Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models.

BACKGROUND: Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. METHODS: Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 µg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. RESULTS: ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. CONCLUSIONS: Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Traumatic brain injury does not disrupt costimulatory blockade-induced immunological tolerance to glial-restricted progenitor allografts.

BACKGROUND: Cell transplantation-based treatments for neurological disease are promising, yet graft rejection remains a major barrier to successful regenerative therapies. Our group and others have shown that long-lasting tolerance of transplanted stem cells can be achieved in the brain with systemic application of monoclonal antibodies blocking co-stimulation signaling. However, it is unknown if subsequent injury and the blood-brain barrier breach could expose the transplanted cells to systemic immune system spurring fulminant rejection and fatal encephalitis. Therefore, we investigated whether delayed traumatic brain injury (TBI) could trigger graft rejection. METHODS: Glial-restricted precursor cells (GRPs) were intracerebroventricularly transplanted in immunocompetent neonatal mice and co-stimulation blockade (CoB) was applied 0, 2, 4, and 6 days post-grafting. Bioluminescence imaging (BLI) was performed to monitor the grafted cell survival. Mice were subjected to TBI 12 weeks post-transplantation. MRI and open-field test were performed to assess the brain damage and behavioral change, respectively. The animals were decapitated at week 16 post-transplantation, and the brains were harvested. The survival and distribution of grafted cells were verified from brain sections. Hematoxylin and eosin staining (HE) was performed to observe TBI-induced brain legion, and neuroinflammation was evaluated immunohistochemically. RESULTS: BLI showed that grafted GRPs were rejected within 4 weeks after transplantation without CoB, while CoB administration resulted in long-term survival of allografts. BLI signal had a steep rise following TBI and subsequently declined but remained higher than the preinjury level. Open-field test showed TBI-induced anxiety for all animals but neither CoB nor GRP transplantation intensified the symptom. HE and MRI demonstrated a reduction in TBI-induced lesion volume in GRP-transplanted mice compared with non-transplanted mice. Brain sections further validated the survival of grafted GRPs and showed more GRPs surrounding the injured tissue. Furthermore, the brains of post-TBI shiverer mice had increased activation of microglia and astrocytes compared to post-TBI wildtype mice, but infiltration of CD45+ leukocytes remained low. CONCLUSIONS: CoB induces sustained immunological tolerance towards allografted cerebral GRPs which is not disrupted following TBI, and unexpectedly TBI may enhance GRPs engraftment and contribute to post-injury brain tissue repair.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

First-in-human study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of ALPN-101, a dual CD28/ICOS antagonist, in healthy adult subjects.

ALPN-101 (ICOSL vIgD-Fc) is an Fc fusion protein of a human inducible T cell costimulatory ligand (ICOSL) variant immunoglobulin domain (vIgD) designed to inhibit the cluster of differentiation 28 (CD28) and inducible T cell costimulator (ICOS) pathways simultaneously. A first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ALPN-101 in healthy adult subjects. ALPN-101 was generally well-tolerated with no evidence of cytokine release, clinically significant immunogenicity, or severe adverse events following single subcutaneous (SC) doses up to 3 mg/kg or single intravenous (IV) doses up to 10 mg/kg or up to 4 weekly IV doses of up to 1 mg/kg. ALPN-101 exhibited a dose-dependent increase in exposure with an estimated terminal half-life of 4.3-8.6 days and SC bioavailability of 60.6% at 3 mg/kg. Minimal to modest accumulation in exposure was observed with repeated IV dosing. ALPN-101 resulted in a dose-dependent increase in maximum target saturation and duration of high-level target saturation. Consistent with its mechanism of action, ALPN-101 inhibited cytokine production in whole blood stimulated by Staphylococcus aureus enterotoxin B ex vivo, as well as antibody responses to keyhole limpet hemocyanin immunization, reflecting immunomodulatory effects upon T cell and T-dependent B cell responses, respectively. In conclusion, ALPN-101 was well-tolerated in healthy subjects with dose-dependent PK and PD consistent with the known biology of the CD28 and ICOS costimulatory pathways. Further clinical development of ALPN-101 in inflammatory and/or autoimmune diseases is therefore warranted.

CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

Predicting immunotherapy response through genomics.

Immune checkpoint inhibitors (ICI) aim to restore the immune system anti-tumor function by blocking two inhibitory axes: CTLA-4/CD28 and PD1/PDL1. ICI is established as a treatment option for multiple cancers, but their remarkable clinical impact is observed only in a fraction of patients. Together with their adverse effects and high cost, it's imperative to identify patients who are likely to benefit from this type of treatment. Genomic features represent promising candidates as predictive biomarkers of response to ICI, with agnostic FDA-approvals of an anti-PD1 drug for tumors with microsatellite instability and tumors with a high mutational burden. Other genomic markers are also emerging to help refine patient selection. In this review, we discuss recent progress in genomic biomarkers development and its challenges, with a focus on alterations in the neoantigen burden, immune, and oncogenic pathways.

Tasmanian devil CD28 and CTLA4 capture CD80 and CD86 from adjacent cells.

Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumors. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species.

CD28 Agonism Improves Survival in Immunologically Experienced Septic Mice via IL-10 Released by Foxp3+ Regulatory T Cells.

Immune dysregulation during sepsis is mediated by an imbalance of T cell costimulatory and coinhibitory signaling. CD28 is downregulated during sepsis and is significantly altered on memory versus naive T cells. Thus, to study the role of CD28 during sepsis in a more physiologically relevant context, we developed a "memory mouse" model in which animals are subjected to pathogen infections to generate immunologic memory, followed by sepsis induction via cecal ligation and puncture. Using this system, we show that agonistic anti-CD28 treatment resulted in worsened survival in naive septic animals but conferred a significant survival advantage in immunologically experienced septic animals. Mechanistically, this differential response was driven by the ability of CD28 agonism to elicit IL-10 production from regulatory T cells uniquely in memory but not naive mice. Moreover, elevated IL-10 released by activated regulatory T cells in memory mice inhibited sepsis-induced T cell apoptosis via the antiapoptotic protein Bcl-xL. Together, these data demonstrate that immunologic experience is an important parameter that affects sepsis pathophysiology and can fundamentally change the outcome of modulating the CD28 pathway during sepsis. This study suggests that testing therapeutic strategies in immunologically experienced hosts may be one way to increase the physiologic relevance of rodent models in sepsis research.

Discovery of an Oxepine-Containing Diketopiperazine Derivative Active against Concanavalin A-Induced Hepatitis.

Varioxepine B (1), an oxepine-containing diketopiperazine derivative, was isolated from a marine-derived Aspergillus terreus strain. The structure of 1 was identified by spectroscopic experiments, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. It is noteworthy that 1 could suppressed murine splenocyte proliferation activated by concanavalin A (Con A) in vitro. More importantly, in Con A-challenged mice, pretreatment with 1 obviously decreased the generation of proinflammatory cytokines and ameliorated liver injury. Meanwhile, 1 also exhibited inhibitory activity in anti-CD3/anti-CD28 monoclonal antibodies (mAbs)-induced murine splenocytes and human T cell proliferation as well as both Th1 and Th2 cytokine production.

Targeting co-stimulatory molecules in autoimmune disease.

Therapeutic targeting of immune checkpoints has garnered significant attention in the area of cancer immunotherapy, in which efforts have focused in particular on cytotoxic T lymphocyte antigen 4 (CTLA4) and PD1, both of which are members of the CD28 family. In autoimmunity, these same pathways can be targeted to opposite effect: to curb the over-exuberant immune response. The CTLA4 checkpoint serves as an exemplar, whereby CTLA4 activity is blocked by antibodies in cancer immunotherapy and augmented by the provision of soluble CTLA4 in autoimmunity. Here, we review the targeting of co-stimulatory molecules in autoimmune diseases, focusing in particular on agents directed at members of the CD28 or tumour necrosis factor receptor families. We present the state of the art in co-stimulatory blockade approaches, including rational combinations of immune inhibitory agents, and discuss the future opportunities and challenges in this field.

Novel DNA-based T-Cell Activator Promotes Rapid T-Cell Activation and Expansion.

Autologous chimeric antigen receptor engineered T-cell therapies are beginning to dramatically change the outlook for patients with several hematological malignancies. Yet methods to activate and expand these cells are limited, often pose challenges to automation, and have biological limitations impacting the output of the injectable dose. This study describes the development of a novel, highly flexible, soluble DNA-based T-cell activation and expansion platform which alleviates the limitations of current technologies and provides rapid T-cell activation and expansion.

Hypersonins A-D, Polycyclic Polyprenylated Acylphloroglucinols with a 1,2-seco-Homoadamantane Architecture from Hypericum wilsonii.

Hypersonins A-D (1-4), four 1,2-seco-homoadamantane type polycyclic polyprenylated acylphloroglucinols (PPAPs) possessing a new bicyclo[4.3.1]decane-3-methoxycarbonyl architecture, were obtained from Hypericum wilsonii. The structures of hypersonins A-D were identified by spectroscopic data, electronic circular dichroism comparison, and X-ray crystallographic data. Hypersonins A-D are the first seco-homoadamantane-type PPAPs with cleavage at the C-1-C-2 bond. Hypersonin A (1) showed moderate inhibitory activity to anti-CD3/anti-CD28 monoclonal antibody-induced proliferation of murine splenocytes, with an IC50 value of 8.3 ± 0.2 µM.

Iron Deprivation in Human T Cells Induces Nonproliferating Accessory Helper Cells.

Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the transcription factors NF-κB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly, protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic T cells that can help and stimulate other immune cells by providing cytokines such as IL-2.

Treatment of mice with a ligand binding blocking anti-CD28 monoclonal antibody improves healing after myocardial infarction.

Both conventional and regulatory CD4+ T-cells rely on costimulatory signals mediated by cell surface receptors including CD28 for full activation. We showed previously that stimulation of CD4+ Foxp3+ regulatory T-cells by superagonistic anti-CD28 monoclonal antibodies (mAb) improves myocardial healing after experimental myocardial infarction (MI). However, the effect of ligand binding blocking anti-CD28 monoclonal antibodies has not yet been tested in this context. We hypothesize that ligand blocking anti-CD28 mAb treatment might favorably impact on healing after MI by limiting the activation of conventional CD4+ T-cells. Therefore, we studied the therapeutic effect of the recently characterized mAb E18 which blocks ligand binding to CD28 in a mouse permanent coronary ligation model. E18 or an irrelevant control mAb was applied once on day two after myocardial infarction to wildtype mice. Echocardiography was performed on day 7 after MI. E18 treatment improved the survival and reduced the incidence of left ventricular ruptures after experimental myocardial infarction. Accordingly, although we found no difference in infarct size, there was significantly less left ventricular dilation after E18 treatment in surviving animals as determined by echocardiography at day 7 after MI. In sham operated control mice neither antibody had an impact on body weight, survival, and echocardiographic parameters. Mechanistically, compared to control immunoglobulin, E18 treatment reduced the number of CD4+ T-cells and monocytes/macrophages within the infarct and periinfarct zone on day 5. This was accompanied by an upregulation of arginase which is a marker for alternatively differentiated macrophages. The data indicate that CD28-dependent costimulation of CD4+ T-cells impairs myocardial healing and anti-CD28 antibody treatment constitutes a potentially clinically translatable approach to improve the outcome early after MI.

CD5 blockade enhances ex vivo CD8+ T cell activation and tumour cell cytotoxicity.

CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.

Expanding CAR T cells in human platelet lysate renders T cells with in vivo longevity.

BACKGROUND: Pre-clinical and clinical studies have shown that the infusion of CAR T cells with a naive-like (TN) and central memory (TCM) phenotype is associated with prolonged in vivo T cell persistence and superior anti-tumor effects. To optimize the maintenance of such populations during the in vitro preparation process, we explored the impact of T cell exposure to both traditional [fetal bovine serum (FBS), human AB serum (ABS)] and non-traditional [human platelet lysate (HPL) - a xeno-free protein supplement primarily used for the production of clinical grade mesenchymal stromal / stem cells (MSCs)] serum supplements. METHODS: Second generation chimeric antigen receptor with CD28 and CD3ζ endodomain targeting prostate stem cell antigen (PSCA) (P28z) or CD19 (1928z) were constructed and used for this study. After retroviral transduction, CAR T cells were divided into 3 conditions containing either FBS, ABS or HPL and expanded for 7 days. To evaluate the effect of different sera on CAR T cell function, we performed a series of in vitro and in vivo experiments. RESULTS: HPL-exposed CAR T cells exhibited the less differentiated T cell phenotype and gene signature, which displayed inferior short-term killing abilities (compared to their FBS- or ABS-cultured counterparts) but superior proliferative and anti-tumor effects in long-term in vitro coculture experiments. Importantly, in mouse xenograft model, HPL-exposed CAR T cells outperformed their ABS or FBS counterparts against both subcutaneous tumor (P28z T cells against Capan-1PSCA) and systemic tumor (1928z T cells against NALM6). We further observed maintenance of less differentiated T cell phenotype in HPL-exposed 1928z T cells generated from patient's PBMCs with superior anti-tumor effect in long-term in vitro coculture experiments. CONCLUSIONS: Our study highlights the importance of serum choice in the generation of CAR T cells for clinical use.

Immune inhibitory proteins and their pathogenic and therapeutic implications in autoimmunity and autoimmune hepatitis.

Key inhibitory proteins can blunt immune responses to self-antigens, and deficiencies in this repertoire may promote autoimmunity. The goals of this review are to describe the key immune inhibitory proteins, indicate their possible impact on the development of autoimmune disease, especially autoimmune hepatitis, and encourage studies to clarify their pathogenic role and candidacy as therapeutic targets. English abstracts were identified in PubMed by multiple search terms. Full length articles were selected for review, and secondary and tertiary bibliographies were developed. Cytotoxic T lymphocyte antigen-4 impairs ligation of CD28 to B7 ligands on antigen presenting cells and inhibits the adaptive immune response by increasing anti-inflammatory cytokines, generating regulatory T cells, and reducing T cell activation and proliferation. Programed cell death antigen-1 inhibits T cell selection, activation, and proliferation by binding with two ligands at different phases and locations of the immune response. A soluble alternatively spliced variant of this protein can dampen the inhibitory signal. Autoimmune hepatitis has been associated with polymorphisms of the cytotoxic T lymphocyte antigen-4 gene, reduced hepatic expression of a ligand of programed cell death antigen-1, an interfering soluble variant of this key inhibitory protein, and antibodies against it. Findings have been associated with laboratory indices of liver injury and suboptimal treatment response. Abatacept, belatacept, CD28 blockade, and induction of T cell exhaustion are management considerations that require scrutiny. In conclusion, deficiencies in key immune inhibitory proteins may promote the occurrence of autoimmune diseases, such as autoimmune hepatitis, and emerging interventions may overcome these deficiencies. Investigations should define the nature, impact and management of these inhibitory disturbances in autoimmune hepatitis.

Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells.

PURPOSE: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 µmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.