Upon structural analysis, JES6-1 was observed to block IL-2R (CD122) binding, but CD25 could peel off the antibody and allow IL-2 to bind heterotrimeric complexes.16 A newly generated anti-hIL-2 antibody F5111 had a similar mode of action and could treat autoimmune disorders, such as type I diabetes in NOD mice, as well as experimental allergic encephalitis and xenogeneic graft-versus-host diseases.33 IL-2/F5111 complex blocked the CD122 binding site with relatively weak Vernakalant (RSD1235) affinity and CD25/IL-2 binding released F5111, allowing them to form IL-2/IL-2R tetrameric complex. inducing a prodigious expansion of host memory phenotype (MP) CD8 T (60-fold) and NK cells (18-fold) with less efficient Treg proliferation (5-fold). As a result, there was an average eightfold increase in the ratio of MP CD8 to Tregs. Accordingly, hIL-2/TCB2c strongly inhibited the growth of B16F10, MC38, and CT26 tumors. More remarkably, hIL-2/TCB2c showed synergy with checkpoint inhibitors such as anti-CTLA-4 or PD1 antibodies, and resulted in almost complete regression of implanted tumors and resistance to secondary tumor challenge. For direct clinical use, we generated a humanized form of TCB2 that had equal immunostimulatory and anti-tumor efficacy as a murine one. Collectively, these results show that TCB2 can provide a potent immunotherapeutic modality either alone or together with checkpoint inhibitors in cancer patients. lifespan and preferentially direct its effects to CD8 T and NK cells, but not Tregs or endothelial cells. In 2006, Boyman and colleagues reported that when a cytokine is complexed with its antibody, it dramatically increases its half-life and efficacy.2 Depending on the structure of the complex, it could also be selective CALCR for different forms of receptors. For example, anti-IL-2 antibodies such as S4B6 (anti-mouse) and MAB602 (anti-human) can preferentially stimulate CD8 T or NK cells, while JES6-1 (anti-mouse) or 5344 (anti-human) favor the proliferation of Tregs. The recently developed anti-hIL-2 antibody, Nara1, can preferentially stimulate the dimeric IL-2 receptor when complexed with hIL-2.11 Structurally, Nara1 binds near the CD25 binding site of IL-2 and abolishes its binding to IL-2, thereby disfavoring Treg activation. In this report, we generated anti-hIL-2 antibodies and screened clones that could preferentially work on the dimeric IL-2 receptor by blocking the CD25 binding motif. We selected three clones, named TCB1C3, and found that TCB2 has a unique CDR sequence compared to TCB1, TCB3, and Nara1. The hIL-2/TCB2 complex (hIL-2/TCB2c) had exceptional efficacy in stimulating CD8 T and NK cells, and could inhibit the growth and spread of various tumors such as MC38, CT26, and B16F10. When hIL-2/TCB2c was combined with checkpoint inhibitors such as anti-PD1 or CTLA4 antibodies, tumors were almost completely regressed in mice. Finally, we generated a humanized form of TCB2 that we confirmed to have equal immunostimulatory and anti-tumor effects as a murine one. Collectively, we showed that TCB2 is a new anti-hIL-2 monoclonal antibody (mAb) that has potential therapeutic effects in human cancer patients. Material and methods Mice BALB/c, C57BL/6 (B6), Thy1.1 Foxp3-eGFP, mice Vernakalant (RSD1235) were maintained at POSTECH Biotech Centre (Korea) under specific pathogen-free condition. For the experiments with anti-tumor effect, B6 or BALB/c mice were purchased from OrientBio (Korea). Unless it is specified, all mice with 6C10 weeks old were used for the Vernakalant (RSD1235) experiments according to the protocols approved by the Animal Experimental and Ethic Committee at the Institute for Basic Science (Korea). Flow cytometry Single-cell suspensions were prepared from indicated organs and FACS Vernakalant (RSD1235) stained Vernakalant (RSD1235) in PBS buffer containing 2% FBS and 0.05% sodium azide. For intracellular staining, single-cell suspensions were surface stained, fixed, and permeabilized with eBioscience Foxp3 staining buffer set. Following antibodies from BD bioscience, BioLegend or ThermoFisher were used for surface or intracellular staining; CD3 (145-2C11), CD4 (GK1.5 or RM4C5), CD8 (53C6.7), CD25 (PC61), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD90.1 (HIS51 or OX-7), CD122 (TM-1), CD132 (TUGm2), TCR (H57-597), NK1.1 (PK136), Granzyme B (GB11), Foxp3 (MF-14), IFN- (XMG1.2), NKp46 (29A1.4), CD11b (M1/70), CD27 (LG.7F9), T-bet (4B1D) and pSTAT5 (47/Stat5(pY694)). For pSTAT5 staining, B6 mice were intravenously injected with PBS, hIL-2 (1 g), hIL-2/5344 (1 g/10 g), hIL-2/MAB602 (1 g/10 g) or hIL-2/TCB2 (1 g/10 g) complexes. After RBC lysis using ACK buffer, blood mononuclear cells were fixed with 1.6% PFA (EMS, 25C) and methanol (DUKSAN, C 20C) for 10 min, respectively, and stained with surface and intracellular markers for 1 h at room temperature.12 Samples were analyzed using a LSR II or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). Generation of anti-hIL-2 mAb producing hybridoma Immunization of mice and fusion of splenocytes with SP2/0 myeloma cell line (ATCC) was performed as described previously.13 In brief, splenocytes from BALB/c mice that were immunized with 20C30 g of human IL-2 (hIL-2, Prospec) intraperitoneally for 3C4 times over several weeks were fused with SP2/0 myeloma cell line. Clones were grown in RPMI1640 (Welgene) supplemented with 10% fetal bovine serum (FBS, Atlas Biological), penicillin-streptomycin (Welgene) and hypoxanthine-aminopterin-thymidine (Gibco). Once colonies of hybridoma were visible, the culture supernatants were subjected to ELISA against hIL-2. The positive clones were sub-cloned through serial dilution to ensure that the cell line is the progeny of a single clone. Purification of anti-hIL-2 mAbs Culture supernatant from hybridoma were concentrated by adding 50/45 (v/v) of saturated ammonium sulfate (DAEJUNG) and.