T-IL2-12 Biological Probe

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Overview

Interleukin 2 (IL-2) is a type of cytokine signaling molecule in the immune system that regulates the activities of white blood cells responsible for immunity. IL-2 is produced by T-cells in response to antigenic or mitogenic stimulation, and the protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. IL-2 can stimulate B-cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells.

Recombinant IL-2 is used as a protein therapeutic, mainly against malignant melanoma [1]. However, IL-2 has a narrow therapeutic window and many side effects. IL-2 may also be involved in psoriasis [2]. In order to facilitate the study of the functions of IL-2, a neutralizing antibody fragment that can be used as a biological probe against this target, has been generated.

Methods

Recombinant IL-2 was produced through bacterial expression and used as an antigen in a phage display selection, utilizing a synthetic human single-chain variable fragment (scFv) library. Several binder candidates were identified following a range of antigen binding assays, including ELISA, HTRF, Luminex and SPR. Successful scFvs were then validated in a cell-based dimerization assay for their ability to block receptor dimerization.

Acknowledgements

This biological probe was created at SGC Karolinska in collaboration with researchers at CMM, Department of Medicine, Karolinska Institutet.

Authors: Charlotta Preger, Edvard Wigren, Elena Ossipova, Helena Persson Lotsholm, Susanne Gräslund.

Antigen Information

Antigen, full length IL-2 excluding signal sequence A21-T153 (Avi tag underlined):

SMAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSSKGGYGLNDIFEAQKIEWHE

IL-2 visualized using PDB: 1M47

Methods

Antigen production:

The DNA sequence encoding amino acids A21-T153 of IL-2 (Uniprot ID P10145) was sub-cloned into the expression vector pNIC-Bio3 (Genbank Acc. No JN792439). The vector was modified by inserting a MBP fragment at the Nde1 site, resulting in a construct with a N-terminal TEV cleavable MBP-His₆ tag. In addition, pNIC-Bio3 carries a C-terminal Avi tag for site-specific biotinylation. The expression plasmid was transformed into E. coli expression strain BL21(DE3) R3 pRARE2 carrying a plasmid for co-expression of BirA ligase.

The clone was grown at 37°C to an OD₆₀₀ of 1.5–2.0 in Terrific broth (TB) medium supplemented with Kanamycin (50 µg/mL), Chloramphenicol (35 µg/mL), Spectinomycin (50 µg/mL) and 100 µM Biotin. After lowering the temperature to 18°C protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM. Cultures were incubated for approximately 20 hours and then harvested by centrifugation at 4500 × g for 15 minutes and re-suspended in buffer containing 50 mM HEPES pH 8.0, 500 mM NaCl, 5% glycerol, 10 mM imidazole, and Complete EDTA-free protease inhibitor. Re-suspended cells were sonicated on ice followed by centrifugation at 44000 × g for 50 minutes. The soluble fraction was decanted, filtered (0.45 um) and subsequently loaded onto a HiTrap Ni-chelating column (GE Healthcare) on an ÄKTA Xpress (GE Healthcare). After washing with 20 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 10 mM imidazole, the protein was eluted in 20 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 250 mM imidazole. The eluate was applied to a HiLoad XK16/60 Superdex 200 column (GE Healthcare) equilibrated with 20 mM HEPES, pH 7.5, and 300 mM NaCl.

Selected peak fractions from size exclusion chromatography purification were pooled and subjected to treatment with His₆-TEV protease for removal of the N-terminal MBP-His₆-tag (~ 1:50 ratio of TEV protease:substrate; w/w). After completion of the reaction, an additional step of Ni^2+-affinity purification using Ni-NTA agarose resin (GE Healthcare) was performed to separate cleaved IL2 from the MBP-His₆-tag, contaminants and the TEV protease. The final batch was analysed for verification of cleaved protein and purity by SDS-PAGE gel and mass spectrometry characterisation.

Antibody Sequence

Sequence of T-IL2- scFv (VH-linker-VL-tags)

EVQLLESGGGLVQPGGSLRLSCAASGFTFGSSGMGWVRQAPGKGLEWVSSIYYGSGGTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIHGYDPPYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVYYPFTFGQGTKLEIKRTDYKDHDGDYKDHDIDYKDDDDKAAALPETGGHHHHHH*

Antibody Kinetics

SPR Single cycle kinetics on Biacore T200:

Concentrations of antigen: 0.32, 1.6, 8, 40, and 200 nM

Methods

Surface Plasmon Resonance:

Affinity measurements for scFv:

Affinity measurement was performed using single cycle kinetics on a Biacore T200 biosensor instrument (GE Healthcare). The anti-FLAG M2 antibody (Sigma-Aldrich, St. Louis, MO, USA), functioning as a capture molecule, was covalently coupled to a Series S CM5 amine sensor chip according to the manufacturer’s instructions. FLAG-tagged scFv, purified from the periplasm, was caught onto the surface, and followed by the addition of 0.32, 1.6, 8, 40, and 200 nM IL-2 at a flow-rate of 30 μl/min. The anti-FLAG antibody surface was regenerated with 10 mM glycine-HCl pH 2.5. The experiment was performed at 25°C and using HBS-EP+ (GE Healthcare) as running buffer. By subtracting the signal of a reference surface, an anti-FLAG antibody-coupled surface, response curves for the scFvs were obtained. The Biacore T200 Evaluation 3.1 software was used for analyses of the response curves and reaction rate kinetics calculated using the predefined 1:1 Langmuir binding model.

Cell-Based Assay

U2OS IL2RB/IL2RG/IL2RA Bioassay in collaboration with DiscoverX/Eurofins

The assay measures ligand-induced dimerization of the IL-2RB receptor subunit with the IL2RG signalling subunit, using exogenous tagged receptors (in the presence of untagged IL2Rα). The IC50 value of T-IL2-12 scFv was measured to approximately 3 µg/ml.

Methods

A vial of frozen ready-to-use U2OS IL2RB/IL2RG/IL2RA bioassay cells were thawed, and resuspended in CP5 at 2.5 x 10⁵ cells/mL. To evaluate responsiveness to anti-IL-2 binders, 20 µL of cells were seeded at 5K/well in a 384 well plate in CP5 plating reagent and allowed to adhere for 24 h in a humidified incubator at 37 °C / 5% CO_2. A dilution series of each antibody (1:3 dilutions, 11pt-DRC) were prepared as 10X stocks in 0.1% BSA / PBS in triplicate for each dose. No antibody was added to the last well (e.g. lowest dose) in each DRC (to serve as vehicle only control). A 10X stock of the stimulation dose of IL-2 (1X dose = 5 ng/mL) was prepared and mixed 1:1 with the antibody dilution series, then incubated for the indicated time (30 minutes) at 37 °C / 5% CO_2.5 mL of the antibody/ligand premix was added to the appropriate wells containing cells, then incubated in a humidified incubator at 37 °C / 5% CO₂ for 6h.

PathHunter FLASH Detection Reagent (25 mL, prepared per package insert) was added to each well. Plates were incubated for 60 minutes at room temperature in the dark prior to reading luminescence signal on an Envision luminometer. Data were plotted using GraphPad Prism 6; EC-50 values were calculated using a sigmoidal dose response curve fit with variable slope (four parameter) with no constraints; fit method = least squares (normal fit).

References

Bhatia S, Tykodi SS, Thompson JA (May 2009). "Treatment of metastatic melanoma: an overview". Oncology. 23 (6): 488–96. PMC 2737459. PMID 19544689.
Reich A, Szepietowski JC (2007). "Mediators of pruritus in psoriasis". Mediators of Inflammation. 2007: 1–6. doi:10.1155/2007/64727. PMC 2221678. PMID 18288273.