Antibody Humanization Protocol and FAQs

Antibody Humanization refers to the modification of the structure of antibodies derived from non-human species (such as mice, rabbits, etc.) by molecular biology to make them closer to the antibody structure of the human immune system while maintaining their original immune activity. This process is to overcome the problem of immune response and therapeutic effect caused by ' heterologous antibody '. With the development of monoclonal antibody technology, antibody humanization has become a key technology in the field of biopharmaceuticals.

Non-human antibodies, such as mouse antibodies, have shown good results in the treatment of certain diseases, but because they are derived from non-human species, they are easy to cause immune rejection when used in humans. These immune responses may reduce the therapeutic effect and even lead to serious side effects. In order to solve this problem, scientists have developed antibody humanization technology, which replaces the structure of non-human antibodies with sequences closer to human antibodies, so that they can be better compatible with the human immune system.

Principle of Antibody Humanization

Antibody is a Y-shaped protein molecule composed of two heavy chains and two light chains. Each chain is composed of a series of amino acids, and the specific amino acid sequence determines the antigen binding characteristics of the antibody. The variable region (V region) of the antibody is responsible for binding to the antigen, while the constant region (C region) determines the biological function of the antibody.

In the antibodies of non-human species, the structure of the antigen binding site is usually quite different from that of human antibodies. This difference may cause the immune system to regard it as an ' exotic substance ' to produce an immune response. The main goal of antibody humanization is to change the amino acid sequence of the antibody, so that the variable region of the antibody retains the antigen binding ability of the original antibody as much as possible, and the structure of the constant region is modified to be closer to the structure of the human antibody.

Preserving Antigen Binding Sites

In the process of humanization, it is necessary to keep the structure of the antigen binding site (i.e., the variable region) of the antibody unchanged to ensure the specificity of the antibody.

Reducing Non-human Components

The amino acid sequences from non-human species in the heavy and light chains of antibodies need to be gradually replaced with human amino acid sequences.

Avoid Immune Response

By optimizing the amino acid sequence of the antibody, reduce or avoid the immune response caused by heterologous.

Dissolution and Complex Stability

The hydrophobic region of membrane protein makes it easy to aggregate or precipitate in water-soluble solutions, so appropriate dissolution buffers and auxiliary stabilizers are needed to maintain the stability of its structure.

The Steps of Antibody Humanization

Select the Appropriate Antibody Source

Usually, the first step in antibody humanization is to select an effective initial antibody. The antibody is generally derived from mice, rabbits or other species, with strong antigen specificity and high affinity. The most commonly used source is mouse monoclonal antibodies, which can recognize and bind to specific antigens.

Determine The Variable Region Sequence of The Antibody

After selecting the appropriate antibody, the researchers need to determine the amino acid sequence of its variable region. This step is usually done by gene sequencing technology, based on the amino acid sequence of the original antibody, to determine which parts need to be replaced by a sequence closer to the human antibody.

Design Humanized Sequences

Based on the known human antibody sequence library, scientists designed suitable ' humanized sequences '. The usual methods include :

CDR replacement method (Complementarity-Determining Region)

Only the complementarity determining region (CDR) of the antibody is modified to replace the CDR sequence of non-human species with the corresponding sequence of humans. CDR is a key region for the binding of antibodies to antigens, so this step should ensure that the specificity and affinity of antibodies are retained as much as possible.

Whole sequence replacement

In some cases, the entire variable region sequence needs to be humanized to replace all non-human amino acids.

Purification of Membrane Protein

Purification of membrane proteins usually uses affinity chromatography, ion exchange chromatography, gel filtration chromatography and other techniques. Affinity chromatography usually uses tags (such as 6xHis tags) or antibody-specific purification of membrane proteins. Gel filtration is used to separate membrane proteins and other small molecular impurities according to molecular size. During the purification process, it is important to maintain the stability of membrane proteins. Stabilizers and low temperature conditions are often required to prevent degradation or aggregation of membrane proteins.

Gene Snthesis and Recombination

After the design is completed, genetic engineering technology is used to synthesize the modified antibody gene. The humanized antibody gene was inserted into the expression vector by gene recombination technology. These vectors are usually viral vectors or plasmids that can express the required antibodies in cells.

Cell Expression and Antibody Production

After gene recombination, the laboratory transferred these vectors into suitable host cells (such as E.coli, mammalian cells or insect cells) for expression. These host cells can produce humanized antibodies on a large scale in culture medium.

Antibody Purification

The antibodies produced need to be purified, usually using affinity chromatography or other separation techniques to extract high purity antibody molecules.

Functional Verification and Evaluation

The purified antibody needs to be functionally verified to ensure that it has good antigen binding ability and biological activity. Commonly used methods include ELISA (enzyme-linked immunosorbent assay), immunofluorescence staining, flow cytometry, etc.

Animal Experiments and Preclinical Studies

After the preliminary verification of humanized antibodies, small animal experiments are usually carried out to evaluate their safety and efficacy in vivo. This stage can determine the potential problems such as antibody toxicity and immune response, and provide data support for clinical trials.

Clinical Trials and Applications

After passing the pre-clinical study, humanized antibody will enter the clinical trial stage. After several rounds of clinical trial verification, if it meets the requirements of safety and effectiveness, it can enter the market application stage.

Figure 1. Schematic overview of the Nanobody humanization strategy. (Reference source: General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold.）

Advantages of Antibody Humanization

1 Reduce immunogenicity : reduce the rejection of the immune system.
2 Improve safety : reduce side effects and allergic reactions.
3 Enhance efficacy : Optimize the binding of antibodies to target antigens and increase affinity.
4 Improve half-life : prolong the action time of the drug in the body.
5 Suitable for long-term treatment : suitable for chronic diseases and long-term treatment, reduce the frequency of injection.
6 Widely used : suitable for cancer, immune diseases and other diseases.

Alpha Lifetech's comprehensive service includes the design, synthesis, expression, and characterization of humanized antibodies, as well as the validation of their function through a variety of in vitro and in vivo assays.

FAQ

1. Even after humanized treatment, some antibodies may still cause rejection of the immune system, resulting in the production of anti-drug antibodies (ADA), affecting efficacy and safety.

Solution : (1) Homology modeling and sequence optimization were used to optimize the antibody framework regions (FRs) and reduce the potential immunogenicity regions. (2) Combined with in vitro immunogenicity prediction tools (such as T cell epitope prediction) for optimization. (3) Use a fully human antibody library (such as Phage Display) or monoclonal antibody engineering to reduce immunogenicity.
2. The process of humanization may lead to a decrease in the affinity of antibodies and antigens, affecting the efficacy.

Solutions : (1) Reverse mutation technology was used to restore key sites and improve affinity. (2) Combined with computer simulation, the structure of complementary determining regions (CDRs) was optimized to improve the affinity. (3) Antibodies are optimized by affinity maturation techniques (such as phage display screening).
3. Humanization of antibodies may affect folding stability, leading to aggregation or degradation, affecting drug development.

Solutions : (1) Stable mutation sites were screened by computer simulation. (2) The directional mutation is used to optimize the key structural regions and improve the stability. (3) In vitro expression and functional verification were performed to ensure that the antibody maintained its original activity and stability.
4. When humanized antibodies are expressed in mammalian cells, low yield or misfolding may occur.

Solutions : (1) The appropriate expression system (such as CHO cells) was selected to optimize antibody expression. (2) Adjust the signal peptide and codon optimization to improve translation efficiency. (3) Protein engineering (such as optimizing glycosylation sites) was used to improve stability and expression levels.
5. Humanized antibodies may show a shorter half-life, affecting drug efficacy.

Solutions : (1) The interaction between antibody and neonatal Fc receptor (FcRn) was optimized by Fc engineering to improve the half-life. (2) PEG modification or fusion such as human serum albumin was used to improve in vivo stability. (3) PK / PD modeling was performed to optimize the dose and dosage regimen.

reference

[1] Safdari Y, Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods - a review and update. Biotechnol Genet Eng Rev. 2013;29:175-186. doi:10.1080/02648725.2013.801235

[2] Jiacomini IG, Beltramino M, Boursin F, et al. An effective strategy for the humanization of antibody fragments under an accelerated timeline. Int J Biol Macromol. 2022;216:465-474. doi:10.1016/j.ijbiomac.2022.06.195

[3] Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem. 2009;284(5):3273-3284. doi:10.1074/jbc.M806889200

[4] Aubrey N, Billiald P. Antibody Fragments Humanization: Beginning with the End in Mind. Methods Mol Biol. 2019;1904:231-252. doi:10.1007/978-1-4939-8958-4_10

[5] Waldmann H. Human Monoclonal Antibodies: The Benefits of Humanization. Methods Mol Biol. 2019;1904:1-10. doi:10.1007/978-1-4939-8958-4_1