Yozo Nakazawa

piggyBac Transposon/Transposase System to Generate CD19-Specific T Cells for the Treatment of B-Lineage Malignancies

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon/transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19(+) artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

PiggyBac-mediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor

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.

Genome-Wide Mapping of PiggyBac Transposon Integrations in Primary Human T Cells

The piggyBac transposon system represents a promising nonviral tool for gene delivery and discovery, and may also be of value for clinical gene therapy. PiggyBac is a highly efficient integrating vector that stably transfects (approximately 40%) of primary human T cells for potential adoptive immunotherapy applications. To evaluate the potential genotoxicity of piggyBac, we compared 228 integration sites in primary human T cells to integrations in 2 other human-derived cell lines (HEK293 and HeLa) and randomly simulated integrations into the human genome. Our results revealed distinct differences between cell types. PiggyBac had a nonrandom integration profile and a preference for transcriptional units (approximately 50% into RefSeq genes in all cell types), CpG islands (18% in T cells and 8% in other human cells), and transcriptional start sites (<5 kb, 16% to 20% in all cell types). PiggyBac also preferred TTAA but not AT-rich regions of the human genome. We evaluated the expression of mapped genes into which piggyBac integrated, and found selection of more active genes in primary human T cells compared with other human cell types, possibly due to concomitant T-cell activation during transposition. Importantly, we found that in comparison to what has been reported for gammaretroviral and human lenitviral vectors, piggyBac had decreased integration frequency into or within 50 kb of the transcriptional start sites of known proto-oncogenes. Hence the piggyBac nonviral gene delivery system seems to represent a promising gene transfer system for clinical applications using human T lymphocytes.

Optimization of the piggybac transposon system for the sustained genetic modification of human T lymphocytes

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.

An Increase in Circulating Mast Cell Colony-Forming Cells in Asthma

We compared a potential to generate mast cells among various sources of CD34+ peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34+ PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34+ PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34+ PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34+ PB cells between the two groups. In the presence of TPO, CD34+ PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34+ PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.

Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination

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.

Exploiting cytokine secretion to rapidly produce multivirus-specific T cells for adoptive immunotherapy

Viral infections remain a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT), and conventional small-molecule therapeutics often have modest benefit, high cost, and adverse effects. Adoptive transfer of donor-derived virus-specific T cells has proved feasible and safe after HSCT and to reconstitute immunity against cytomegalovirus, Epstein-Barr virus, and adenovirus. Current protocols to generate these cytotoxic T cell lines are lengthy, taking up to 12 weeks. As viral infections often occur <30 days after HSCT, speedy production of virus-specific cytotoxic T cells lacking alloreactivity is highly desirable. We now describe a modified rapid selection method for production and characterization of CD4+ and CD8+ T cells specific for cytomegalovirus, Epstein-Barr virus, and adenovirus in a single infusate. We use Ad5f35-pp65/latent membrane protein 2 vectors in a single procedure over a 48-hour time period and manufacture a product suited for clinical use. By simultaneously expanding a portion of the selected product, we can characterize phenotype and function of the infused product and link them with subsequent in vivo outcome.

Significance of chemokine receptor expression in aggressive NK cell leukemia

Natural killer (NK) cell-type lymphoproliferative diseases of granular lymphocytes can be subdivided into aggressive NK cell leukemia (ANKL) and chronic NK cell lymphocytosis (CNKL). One reason for the poor outcome in ANKL is leukemic infiltration into multiple organs. The mechanisms of cell trafficking associated with the chemokine system have been investigated in NK cells. To clarify the mechanism of systemic migration of leukemic NK cells, we enrolled nine ANKL and six CNKL cases, and analyzed the expression profiles and functions of chemokine receptors by flowcytometry and chemotaxis assay. CXCR1 was detected on NK cells in all groups, and CCR5 was positive in all ANKL cells. Proliferating NK cells were simultaneously positive for CXCR1 and CCR5 in all ANKL patients examined, and NK cells with this phenotype did not expand in CNKL patients or healthy donors. ANKL cells showed enhanced chemotaxis toward the ligands of these receptors. These results indicated that the chemokine system might play an important role in the pathophysiology of ANKL and that chemokine receptor profiling might be a novel tool for discriminating ANKL cells from benign NK cells.

Sustained elevation of serum interleukin-18 and its association with hemophagocytic lymphohistiocytosis in XIAP deficiency

X-linked lymphoproliferative syndrome (XLP) is a rare primary immunodeficiency characterized by increased vulnerability to Epstein-Barr virus infection. XLP type 1 is caused by mutations in SH2D1A, whereas X-linked inhibitor of apoptosis (XIAP) encoded by XIAP/BIRC4 is mutated in XLP type 2. In XIAP deficiency, hemophagocytic lymphohistiocytosis (HLH) occurs more frequently and recurrence is common. However, the underlying mechanisms remain mostly unknown. We describe the characteristics of the cytokine profiles of serum samples from 10 XIAP-deficient patients. The concentration of interleukin (IL)-18 was strikingly elevated in the patients presented with HLH, and remained high after the recovery from HLH although levels of other pro-inflammatory cytokines approached the normal range. Longitudinal examination of two patients demonstrated marked exacerbation of IL-18 levels during every occasion of HLH. These findings may suggest the association between HLH susceptibility and high serum IL-18 levels in XIAP deficiency.

STI571 inhibits growth and adhesion of human mast cells in culture

Stem cell factor (SCF)/c‐kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c‐kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10−6 M or higher almost completely abolished the SCF‐dependent progeny generation from cord blood‐derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c‐kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two‐hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.