Monitoring malignant T-cell clones by direct TCR expression assay in patients with leukemic cutaneous T-cell lymphoma during extracorporeal photopheresis.

Background/purpose: Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated.

Very rapid cloning, expression and identifying specificity of T-cell receptors for T-cell engineering.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells.

Percutaneous BCG enhances innate effector antitumor cytotoxicity during treatment of bladder cancer: a translational clinical trial.

: Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG.

Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages.

Targeting B-cell malignancies through human B-cell receptor specific CD4 T cells.

The B-cell receptor (BCR) expressed by a clonal B cell tumor is a tumor specific antigen (idiotype). However, the T-cell epitopes within human BCRs which stimulate protective immunity still lack detailed characterization. In this study, we identified 17 BCR peptide-specific CD4 T-cell epitopes derived from BCR heavy and light chain variable region sequences.

Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (T) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR T cells with preserved T potential using the Sleeping Beauty platform.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

Background: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR.

Methods: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines.

Measurement of Average Telomere Length in Ex Vivo Expanded Natural Killer Cells by Fluorescence In Situ Hybridization (FISH) and Flow Cytometry.

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that play a critical role in innate immune surveillance against infections and tumors through cytokine secretion and target cell lysis. NK cells function without any need for prior antigen exposure. Thus, more recently NK cells are considered a promising source of lymphocytes for adoptive tumor therapy.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody.

The beneficial effects of a gas-permeable flask for expansion of Tumor-Infiltrating lymphocytes as reflected in their mitochondrial function and respiration capacity.

Adoptive transfer of autologous expanded tumor-infiltrating lymphocytes (TIL) is a highly successful cell therapy approach in the treatment of late-stage melanoma. Notwithstanding the success of this therapy, only very few centers worldwide can provide it. To make this therapy broadly available, one of the major obstacles to overcome is the complexity of culturing the TIL.

A miR-192-EGR1-HOXB9 regulatory network controls the angiogenic switch in cancer.

A deeper mechanistic understanding of tumour angiogenesis regulation is needed to improve current anti-angiogenic therapies. Here we present evidence from systems-based miRNA analyses of large-scale patient data sets along with in vitro and in vivo experiments that miR-192 is a key regulator of angiogenesis. The potent anti-angiogenic effect of miR-192 stems from its ability to globally downregulate angiogenic pathways in cancer cells through regulation of EGR1 and HOXB9.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages.

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism.

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells.

Genetic editing of HLA expression in hematopoietic stem cells to broaden their human application.

Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C, and -DRB1 regardless of a patient's race.

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

Purpose: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma.

Experimental Design: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis.

Activation and propagation of tumor-infiltrating lymphocytes on clinical-grade designer artificial antigen-presenting cells for adoptive immunotherapy of melanoma.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as "feeders" to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders.

Plasma circulating-microRNA profiles are useful for assessing prognosis in patients with cytogenetically normal myelodysplastic syndromes.

Myelodysplastic syndromes are a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Chromosomal abnormalities and gene mutations have been shown to have essential roles in pathogenesis and correlate with prognosis. Molecular markers, however, are not integrated into currently used prognostic systems.

Recurrent ESR1-CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers.

Characterizing the genetic alterations leading to the more aggressive forms of oestrogen receptor-positive (ER+) breast cancers is of critical significance in breast cancer management. Here we identify recurrent rearrangements between the oestrogen receptor gene ESR1 and its neighbour CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal B tumours, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170-positive tumours.