Efficacy and antitumor activity of a mutant type of interleukin 2

Ethics statement

All experiments and procedures were approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1400.034) and performed in accordance with the approved guidelines and regulations. Animal studies were performed in accordance with the ARRIVE reporting guidelines³³. Human blood samples were obtained from one of the authors as a volunteer according to the same proposal approved by the ethics committee of Pasteur Institute of Iran and in accordance with the Declaration of Helsinki and other relevant guidelines, including the ethical guidelines for medical and health research on human subjects by the Iranian government. The informed consent was obtained before human blood sampling.

Cloning, expression and purification of wild and mutant hIL-2

The amino acid sequence of the wild IL-2 was obtained from drug bank (Accession No. DB00041). In our previous study, IL-2 mutant molecules were designed through computational approaches. In brief, the amino acid residues involved in electrostatic interactions between IL-2 and IL-2Rα subunit were identified and alanine mutations were applied at selected positions and the variant with the lowest affinity towards IL-2Rα (M2 molecule, K35A, E61A, and F42A) was identified using docking, molecular dynamics simulation, umbrella sampling, and Gibbs energy calculations²³. The two IL-2 encoding gene fragments were separately subcloned into pET28a expression vector at NcoI and HindIII restriction sites. The identity of the gene fragments was confirmed through restriction digestion and sequencing.

E. coli BL21 (DE3) strain was transformed with recombinant vectors and inoculated into 200 ml Luria Bertani (LB) medium. The expression of wild and mutant hIL-2 proteins was induced with 0.5 mM isopropyl β-D-1-thiogalactoside (IPTG) at OD600nm of 0.5 and incubated for additional 6 h. The bacterial pellet was resuspended in lysis buffer I (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole; pH8.0). The cells were sonicated at 100% amplitude (30 s pulses with 10 s intervals) and centrifuged at 10,000×g for 20 min. The pellet, containing inclusion bodies, was solubilized in lysis buffer II (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole, 8 M Urea; pH8.0) and placed under agitation for 1 h. After centrifugation (10,000×g for 30 min), the supernatant was loaded to Ni–NTA agarose column (ABT, Spain) at a flow rate of 1 ml/min. Refolding of the proteins was carried out through a gradient of urea concentration (from 8 M to 0) using the refolding buffer (50 mM NaH₂PO₄, 500 mM NaCl, 20 mM Imidazole; pH8.0). Histidine-tagged protein was eluted by the elution buffer (50 mM NaH₂PO₄, 500 mM NaCl, 250 mM imidazole; pH8.0). Protein concentration was determined by UV adsorption at 280 nm using a spectrophotometer (BioTeK, USA). The purity of the eluted protein was analyzed on 12% SDS-PAGE and Coomassie Brilliant Blue staining.

For western blotting, proteins were transferred to nitrocellulose membrane. After blocking the membrane with 3% w/v skim milk in phosphate-buffered saline (PBS) overnight (o/n) at 4 °C, the membrane was washed with PBS supplemented with 1% Tween-20 for 5 min (3 times). The membrane was treated with 1:2000 dilution of HRP-conjugated anti-His antibody, and the protein bands were visualized using 3,3 diaminobenzidine tetrahydrochloride (DAB) solution as the substrate.

PBMC proliferation assay

Alamarblue assay was used to evaluate the proliferative responses of lymphocytes to the recombinant proteins. Heparinized peripheral blood was obtained from a healthy volunteer after approval by the Ethics committee of Pasteur Institute of Iran (IR.PII.REC.1400.034) according to the declaration of Helsinki and other relevant guidelines. PBMCs were isolated on a Ficoll-Hypaque (Lymphoprep, Nyegaard, Norway) density gradient approach¹⁷. PBMCs were washed and resuspended in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, UK), 2 mM L-glutamine, and 10% fetal calf serum (FCS, Sigma). The cells were seeded at a density of 2 × 10⁵ cells/well and stimulated with 2 µg/well of concanavalin A (Con A, Sigma). Negative control cells were treated with medium without any mitogen. The cells were incubated at 37 °C, 5% CO₂ for 24 h. Subsequently, wIL-2 or mutant IL-2 proteins were added to the cells in a range of 0.01 nM to 10 µM and incubation continued for 48 h. Then, Alamarblue reagent was added to the wells and incubated for 16 h. Replicates of each experiment were analyzed on a microplate reader (BioTek ELx808, USA) at 570 and 600 nm. The percentage of reduction in Alamar blue was calculated according to the Manufacturer’s recommendations (Thermo Fisher Scientific, USA).

IFN-γ assay

Isolated PBMCs were seeded at a density of 2 × 10⁶ cells/ml in 24-well plates, treated with 1 µg IL-2 proteins and incubated at 37 °C for 48 h. The supernatants were harvested and IFN-γ secretion level was measured using the Human IFN-γ ELISA kit (Karmania Pars Gene, IRAN).

Cytotoxicity assay

Cytotoxicity assay was performed using Calcein-AM bio-detergent. Isolated PBMCs from Healthy donor were served as effector cells (E) against the target cell line (T), K562 (Human leukemia cell). In brief, PBMCs were stimulated with 1 µg mutant IL-2 or wtIL-2 for 48 h. Then, 1 × 10⁶ K562 cells/ml were labeled with 2 µM Calcein-AM (R&D Systems, USA) in complete RPMI-1640 medium, washed twice with the medium and incubated for 30 min at 37 °C. PBMCs were co-cultured with labeled K562 cells in specific cell ratios (T/E ratios of 1:1, 1:5, 1:10, 1:20) 96-well plates and incubated under 5% CO₂ at 37 °C for 12 h in triplicate. The culture supernatants were transferred into a black 96-well plate and fluorescence was measured using a fluorometer (BioTeK, USA) at 490 nm excitation and 520 nm emission wavelengths. Target cells within complete medium and the cells treated with 1% Triton X-100 were considered as negative and positive controls representing spontaneous and maximum fluorescence release, respectively. Cytotoxicity was calculated according to the following formula³⁴:

Antitumor activity

Fifteen female C57BL/6J mice (6–8 weeks, 20 g) were purchased from the animal resource center (Pasteur Institute of Iran) and maintained under standard housing conditions. The research protocols and all animal studies were approved by the Ethics committee of Pasteur Institute of Iran (IR.PII.REC.1400.034) and followed ARRIVE reporting guidelines³³. Lymphoblast B lymphocyte TC-1 (#ATCC: CRL-2785) cell line was obtained from the department of Cell Bank (Pasteur Institute of Iran) and cultured in DMEM medium supplemented with 10% Fetal bovine serum (FBS, Sigma) at 37 °C under 5% CO₂ atmosphere. On day 0, 1 × 10⁶ cells in PBS were injected subcutaneously (s.c) into the right flank of the mice and tumor growth was daily examined until the tumor dimension reached 50 mm³. After scarification of the mice by cervical dislocation, solid tumor samples were thinly sliced (2 mm thick) and subcutaneously transplanted into the shaved right flank of 15 mice which were anesthetized with intraperitoneal (i.p) ketamine (10 mg/ml)/xylazine (10 mg/ml) (Sigma, USA) formulated in water and randomly divided into three groups (5 mice per group). Mutant and wild type groups were injected by the recombinant purified IL-2 proteins diluted in sterile PBS to a final concentration of 1 mg/kg at the tumor site, two times per week for one month. The control group received 200 µl PBS. Tumor size in each group was measured using a caliper and tumor volume was calculated by the following formula: 0.5 × length × width² of the tumor³⁵.

Statistical analysis

Data analysis was performed with GraphPad Prism (v. 8.0). The significance level between two or more groups was determined by two-tailed unpaired t-test and One-way ANOVA analysis of the variance.