Design and characterization of a protein superagonist of IL‐15 fused with IL‐15Rα and a high‐affinity T cell receptor

To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (K_d = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐K^b (SIY/K^b) with nanomolar affinity (K_d = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/K^b with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Coiled coil miniprotein randomization on phage leads to charge pattern mimicry of the receptor recognition determinant of interleukin 5

Phage display was used to identify sequences that mimic structural determinants in interleukin5 (IL5) for IL5 receptor recognition. A coiled coil stem loop (CCSL) miniprotein scaffold library was constructed with its turn region randomized and panned for binding variants against human IL5 receptor alpha chain (IL5Ralpha). Competition enzyme-linked immunosorbent assays identified CCSL-phage selectants for which binding to IL5Ralpha was competed by IL5. The most frequently selected and IL5-competed CCSL-phage contain charged residues Arg and Glu in their turn sequences, in this regard resembling a beta strand sequence in the 'CD turn' region, of IL5, that has been proposed to present a key determinant for IL5 receptor alpha chain recognition. The most dominant CCSL-phage selectant sequence, PVEGRV, contains a negative/positive charge pattern similar to that seen in the original CD turn. To test the relatedness of CCSL-phage selectant sequences to the IL5 receptor recognition epitope, PVEGRV was grafted into the sequence 87--92 of a monomeric IL5. The resulting IL5 variant, [(87)PVEGRV(92)]GM1, was able to bind to IL5Ralpha in biosensor assays, to elicit TF-1 cell proliferation and to induce STAT5 phosphorylation in TF-1 cells. The results help discern sequence patterns in the IL5 CD turn region which are key in driving receptor recognition and demonstrate the utility of CCSL miniprotein scaffold phage display to identify local IL5 mimetic sequence arrangements that may ultimately lead to IL5 antagonists.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Relative roles of T‐cell receptor ligands and interleukin‐2 in driving T‐cell proliferation

Stimulation of T cells by the T‐cell receptor (TCR)/CD3 complex results in interleukin‐2 (IL‐2) synthesis and surface expression of the IL‐2 receptor (IL‐2R), which in turn drive T‐cell proliferation. However, the significance of the requirement of IL‐2 in driving T‐cell proliferation, when TCR stimulation itself delivers potential mitogenic signals, is unclear. We show that blocking of IL‐2 synthesis by Cyclosporin A (CsA) suppressed both the Concanavalin A (Con A)‐ and phorbol myristate acetate (PMA)/ionomycin‐induced proliferation of T cells. The latter is also inhibited by anti‐IL‐2R. Kinetic studies showed that T‐cell proliferation begins to become resistant to CsA inhibition by about 12 h and became largely resistant by 18 h of stimulation. PMA, the protein kinase C activator, enhanced Con A‐induced T‐cell proliferation if added only within first 12 h of stimulation, and not after that. Given the fact that, in the present study, TCR is downregulated within 2 h of Con A stimulation and T cells entered the S phase of cell cycle by about 18 h of stimulation, the above results suggest that TCR stimulation provides the initial trigger to the resting T cells, which allows the cells to traverse the first two third portions of G1 phase of cell cycle and become proliferation competent. IL‐2 action begins afterward, delivering the actual proliferation signal(s), allowing the cells to traverse the rest of G1 phase and enter the S phase of the cell cycle. J. Cell. Biochem. 76:37–43, 1999. © 1999 Wiley‐Liss, Inc.     

Anti‐interleukin‐5‐neutralizing antibody attenuates caradiac injury and cadiac dysfunction by aggravating the inflammatory response in doxorubicin‐treated mice

Previous studies have demonstrated that interleukins (ILs) are closely associated with doxorubicin (DOX)‐induced cardiac injury. IL‐5 is an important member of the IL family, and this study was performed to investigate whether IL‐5 affects DOX‐induced cardiac injury and its underlying mechanisms. The cardiac IL‐5 expression was first detected and the results showed that cardiac IL‐5 levels were significantly lower in DOX‐treated mice, and IL‐5 was mainly derived from cardiac macrophage (Mø). In addition, some DOX‐treated mice received an injection of anti‐IL‐5‐neutralizing antibody (nAb), and we found that treatment with a mouse anti‐IL‐5 nAb significantly upregulated the levels of myocardial injury markers, aggravated cardiac dysfunction, increased M1 macrophage (Mø1) and decreased M2 macrophage (Mø2) differentiation, and promoted apoptotic marker expression. Furthermore, the effect of mouse IL‐5 nAb on DOX‐induced Mø differentiation and its role on mouse cardiomyocyte (MCM) cells apoptosis were detected in vitro, and the results exhibited that mouse IL‐5 nAb promoted Mø1 differentiation but inhibited Mø2 differentiation in vitro and alleviated apoptosis in MCM cells. Our results found a mouse anti‐IL‐5 nAb‐aggravated DOX‐induced cardiac injury and dysfunction by alleviating the inflammatory response and myocardial cell apoptosis.     

NKG2D ligand RAE1ε induces generation and enhances the inhibitor function of myeloid‐derived suppressor cells in mice

Expression of surface NKG2D ligands on tumour cells, which activates nature killer (NK) cells and CD8⁺ T cells, is crucial in antitumour immunity. Some types of tumours have evolved mechanisms to suppress NKG2D‐mediated immune cell activation, such as tumour‐derived soluble NKG2D ligands or sustained NKG2D ligands produced by tumours down‐regulate the expression of NKG2D on NK cells and CD8⁺ T cells. Here, we report that surface NKG2D ligand RAE1ε on tumour cells induces CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cell (MDSC) via NKG2D in vitro and in vivo. MDSCs induced by RAE1ε display a robust induction of IL‐10 and arginase, and these MDSCs show greater suppressive activity by inhibiting antigen‐non‐specific CD8⁺ T‐cell proliferation. Consistently, upon adoptive transfer, MDSCs induced by RAE1ε significantly promote CT26 tumour growth in IL‐10‐ and arginase‐dependent manners. RAE1ε moves cytokine balance towards Th2 but not Th1 in vivo. Furthermore, RAE1ε enhances inhibitory function of CT26‐derived MDSCs and promotes IL‐4 rather than IFN‐γ production from CT26‐derived MDSCs through NKG2D in vitro. Our study has demonstrated a novel mechanism for NKG2D ligand⁺ tumour cells escaping from immunosurveillance by facilitating the proliferation and the inhibitory function of MDSCs.     

Induced conformational change in human IL‐4 upon binding of a signal‐neutralizing DARPin

The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97 , 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ_c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Role of IL‐18 in atopic asthma is determined by balance of IL‐18/IL‐18BP/IL‐18R

It is recognized that IL‐18 is related to development of asthma, but role of IL‐18 in asthma remains controversial and confusing. This is largely due to lack of information on expression of IL‐18 binding protein (BP) and IL‐18 receptor (R) in asthma. In this study, we found that plasma levels of IL‐18 and IL‐18BP were elevated in asthma. The ratio between plasma concentrations of IL‐18 and IL‐18BP was 1:12.8 in asthma patients. We demonstrated that 13‐fold more monocytes, 17.5‐fold more neutrophils and 4.1‐fold more B cells express IL‐18BP than IL‐18 in asthmatic blood, suggesting that there is excessive amount of IL‐18BP to abolish actions of IL‐18 in asthma. We also discovered that more IL‐18R+ monocytes, neutrophils and B cells are located in asthmatic blood. Once injected, IL‐18 eliminated IL‐18R+ monocytes in blood, but up‐regulated expression of IL‐18R in lung macrophages of OVA‐sensitized mice. Our data clearly indicate that the role of IL‐18 in asthma is very likely to be determined by balance of IL‐18/IL‐18BP/IL‐18R expression in inflammatory cells. Therefore, IL‐18R blocking or IL‐18BP activity enhancing therapies may be useful for treatment of asthma.     

Identification of interleukin‐16 (IL‐16) and interleukin‐17D (IL‐17D) genes from Dabry's sturgeon (Acipenser dabryanus)

Dabry's sturgeon (Acipenser dabryanus) represents an ancient Actinopterygian lineage that are termed “living fossils”. Many diseases have been found in Dabry's sturgeon. In the present study, genes encoding interleukin (IL)‐16 and IL‐17D in Dabry's sturgeon were identified by RNA‐sequencing. Phylogenetic tree analysis suggested that they clustered together with the corresponding pro‐IL‐16 proteins and IL‐17D proteins from other fish. Sequence analysis revealed that IL‐17D protein was more conserved than pro‐IL‐16. Dabry's sturgeon pro‐IL‐16 contains four putative PDZ domains and do not include signal peptides, while IL‐17D only possesses signal peptides (1–25 aa). The expression patterns of IL‐16 and IL‐17D genes were investigated in Dabry's sturgeon to reveal their functions in disease. The expression level of IL‐16 showed no significant changes in embryos; however, the high expression level of IL‐17D at 0–14 hpf (hours post fertilization) implied the existence of maternal expression in the oocyte and an association with embryonic development. Tissue distribution analysis revealed that IL‐16 and IL‐17D proteins have potential functions in immune and non‐immune tissue compartments. IL‐16 and IL‐17D had different fold changes in primary spleen leukocytes after polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) administration, which suggested that IL‐16 has a stronger antiviral capability compared with its antibacterial response, and IL‐17D has a stronger antibacterial capability compared with its antiviral response. IL‐16 showed an earlier response to virus compared with IL‐17D, and IL‐17D showed earlier and shorter response to bacteria compared with IL‐16. Our findings suggested the roles of IL‐16 and IL‐17D in Dabry's sturgeon, and provided the theoretical basis for the prevention and control of diseases of Dabry's sturgeon.     

Lipid‐dependent cytotoxicity by the lipase PLRP2 and by PLRP2‐positive cytotoxic T lymphocytes (CTLs)

IL‐4 induces a lipase, pancreatic lipase related protein 2 (PLRP2), in cytotoxic T lymphocytes (CTLs). Because PLRP2 in semen can mediate lipid‐dependent toxicity to sperm, we questioned whether CTL‐derived PLRP2 could support similar cytotoxicity toward tumor cells. Recombinant PLRP2 was toxic to P815 tumor cells in 48 h when lipid and another protein, colipase, were present. However, PLRP2‐positive CTLs (induced with many lots of IL‐4) were unable to mediate lipid‐dependent cytotoxicity. Notably, CTLs induced with only one lot of IL‐4 had lipid‐dependent cytotoxicity. The exceptional lot of IL‐4 was effective in multiple experiments at inducing lipid‐dependent cytotoxicity. The lipid‐dependent cytotoxicity it induced was determined to be perforin‐independent. CTLs induced with IL‐4 that was unable to induce lipid‐dependent cytotoxicity had mRNA for PLRP2 but not mRNA for colipase. Therefore, we added exogenous colipase to the CTL assays but still cytotoxicity was unchanged. We conclude (1) that lipid‐dependent cytotoxicity, promoted by the lipase PLRP2 and colipase, will kill tumor cells and (2) that more than PLRP2 alone is required for lipid‐dependent cytotoxicity mediated by CTLs. Copyright © 2009 John Wiley & Sons, Ltd.     

Association of IL‐37 gene polymorphisms with susceptibility to tuberculosis in Saudi subjects

Tuberculosis (TB) is one of the most common infectious diseases worldwide. IL‐37, a novel member of the IL‐1 family, has anti‐inflammatory activity. Various cytokine genes polymorphisms are reportedly associated with susceptibility to TB infection. However, an association between genetic variations in the IL‐37 gene and susceptibility to TB infection has not been investigated. The aim of this case‐control study was therefore to identify such an association in Saudi subjects, in which five single‐nucleotide polymorphisms (SNPs) in the IL‐37 gene were assessed. Serum concentrations of IL‐37 were evaluated using ELISA, and genetic variants genotyped by multiplex PCR and ligase detection reaction. It was found that the C/C genotype of rs2723176 (–6962 A/C) occurs significantly more frequently in patients with active TB and that the C allele of this SNP is associated with TB. In addition, the C allele of rs2723176 SNP was associated with high circulating concentrations of IL‐37. However, the genotype and allele frequency of the other four SNPs (rs3811046, rs3811047, rs2723186 and rs2723187) were not significantly associated with TB infection. In conclusion, the present data suggest that rs2723176 SNP of IL‐37 is involved in the development of TB infection. Furthermore, high circulating concentrations of IL‐37 may have a negative effect on protective immunity against TB infection.     

rIL‐10 enhances IL‐10 signalling proteins in foetal alveolar type II cells exposed to hyperoxia

Although the mechanisms by which hyperoxia promotes bronchopulmonary dysplasia are not fully defined, the inability to maintain optimal interleukin (IL)‐10 levels in response to injury secondary to hyperoxia seems to play an important role. We previously defined that hyperoxia decreased IL‐10 production and pre‐treatment with recombinant IL‐10 (rIL‐10) protected these cells from injury. The objectives of these studies were to investigate the responses of IL‐10 receptors (IL‐10Rs) and IL‐10 signalling proteins (IL‐10SPs) in hyperoxic foetal alveolar type II cells (FATIICs) with and without rIL‐10. FATIICs were isolated on embryonic day 19 and exposed to 65%‐oxygen for 24 hrs. Cells in room air were used as controls. IL‐10Rs protein and mRNA were analysed by ELISA and qRT‐PCR, respectively. IL‐10SPs were assessed by Western blot using phospho‐specific antibodies. IL‐10Rs protein and mRNA increased significantly in FATIICs during hyperoxia, but JAK1 and TYK2 phosphorylation showed the opposite pattern. To evaluate the impact of IL‐8 (shown previously to be increased) and the role of IL‐10Rs, IL‐10SPs were reanalysed in IL‐8‐added normoxic cells and in the IL‐10Rs’ siRNA‐treated hyperoxic cells. The IL‐10Rs’ siRNA‐treated hyperoxic cells and IL‐8‐added normoxic cells showed the same pattern in IL10SPs with the hyproxic cells. And pre‐treatment with rIL‐10 prior to hyperoxia exposure increased phosphorylated IL‐10SPs, compared to the rIL‐10‐untreated hyperoxic cells. These studies suggest that JAK1 and TYK2 were significantly suppressed during hyperoxia, where IL‐8 may play a role, and rIL‐10 may have an effect on reverting the suppressed JAK1 and TYK2 in FATIICs exposed to hyperoxia.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

IL‐6 Trans‐Signaling Plays Important Protective Roles in Acute Liver Injury Induced by Acetaminophen in Mice

Our study was undertaken to evaluate the important role of interleukin‐6 (IL‐6) trans‐signaling in acetaminophen (AAP)‐induced liver injury. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL‐6 trans‐signaling, whereas an IL‐6/soluble IL‐6 receptor (sIL‐6R) fusion protein (hyper‐IL‐6) mimics IL‐6 trans‐signaling. Using these tools, we investigated the role of IL‐6 trans‐signaling in AAP‐induced liver injury. Blockade of IL‐6 trans‐signaling during AAP‐induced liver injury remarkably increased the levels of serum aspartate aminotransferase and alanine aminotransferase; lowered the level of serum sIL‐6R; aggravated liver injury; inhibited the expression of phosphorylation of STAT3 (pSTAT3), proliferating cell nuclear antigen, vascular endothelial growth factor, and glycogen synthesis; and induced the expression of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocyte apoptosis in the liver of mice. In summary, our study suggested that IL‐6 trans‐signaling plays important protective roles by regulating the hepatocyte proliferation and apoptosis, angiogenesis, CYP2E1 expression, and glycogen metabolism during AAP‐induced liver injury in mice.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.