The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling.

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8(+) T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8(+) T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8(+) T cells, as miR-155-deficient CD8(+) T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8(+) T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8(+) T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon-associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8(+) T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8(+) T cell responses to pathogens in vivo.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

CAR T Cells Targeting MISIIR for the Treatment of Ovarian Cancer and Other Gynecologic Malignancies.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation.

Influenza virus outbreaks remain a serious threat to public health. A greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo-infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus-infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes compared to AEC from uninfected animals, directly infected cells produce more interferon and express lower levels of Wnt signaling-associated transcripts, while concurrently expressing more transcripts associated with cell death pathways, than bystander uninfected AEC. The Wnt signaling pathway was downregulated in both in vivo-infected AEC and in vitro-infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein-expressing influenza virus, we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis, as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.

Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRß high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vß family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vß gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

Phosphatidylinositol 3-Kinase p110δ Isoform Regulates CD8+ T Cell Responses during Acute Viral and Intracellular Bacterial Infections.

The p110δ isoform of PI3K is known to play an important role in immunity, yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells, we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation, proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases, our study suggests caution in using these drugs in patients, as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens.

Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/ß on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/ß stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo.

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.

The inflammasome activating caspase 1 mediates fibrosis and myofibroblast differentiation in systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a chronic idiopathic disease of unknown etiology that is characterized by fibrosis of the skin and visceral organs mediated by activated myofibroblasts. The recently identified inflammasomes are cytosolic receptors that tightly regulate the activity of caspase 1 and downstream signaling molecules such as interleukin-1ß (IL-1ß) and IL-18. The nucleotide-binding oligomerization domain (NOD)-like receptor 3 (NLRP3) inflammasome has been implicated in the development of idiopathic pulmonary fibrosis. This study was undertaken to assess the role of the inflammasome in SSc-related dermal or pulmonary fibrosis. METHODS: Inflammasome gene transcripts were assayed in fibroblasts obtained from patients with SSc. Caspase 1 activation in SSc primary dermal and lung fibroblasts was inhibited, and the levels of hydroxyproline, COL1A1, COL3A1, IL-1ß, IL-18, and α-smooth muscle actin (α-SMA) were measured. The role of the inflammasome in dermal fibrosis was investigated in NLRP3(-/-) and ASC(-/-) mice. RESULTS: We identified increased expression of 40 genes associated with the inflammasome or downstream signaling molecules in SSc fibroblasts. Inhibition of caspase 1 in SSc dermal and lung fibroblasts abrogated the secretion of collagens, IL-1ß, and IL-18. In addition, we observed decreased expression of the myofibroblast protein α-SMA in SSc dermal fibroblasts treated with a caspase 1 inhibitor. Furthermore, NLRP3(-/-) mice and ASC(-/-) mice were resistant to bleomycin-induced skin fibrosis, which suggests a key role for the inflammasome in in vivo fibrosis. CONCLUSION: Innate immune signaling contributes to SSc fibrosis via activation of the inflammasome and caspase 1. These results suggest that inflammasome activation may play an important role in the pathogenesis of SSc.

BLIMP1 and NR4A3 transcription factors reciprocally regulate antitumor CAR T cell stemness and exhaustion.

Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]-expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.

Chronic antigen stimulation alone is sufficient to drive CD8+ T cell exhaustion.

The failure of CD8(+) T cells to respond to chronic infection has been termed "exhaustion" and describes the condition in which CD8(+) T cells exhibit reduced differentiation, proliferation, and effector function. CD8(+) T cell exhaustion has been extensively studied in the murine model of chronic infection, lymphocytic choriomeningitis virus (LCMV). Although LCMV-based studies have yielded many interesting findings, they have not allowed for discrimination between the roles of cytokine- and Ag-driven exhaustion. We have created a system of chronic Ag stimulation using murine influenza A virus that leads to exhaustion and functional disability of virus-specific CD8(+) T cells, in the absence of high viral titers, sustained proinflammatory cytokine production and lymphocyte infection. Our findings show that Ag alone is sufficient to drive CD8(+) T cell impairment, that Ag-driven loss of virus-specific CD8(+) T cells is TRAIL mediated, and that removal of Ag reverses exhaustion. Although programmed death 1 was up-regulated on chronic Ag-stimulated CD8(+) T cells, it played no role in the exhaustion. These findings provide a novel insight into the mechanisms that control functional exhaustion of CD8(+) T cells in chronic infection.

Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4.

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC.

Phosphorothioate 2' deoxyribose oligomers as microbicides that inhibit human immunodeficiency virus type 1 (HIV-1) infection and block Toll-like receptor 7 (TLR7) and TLR9 triggering by HIV-1.

Topical microbicides may prove to be an important strategy for preventing human immunodeficiency virus type 1 (HIV-1) transmission. We examined the safety and efficacy of sequence-nonspecific phosphorothioate 2' deoxyribose oligomers as potential novel microbicides. A short, 13-mer poly(T) phosphorothioate oligodeoxynucleotide (OPB-T) significantly inhibited infection of primary peripheral blood mononuclear cells (PBMC) by high-titer HIV-1(Ba-L) and simian immunodeficiency virus mac251 (SIV(mac251)). Continuous exposure of human vaginal and foreskin tissue explants to OPB-T showed no toxicity. An abasic 14-mer phosphorothioate 2' deoxyribose backbone (PDB) demonstrated enhanced anti-HIV-1 activity relative to OPB-T and other homo-oligodeoxynucleotide analogs. When PDB was used to pretreat HIV-1, PDB was effective against R5 and X4 isolates at a half-maximal inhibitory concentration (IC(50)) of <1 µM in both PBMC and P4-R5 MAGI cell infections. PDB also reduced HIV-1 infectivity following the binding of virus to target cells. This novel topical microbicide candidate exhibited an excellent in vitro safety profile in human PBMC and endocervical epithelial cells. PDB also retained activity in hydroxyethylcellulose gel at pH 4.4 and after transition to a neutral pH and was stable in this formulation for 30 days at room temperature. Furthermore, the compound displayed potent antiviral activity following incubation with a Lactobacillus strain derived from normal vaginal flora. Most importantly, PDB can inhibit HIV-1-induced alpha interferon production. Phosphorothioate 2' deoxyribose oligomers may therefore be promising microbicide candidates that inhibit HIV-1 infection and also dampen the inflammation which is critical for the initial spread of the virus.

Costimulation signals for memory CD8+ T cells during viral infections.

Costimulation signals have been recognized as critical for optimal T-cell responses and result from important interactions between receptors on the surface of T cells and their ligands on antigen-presenting cells. Two families of receptors, the CD28 family and the tumor necrosis factor receptor (TNFR) family, have been found to be major players in providing costimulation to CD8+ T cells. Recent studies using viral infection models have highlighted the importance of CD28 costimulation signals during memory responses against viruses. Programmed death-1 (PD-1), another member of the CD28 family, may contribute to functional defects of helpless memory CD8+ T cells. Members of the TNFR family, such as CD27, 4-1BB, CD40, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), and OX40, have also been implicated in the survival, generation, maintenance, and quality of virus-specific memory CD8+T cells. The delivery of costimulatory molecules such as CD28, 4-1BB, and OX40 can help boost the generation and function of virus-specific memory CD8+ T cells. The use of costimulatory molecules as adjuvants, along with viral antigens in vaccines, may facilitate the generation of effective antigen-specific memory CD8+ T-cell responses. Understanding the costimulatory requirements of memory CD8+ T cells, therefore, may lead to improved vaccines that target anti-viral CD8+ T-cell memory.

CAR T Cell Therapy of Non-hematopoietic Malignancies: Detours on the Road to Clinical Success.

Chimeric antigen receptor (CAR)-engineered T cells represent a breakthrough in personalized medicine. In this strategy, a patient's own T lymphocytes are genetically reprogrammed to encode a synthetic receptor that binds a tumor antigen, allowing T cells to recognize and kill antigen-expressing cancer cells. As a result of complete and durable responses in individuals who are refractory to standard of care therapy, CAR T cells directed against the CD19 protein have been granted United States Food and Drug Administration (FDA) approval as a therapy for treatment of pediatric and young adult acute lymphoblastic leukemia and diffuse large B cell lymphoma. Human trials of CAR T cells targeting CD19 or B cell maturation antigen in multiple myeloma have also reported early successes. However, a clear and consistently reproducible demonstration of the clinical efficacy of CAR T cells in the setting of solid tumors has not been reported to date. Here, we review the history and status of CAR T cell therapy for solid tumors, potential T cell-intrinsic determinants of response and resistance as well as extrinsic obstacles to the success of this approach for much more prevalent non-hematopoietic malignancies. In addition, we summarize recent strategies and innovations that aim to augment the potency of CAR T cells in the face of multiple immunosuppressive barriers operative within the solid tumor microenvironment. Advances in the field of CAR T cell biology over the coming years in the areas of safety, reliability and efficacy against non-hematopoietic cancers will ultimately determine how transformative adoptive T cell therapy will be in the broader battle against cancer.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).