Humanization of Therapeutic Antibodies

Duration: 3 min

Jeffery Shi

Protein and Antibody Product Marketing

Jeffrey Shi, Head of Protein and Antibody Product Marketing Team of Marketing Department. He and his team are responsible for customer-centric development of full product life cycle management for Protein and Antibody, and drive the sustainable development of the protein antibody business.

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CONTENTS

• CDR Grafting
• Back Mutation
• Screening and Optimization
• Expression and Validation

Most therapeutic antibodies originate from non-human species, particularly mice. However, because the human immune system recognizes non-human antibodies as foreign, unmodified animal antibodies can trigger an immune response when administered to patients, leading to reduced efficacy or adverse reactions. This immune response can include the production of human anti-mouse antibodies (HAMA), which can rapidly clear the therapeutic antibody from circulation, neutralize its activity, or cause allergic reactions and other serious side effects. To overcome these challenges, the antibody must undergo a process known as humanization ^[1].

Humanization involves modifying the non-human antibody to resemble a human antibody as closely as possible, while retaining its ability to bind to the target antigen. This is typically done by grafting the antigen-binding regions (complementarity-determining regions or CDRs) from the animal antibody onto a human antibody framework. The main steps involved in antibody humanization include:

CDR Grafting: The most common method, known as complementarity-determining region (CDR) grafting, involves transferring the CDRs—the regions responsible for antigen binding—from the non-human antibody to a human antibody framework. These CDRs, derived from the variable regions of the original murine antibody, are critical for maintaining the specificity and affinity for the antigen.

Back Mutation: During CDR grafting, some key framework residues (outside the CDRs) in the original murine antibody may also be essential for maintaining the proper folding, stability, or antigen-binding conformation. In such cases, back mutation is performed, where specific murine framework residues are reintroduced into the humanized antibody to restore or enhance the binding affinity and overall stability. This step helps preserve the structure and function of the humanized antibody, especially if the human framework alone cannot support the optimal conformation.

Screening and Optimization: After CDR grafting and back mutations, the humanized antibody is tested for antigen binding and functionality. Further optimization may be required to improve its affinity, stability, and reduce immunogenicity. This could involve additional mutations in the framework regions, sequence engineering, or fine-tuning of the CDRs.

Expression and Validation: The humanized antibody is expressed in a suitable system (e.g., mammalian cells), followed by validation through biophysical and biochemical assays to ensure that the antibody retains its binding activity, stability, and therapeutic potential ^[2].

The resulting humanized antibody is less likely to be rejected by the patient’s immune system and is more suitable for therapeutic use in humans. This process not only reduces the risk of adverse immune reactions but also improves the pharmacokinetic properties of the antibody, allowing it to remain in the bloodstream for a longer period and maintain its therapeutic effect. Humanized antibodies have become a cornerstone of modern biopharmaceuticals, offering targeted treatments for a wide range of diseases, including cancer, autoimmune disorders, and infectious diseases ^[3].

References

[1] Wang Y, Chen Y L, Xu H, et al. Comparison of "framework Shuffling" and "CDR Grafting" in humanization of a PD-1 murine antibody. Front Immunol. 2024 Jul 15;15:1395854. doi: 10.3389/fimmu.2024.1395854.

[2] Gupta P, Horspool A M, Trivedi G, et al. Matrixed CDR grafting: A neoclassical framework for antibody humanization and developability. J Biol Chem. 2024 Jan;300(1):105555. doi: 10.1016/j.jbc.2023.105555.

[3] Ling W L, Lua W H, Gan S K E. Sagacity in antibody humanization for therapeutics, diagnostics and research purposes: considerations of antibody elements and their roles. Antib Ther. 2020 Apr 18;3(2):71-79. doi: 10.1093/abt/tbaa005.