Bone marrow-derived mesenchymal stromal cells obstruct AML-targeting CD8+ clonal effector and CAR T-cell function while promoting a senescence-associated phenotype.

Bone marrow mesenchymal stromal cells (MSCs) have been described as potent regulators of T-cell function, though whether they could impede the effectiveness of immunotherapy against acute myeloid leukemia (AML) is still under investigation. We examine whether they could interfere with the activity of leukemia-specific clonal cytotoxic T-lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells, as well as whether the immunomodulatory properties of MSCs could be associated with the induction of T-cell senescence. Co-cultures of leukemia-associated Wilm's tumor protein 1 (WT1) and tyrosine-protein kinase transmembrane receptor 1 (ROR1)-reactive CTLs and of CD123-redirected switchable CAR T cells were prepared in the presence of MSCs and assessed for cytotoxic potential, cytokine secretion, and expansion. T-cell senescence within functional memory sub-compartments was investigated for the senescence-associated phenotype CD28-CD57+ using unmodified peripheral blood mononuclear cells. We describe inhibition of expansion of AML-redirected switchable CAR T cells by MSCs via indoleamine 2,3-dioxygenase 1 (IDO-1) activity, as well as reduction of interferon gamma (IFNγ) and interleukin-2 (IL-2) release. In addition, MSCs interfered with the secretory potential of leukemia-associated WT1- and ROR1-targeting CTL clones, inhibiting the release of IFNγ, tumor necrosis factor alpha, and IL-2. Abrogated T cells were shown to retain their cytolytic activity. Moreover, we demonstrate induction of a CD28loCD27loCD57+KLRG1+ senescent T-cell phenotype by MSCs. In summary, we show that MSCs are potent modulators of anti-leukemic T cells, and targeting their modes of action would likely be beneficial in a combinatorial approach with AML-directed immunotherapy.

64Cu tumor labeling with hexadentate picolinic acid-based bispidine immunoconjugates.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities >200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed specific accumulation of the 64Cu-labeled TMs in PSCA- and FAP-overexpressing tumors (standardized uptake value (SUV) for PC3: 2.7±0.6 and HT1080: 7.2±1.25) with almost no uptake in wild type tumors.

UniCAR T-Cell Potency-A Matter of Affinity between Adaptor Molecules and Adaptor CAR T-Cells?

Although Chimeric Antigen Receptor (CAR) T-cells have shown high efficacy in hematologic malignancies, they can cause severe to life-threatening side effects. To address these safety concerns, we have developed adaptor CAR platforms, like the UniCAR system. The redirection of UniCAR T-cells to target cells relies on a Target Module (TM), containing the E5B9 epitope and a tumor-specific binding moiety. Appropriate UniCAR-T activation thus involves two interactions: between the TM and the CAR T-cell, and the TM and the target cell. Here, we investigate if and how alterations of the amino acid sequence of the E5B9 UniCAR epitope impact the interaction between TMs and the UniCAR. We identify the new epitope E5B9L, for which the monoclonal antibody 5B9 has the greatest affinity. We then integrate the E5B9L peptide in previously established TMs directed to Fibroblast Activation Protein (FAP) and assess if such changes in the UniCAR epitope of the TMs affect UniCAR T-cell potency. Binding properties of the newly generated anti-FAP-E5B9L TMs to UniCAR and their ability to redirect UniCAR T-cells were compared side-by-side with the ones of anti-FAP-E5B9 TMs. Despite a substantial variation in the affinity of the different TMs to the UniCAR, no significant differences were observed in the cytotoxic and cytokine-release profiles of the redirected T-cells. Overall, our work indicates that increasing affinity of the UniCAR to the TM does not play a crucial role in such adaptor CAR system, as it does not significantly impact the potency of the UniCAR T-cells.

Multivalent adaptor proteins specifically target NK cells carrying a universal chimeric antigen receptor to ErbB2 (HER2)-expressing cancers.

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

Preclinical Characterization of the 177Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Targeting CD10 on B-Cell Leukemia Using the Universal CAR T-Cell Platform (UniCAR).

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells.

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL-2, TNF and interferon-γ and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells.

Tonic Signaling and Its Effects on Lymphopoiesis of CAR-Armed Hematopoietic Stem and Progenitor Cells.

Long-term survival of adoptively transferred chimeric Ag receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multicell lineages (e.g., T cells, NK cells). In dependence on the CAR construct, a variable extent of tonic signaling in CAR T cells was reported; thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess the effects of tonic signaling, two CAR constructs were established and analyzed 1) a signaling CAR inducing a solid Ag-independent tonic signaling termed CAR-28/ζ and 2) a nonstimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow cells from immunocompetent mice were isolated, purified for HSC-containing Lin-cKit+ cells or the Lin-cKit+ Sca-1+ subpopulation (Lin-Sca-1+cKit+), and transduced with both CAR constructs. Subsequently, modified bone marrow cells were transferred into irradiated mice, in which they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ζ led to elimination of mature CAR+ T and B cells, suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.

Nanocytometer for smart analysis of peripheral blood and acute myeloid leukemia: a pilot study.

We realize an ultracompact nanocytometer for real-time impedimetric detection and classification of subpopulations of living cells. Nanoscopic nanowires in a microfluidic channel act as nanocapacitors and measure in real time the change of the amplitude and phase of the output voltage and, thus, the electrical properties of living cells. We perform the cell classification in the human peripheral blood (PBMC) and demonstrate for the first time the possibility to discriminate monocytes and subpopulations of lymphocytes in a label-free format. Further, we demonstrate that the PBMC of acute myeloid leukemia and healthy samples grant the label free identification of the disease. Using the algorithm based on machine learning, we generated specific data patterns to discriminate healthy donors and leukemia patients. Such a solution has the potential to improve the traditional diagnostics approaches with respect to the overall cost and time effort, in a label-free format, and restrictions of the complex data analysis.

A Small Step, a Giant Leap: Somatic Hypermutation of a Single Amino Acid Leads to Anti-La Autoreactivity.

The anti-La mab 312B, which was established by hybridoma technology from human-La transgenic mice after adoptive transfer of anti-human La T cells, immunoprecipitates both native eukaryotic human and murine La protein. Therefore, it represents a true anti-La autoantibody. During maturation, the anti-La mab 312B acquired somatic hypermutations (SHMs) which resulted in the replacement of four aa in the complementarity determining regions (CDR) and seven aa in the framework regions. The recombinant derivative of the anti-La mab 312B in which all the SHMs were corrected to the germline sequence failed to recognize the La antigen. We therefore wanted to learn which SHM(s) is (are) responsible for anti-La autoreactivity. Humanization of the 312B ab by grafting its CDR regions to a human Ig backbone confirms that the CDR sequences are mainly responsible for anti-La autoreactivity. Finally, we identified that a single amino acid replacement (D > Y) in the germline sequence of the CDR3 region of the heavy chain of the anti-La mab 312B is sufficient for anti-La autoreactivity.

And Yet It Moves: Oxidation of the Nuclear Autoantigen La/SS-B Is the Driving Force for Nucleo-Cytoplasmic Shuttling.

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens' syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

Midostaurin abrogates CD33-directed UniCAR and CD33-CD3 bispecific antibody therapy in acute myeloid leukaemia.

Combinatory therapeutic approaches of different targeted therapies in acute myeloid leukaemia are currently under preclinical/early clinical investigation. To enhance anti-tumour effects, we combined the tyrosine kinase inhibitor (TKI) midostaurin and T-cell mediated immunotherapy directed against CD33. Clinically relevant concentrations of midostaurin abrogated T-cell mediated cytotoxicity both after activation with bispecific antibodies and chimeric antigen receptor T cells. This information is of relevance for clinicians exploring T-cell mediated immunotherapy in early clinical trials. Given the profound inhibition of T-cell functionality and anti-tumour activity, we recommend specific FLT3 TKIs for further clinical testing of combinatory approaches with T-cell based immunotherapy.

Conventional CARs versus modular CARs.

The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.

Anti-CAR-engineered T cells for epitope-based elimination of autologous CAR T cells.

Although CAR T-cell therapy has demonstrated tremendous clinical efficacy especially in hematological malignancies, severe treatment-associated toxicities still compromise the widespread application of this innovative technology. Therefore, developing novel approaches to abrogate CAR T-cell-mediated side effects is of great relevance. Several promising strategies pursue the selective antibody-based depletion of adoptively transferred T cells via elimination markers. However, given the limited half-life and tissue penetration, dependence on the patients' immune system and on-target/off-side effects of proposed monoclonal antibodies, we sought to exploit αCAR-engineered T cells to efficiently eliminate CAR T cells. For comprehensive and specific recognition, a small peptide epitope (E-tag) was incorporated into the extracellular spacer region of CAR constructs. We provide first proof-of-concept for targeting this epitope by αE-tag CAR T cells, allowing an effective killing of autologous E-tagged CAR T cells both in vitro and in vivo whilst sparing cells lacking the E-tag. In addition to CAR T-cell cytotoxicity, the αE-tag-specific T cells can be empowered with cancer-fighting ability in case of relapse, hence, have versatile utility. Our proposed methodology can most probably be implemented in CAR T-cell therapies regardless of the targeted tumor antigen aiding in improving overall safety and survival control of highly potent gene-modified cells.

Theranostic CAR T cell targeting: A brief review.

More than hundred years ago, Paul Ehrlich postulated that our immune system should be able to recognize tumor cells. Just recently, the development of check point inhibitors, bispecific antibodies, and T cells genetically modified to express chimeric antigen receptors (CARs) underlines the true power of our immune system. T cells genetically modified with CARs can lead to complete remission of malignant hematologic diseases. However, they can also cause life-threatening side effects. In case of cytokine release syndrome, tumor lysis syndrome, or deadly side effects on the central nervous system, an emergency shut down of CAR T cells is needed. Targeting of tumor-associated antigens that are also expressed on vital tissues require a possibility to repeatedly switch the activity of CAR T cells on and off on demand and to follow the treatment by imaging. Theranostic, modular CARs such as the UniCAR system may help to overcome these problems.

Engrafting human regulatory T cells with a flexible modular chimeric antigen receptor technology.

As regulatory T cells (Tregs) play a fundamental role in immune homeostasis their adoptive transfer emerged as a promising treatment strategy for inflammation-related diseases. Preclinical animal models underline the superiority of antigen-specific Tregs compared to polyclonal cells. Here, we applied a modular chimeric antigen receptor (CAR) technology called UniCAR for generation of antigen-specific human Tregs. In contrast to conventional CARs, UniCAR-endowed Tregs are indirectly linked to their target cells via a separate targeting module (TM). Thus, transduced Tregs can be applied universally as their antigen-specificity is easily adjusted by TM exchange. Activation of UniCAR-engrafted Tregs occurred in strict dependence on the TM, facilitating a precise control over Treg activity. In order to augment efficacy and safety, different intracellular signaling domains were tested. Both 4-1BB (CD137) and CD28 costimulation induced strong suppressive function of genetically modified Tregs. However, in light of safety issues, UniCARs comprising a CD137-CD3ζ signaling domain emerged as constructs of choice for a clinical application of redirected Tregs. In that regard, Tregs isolated from patients suffering from autoimmune or inflammatory diseases were, for the first time, successfully engineered with UniCAR 137/ζ and efficiently suppressed patient-derived effector cells. Overall, the UniCAR platform represents a promising approach to improve Treg-based immunotherapies for tolerance induction.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

BACKGROUND: Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. METHODS: In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. RESULTS: Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. CONCLUSIONS: Overall, the novel modular system represents a promising tool for multiple tumor targeting.