Regression of renal cell carcinoma by T cell receptor-engineered T cells targeting a human endogenous retrovirus.

BACKGROUND: We discovered a novel human endogenous retrovirus (CT-RCC HERV-E) that was selectively expressed in most clear cell renal cell carcinomas (ccRCC) and served as a source of antigens for T cell-mediated killing. Here, we described the cloning of a novel T cell receptor (TCR) targeting a CT-RCC HERV-E-derived antigen specific to ccRCC and characterized antitumor activity of HERV-E TCR-transduced T cells (HERV-E T cells). METHODS: We isolated a CD8+ T cell clone from a patient with immune-mediated regression of ccRCC post-allogeneic stem cell transplant that recognized the CT-RCC-1 HERV-E-derived peptide in an HLA-A11-restricted manner. We used 5'Rapid Amplification of cDNA Ends (RACE) to clone the full length HERV-E TCR and generated retrovirus encoding this TCR for transduction of T cells. We characterized HERV-E T cells for phenotype and function in vitro and in a murine xenograft model. Lastly, we implemented a good manufacturing practice-compliant method for scalable production of HERV-E T cells. RESULTS: The HLA-A11-restricted HERV-E-reactive TCR exhibited a CD8-dependent phenotype and demonstrated specific recognition of the CT-RCC-1 peptide. CD8+ T cells modified to express HERV-E TCR displayed potent antitumor activity against HLA-A11+ ccRCC cells expressing CT-RCC HERV-E compared with unmodified T cells. Killing by HERV-E T cells was lost when cocultured against HERV-E knockout ccRCC cells. HERV-E T cells induced regression of established ccRCC tumors in a murine model and improved survival of tumor-bearing mice. Large-scale production of HERV-E T cells under good manufacturing practice conditions generated from healthy donors retained specific antigen recognition and cytotoxicity against ccRCC. CONCLUSIONS: This is the first report showing that human ccRCC cells can be selectively recognized and killed by TCR-engineered T cells targeting a HERV-derived antigen. These preclinical findings provided the foundation for evaluating HERV-E TCR-transduced T cell infusions in patients with metastatic ccRCC in a clinical trial (NCT03354390).

Nonactivated and IL-7 cultured CD19-specific CAR T cells are enriched in stem cell phenotypes and functionally superior.

ABSTRACT: CD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.

Melanoma reactive TCR-modified T cells generated without activation retain a less differentiated phenotype and mediate a superior in vivo response.

Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

HDAC inhibition prevents transgene expression downregulation and loss-of-function in T-cell-receptor-transduced T cells.

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.

Endothelin-1-Mediated Drug Resistance in EGFR-Mutant Non-Small Cell Lung Carcinoma.

Progression on therapy in non-small cell lung carcinoma (NSCLC) is often evaluated radiographically, however, image-based evaluation of said therapies may not distinguish disease progression due to intrinsic tumor drug resistance or inefficient tumor penetration of the drugs. Here we report that the inhibition of mutated EGFR promotes the secretion of a potent vasoconstrictor, endothelin-1 (EDN1), which continues to increase as the cells become resistant with a mesenchymal phenotype. As EDN1 and its receptor (EDNR) is linked to cancer progression, EDNR-antagonists have been evaluated in several clinical trials with disappointing results. These trials were based on a hypothesis that the EDN1-EDNR axis activates the MAPK-ERK signaling pathway that is vital to the cancer cell survival; the trials were not designed to evaluate the impact of tumor-derived EDN1 in modifying tumor microenvironment or contributing to drug resistance. Ectopic overexpression of EDN1 in cells with mutated EGFR resulted in poor drug delivery and retarded growth in vivo but not in vitro. Intratumoral injection of recombinant EDN significantly reduced blood flow and subsequent gefitinib accumulation in xenografted EGFR-mutant tumors. Furthermore, depletion of EDN1 or the use of endothelin receptor inhibitors bosentan and ambrisentan improved drug penetration into tumors and restored blood flow in tumor-associated vasculature. Correlatively, these results describe a simplistic endogenous yet previously unrealized resistance mechanism inherent to a subset of EGFR-mutant NSCLC to attenuate tyrosine kinase inhibitor delivery to the tumors by limiting drug-carrying blood flow and the drug concentration in tumors. SIGNIFICANCE: EDNR antagonists can be repurposed to improve drug delivery in VEGFA-secreting tumors, which normally respond to TKI treatment by secreting EDN1, promoting vasoconstriction, and limiting blood and drug delivery.

Thioredoxin-1 improves the immunometabolic phenotype of antitumor T cells.

Adoptive transfer of tumor epitope-reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)-Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62Lhi (CD62Lhi) central memory-like phenotype with reduced glucose uptake (2-NBDGlo) and decreased effector function (interferon γlo). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.

Potassium channel activity controls breast cancer metastasis by affecting ß-catenin signaling.

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K+ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial-mesenchymal transition via attenuation of Wnt/ß-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier.

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocin-induced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2AKITA) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes.

Inhibition of insulin-like growth factor 1 receptor enhances the efficacy of sorafenib in inhibiting hepatocellular carcinoma cell growth and survival.

Hepatocellular carcinoma (HCC) is the fifth most common primary cancer and second largest cause of cancer-related death worldwide. The first-line oral chemotherapeutic agent sorafenib only increases survival in patients with advanced HCC by less than 3 months. Most patients with advanced HCC have shown limited response rates and survival benefits with sorafenib. Although sorafenib is an inhibitor of multiple kinases, including serine/threonine-protein kinase c-Raf, serine/threonine-protein kinase B-Raf, vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor ß, HCC cells are able to escape from sorafenib treatment using other pathways that the drug insufficiently inhibits. The aim of this study was to identify and target survival and proliferation pathways that enable HCC to escape the antitumor activity of sorafenib. We found that insulin-like growth factor 1 receptor (IGF1R) remains activated in HCC cells treated with sorafenib. Knockdown of IGF1R sensitizes HCC cells to sorafenib treatment and decreases protein kinase B (AKT) activation. Overexpression of constitutively activated AKT reverses the effect of knockdown of IGF1R in sensitizing HCC cells to treatment with sorafenib. Further, we found that ceritinib, a drug approved by the U.S. Food and Drug Administration for treatment of non-small cell lung cancer, effectively inhibits the IGF1R/AKT pathway and enhances the inhibitory efficacy of sorafenib in human HCC cell growth and survival in vitro, in a xenograft mouse model and in the c-Met/ß-catenin-driven HCC mouse model. Conclusion: Our study provides a biochemical basis for evaluation of a new combination treatment that includes IGF1R inhibitors, such as ceritinib and sorafenib, in patients with HCC. (Hepatology Communications 2018;2:732-746).