Engineering and Translational Evaluation of A Novel Albumin-binding VHH for Half-life Extension
Description
This dataset supports the manuscript “Engineering and Translational Evaluation of A Novel Albumin-binding VHH for Half-life Extension”, containing raw data for key experimental/computational analyses to ensure reproducibility of albumin-binding VHH-mediated protein drug half-life extension findings. 1. Image-Associated Raw Data Includes raw data for all manuscript figures: Surface Plasmon Resonance (SPR) files (Biacore T200 sensorgrams, CSV) for anti-HSA VHH (e.g., clone H2) binding to HSA/CSA, plus alanine-scanning mutagenesis data (key HSA mutants) validating binding residues. Pharmacokinetic (PK)/Pharmacodynamic (PD) data (Excel/CSV) covers serum concentration-time profiles (hFcRn transgenic mice, HSA/hFcRn double-transgenic mice, cynomolgus monkeys: VHH candidates D6/A1/H2, FSH-m7, wild-type FSH) and juvenile Sprague-Dawley rat ovarian weight/serum estradiol (LC-MS/MS). Dynamic Light Scattering (DLS)/nanoDSF data (CSV) includes FSH-m7’s hydrodynamic size, PDI, Tm, and Tagg from Prometheus Panta. 2. In Silico Immunogenicity Prediction Raw Data Raw CSV/TXT files for H2-VHH4 and FSH-m7: NetMHCpan EL 4.1 (9-mer MHC-I) and NetMHCIIpan EL 4.1 (15-mer MHC-II) outputs (binding scores, strong/weak binder classification, 43 HLA-I/30 HLA-II alleles for global coverage). ABCpred data (16-residue window, threshold 0.51) provides residue-wise scores for 348-aa FSH-m7, annotating VHH-FSH junction and GGGGS linkers. 3. H2-HSA Complex 3D Structure Prediction Data AlphaFold3 raw files for HSA-VHH (H2) heterodimer: CIF/JSON 3D structure files (residue coordinates, intermolecular interactions) and Excel/CSV assessment data (iptm=0.87, overall ranking score=0.87, residue-wise interaction annotations validated by alanine scanning). Data Reuse Value Enables validating VHH-HSA binding, reproducing FSH-m7/VHH PK/PD profiles, extending albumin-binding biologic immunogenicity analyses, and optimizing VHH-albumin structures. Data is compatible with GraphPad Prism/Discovery Studio 2025 and adheres to FAIR principles.
Files
Steps to reproduce
To reproduce the data, follow these key steps: 1 VHH Screening & CloningUse a naïve alpaca VHH phage display library. Perform biopanning against human serum albumin (HSA) and cynomolgus serum albumin (CSA). Identify positive VHH clones via ELISA, sequence them, and clone selected VHH genes into expression vectors. 2 Protein Expression & PurificationExpress VHH, wild-type FSH, and FSH-VHH fusion protein (FSH-m7) in E. coli or mammalian cell systems. Purify proteins via affinity chromatography, then verify purity/integrity using SDS-PAGE and SEC-HPLC. 3 Pharmacokinetic (PK) ExperimentAdminister FSH-m7 or wild-type FSH to female HSA/hFcRn double-transgenic mice (6–8 weeks old) via intravenous injection. Collect blood samples at time points: 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 h. Measure drug concentrations by ELISA, and calculate PK parameters (half-life, AUC, clearance) via non-compartmental analysis. 4 Pharmacodynamic (PD) ExperimentInject FSH-m7 or vehicle control into juvenile female SD rats. Harvest ovaries to measure ovarian weight, and use commercial kits to assess serum estradiol (E2) levels. Ensure reagents (e.g., antibodies, ELISA kits) match specified suppliers, and instruments (HPLC, ELISA reader) follow standard operating protocols.