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

SASDJQ8 – IL12-L19L19 immunocytokine monomer

IL12-L19L19 immunocytokine
MWexperimental 115 kDa
MWexpected 112 kDa
VPorod 224 nm3
log I(s) 1.20×10-1 1.20×10-2 1.20×10-3 1.20×10-4
IL12-L19L19 immunocytokine small angle scattering data  s, nm-1
ln I(s)
IL12-L19L19 immunocytokine Guinier plot ln 1.20×10-1 Rg: 4.5 nm 0 (4.5 nm)-2 s2
(sRg)2I(s)/I(0)
IL12-L19L19 immunocytokine Kratky plot 1.104 0 √3 sRg
p(r)
IL12-L19L19 immunocytokine pair distance distribution function Rg: 4.6 nm 0 Dmax: 14.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
IL12-L19L19 immunocytokine GASBOR model
log I(s)
 s, nm-1
IL12-L19L19 immunocytokine ALLOSMOD model
Synchrotron SAXS data from solutions of the IL12-L19L19 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 IL12-L19L19 concentrated at 3.5 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. 36 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.

IL12-L19L19 immunocytokine (IL12-L19L19)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   111.7 kDa
Sequence   FASTA