Definition and Significance of Antibody Humanization

Antibodies have become essential tools for the diagnosis and treatment of numerous human diseases. However, non-human antibodies, such as those derived from murine sources, often provoke human anti-mouse antibody (HAMA) responses. This immunogenicity leads to rapid clearance and neutralization, limiting their clinical use.

To address this challenge, antibody humanization strategies have been developed to reduce the immunogenicity of therapeutic antibodies. These methods aim to preserve antigen specificity and binding affinity while replacing immunogenic sequences with human counterparts. Over the past decades, several humanization techniques have emerged, each with varying success in preserving function and minimizing immune responses.

However, despite the development of alternative discovery platforms such as transgenic mice and phage display libraries containing fully human gene fragments, most antibody therapeutics continue to be derived from non-human sources. The subsequent humanization of these antibodies remains critical to improve their compatibility with the human immune system and minimize anti-drug antibody (ADA) responses.

CDR Grafting Based on Framework Region Homology and the Role of Vernier Zone Residues

Complementarity-determining region (CDR) grafting remains the most widely used humanization technique. It involves transplanting the CDRs of non-human antibodies onto human antibody framework regions (FWRs) that share high sequence homology. This method was originally established by selecting human frameworks that most closely resemble the murine antibody's FWRs to preserve the structural support of the antigen-binding loops.

One limitation of this approach is the potential loss of antigen-binding affinity, often due to structural differences between the donor and recipient frameworks. Critical framework residues known as vernier zone residues, located within β-sheet regions adjacent to CDRs, play essential roles in maintaining CDR loop conformation. Inadequate retention of these residues can compromise antibody function. Early CDR grafting strategies often failed to consider the broader structural and sequence context of individual antibodies, limiting their success rate. This has led to a shift toward more nuanced humanization methods that evaluate the role of framework and surface residues beyond just the CDR loops.

For example, in the murine D1.3 antibody, 30 vernier zone residues have been identified, including 16 in the heavy chain and 14 in the light chain. Humanized variants retaining these residues have shown restored or preserved affinity. Herceptin (trastuzumab), developed via CDR grafting with retained vernier residues, exemplifies the successful application of this method.

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Germline Humanization

Germline sequences, representing unmutated human antibody gene segments, offer an alternative framework for humanization. These frameworks have fewer somatic hypermutations than mature IgG sequences and are thus potentially less immunogenic.

For example, the murine antibody WO-2, targeting Aβ peptide, was humanized using human germline frameworks while retaining murine vernier residues. Although the humanized single-chain and Fab fragments exhibited reduced affinity relative to papain-cleaved parental Fab, the nanomolar-range binding remained therapeutically relevant. Similarly, multiple Fab formats of an anti-CD28 antibody were constructed using germline sequences, with humanized variants showing moderate reductions in avidity but retaining immunosuppressive activity. Comparable outcomes have been observed with other antibodies using this strategy, where affinity loss was mitigated through targeted CDR and framework mutations.

IgG- or Germline-Based Humanization: Advantages and Disadvantages

While germline frameworks are generally less immunogenic, IgG-derived sequences can provide superior functional characteristics, particularly through Fc-mediated effector functions. For instance, a chimeric IgG1 antibody based on murine 2C9 effectively neutralizes yellow fever virus, whereas the IgM variant was ineffective. The critical role of the Fc domain was further highlighted by differences in antibody-dependent cellular cytotoxicity (ADCC) observed between IgG1 and IgG4 variants of the anti-GD2 antibody hu3F8.

Such findings illustrate the importance of Fc region selection, not only in effector function but also in pharmacokinetics and stability, highlighting a trade-off between immunogenicity and therapeutic performance.

Affinity Maturation of Humanized Antibodies

Affinity maturation strategies are often required to restore or enhance the antigen-binding properties of humanized antibodies. These methods typically involve mutagenesis within CDRs, especially CDR-H3, due to its high variability and central role in antigen interaction.

Yang et al. (2017) generated affinity-matured variants of the hHzKR127 antibody against hepatitis B virus by introducing mutations in CDR-H3. Some clones showed up to six-fold higher affinity than the parental humanized antibody, illustrating the significant impact of targeted modifications. Notably, differences in constant regions also affected binding affinity, highlighting inter-domain interactions.

An alternative approach, known as specificity-determining residue (SDR) grafting, focuses only on the antigen-contacting residues within CDRs. This method can reduce the number of murine residues retained, potentially lowering immunogenicity. 

Efforts to enhance antibody affinity must be balanced with the need to preserve other drug-like properties such as solubility, stability, and reduced immunogenicity. This introduces a multi-parameter optimization problem, where improvements in one property may compromise another. Emerging computational approaches are being developed to address this complexity systematically.

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Humanization via Resurfacing

Resurfacing, also referred to as superhumanization, involves replacing potentially antigenic surface-exposed framework residues with the most common human counterparts, while leaving buried residues and CDRs intact. This approach was developed based on the observation that HAMA responses are primarily directed against solvent-accessible residues. In one example, the 82D6A3 antibody was humanized using this method, resulting in minimal affinity loss. Another application involved the humanization of the murine anti-TNF-α antibody m357 by modifying six of seventeen non-conserved surface residues to match human sequences. The resulting antibody, h357 IgG1, maintained high antigen-binding affinity and demonstrated functional bioactivity, including ADCC and complement-dependent cytotoxicity.

Humanization Based on CDR Homology

In contrast to framework-based grafting, the CDR homology approach focuses on identifying human frameworks with similar CDR loop structures, irrespective of framework homology. This method avoids retention of murine vernier residues, reducing the risk of immunogenicity. Structurally compatible human frameworks can support murine CDRs without backmutation, making this approach particularly attractive for minimizing immunogenic elements. 

Mutation in the Constant Region of Antibodies

Beyond variable regions, modifications in the constant region (Fc) can influence antibody safety and function. Such targeted Fc engineering has been employed in HIV and Ebola antibody development, where preventing immune-mediated enhancement of infection is crucial.

Improving Specificity and Affinity

Antibody specificity and affinity can be enhanced through rational design and directed evolution. Common strategies include the insertion or substitution of amino acid residues in CDRs, particularly CDR-H3, to optimize binding interactions. Site-directed mutagenesis may be used to introduce specific chemical interactions such as salt bridges or hydrophobic contacts. Mutagenesis of framework or light chain regions can also influence binding characteristics and paratope configuration.

Insertional mutations within CDR loops are another method to increase antigen-binding affinity. Modifications may also involve substituting residues to reduce off-target binding and improve selectivity. Framework region mutations, including those that preserve structural integrity while modulating paratope conformation, are frequently used to optimize therapeutic antibodies. Additionally, mutational lineage-guided approaches can be applied during humanization to retain beneficial characteristics while minimizing non-human content.

Immunogenicity of Humanized Antibodies

Despite advances in humanization, some degree of immunogenicity may persist, even in fully human antibodies. Framework-homology-based humanized antibodies carry murine CDRs and possibly vernier zone residues, which can be recognized as foreign. In contrast, CDR-homology-based antibodies, with fully human frameworks and no murine vernier residues, may exhibit lower immunogenicity.

Immunogenicity is influenced by both the antibody framework and the antigen-binding site. Modifying CDR residues is a potential strategy to further reduce immunogenicity. Antibody responses in patients can be broadly categorized based on the incidence of anti-drug antibodies: negligible, tolerable, or marked. Most humanized antibodies exhibit low to moderate immunogenicity, but exceptions occur. Even fully human antibodies can elicit immune responses, demonstrating that humanization reduces but does not completely eliminate the risk of immunogenicity.

While humanness metrics are frequently used to predict immunogenic potential, they do not provide a definitive safeguard against ADA responses. These metrics often rely on sequence similarity to human germline antibodies, but even genetically human sequences can provoke immunogenicity due to patient-specific factors such as HLA variability and immune status.

Linkers in Single-Chain Antibodies

Single-chain variable fragments (scFvs) require peptide linkers to covalently connect the VH and VL domains into a single polypeptide chain. These linkers play a critical role in maintaining proper domain orientation and ensuring functional antigen binding. The most commonly used linker sequence is (Gly₄Ser)₃, chosen for its flexibility and minimal impact on structural integrity.

Linker length and composition directly influence the conformation, oligomerization state, and antigen-binding affinity of scFvs. Longer linkers typically promote the formation of monomeric scFvs, whereas shorter linkers can lead to dimerization or trimerization through inter-domain interactions. Optimal linker design is essential to ensure correct folding, stability, and binding efficiency of the engineered antibody fragment.

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References:

1. Safdari, Y., Farajnia, S., Asgharzadeh, M., & Khalili, M. (2013). Antibody humanization methods – a review and update. Biotechnology and Genetic Engineering Reviews. https://doi.org/10.1080//02648725.2013.801235

2. Yang, G. H., Yoon, S. O., Jang, M. H., & Hong, H. J. (2007). Affinity maturation of an anti-hepatitis B virus PreS1 humanized antibody by phage display. Journal of microbiology (Seoul, Korea), 45(6), 528–533.