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