Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Sevenday coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56 + CD3 -NK cells from peripheral blood (range, 5.1-to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3 + lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer. [Cancer Res 2009;69(9):4010-7]

BACKGROUND Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS We performed a dual-center, phase 1–2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four in-fants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, .)

IntroductionHIV-based lentiviral vectors are rapidly becoming the retrovirus vector system of choice for research and clinical gene transfer applications. The enhanced ability of lentiviral vectors to transduce both quiescent stem cells 1 and nondividing terminally differentiated cells 2 has led to the development of a wide range of therapeutic gene delivery vectors, 3 as well as promising research tools such as short hairpin RNA gene knockdown libraries 4 and vectors for induction of pluripotency in terminally differentiated cells. 5 Early gamma-retroviral clinical gene therapy vectors restored immune function in patients with X-linked severe combined immunodeficiency (SCID-X1), but they were subsequently found to cause proliferative disorders via transactivation of protooncogenes. 6,7 Newer lentiviral vector designs may significantly reduce that risk, and they await clinical testing for final validation of their predicted safety. Clinical-scale production of these vectors, however, is problematic, as the generation of stable producer cell lines is made significantly more difficult by their self-inactivating (SIN) long terminal repeats (LTRs). As a result, most clinical-grade production of lentiviral vectors is currently being performed using cumbersome transient transfection processes.Insertional mutagenesis by previous gamma-retroviral gene therapy vectors occurred when strong viral enhancers within the LTR activated genes (eg, LMO2) surrounding the integrated vector. 6,7 SIN vector designs completely eliminate the viral enhancers and promoters in the LTR, and when coupled with appropriate internal promoters having less or no enhancer activity, they have been shown to significantly reduce oncogene activation. [8][9][10] Chromatin insulator sequences have also been inserted into SIN LTRs and appear to protect neighboring genes from residual transactivation from the internal promoters. 11 When inserted into the LMO2 locus in Jurkat cells, lentiviral vector genomes containing an internal EF1␣ promoter flanked by SIN LTRs and chicken HS4 chromatin insulators caused only minimal transactivation of the LMO2 promoter. 12 Clinical-scale production of such safety-modified vectors would be greatly facilitated by stable producer cell lines, which allow convenient generation of standardized, large-volume supernatants for downstream process optimization and preclinical studies. Although there have been numerous reports of lentiviral packaging cell lines, 13-22 all high-titer (Ͼ 10 7 transducing units per milliliter [TU/mL]) stable producer lines described in these publications were created by the traditional method of viral transduction of packaging cell lines using non-SIN vector supernatants, which efficiently creates populations of cells with vector genomes integrated at sites favorable for active transcription, and in multiple copies per cell. SIN vector genomes, by virtue of the inactivating deletion in the LTR, are thus incompatible with this method. "Conditional SIN" vectors, 22 which contain regulatable enhancers ...

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The spectrum of immunogenic epitopes presented by the H2-IA b MHC class II molecule to CD4 ؉ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor V␣ and V␤ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (Ϸ20-80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.T he primary role of any vaccine is to generate sustained immune memory to antigenic epitopes that are expressed on, or by, the pathogen in question. Vaccines designed to prevent the development of virus-induced pathology must promote the clonal expansion of CD4 ϩ T cells specific for those complexes of nonself peptide and self MHC class II glycoprotein that will be encountered again as a consequence of natural virus challenge. Experiments in a variety of mouse model systems have established that the virus-specific CD4 ϩ (T h ) subset functions both to enhance antibody production by cognate interaction with B cells (1, 2) and to promote the development of effector CD8 ϩ T cells (3, 4). Studies of readily eliminated viruses further indicate that the development of long-term memory in both lymphocyte compartments depends substantially on a concurrent T h response (5), and CD8 ϩ T cell-mediated control of persistent infections requires the continued presence of virus-specific CD4 ϩ T cells (6, 7). This CD4 ϩ T h for the CD8 ϩ subset is thought to operate via the intermediary of the activated dendritic cell (8).In some infections, particularly with the large DNA viruses, INF-␥-producing CD4 ϩ T cells are also important effectors of immunity (9-11). An ongoing CD4 ϩ T cell response also is thought to be important for the CD8 ϩ T cell-mediated control of HIV infection (12). Effective priming of the CD4 ϩ T cell response would thus seem to be a priority for any HIV vaccine.What is known about the antigenic epitopes recognized by HIV-specific CD4 ϩ T cells? Previous studies have sought to characterize immunogenic peptides from the HIV envelope (env) protein (13-20) for a variety of mammalian species expressing a spectrum of MHC class II phenotypes. Much of the available information was generated by scoring heterogeneous CD4 ϩ T cell respons...