Techniques for Humanizing Antibodies: CDR Grafting, Framework Substitution, and Affinity Maturation

Introduction to Antibody Humanization

Antibodies are one of the fastest-growing fields of drugs in the world. However, non-human antibodies, particularly those derived from murine sources, can elicit immune responses in humans that result in their rapid clearance and diminished therapeutic efficacy. Humanization technologies were developed to mitigate this immunogenicity by reducing the presence of foreign sequences while retaining antigen specificity and biological activity.

Over the years, several humanization strategies have been introduced, including CDR grafting, germline or IgG-based framework substitution, and techniques for affinity maturation. This article focuses on recent findings that have shaped the current landscape of antibody humanization.

CDR Grafting Based on Framework Homology and Vernier Zone Residues

CDR grafting remains one of the most applied methods in antibody humanization. In this approach, the complementarity-determining regions (CDRs) from a non-human antibody are transplanted onto the framework regions (FWRs) of a human antibody. Frameworks with the highest sequence homology to the original murine antibody are typically selected as the acceptor scaffold.

However, loss of antigen-binding affinity after humanization can happen. Vernier zone residues play a critical role in supporting the correct conformation of CDR loops. These vernier zone residues are located in β-sheet regions underlying the CDRs and can significantly influence binding characteristics.

Several therapeutic antibodies, including trastuzumab, have been developed using this method, with selective retention of vernier zone residues to balance affinity and reduced immunogenicity.

Related: Affinity Humanization

Framework Substitution Using Germline and IgG-Derived Sequences

Framework substitution involves replacing non-human framework regions (FWRs) with human sequences to reduce immunogenicity while preserving antigen-binding functionality. Human frameworks used for substitution typically derive from either germline genes or mature IgG sequences.

Human germline sequences are preferred for minimizing immunogenicity due to their lower degree of somatic hypermutation. However, they may lack optimal structural compatibility with non-human CDRs, which can lead to reduced affinity or compromised stability. This issue has been observed in multiple cases, where affinity loss following germline substitution was partially restored by introducing targeted mutations in both CDR and framework residues.

The Fc region also plays a key role in maintaining antibody function. Differences in Fc isotype can influence effector functions, including antibody-dependent cellular cytotoxicity (ADCC). Antibodies with identical variable regions but differing Fc domains, such as IgG1 versus IgG4, have been shown to vary significantly in their ability to mediate ADCC, reflecting the importance of constant region selection in antibody engineering.

Framework shuffling presents an alternative approach that addresses many of the structural limitations associated with direct framework substitution or CDR grafting. This method combines CDRs with a large panel of human germline frameworks, allowing for the identification of structurally favorable and biophysically robust combinations. Humanized antibodies generated through framework shuffling typically exhibit high thermal stability, maintain antigen-binding function after thermal stress, and display lower aggregation tendencies during expression and purification.

Related: Affinity Maturation

Affinity Maturation Strategies

Antibody humanization can reduce binding affinity due to structural changes affecting the antigen-binding site. To address this, affinity maturation is commonly performed through targeted mutagenesis, particularly in CDR-H3, which plays a central role in antigen recognition.

An alternative method, SDR grafting, involves transferring only the antigen-contacting residues within the CDRs. This can improve affinity while limiting the number of non-human residues retained, potentially reducing immunogenicity.

Affinity enhancement can also be achieved through rational modifications, such as amino acid insertions or substitutions that introduce stabilizing interactions or improve specificity. Targeted changes in other CDRs or adjacent regions have been shown to reduce cross-reactivity and strengthen binding.

These approaches help recover or improve antibody function following humanization and support downstream development by optimizing key biophysical and binding properties.

Humanization via Resurfacing

Resurfacing is a strategy for reducing the immunogenicity of non-human antibodies by replacing solvent-accessible framework residues with their human equivalents. Unlike CDR grafting, which transfers entire antigen-binding loops, resurfacing retains the original CDRs and focuses only on altering surface-exposed residues that are likely to be recognized by the human immune system.

This method relies on structure-based modeling to identify antigenic residues while preserving those that maintain CDR conformation. Typically, internal framework residues and those critical for structural support are left unchanged to retain binding function.

Resurfaced antibodies often show minimal changes in affinity and stability. In several cases, selective replacement of surface residues has resulted in antibodies with reduced immunogenic potential while preserving antigen-binding activity and effector functions such as ADCC and CDC. Resurfacing offers a targeted approach to humanization, with limited impact on overall structure and function.

Humanization Based on CDR Homology

CDR homology-based humanization selects human frameworks that share high similarity with the murine CDRs, rather than focusing on overall framework sequence identity. This strategy assumes that CDR-homologous frameworks are more likely to support native loop conformations, reducing structural disruption during humanization.

Unlike traditional CDR grafting, this method does not require back-mutation of vernier zone residues, thereby minimizing the number of non-human residues retained. As a result, it reduces potential immunogenicity while maintaining functional integrity.

This approach simplifies the design process by avoiding extensive structural modeling and empirical adjustments. It produces fully human frameworks compatible with diverse CDRs, offering an efficient path to generate therapeutically viable antibodies with lower risk of immunogenic responses.

Mutation in Constant Regions to Modify Effector Function

In addition to engineering the variable regions, modifications to the antibody constant domains can be employed to modulate immune effector functions. Changes in the Fc region can suppress undesired activities or enhance therapeutic mechanisms such as ADCC, CDC, and serum persistence.

For example, reducing Fc receptor interactions through targeted mutations in the CH2 domain has been used to eliminate antibody-dependent enhancement (ADE), a known risk in certain viral infections. These modifications can preserve antigen-binding while preventing immune activation that may exacerbate disease.

Fc engineering also enables selective enhancement of immune functions. Adjustments to specific Fc residues can increase or diminish effector responses, depending on the therapeutic goal. This makes constant region optimization a valuable complement to variable region humanization, particularly for antibodies intended for clinical use where precise control of immune activity is critical.

Linkers in Single-Chain Antibodies

In single-chain variable fragments (scFvs), the variable heavy (VH) and light (VL) domains are connected by peptide linkers that enable proper domain pairing and folding. The length and amino acid composition of these linkers can significantly influence the structural stability, folding efficiency, and antigen-binding performance of the scFv.

A commonly used linker is the 15-amino-acid sequence (Gly₄Ser)₃, which provides high flexibility without disrupting the conformation of the VH and VL domains. Structural analyses have shown that such linkers do not interfere with domain orientation and maintain native folding.

Linker length directly affects oligomerization behavior. Linkers longer than 12 residues typically support proper monomeric scFv formation. Intermediate-length linkers between 3 and 12 residues can lead to dimer formation through interchain interactions, while linkers shorter than 3 residues often result in higher-order multimers such as trimers. These effects must be considered during scFv design, as oligomerization can impact binding kinetics, solubility, and overall therapeutic performance.

Implications for Biotech Development and Antibody CROs

Antibody CROs like Biointron offer comprehensive humanization services to support the development of next-generation antibody therapeutics.

References:

1. Safdari, Y., Farajnia, S., Asgharzadeh, M., & Khalili, M. (2013). Antibody humanization methods – a review and update. Biotechnology and Genetic Engineering Reviews, 29(2), 175–186. https://doi.org/10.1080/02648725.2013.801235

2. Wang, Y., Chen, Y., Xu, H., Rana, G. E., Tan, X., He, M., Jing, Q., Wang, Q., Wang, G., Xie, Z., & Wang, C. (2024). Comparison of “framework Shuffling” and “CDR Grafting” in humanization of a PD-1 murine antibody. Frontiers in Immunology, 15, 1395854. https://doi.org/10.3389/fimmu.2024.1395854

3. Apgar, J. R., Mader, M., Agostinelli, R., Benard, S., Bialek, P., Johnson, M., Gao, Y., Krebs, M., Owens, J., Parris, K., Andre, M. S., Svenson, K., Morris, C., & Tchistiakova, L. (2016). Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody. MAbs, 8(7), 1302. https://doi.org/10.1080/19420862.2016.1215786