Monoclonal antibodies are the most shining and mature technology in the field of modern biomedicine. Whether it is tumor targeting drugs, immunological checkpoint blocking drugs, or the hot market CAR-T technology, it is based on monoclonal antibodies and their downstream basis. Above. In general, a humanized mouse monoclonal antibody represented by a murine monoclonal antibody, after demonstrating its pharmaceutical value, requires a series of engineering modifications to improve its pharmacokinetics in vivo. Common antibody engineering strategies include antibody humanization, antibody affinity maturation and FC modification, or reduced antibody aggregation.
Humanization of antibodies
Mouse monoclonal antibody is the first monoclonal antibody to enter the clinic, but early clinical application found that because mouse monoclonal antibody is a heterologous protein, it will gradually cause HAMA response in clinical use, and the infused mouse monoclonal antibody will gradually be Neutralizing antibodies were neutralized, so that the murine monoclonal antibody was ineffective after infusion. Grafting the CDR regions of the murine monoclonal antibody humanization into the FRAME WORK region of the human antibody by antibody engineering techniques can effectively reduce the HAMA response induced by the antibody in clinical use. This approach is more advantageous than human-chimeric chimeric antibodies in terms of humanization. In fact, the most widely used antibody drugs in clinical practice are humanized mab or human and mouse chimeric antibodies.
With the development of bioinformatics, antibody humanization technology has been rapidly updated, and it is usually possible to recognize that some of the CDRs of the antibody antigen-binding region are grafted into the framework region of human antibodies, and some also have mouse anti-CDR regions and framework regions. The surface residues are replaced to be closer in sequence to human antibodies; in addition, the key amino acid sequence of the antibody antigen recognition region is replaced by the antibody antigen recognition region of the human antibody. In addition, different strategies have been proposed, such as CDR compensation, positioning reservation, and template replacement.
At the same time, full-human antibodies obtained by phage display library technology, or fully human antibodies expressed by humanized mice have gradually become an important direction in the field of antibody research and development, but different strategies have certain advantages and disadvantages in evaluating patients. The risk-return rate should always be considered when considering this issue.
Different companies have roughly similar technical pathways in the field of antibody humanization, but how to obtain the most humanized monoclonal antibodies while maintaining their high affinity for target proteins, for each company's technicians It is a big test. The technical team of Elken Bio has accumulated considerable experience in the field of antibody humanization. Based on years of experience in antibody drug research and development, it has provided antibody humanization services for many pharmaceutical companies. Some antibodies have entered the clinical trial stage. .
In vitro affinity maturation
Usually, the murine monoclonal antibody has a good affinity for the target antigen, but the affinity of the antibody obtained by humanization or by the phage display library technology to the target antigen may be unsatisfactory, which is not conducive to reducing the clinical use of the therapeutic antibody. The amount and toxic side effects, at this time the affinity maturation of the antibody, will contribute to future clinical use. The theoretical basis for antibody affinity maturation in vitro is the process of mimicking the affinity of antibodies in vivo. High-affinity antibodies can be screened by constructing a random mutagenesis library that mimics high-frequency mutations in B cells in vivo. In fact, in the course of monoclonal screening, affinity-maturation of any one of the antibodies results in an antibody with improved affinity.
Fc modification enhances effector function and half-life
FC receptors play an important role in the process of cellular immunity, and the modification of the FC region can produce profound effects on monoclonal antibodies with specific pharmacological effects. Monoclonal antibodies can mediate immune responses via Fcγ receptor binding (FcγR). The binding of different types of FcγR to IgG enhances/inhibits the immune response, and therefore preferentially enhances binding to FcγRIII while reducing binding to FcγRIIB to enhance clinical efficacy. At the same time, the half-life of the antibody can be prolonged by modification of the Fc. Since the binding of the antibody to FcRn is a pH-sensitive form, the antibody enters the cell by pinocytosis, in the endosome of acidic pH, the antibody binds to FcRn, and the FcRn mediates the antibody back to the extracellular, in extracellular neutral pH conditions. Next, dissociate from FcRn. In this way, FcRn avoids the fate of other proteins in the blood to be degraded by lysosomes into the cells through the pinocytosis, thereby achieving long-term effects, and the half-life is as long as several weeks (IgG1, IgG2, IgG4 half-life three weeks, IgG3 is 9 days). A number of pharmaceutical companies have attempted to modify antibodies to specifically increase the affinity for FcRn under low pH conditions for further long-acting effects. Although Fc modifications to enhanced cycle time may affect the effector function of Fc, studies have shown that mutations in different AAs of bevacizumab and cetuximab lead to more potent antitumor activity in mouse cancer models, Thus a strategy to increase the half-life of therapeutic antibodies was verified. Since then, a large number of favorable mutations have been reported. Unfortunately, the results of animal models are not always related to humans. However, an increase in affinity with FcRn by a factor of 5-10 usually results in a 2-4 fold increase in half-life.
The binding of the antibody to FcRn is a pH-sensitive form, and the antibody enters the cell by pinocytosis. In the acidic pH endosome, the antibody binds to FcRn, and the FcRn mediates the antibody back to the extracellular, under extracellular neutral pH conditions. , dissociation from FcRn
Improve stability and reduce polymerizability
Since the production of therapeutic antibodies is free from the physiological environment of the body, its thermal stability and stability of the colloids are limited, which in turn causes certain problems in the production and preservation of the drug. By technical means, improving the physical and chemical properties of the antibody helps to improve the overall efficacy and productivity of the antibody. By altering the antibody framework structure, the antigen binding domain of the antibody helps to reduce the polymerization of the antibody. It is usually possible to reduce the polymerization of the antibody by: 1. by changing the antibody dosage form; 2 by changing the framework and CDR regions, 3; adding additional disulfide bonds to the CH2-CH3 region; 4, changing the Fab The disulfide bond structure of the region; 5 increases the fusion tag with dots. In fact, with the development of computer simulation technology, the polymerization of antibodies can be offset by different designs and in vitro screening methods, and the chemical degradation rate and serum clearance rate of antibodies can be predicted.
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