Soluble and membrane-bound interleukin (IL)-15 Rα/IL-15 complexes mediate proliferation of high-avidity central memory CD8+ T cells for adoptive immunotherapy of cancer and infections.

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)-15 can sustain memory T cell expansion when presented in complex with IL-15Rα (15Rα/15). We developed a novel in-vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL-15 were obtained when both IL-15Rα and IL-15 genes were co-transduced in NIH 3T3 fibroblast-based artificial antigen-presenting cells expressing human leucocyte antigen (HLA) A:0201, ß2 microglobulin, CD80, CD58 and CD54 [A2-artificial antigen presenting cell (AAPC)] and a murine pro-B cell line (Baf-3) (A2-AAPC15Rα/15 and Baf-315Rα/15 ). Transduction of cells with IL-15 alone resulted in only transient expression of IL-15, with minimal amounts of immunologically detectable IL-15. In comparison, cells transduced with IL-15Rα alone (A2-AAPCRα ) demonstrated stable expression of IL-15Rα; however, when loaded with soluble IL-15 (sIL-15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL-15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high-avidity CMVpp65 epitope-specific T cells [cytomegalovirus-cytotoxic T lymphocytes (CMV-CTLs)] responding to ≤ 10- 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL-15, sIL-2 or sIL-7 CMV-CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL-15, sustained in-vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV-CTLs (TCM ). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell-bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.

Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic.

Xenogeneic murine tyrosinase DNA vaccine for malignant melanoma of the digit of dogs.

BACKGROUND: Malignant melanoma of dogs is a highly aggressive neoplasm and is the 2nd most common digit tumor. Metastatic disease is a common sequela for which few effective treatment options exist. Studies show that xenogeneic tyrosinase DNA vaccination yields immune responses and prolongation of survival in dogs with oral malignant melanoma. OBJECTIVES/HYPOTHESIS: Describe clinical findings and tumor characteristics of a cohort of dogs with digit malignant melanoma, and evaluate the prognostic utility of a proposed staging system. Determine if a novel xenogeneic DNA vaccine is safe and potentially effective for treatment of dogs with digit melanoma. ANIMALS: Fifty-eight dogs with digit malignant melanoma treated at the Animal Medical Center between 2004 and 2007. METHODS: Retrospective, medical records review of dogs with digit melanoma treated with xenogeneic DNA vaccine. RESULTS: Overall median survival time (MST) for dogs treated with loco-regional control and xenogeneic DNA vaccine was 476 days with a 1-year survival rate of 63%. MST for dogs presenting with metastasis was 105 days versus 533 days for dogs presenting without metastasis (P < .0001). Forty-eight percent of the dogs in the latter group were alive at 2 and 3 years. A proposed staging system proved prognostic with stages I-IV dogs surviving >952, >1,093, 321, and 76 days, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The xenogeneic murine tyrosinase DNA vaccine was safe and appears effective when used in conjunction with local and regional disease control. The proposed staging system was prognostic in this study and future studies might benefit from utilizing this staging system.

Quantitative analysis of clinically relevant mutations occurring in lymphoid cells harboring gamma-retrovirus-encoded hsvtk suicide genes.

The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that gamma-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovir-mediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in gamma-retroviral or lentiviral vectors.

Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in cell factories.

The clinical implementation of gene therapy requires large-scale production of viral vector stocks (VS) derived from packaging cell lines. Upon scaling-up, maintenance of high viral titers and filtration of the VS become significantly challenging. Thus, production schemes amenable to straightforward validation must be developed. To this end, we have established a semi-closed process to manufacture batches of 7 l or more of clinical-grade oncoretroviral VS using 10-tray Cell Factories. Using a peristaltic pump, the VS are collected on 3 consecutive days, filtered, pooled and stored frozen. To ensure the absence of viable vector-producing cells (VPCs) from each VS unit-dose, we undertook an orthogonal log-removal validation study to demonstrate the ability of both the filtration system to remove viable cells and the VS freezing process to inactivate them. We demonstrate a total VPC-reduction of 11.6 log, thus insuring the absence of contaminating VPCs in transduced clinical samples. We also show that this production process generates stable VS that can be stored at -80 degrees C for more than 3 years. Importantly, this relatively simple and affordable process can be customized to generating large volume of VS for small animal or non-human primate studies. This methodology is not limited to the generation of cell-free clinical oncoretroviral VS, and can be applied to other types of vectors produced in packaging cell lines, such as lentiviral vectors.

Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center.

INTRODUCTION: Canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Preclinical and clinical studies by our laboratory and others have shown that xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. These studies provided the impetus for development of a xenogeneic DNA vaccine program in CMM. MATERIALS AND METHODS: Cohorts of three dogs each received increasing doses of xenogeneic plasmid DNA encoding either human tyrosinase (huTyr; 100/500/1500 mcg), murine GP75 (muGP75; 100/500/1500 mcg), murine tyrosinase (muTyr; 5 dogs each at 100/500 mcg), muTyr+/-HuGM-CSF (9 dogs at 50 mcg muTyr, 3 dogs each at 100/400/800 mcg HuGM-CSF, or 3 dogs each at 50 mcg muTyr with 100/400/800 mcg HuGM-CSF), or 50 mcg MuTyr intramuscularly biweekly for a total of four vaccinations. RESULTS: The Kaplan-Meier median survival time (KM MST) for all stage II-IV dogs treated with huTyr, muGP75 and muTyr are 389, 153 and 224 days, respectively. Preliminarily, the KM MST for stage II-IV dogs treated with 50 mcg MuTyr, 100/400/800 mcg HuGM-CSF or combination MuTyr/HuGM-CSF are 242, 148 and >402 (median not reached) days, respectively. Thirty-three stage II-III dogs with loco-regionally controlled CMM across the xenogeneic vaccine studies have a KM MST of 569 days. Minimal to mild pain was noted on vaccination and one dog experienced vitiligo. We have recently investigated antibody responses in dogs vaccinated with HuTyr and found 2- to 5-fold increases in circulating antibodies to human tyrosinase. CONCLUSIONS: The results of these trials demonstrate that xenogeneic DNA vaccination in CMM: (1) is safe, (2) leads to the development of anti-tyrosinase antibodies, (3) is potentially therapeutic, and (4) is an attractive candidate for further evaluation in an adjuvant, minimal residual disease Phase II setting for CMM.

[Abnormalities of the basal ganglia and thalami in adults]. / Atteintes bilatérales des noyaux gris chez l'adulte.

Several diseases may cause non-specific MR signal abnormalities of the bilateral basal ganglia and thalami. As such, diagnosis of the underlying etiology may be difficult to achieve at imaging. In this review, we will present interpretative guidelines based on clinical data (mode of presentation, previous history, clinical symptoms, and evolution) and imaging data (type of signal abnormalities, location of lesions, and associated abnormalities). The main categories of diseases causing MR signal abnormalities of the bilateral basal ganglia and thalami in adults are reviewed: toxic, metabolic, vascular, tumoral, infectious and inflammatory diseases.

Issues in the manufacture and transplantation of genetically modified hematopoietic stem cells.

The advent of safe and practical means to correct, enhance or protect blood cells at the genetic level offers tantalizing therapeutic perspectives. At present, gene delivery using a replication-defective retrovirus is the most efficient method to stably transduce hematopoietic cells. The successful adaptation of retroviral infection to hematopoietic stem cells requires optimized transduction conditions that maximize gene transfer while preserving the cells' potential for engraftment and longterm hematopoiesis. The successful establishment of effective transduction protocols hinges on retrovirus biology as well as stem cell and transplantation biology. Interestingly, the genetic approach could permit novel strategies to promote host repopulation by transplanted stem cells. However, regulated and predictable expression of any transgene integrated at random chromosomal locations cannot be taken for granted. Investigation of the control of transgene expression and prevention of vector silencing will become increasingly important.

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes.

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells' progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral-mediated gene transfer efficiency in primary T-lymphocytes, in particular, T-cell activation status, viral receptor expression, and culture conditions.

Activation conditions determine susceptibility of murine primary T-lymphocytes to retroviral infection.

BACKGROUND: Genetically modified T-lymphocytes are potential therapeutic agents for the treatment of various disorders. Successful retroviral infection of primary murine T-lymphocytes is a prerequisite to the study of adoptive cell therapies in a small animal model. The definition of factors controlling retroviral infection of T-lymphocytes would also be useful to better understand retroviral diseases. However, retroviral-mediated gene transfer into murine primary T-cells has remained elusive. METHODS: In order to define the requirements for stable and efficient gene transfer in primary murine T-lymphocytes, we investigated factors capable of affecting retroviral infection. These include activation conditions (using mitogens or monoclonal antibodies), culture conditions (including media composition and cytokine addition), timing of viral exposure, retroviral receptor selection (ecotropic, amphotropic, or vesicular stomatitis virus G (VSV-G) glycoprotein receptor), and viral titer. RESULTS: We show that efficient gene transfer can be achieved in murine T-lymphocytes, provided that a number of favorable conditions are met, in particular optimized T-cell activation conditions, optimal timing of infection, adequate interleukin-2 concentration and T-cell density, and a high viral titer. On a particulate basis, we find that ecotropic particles are more effective than amphotropic or VSV-G-pseudotyped particles, and recommend the use of a specifically selected retroviral packaging cell line. CD4+ T-cells are equally or more infectible than CD8+ lymphocytes, depending on the activation conditions. The Th1 and Th2 subsets are comparably susceptible to retroviral infection, in contrast to what has been reported in some instances of human T-cell infection by human immunodeficiency virus (HIV). CONCLUSIONS: We establish conditions that enable efficient retroviral-mediated gene transfer in murine primary T-lymphocytes. T-lymphocytes are not uniformly susceptible to retroviral infection depending on the mode of T-cell activation. These findings have implications for devising approaches to the study of T-cell biology, adoptive cell therapies, and the pathophysiology of retroviral diseases in mouse models.

Regulation of IL-4 expression by activation of individual alleles.

To study the in vivo role of IL-4-expressing cells, we developed a strategy to tag these cells, by generating mice in which one IL-4 allele was replaced with a cDNA encoding the human CD2 (huCD2) cell-surface molecule. Expression of the huCD2 reporter was, like IL-4, restricted to the appropriately polarized T helper 2 cells. However, most of the cells expressed only the IL-4 or the targeted allele. Analysis of the frequency of monoallelic versus biallelic expression suggests that the activation of each individual allele is regulated by a stochastic process whose probability can be augmented by increasing the strength of signal delivered through the TCR. Allele-specific activation may be a general feature of cytokine regulation that contributes to the functional diversity within T helper cell subpopulations.

Long-term in vivo expression of the MFG-ADA retroviral vector in rhesus monkeys transplanted with transduced bone marrow cells.

We have tested the recombinant human adenosine deaminase (hADA) retroviral vector MFG-ADA for its efficacy in transducing hemopoietic stem cells of nonhuman primates and its expression level in the hematopoietic system. The percentage of provirus-positive granulocytes 1 year after transplantation of bone marrow transduced with MFG-ADA was 0.1%, which was equivalent to previously obtained results with the hADA virus-producing cell line POC-1. However, in MFG-ADA monkeys, significantly more peripheral blood mononuclear cells carried the hADA gene (1% versus 0.1%). Human ADA expression levels in peripheral blood mononuclear cells were different between POC-1 and MFG-ADA monkeys using samples with equal numbers of provirus copies per cell. In contrast, in total red blood cell lysates of MFG-ADA monkeys, the hADA expression was higher (approximately 10-fold) and could be detected longer (20 weeks and up to more than 1 year after bone marrow transplantation in 2 monkeys) than in POC-1 monkeys that were only positive for up to 12 weeks at the most. At 3 years after bone marrow transplantation, the MFG-ADA provirus could still be detected in 0.1% of bone marrow cells and peripheral blood cells and in 1% of cultured T cells. These results show that MFG-ADA virus can give rise to long-term in vivo expression of hADA in the primate hematopoietic system. However, transduction efficiencies remain low.

Methods for the construction of retroviral vectors and the generation of high-titer producers.

Retroviral vectors are powerful tools for gene transfer that are useful in the context of experimental as well as clinical applications. Defective recombinant retroviruses allow for efficient gene transfer into a broad range of mammalian cells derived from different species and different tissues. Safe gene transfer can be achieved using helper-free viral stocks, owing to the development of vectors and packaging cell lines that minimize the probability of hazardous recombination events. Retrovirus-mediated gene transfer leads to stable proviral integration in the target-cell genome, a key feature when the foreign genetic material must be faithfully transmitted to the progeny of the transduced parental cell, murine leukemia virus (MLV)-based vectors accommodate numerous modifications, thus providing a plastic tool that can be tailored for very diverse applications.

gamma-c gene transfer into SCID X1 patients' B-cell lines restores normal high-affinity interleukin-2 receptor expression and function.

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL-7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL-2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells.

A persistent obstacle in the use of vector systems for gene therapy has been the inability to attain high-level expression of the target gene in primary cells in vivo. The MFG retroviral vector was designed to yield improved expression over the widely used N2 or LN vectors; however, the molecular basis for this effect has not been examined. Using the human glucocerebrosidase (GC) enzyme as a reporter, we compared expression from the MFG and N2 vector backbones in transduced murine hematopoietic cells after syngeneic bone marrow transplantation. Reporter enzyme activities in primary spleen colonies of transplanted mice were seven-fold higher per vector copy in cells transduced with the (MFG-based) MGC vector than in cells bearing the (N2-based) G2 vector. In spleen colonies harboring the MGC vector, the ratio of spliced to unspliced vector RNA was increased four-fold relative to the G2 vector transcripts in Northern blot analyses. Further analyses indicated that MGC-transduced cells contained five-fold higher levels of spliced RNA per vector copy. Since translation of spliced RNA species (in which the complex secondary structure of the packaging signal has been excised) is likely to proceed with enhanced efficiency, the augmented levels of spliced RNA produced by MFG may represent the key element of increased protein expression from this vector. These findings suggest that the MFG retroviral vector may provide higher level expression of target genes used in human gene therapy.

Antiviral activity of autocrine interferon-beta requires the presence of a functional interferon type I receptor.

We and others have previously observed that the antiviral effects of autocrine interferon (IFN)-alpha/beta activity cannot be abolished by neutralizing antibodies, even when present to a large excess. This raises the possibility that the major part of autocrine activity is triggered intracellularly, possibly bypassing the transmembrane IFN-alpha/beta receptor. To examine this possibility, cells derived from IFN-alpha/beta Ro/o knockout mice lacking a functional IFN-alpha/beta receptor were stably transformed with pHMB-KbMuIFN beta or pMFG-MuIFN beta plasmids encoding a constitutively expressed murine IFN-beta gene. Four different clones were isolated and examined for resistance to a retrovirus, MFG-LacZ, and to Semliki Forest virus. Despite the production of autocrine IFN-beta at levels inducing high antiviral resistance in control cells, none of the clones displayed antiviral resistance. Thus, despite its failure to be neutralized by potent antiserum, the antiviral activity of autocrine IFN-beta takes place via the transmembrane IFN-alpha/beta receptor, and no additional pathway is involved.

Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.

To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression in vivo. Murine bone marrow cells were transduced by each of the recombinant viruses and subsequently used to reconstitute the hematopoietic system of lethally irradiated recipients. Five to seven months after transplantation, analysis of the peripheral blood of animals transplanted with cells transduced by vectors which employ viral long terminal repeats (LTRs) for gene expression indicated that in 83% (77/93) of these animals, the level of human enzyme was equal to or greater than the level of endogenous murine enzyme. Even in bone marrow transplant recipients reconstituted for over 1 year, significant levels of gene expression were observed for each of the vectors in their bone marrow, spleen, macrophages, and B and T lymphocytes. However, derivatives of the parental MFG-ADA vector which possess either a single base mutation (termed B2 mutation) or myeloproliferative sarcoma virus LTRs rather than the Moloney murine leukemia virus LTRs led to significantly improved gene expression in all lineages. These studies indicate that retroviral vectors which employ viral LTRs for the expression of inserted sequences make it possible to obtain high levels of a desired gene product in most hematopoietic cell lineages for close to the lifetime of bone marrow transplant recipients.

Development of methods for somatic cell gene therapy directed against viral diseases, using retroviral vectors carrying the murine or human interferon-beta coding sequence: establishment of the antiviral state in human cells.

We are developing methods for somatic cell gene therapy directed against chronic and fatal virus infections, such as acquired immunodeficiency (AIDS), by transforming cells with a constitutively expressed interferon (IFN) coding sequence. Previous work from our laboratory has shown that stable antiviral expression (SAVE) can be obtained in murine BALB/c 3T3 cells and human U937 cells transformed with plasmids carrying either the murine or the human IFN-beta coding sequence placed under the expression control of a 0.6-kb Xho II-Nru I promoter region of the murine H-2Kb major histocompatibility complex (MHC) gene (Macé et al., 1991; Seif et al., 1991). In the present paper, we report the construction of murine (Mu) and human (Hu) IFN-beta-expressing retroviral vectors (pMPZen-MuIFN beta, pHMB-KbMuIFN beta) and the problems encountered. Because of the murine origin of commonly used packaging cells and the species specificity of IFN, it was evident that placing the murine IFN-beta sequence under constitutive expression control could result in the production of Mu IFN in the murine packaging system, and thereby lead to decreased vector production and also to enhanced resistance of target cells. Using a packaging cell line that releases a beta-galactosidase-expressing vector, we show that, as expected, Mu IFN-alpha/beta decreases vector production of murine packaging cells and also inhibits the transformation of target NIH-3T3 cells with this vector, but the presence of anti-Mu IFN antibodies rescues the viral titer of the packaging cells and restores the sensitivity of target cells to virus transformation. However, the same antibody treatment is unable to rescue the viral titer of psi-2 packaging cells producing autocrine Mu IFN-beta encoded by the pMPZen-MuIFN beta and pHMB-KbMuIFN beta vectors. Because of the species specificity of IFN, this problem is circumvented with the pMFG-HuIFN beta vector carrying the human IFN-beta sequence. In spite of the production of Hu IFN, murine psi-CRIP packaging cells are able to release retroviral vectors expressing Hu IFN-beta, and these amphotropic vectors can transform human MRC-5 cells and confer to these cells an enhanced resistance to vesicular stomatitis virus (VSV) infection.