Inference of molecular structure for characterization and improvement of clinical grade immunocytokines

Ongaro T, Guarino S, Scietti L, Palamini M, Wulhfard S, Neri D Villa A, Forneris F, Journal of Structural Biology :107696 (2021) DOI

SASDJN8 – L19-IL2 dimeric immunocytokine

L19-IL2 dimeric immunocytokine
MWI(0) 64 kDa
MWexpected 84 kDa
VPorod 128 nm3
log I(s) 6.62×101 6.62×100 6.62×10-1 6.62×10-2
L19-IL2 dimeric immunocytokine small angle scattering data  s, nm-1
ln I(s)
L19-IL2 dimeric immunocytokine Guinier plot ln 6.62×101 Rg: 4.1 nm 0 (4.1 nm)-2 s2
(sRg)2I(s)/I(0)
L19-IL2 dimeric immunocytokine Kratky plot 1.104 0 √3 sRg
p(r)
L19-IL2 dimeric immunocytokine pair distance distribution function Rg: 4.1 nm 0 Dmax: 11.9 nm

Data validation

Fits and models

log I(s)
 s, nm-1
L19-IL2 dimeric immunocytokine ALLOSMOD model
log I(s)
 s, nm-1
L19-IL2 dimeric immunocytokine GASBOR model
Synchrotron SAXS data from solutions of the L19-IL2 immunocytokine in 25 mM HEPES/NaOH, 0.2 M NaCl, pH 8 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.099 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Size exclusion chromatography coupled to SAXS (SEC-SAXS) was carried out at at 20 °C using Nexera High Pressure Liquid Chromatography system (HPLC; Shimadzu). 50 μL of L19-IL2 concentrated at 4 mg/mL were injected into a Superdex 200 3.2/300 PC (GE Healthcare), pre-equilibrated with 25 mM HEPES/NaOH, 200 mM NaCl, pH 8.0. 101 successive 1 second frames were collected through the sample elution peak and processed using CHROMIXS that included the identification and subtraction of an appropriate solvent-blank.

L19-IL2 dimeric immunocytokine (L19-IL2)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Dimer
Mon. MW   42.0 kDa
Sequence   FASTA