Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists
Abstract
1. Introduction
1.1. Heavy Chain Single Domain Antibodies in Protein Therapeutics
1.2. Cytokines as Central Regulators of the Immune Response
1.3. Antibody-Based Cytokine Mimics
2. Results
2.1. Discovery and Characterization of IL-18Rα and IL-18Rβ Binders as Surrogate Cytokine Agonists
2.2. Generation and Screening of IL-2Rβ and IL-2Rγ Surrogate Cytokine Agonists
2.3. IL-18 and IL-2 SCA Activity Can Be Tuned via Modulation of Linker Length
2.4. SCAs Can Adopt Multiple Dimeric Conformations
2.5. Engineered Disulfide Bonds Between the Two VHHs of an IL-18 SCA Result in Higher Thermostability and Different Activity Levels
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Immunization of Llamas (IL-18 VHHs)
5.2. Library Construction (IL-18 VHHs)
5.3. Phage Display (IL-18 VHHs)
5.4. Immunization of Camels (IL-2 VHHs)
5.5. Library Construction (IL-2 VHHs)
5.6. Phage Display (IL-2 VHHs)
5.7. Protein Design, Expression, and Purification
5.8. IL-18 SCA HEK-Blue Reporter Assay
5.9. IL-18 SCA IFNγ Secretion Assay
5.10. IL-2 SCA STAT5 Phosphorylation, Proliferation, and IFNγ Production on NK and CD8 T Cells
5.11. IL-2 SCA Linker Variant STAT5 Phosphorylation on Total T Cells
5.12. Crystallography
5.13. DR3097 Disulfide Stapling, Stability Test, and Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
AHC | Anti-Human Capture |
CDRs | Complementary determining regions |
CFA | Complete Freund’s Adjuvant |
DLS | Dynamic light scattering |
DSF | Differential scanning fluorimetry |
ECD | Extracellular domain |
IFA | Incomplete Freund’s Adjuvant |
IFNγ | Interferon γ |
HEK | Human embryonic kidney |
IgG | Immunoglobulin G |
IL-2 | Interleukin 2 |
IL-2Rβ | Interleukin-2 receptor β |
IL-2Rγ | Interleukin-2 receptor γ |
IL-18 | Interleukin 18 |
IL-18BP | IL-18 binding protein |
IL-18Rα | Interleukin-18 receptor α |
IL-18Rβ | Interleukin-18 receptor β |
LCMS | Liquid chromatography-mass spectrometry |
mAb | Monoclonal antibody |
MSA | Mouse serum albumin |
NF-kB | Nuclear factor kappa B |
PBMC | Peripheral blood mononuclear cell |
PBS | Phosphate-buffered saline |
SCA | Surrogate cytokine agonist |
scFv | Single-chain fragment variable |
sdAbs | Single domain antibodies |
SEAP | Secreted embryonic alkaline phosphatase |
SEC | Size exclusion chromatography |
SPR | Surface plasmon resonance |
STAT | Signal transducer and activator of transcription |
TIR | Toll/interleukin-1 receptor |
Tm | Melting temperature |
VHH | Variable heavy domain of Heavy chain |
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Linker Length (Residues) | |||||||
---|---|---|---|---|---|---|---|
0 | 2 | 4 | 10 | 15 | 20 | ||
DR3085 | EC50 (nM) | 0.643 | 0.148 | 0.121 | 0.064 | 0.055 | 0.085 |
Emax (ng/mL) | 3.4 | 12.6 | 80.4 | 152.6 | 112.6 | 90.6 | |
DR3087 | EC50 (nM) | 1.491 | 2.861 | 0.771 | 1.157 | ||
Emax (ng/mL) | 109.9 | 90.8 | 162.5 | 86.7 | |||
DR3097 | EC50 (nM) | 0.088 | 0.012 | 0.002 | 0.001 | 0.002 | 0.031 |
Emax (ng/mL) | 1.7 | 37.0 | 156.0 | 246.4 | 190.6 | 180.5 |
SCA Name PDB ID | DR638 9O99 | DR3085 9O9C | DR3087 9O9A | DR3097 9O9B |
---|---|---|---|---|
Data collection | ||||
Space group | P 21 21 21 | P 65 | C 1 2 1 | P 3 |
Cell dimensions | ||||
a, b, c (Å) | 49.21, 63.13, 87.45 | 92.64, 92.64, 181.85 | 91.81, 172.66, 59.29 | 120.1, 120.1, 29.27 |
a, b, γ (°) | 90, 90, 90 | 90, 90, 120 | 90, 90.01, 90 | 90, 90, 120 |
Resolution (Å) * | 38.81–1.80 (1.84–1.80) | 39.17–3.80 (4.25–3.80) | 37.66–2.4 (2.49–2.4) | 39.31–2.8 (2.95–2.80) |
Rsym or Rmerge * | 0.067 (0.774) | 0.34 (0.748) | 0.055 (0.788) | 0.112 (0.672) |
I/sI | 12.8 (3.1) | 3.3 (1.6) | 11.8 (1.6) | 6.7 (1.6) |
Completeness (%) * | 99.6 (99.9) | 99.2 (100.0) | 95.6 (97.1) | 96.3 (97.9) |
Redundancy * | 4.5 (4.5) | 3.0 (3.1) | 3.7 (3.7) | 2.8 (2.8) |
Refinement | ||||
Resolution (Å) | 1.8 | 3.80 | 2.4 | 2.8 |
No. reflections | 24498 | 8208 | 32612 | 11206 |
Rwork/Rfree | 0.180/0.211 | 0.238/0.291 | 0.241/0.280 | 0.246/0.283 |
No. nonhydrogen atoms | 2088 | 3447 | 3533 | 3263 |
Protein | 1820 | 3447 | 3486 | 3263 |
Ligand/ion | 0 | 0 | 0 | 0 |
Water | 268 | 0 | 47 | 0 |
B-factors | ||||
Protein | 26.6 | 87.7 | 74.9 | 64.4 |
Ligand/ion | ||||
Water | 40.1 | 58.1 | ||
R.m.s. deviations | ||||
Bond lengths (Å) | 0.009 | 0.004 | 0.007 | 0.007 |
Bond angles (°) | 1.767 | 1.283 | 1.718 | 1.331 |
Ramachandran | ||||
Favored (%) | 97.95 | 91.91 | 94.77 | 91.5 |
Allowed (%) | 2.05 | 8.09 | 5.00 | 8.5 |
Outliers (%) | 0 | 0 | 0.23 | 0 |
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Trenker, R.; Rokkam, D.; Morin, A.; Balasubrahmanyam, P.; Paredes, V.; Cheng, I.; de Waal Malefyt, R.; Oft, M.; Lupardus, P.; Vivona, S. Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists. Antibodies 2025, 14, 74. https://doi.org/10.3390/antib14030074
Trenker R, Rokkam D, Morin A, Balasubrahmanyam P, Paredes V, Cheng I, de Waal Malefyt R, Oft M, Lupardus P, Vivona S. Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists. Antibodies. 2025; 14(3):74. https://doi.org/10.3390/antib14030074
Chicago/Turabian StyleTrenker, Raphael, Deepti Rokkam, Andrew Morin, Priyanka Balasubrahmanyam, Verenice Paredes, Ivan Cheng, Rene de Waal Malefyt, Martin Oft, Patrick Lupardus, and Sandro Vivona. 2025. "Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists" Antibodies 14, no. 3: 74. https://doi.org/10.3390/antib14030074
APA StyleTrenker, R., Rokkam, D., Morin, A., Balasubrahmanyam, P., Paredes, V., Cheng, I., de Waal Malefyt, R., Oft, M., Lupardus, P., & Vivona, S. (2025). Structure-Guided Stapling of Dimeric Conformations and Linker Engineering Enhance Thermostability and Fine-Tune Activity of Bispecific VHH Cytokine Agonists. Antibodies, 14(3), 74. https://doi.org/10.3390/antib14030074