Leslie E. Huye
Baylor College of Medicine
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Featured researches published by Leslie E. Huye.
Molecular Therapy | 2012
Ulrike Gerdemann; Jacqueline M. Keirnan; Usha L. Katari; Ryu Yanagisawa; Anne Christin; Leslie E. Huye; Serena K. Perna; Sravya Ennamuri; Stephen Gottschalk; Malcolm K. Brenner; Helen E. Heslop; Cliona M. Rooney; Ann M. Leen
Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein–Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4+ and CD8+) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.
Molecular Therapy | 2011
Yozo Nakazawa; Leslie E. Huye; Vita S. Salsman; Ann M. Leen; Nabil Ahmed; Lisa Rollins; Gianpietro Dotti; Stephen Gottschalk; Matthew H. Wilson; Cliona M. Rooney
Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.
Blood | 2015
Soranobu Ninomiya; Neeharika Narala; Leslie E. Huye; Shigeki Yagyu; Barbara Savoldo; Gianpietro Dotti; Helen E. Heslop; Malcolm K. Brenner; Cliona M. Rooney; Carlos A. Ramos
Although T cells expressing CD19-specific chimeric antigen receptors (CARs) are a promising new therapy for B-cell malignancies, objective responses are observed at lower frequencies in patients with lymphoma than in those with acute B-cell leukemia. We postulated that the tumor microenvironment suppresses CAR-expressing T cells (CARTs) through the activity of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that converts tryptophan into metabolites that inhibit T -: cell activity. To investigate the effects of tumor IDO on CD19-CART therapy, we used a xenograft lymphoma model expressing IDO as a transgene. CD19-CARTs inhibited IDO-negative tumor growth but had no effect on IDO-positive tumors. An IDO inhibitor (1-methyl-tryptophan) restored IDO-positive tumor control. Moreover, tryptophan metabolites inhibited interleukin (IL)-2-, IL-7-, and IL-15-dependent expansion of CARTs; diminished their proliferation, cytotoxicity, and cytokine secretion in vitro in response to CD19 recognition; and increased their apoptosis. Inhibition of CD19-CARTs was not mitigated by the incorporation of costimulatory domains, such as 4-1BB, into the CD19-CAR. Finally, we found that fludarabine and cyclophosphamide, frequently used before CART administration, downregulated IDO expression in lymphoma cells and improved the antitumor activity of CD19-CART in vivo. Because tumor IDO inhibits CD19-CARTs, antagonizing this enzyme may benefit CD19-CART therapy.
Journal of Immunotherapy | 2009
Yozo Nakazawa; Leslie E. Huye; Gianpietro Dotti; Aaron E. Foster; Juan F. Vera; Pallavi R. Manuri; Carl H. June; Cliona M. Rooney; Matthew H. Wilson
Optimal implementation of adoptive T-cell therapy for cancer will likely require multiple and maintained genetic modifications of the infused T cells and their progeny so that they home to tumor sites and recognize tumor cells, overcome tumor immune evasion strategies, and remain safe. Retroviral vectors readily transduce T cells and integrate into the host cell genome, but have a limited capacity for multigene insertion and cotransduction and are prohibitively expensive to produce at clinical grade. Genetic modification of T cells using transposons as integrating plasmids is an attractive alternative because of the increased simplicity and cost of production. Of available transposons, piggyBac has the higher transposase activity and larger cargo capacity, and we now evaluate piggyBac for potential adoptive therapies with primary T cells. PiggyBac transposons mediated stable gene expression in approximately 20% of primary T cells without selection. Treatment and maintenance of T cells with interleukin-15 increased stable transgene expression up to approximately 40% and expression was sustained through multiple logs of expansion for over 9 weeks in culture. We demonstrate simultaneous integration of 2 independent transposons in 20% of T cells, a frequency that could be increased to over 85% by selection of a transgenic surface marker (truncated CD19). PiggyBac could also deliver transposons of up to 13 kb with 10,000-fold expansion of transduced T cells in culture and finally we demonstrate delivery of a functional suicide gene (iCasp9). PiggyBac transposons may thus be used to express the multiple integrated transgenes that will likely be necessary for the broader success of T-cell therapy.
Blood | 2009
Biagio De Angelis; Gianpietro Dotti; Concetta Quintarelli; Leslie E. Huye; Lan Zhang; Ming Zhang; Fabrizio Pane; Helen E. Heslop; Malcolm K. Brenner; Cliona M. Rooney; Barbara Savoldo
Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) to solid organ transplant (SOT) recipients has been shown safe and effective for the treatment of EBV-associated posttransplantation lymphoproliferative disorders (PTLDs). SOT recipients, however, require the continuous administration of immunosuppressive drugs to prevent graft rejection, and these agents may significantly limit the long-term persistence of transferred EBV-CTLs, precluding their use as prophylaxis. Tacrolimus (FK506) is one of the most widely used immunosuppressive agents in SOT recipients, and its immunosuppressive effects are largely dependent on its interaction with the 12-kDa FK506-binding protein (FKBP12). We have knocked down the expression of FKBP12 in EBV-CTLs using a specific small interfering RNA (siRNA) stably expressed from a retroviral vector and found that FKBP12-silenced EBV-CTLs are FK506 resistant. These cells continue to expand in the presence of the drug without measurable impairment of their antigen specificity or cytotoxic activity. We confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high risk of PTLD after transplantation.
Molecular and Cellular Biology | 2002
Leslie E. Huye; Mary M. Purugganan; Ming-Ming Jiang; David Roth
ABSTRACT Although both RAG-1 and RAG-2 are required for all steps of V(D)J recombination, little is known about the specific contribution of either protein to these steps. RAG-1 contains three acidic active-site amino acids that are thought to coordinate catalytic metal ions. To search for additional catalytic amino acids and to better define the functional anatomy of RAG-1, we mutated all 86 conserved basic amino acids to alanine and evaluated the mutant proteins for DNA binding, nicking, hairpin formation, and joining. We found several amino acids outside of the canonical nonamer-binding domain that are critical for DNA binding, several step arrest mutants with defects in nicking or hairpin formation, and four RAG-1 mutants defective specifically for joining. Analysis of coding joints formed by some of these mutants revealed excessive deletions, frequent use of short sequence homologies, and unusually long palindromic junctional inserts, known as P nucleotides, that result from aberrant hairpin opening. These features characterize junctions found in scid mice, which are deficient for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), suggesting that the RAG proteins and DNA-PKcs perform overlapping functions in coding joint formation. Interestingly, the amino acids that are altered in 12 of our mutants are also mutated in human inherited immunodeficiency syndromes. Our analysis of these mutants provides insights into the molecular mechanisms underlying these disorders.
Human Gene Therapy | 2012
Joseph E. Doherty; Leslie E. Huye; Kosuke Yusa; Liqin Zhou; Nancy L. Craig; Matthew H. Wilson
We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured human cells. Native pB and SB100X, the most active transposase of the Sleeping Beauty transposon system, exhibited similar transposition efficiency in cultured human cell lines. When delivered to primary human T cells ex vivo, 7pB increased gene delivery two- to threefold compared with piggyBac and SB100X. The activity of hyperactive 7pB transposase was not affected by the addition of a 24-kDa N-terminal tag, whereas SB100X manifested a 50% reduction in transposition. Hyperactive 7pB was compared with native pB and SB100X in vivo in mice using hydrodynamic tail-vein injection of a limiting dose of transposase DNA combined with luciferase reporter transposons. We followed transgene expression for up to 6 months and observed approximately 10-fold greater long-term gene expression in mice injected with a codon-optimized version of 7pB compared with mice injected with native pB or SB100X. We conclude that hyperactive piggyBac elements can increase gene transfer in human cells and in vivo and should enable improved gene delivery using the piggyBac transposon system in a variety of cell and gene-therapy applications.
Molecular Therapy | 2010
Jiali Sun; Gianpietro Dotti; Leslie E. Huye; Aaron E. Foster; Barbara Savoldo; Maria Monica Gramatges; David M. Spencer; Cliona M. Rooney
Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes.
Molecular Cell | 2002
Matthew B. Neiditch; Gregory S. Lee; Leslie E. Huye; Vicky L. Brandt; David Roth
V(D)J recombination generates two types of products: coding joints, which constitute the rearranged variable regions of antigen receptor genes, and signal joints, which often form on immunologically irrelevant, excised circular molecules that are lost during cell division. It has been widely believed that signal joints simply convert reactive broken DNA ends into safe, inert products. Yet two curious in vivo observations made us question this assumption: signal ends are far more abundant than coding ends, and signal joints form only after RAG expression is downregulated. In fact, we find that signal joints are not at all inert; they are cleaved quite efficiently in vivo and in vitro by a nick-nick mechanism and form an excellent substrate for RAG-mediated transposition in vitro, possibly explaining how genomic stability in lymphocytes may be compromised.
Molecular Therapy | 2011
Leslie E. Huye; Yozo Nakazawa; Mayuri P Patel; Eric Yvon; Jiali Sun; Barbara Savoldo; Matthew H. Wilson; Gianpietro Dotti; Cliona M. Rooney
Despite activity as single agent cancer therapies, Rapamycin (rapa) and its rapalogs may have their greatest effects when combined with other therapeutic modalities. In addition to direct antitumor activity, rapalogs reverse multiple tumor-intrinsic immune evasion mechanisms. These should facilitate tumor-specific T cell activity, but since rapa directly inhibits effector T cells, this potential immune enhancement is lost. We hypothesized that if T cells were rendered resistant to rapa they could capitalize on its downregulation of tumor immune evasion. We therefore modified T cells with a rapa-resistant mutant of mTor, mTorRR, and directed them to B lymphomas by coexpressing a chimeric antigen receptor (CAR) for CD19 (CAR.CD19-28ζ). T cells expressing transgenic mTorRR from a piggyBac transposon maintain mTor signaling, proliferate in the presence of rapa and retain their cytotoxic function and ability to secrete interferon-γ (IFNγ) after stimulation, effector functions that were inhibited by rapa in control T cells. In combination, rapa and rapa-resistant-CAR.CD19-28ζ-expressing T cells produced greater antitumor activity against Burkitts lymphoma and pre-B ALL cell lines in vitro than CAR.CD19-28ζ T cells or rapa alone. In conclusion, the combination of rapa and rapa-resistant, CAR.CD19-28ζ-expressing T cells may provide a novel therapy for the treatment of B cell malignancies and other cancers.