The N162 Glycan on FcγRIIIa/CD16a Regulates Antibody Binding Affinity and Natural Killer Cell ADCC

Centrala begrepp

The N-glycan at position 162 of the FcγRIIIa receptor plays a crucial role in regulating antibody binding affinity and the potency of natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC).

Sammanfattning

• Bibliographic Information: Kulp, P. G., & Borrok, A. W. (2023). One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure. eLife, 12, e86014. https://doi.org/10.7554/eLife.86014
• Research Objective: This study investigates the role of the N-glycan at position 162 (N162) of the Fc gamma receptor IIIa (FcγRIIIa/CD16a) in antibody binding affinity and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC).
• Methodology: The researchers employed a multi-faceted approach involving site-directed mutagenesis to generate FcγRIIIa variants, surface plasmon resonance (SPR) to measure antibody binding affinities, lentiviral transduction to express FcγRIIIa variants in NK cells, and ADCC assays to assess the functional impact of these variants. Additionally, they used solution NMR spectroscopy and molecular dynamics (MD) simulations to investigate the structural basis for the observed effects.
• Key Findings:
  • The N162 glycan on FcγRIIIa is essential for the increased ADCC potency observed when NK cell N-glycan processing is inhibited.
  • Truncating the N162 glycan to a single N-acetylglucosamine (GlcNAc) residue results in the highest antibody binding affinity among the tested glycoforms.
  • The N162 glycan modulates the conformation of the FcγRIIIa FG loop, which contains the antibody binding site.
  • Removing the N162 glycan eliminates the synergistic effect of afucosylated antibodies and kifunensine treatment on NK cell ADCC.
• Main Conclusions: The N162 glycan on FcγRIIIa acts as a critical regulator of antibody binding affinity and NK cell ADCC. This regulation is mediated by the glycan's influence on the conformational dynamics of the FcγRIIIa FG loop.
• Significance: This study provides a deeper understanding of the molecular mechanisms underlying NK cell ADCC and highlights the importance of FcγRIIIa glycosylation in this process. These findings have implications for the development of improved antibody-based therapies that leverage NK cell-mediated cytotoxicity.
• Limitations and Future Research: The study primarily focused on the N162 glycan and its impact on FcγRIIIa function. Further research is needed to explore the potential roles of other N-glycans on FcγRIIIa or other NK cell surface receptors in ADCC. Additionally, investigating the in vivo relevance of these findings and the potential for manipulating N-glycan composition to enhance NK cell-based therapies is crucial.

Statistik

FcγRIIIa V158 binds approximately 4-fold tighter than the V158F allotype.
Removing the core α-linked mannose or sialic acid residues from the N-glycan increased antibody binding affinity by 5-fold or 2-fold, respectively.
Overall, N-glycan truncation increased antibody binding affinity by 12-fold.
The FcγRIIIa R155S (1)GlcNAc glycoform bound IgG1 Fc with a 5-fold weaker affinity compared to the wild type in the same glycoform.

Citat

"These results indicate that greater FcγRIIIa antibody-binding affinity on NK cells increases ADCC and FcγRIIIa engineering represents an unexplored avenue to improve NK cell-mediated immunotherapies."
"This result indicates that the N162 glycan is a critical mediator of increased ADCC responses following inhibiting both NK cell glycan remodeling and antibody fucosylation."
"Finally, we revealed new structural insights into FcγRIIIa that may lead to NK cells engineered to bind antibody through FcγRIIIa with high affinity as a novel strategy to improve immunotherapies."

Viktiga insikter från

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure

by Kremer,P. G.... på www.biorxiv.org 06-18-2024

https://www.biorxiv.org/content/10.1101/2024.06.17.599285v2

Djupare frågor

How can the understanding of N-glycan regulation of FcγRIIIa be leveraged to develop more effective NK cell-based therapies for cancer or infectious diseases?

Answer:
The discovery of the N162 glycan's regulatory role in FcγRIIIa activity opens exciting avenues for enhancing NK cell-based therapies. Here's how this understanding can be translated into therapeutic strategies:

Engineering NK Cells with Enhanced ADCC:

Ex vivo Glycan Engineering: NK cells could be engineered ex vivo to express predominantly high-affinity FcγRIIIa glycoforms. This could involve manipulating the glycosylation machinery within NK cells during cell expansion, for instance, by overexpressing enzymes favoring oligomannose-type glycans or knocking down enzymes involved in complex-type glycan formation.
S164A Mutation for Consistent ADCC: Introducing the S164A mutation in FcγRIIIa during NK cell engineering could provide a consistent and potentially safer approach. While this eliminates the ADCC boost from kifunensine or afucosylated antibodies, it ensures a predictable ADCC response, which might be beneficial in settings where uncontrolled ADCC could be detrimental.
CAR-NK Cells with Tuned Glycosylation: Chimeric antigen receptor (CAR)-NK cells could be engineered to express CARs with modified glycosylation sites near the antibody binding domain. This could enhance the binding affinity of the CAR to tumor-associated antigens, leading to improved tumor cell killing.

Optimizing Therapeutic Antibodies:

Afucosylation for Enhanced ADCC: The synergistic effect of afucosylated antibodies and NK cells with less-processed N-glycans could be exploited. This combination could lead to highly potent ADCC responses, potentially requiring lower antibody doses and minimizing side effects.

Personalized NK Cell Therapies:

Glycoprofiling for Patient Stratification: Patients could be stratified based on their NK cell N-glycan profiles. Individuals with a higher proportion of high-affinity FcγRIIIa glycoforms might respond better to certain antibody therapies or require lower doses.
Tailored Glycan Engineering: NK cell therapies could be personalized by tailoring the N-glycan profile to the patient's specific needs. This could involve modifying the glycosylation of infused NK cells to match the patient's antibody repertoire or disease state.

Could other mechanisms, beyond the N162 glycan's impact on conformational entropy, contribute to the observed differences in antibody binding affinity and ADCC potency?

Answer:
While the study highlights the significant role of the N162 glycan's conformational entropy in modulating FcγRIIIa affinity and ADCC, other mechanisms could contribute to the observed differences:

Direct Glycan-Glycan Interactions: Although the study suggests minimal contribution, direct interactions between the N162 glycan and the antibody Fc glycans cannot be entirely ruled out. Subtle variations in glycan composition could influence these interactions, impacting binding affinity.
Glycan-Mediated Receptor Clustering: N-glycans can influence receptor clustering on the cell surface. Different N-glycan compositions on FcγRIIIa might lead to variations in receptor density and clustering, affecting the avidity of the interaction with antibody-coated target cells.
Altered Receptor Dynamics and Signaling: N-glycans can influence receptor conformation and dynamics, impacting downstream signaling events. Variations in N-glycan composition could affect the efficiency of signal transduction upon antibody binding, influencing ADCC potency.
Impact on Receptor Expression and Turnover: N-glycosylation plays a role in protein folding, stability, and trafficking. Different N-glycan structures on FcγRIIIa might influence its expression levels, half-life, and turnover rate on the cell surface, indirectly affecting ADCC.

What are the broader implications of discovering a specific glycan modification that acts as a "tuning knob" for immune cell function, and could similar mechanisms exist for other immune receptors?

Answer:
The identification of the N162 glycan as a "tuning knob" for FcγRIIIa function has profound implications for our understanding of immune regulation and therapeutic development:

Glycans as Key Regulators of Immune Responses: This study underscores the importance of glycosylation in fine-tuning immune responses. It highlights that even subtle changes in glycan composition can significantly impact immune cell function, adding another layer of complexity to immune regulation.
Potential for Broad Applicability: Given the prevalence of glycosylation in immune receptors, it's highly likely that similar glycan-mediated regulatory mechanisms exist for other receptors. This opens a new avenue for investigating how glycans modulate immune cell signaling, activation, and effector functions.
New Therapeutic Targets: Targeting glycan-modifying enzymes or engineering specific glycoforms of immune receptors could offer novel therapeutic strategies for various immune-related diseases. This could involve enhancing or suppressing immune responses by manipulating the glycosylation of key immune receptors.
Challenges and Opportunities in Glycobiology: This discovery emphasizes the need for advanced tools and techniques in glycobiology to decipher the complex interplay between glycans and immune function. Understanding the specific enzymes and pathways involved in N-glycan regulation will be crucial for translating this knowledge into therapeutic applications.
In conclusion, the study not only provides valuable insights into FcγRIIIa regulation but also highlights the broader significance of glycosylation in immune cell biology. It paves the way for exploring glycan-mediated mechanisms in other immune receptors and developing novel therapeutic strategies targeting this intricate regulatory layer.