of antibody and FC γ RIIA and increase ADCC activity, there is no fucosylation in high mannose type. Therefore, it is not clear whether it is high mannose or de fucosylation that enhance ADCC activity.

The influence of different glycosylation modifications on antibody activity has been mentioned above, but what if there is no glycosylation modification at asn-297? Up to now, high levels of non glycosylated heavy chain (NGHC) have not been found to have immunogenicity and no biological activity. NGHC type antibody completely lost the binding ability of FC γ RIII, and the binding ability of nghc type antibody to C1q is also reduced by 10 times, resulting in the loss of ADCC and CDC activity of this type of antibody. In addition,researchers have compared the biological activities of the hemiglycosylated McAb (one sugar chain) and the complete glycosylated McAb (two sugar chains). Compared with the complete glycosylated McAb, the ADCC and CDC activities of the hemiglycosylated McAb decreased.

Conformation determines performance. The effects of glycosylation on the stability, half-life, safety and biological activity of antibody drugs are mutual. The best choice is to control the glycosylation of antibody in a proper range and make balance among the three.

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