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

SASDJP8 – L19L19-IL2 immunocytokine monomer

L19L19-IL2 immunocytokine
MWI(0) 60 kDa
MWexpected 69 kDa
VPorod 93 nm3
log I(s) 2.10×10-2 2.10×10-3 2.10×10-4 2.10×10-5
L19L19-IL2 immunocytokine small angle scattering data  s, nm-1
ln I(s)
L19L19-IL2 immunocytokine Guinier plot ln 2.10×10-2 Rg: 3.6 nm 0 (3.6 nm)-2 s2
(sRg)2I(s)/I(0)
L19L19-IL2 immunocytokine Kratky plot 1.104 0 √3 sRg
p(r)
L19L19-IL2 immunocytokine pair distance distribution function Rg: 3.6 nm 0 Dmax: 10.8 nm

Data validation

Fits and models

log I(s)
 s, nm-1
L19L19-IL2 immunocytokine GASBOR model
log I(s)
 s, nm-1
L19L19-IL2 immunocytokine ALLOSMOD model
Synchrotron SAXS data from solutions of the L19L19-IL2 immunocytokine in 25 mM HEPES/NaOH, 0.2 M NaCl, pH 8 were collected on the B21 beam line at Diamond (Didcot, UK) at a sample-detector distance of 4.01 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 an Agilent HPLC system. 50 μL of L19L19-IL2 concentrated at 1 mg/mL were injected into a Superdex 75 3.2/300 PC (GE Healthcare), pre-equilibrated with 25 mM HEPES/NaOH, 200 mM NaCl, pH 8.0. 30 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.

L19L19-IL2 immunocytokine (L19L19-IL2)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   68.7 kDa
Sequence   FASTA