Optimization methods of therapeutic antibodies

 

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Optimization methods of therapeutic antibodies

Since FDA approved the first antibody, Orthoclone OKT3, in 1986, more than 70 recombinant antibodies have been approved. There are still hundreds of therapeutic antibodies in clinical research, and the competition in the antibody field is becoming more intense. Therefore, it is necessary to deeply optimize therapeutic antibodies. This article summarizes some methods that have been used for antibody optimization.


1. Humanization of antibodies

Monoclonal antibodies produced by mice or other animals can stimulate the immune response in humans due to their immunogenicity. To reduce this immunogenicity, the variable region of murine antibodies can be fused to the constant region of human antibodies, and then a humanized chimeric antibody is produced. The variable region of the chimeric antibody can be further modified to increase its similarity to human body antibodies, thereby reducing its immunogenicity. Humanization of antibodies is typically designed by studying the differences between mouse antibody protein sequences and homologous human antibody sequences. At each different amino acid position between mouse and human, a choice must be made: if the antigen binding affinity of the antibody is not affected or the basic properties of the antibody is not significantly reduced by human amino acid, a human amino acid can be selected; otherwise, the mouse amino acid is maintained. A variety of methods are available for antibody humanization design, including computer-aided design, phage display, and yeast display (three widely used methods).

2. Increase tolerance to chimeric antibodies and reduce antibody immunogenicity

Even humanization of chimeric antibodies to a large extent or 100 percent, the immunogenicity is somewhat present. Among them, the content of T cell epitopes is the main factor causing the immunogenicity of therapeutic antibodies. Although there are currently a number of tools available to predict T cell epitopes in antibody sequences, most of these tools do not show whether the predicted epitope is displayed on the surface of the protein (which will be detected by the human immune system), so the information predicted by the sequence from linearity should be combined with 3D model for analysis, preferably by experimentation. In addition, a new method of reducing the immunogenicity of antibodies is to induce immune tolerance in the body by introducing a Treg epitope into the antibody.

3. Improve antibody affinity

Binding affinity is one of the important factors affecting the function of therapeutic antibodies. Therefore, it is meaningful to increase the binding affinity of antibodies. It can be achieved by random mutagenesis, targeted mutagenesis, heavy-light-chain shuffling and computer simulation design. The first three methods are usually performed using display technology, and computer simulation is a relatively new method with computer aided design.

4. Antibody specificity optimization

To increase the targeting of antibodies, antibodies need to be engineered to optimize their specificity. Random mutagenesis and targeted mutagenesis are two commonly used methods to optimize antibody specificity.

5. Improve efficacy of the Fc end of the antibody

In addition to increasing the antigen binding ability of the antibody by altering the variable region, the biological effect of the antibody can also be enhanced by engineering the constant region (Fc) of the antibody. One way is to introduce mutations into the Fc domain of an antibody as needed to improve or reduce antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis and antibody-induced complement-dependent cytotoxicity. Another way is to improve the interaction between Fc and FcγRs is glycosylation of the Fc domain. Because FcγR interacts with carbohydrates on the CH2 domain, and the composition of these glycans has a substantial effect on antibody functional activity.

6. Improve antibody pharmacokinetic

Improving the pharmacokinetic properties of the therapeutic antibody can reduce the dosage and reduce the cost while extending the dosing interval, which is more convenient.

The clearance of therapeutic antibodies in humans is usually caused by two mechanisms: non-specific clearance: antibodies are non-specifically endocytosed by cells; and specific clearance: antigen binding mediates internalization and clearance.

To reduce non-specific clearance, experiments have shown that lowering the isoelectric point of an antibody can increase the half-life of the antibody in the study. Thus, it can be achieved by selecting a low isoelectric point antibody from a library of antibody variants (usually produced by random site mutagenesis) or from libraries of different humanized designs. To improve the second problem, antibodies that are sensitive to pH can be selected or developed. Such antibodies bind to the antigen under physiological conditions and dissociate from the antigen at low pH (the pH of the endosomal is 6.0).

7. Improvement in antibody medicinal properties

Therapeutic antibodies need to have good pharmaceutical properties, including: thermal stability, solubility, chemical stability, and less heterogeneity.

(1). Improve thermal stability

Poor thermal stability can result in antibody aggregation and low expression. The experimental results of Vogt et al. show that the number of hydrogen bonds and the fraction of polar surface area increase uniformly with the increase of thermal stability. Therefore, improving the thermal stability of antibodies can be optimized by optimizing hydrophobic cores and charged cluster residues, which can be obtained by point mutation studies using databases and computer design.

(2). Improve solubility

Since the volume of a single subcutaneous injection is usually limited to less than 1.5 mL, subcutaneous injection usually requires the use of a high concentration of antibody preparation. This also requires that antibodies as clinical drug candidates should have good solubility and viscosity. The primary method of increasing the solubility of antibodies is to remove surface hydrophobicity.

(3). Improve chemical stability

Chemical degradation in the body often leads to reduced efficacy of therapeutic antibodies and may be an issue requiring additional control in pharmaceutical research, so antibody clinical development should try to avoid these factors. In addition, the enzymatic site on the surface of the antibody protein structure also affects the chemical stability of the antibody. For this, a crystal structure or a computer-built 3D model structure can be used to study and improve the surface properties of the antibody structure, or by phage/yeast/mammalian libraries performing random or targeted mutations.

(4). Reduce heterogeneity

Another problem often encountered in antibody engineering is the glycosylation of proteins after translation, and the heterogeneity of antibody proteins caused by N-pyroglutamine cyclization, which also causes inconsistent antibody quality between different production batches. Antibodies with these modifications require additional quality control in manufacturing, which not only increases the workload but also increases the cost of the commodity. If such heterogeneity cannot be avoided by temperature control, "target site mutation" can be used to remove amino acids that may cause antibody heterogeneity.

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