AAV-mediated gene transfer of a checkpoint inhibitor in combination with HER2-targeted CAR-NK cells as experimental therapy for glioblastoma.

Glioblastoma (GB) is the most common primary brain tumor, which is characterized by low immunogenicity of tumor cells and prevalent immunosuppression in the tumor microenvironment (TME). Targeted local combination immunotherapy is a promising strategy to overcome these obstacles. Here, we evaluated tumor-cell specific delivery of an anti-PD-1 immunoadhesin (aPD-1) via a targeted adeno-associated viral vector (AAV) as well as HER2-specific NK-92/5.28.z (anti-HER2.CAR/NK-92) cells as components for a combination immunotherapy. In co-culture experiments, target-activated anti-HER2.CAR/NK-92 cells modified surrounding tumor cells and bystander immune cells by triggering the release of inflammatory cytokines and upregulation of PD-L1. Tumor cell-specific delivery of aPD-1 was achieved by displaying a HER2-specific designed ankyrin repeat protein (DARPin) on the AAV surface. HER2-AAV mediated gene transfer into GB cells correlated with HER2 expression levels, without inducing anti-viral responses in transduced cells. Furthermore, AAV-transduction did not interfere with anti-HER2.CAR/NK-92 cell-mediated tumor cell lysis. After selective transduction of HER2+ cells, aPD-1 expression was detected at the mRNA and protein level. The aPD-1 immunoadhesin was secreted in a time-dependent manner, bound its target on PD-1-expressing cells and was able to re-activate T cells by efficiently disrupting the PD-1/PD-L1 axis. Moreover, high intratumoral and low systemic aPD-1 concentrations were achieved following local injection of HER2-AAV into orthotopic tumor grafts in vivo. aPD-1 was selectively produced in tumor tissue and could be detected up to 10 days after a single HER2-AAV injection. In subcutaneous GL261-HER2 and Tu2449-HER2 immunocompetent mouse models, administration of the combination therapy significantly prolonged survival, including complete tumor control in several animals in the GL261-HER2 model. In summary, local therapy with aPD-1 encoding HER2-AAVs in combination with anti-HER2.CAR/NK-92 cells may be a promising novel strategy for GB immunotherapy with the potential to enhance efficacy and reduce systemic side effects of immune-checkpoint inhibitors.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

Donor vigilance data of a blood transfusion service: A multicenter analysis.

BACKGROUND: Donor vigilance is an important part of the quality management system of blood transfusion services. The evaluation of donor side effects helps to improve the donation process and donor compliance. The aim of the present study was to evaluate donor vigilance data in whole blood and plasmapheresis donors of a blood donor service. MATERIALS AND METHODS: Donors fulfilling current national and European eligibility criteria underwent whole blood and plasmapheresis donation (PCS and MCS+ (Haemonetics, Braintree, USA), A 200 (Fenwal, Round Lake, USA). Whole blood was collected at fixed and mobile sites while plasmaphereses were performed at 8 plasma centers. From 2011 to 2013 donor information was provided for gender, age, body weight, height, first and repeat donation. Donors were monitored for venipuncture and circulatory associated side effects. RESULTS: The total incidences of adverse events were 5004 (0.56%) in repeat donors and 2111 (2.78%) in first time donors for whole blood donation and 3323 (1.01%) and 514 (7.96%) for plasmaphereses, respectively. Circulatory associated events were 2679 (0.30%) for whole blood donation and 1624 (0.49%) for plasmaphereses. CONCLUSION: Our donor vigilance data of a blood transfusion service show that whole blood and plasmapheresis are safe with low incidences of adverse events. Repeat donation and age are predictors for low rates of adverse events. On the other hand, first time donation and female gender were associated with higher incidences of adverse events.

Validation of cord blood split products prepared by an automated method.

OBJECTIVES: In this study, we studied whether the contents of the two compartments of automatically processed cord blood (CB) units are comparable with respect to cell counts and viability and therefore suitable for clinical therapy. BACKGROUND: CB-derived stem cells are increasingly used for allogeneic transplantation. Many centres prepare the transplants by automated methods allowing to split the product into two portions. METHODS: CB was collected at different sites in Germany and transported to a single centre for processing. Before and after cryopreservation laboratory analyses were performed to compare the quality of the two CB segments. RESULTS: In total, 33 products were processed [mean collection volume: 18·6 ± 1·2 mL (range 15·2-20·2 mL) segment A; mean: 4·7 ± 0·3 mL (range 4·2-5·2 mL) segment B]. CD34+ cell counts, viability of CD34+ cells and many other haematological parameters showed a good comparibility between the two segments. However, lymphocyte counts and results of clonogenic assays were significantly different between the two segments of the split product. CONCLUSION: We conclude that the preparation of the cord blood unit by the automated process results in a homogenous distribution of stem and progenitor cells. However, our findings show that the clonogenic capacity differs between the two segments.

Safety and efficacy of healthy volunteer stem cell mobilization with filgrastim G-CSF and mobilized stem cell apheresis: results of a prospective longitudinal 5-year follow-up study.

BACKGROUND AND OBJECTIVES: G-CSF-mobilized peripheral blood stem cells have long replaced marrow as the major source for allogeneic transplants. Conclusive evidence questioning the long-term safety of G-CSF for donors has not been provided, but the cumulative number of followed donors remains insufficient to rule out rare adverse events. A long-term active follow-up study of G-CSF-mobilized healthy volunteer donors was therefore performed. PATIENTS AND METHODS: Two hundred and three successive donors were evaluated pre-apheresis, subjected to G-CSF-mobilization/apheresis, and actively followed for 5 years by the same physicians and laboratories. Follow-up laboratory work included standard biochemical/haematological tests and T-cell phenotyping. RESULTS: Donor epidemiology was typical for reported stem cell donor cohorts. Acute adverse effects of G-CSF and apheresis were mild and transient, consistent with the previous reports. Mean circulating CD34(+) cells after nine doses of G-CSF were 124 per µl. Other biochemical/haematological parameters were also altered, consistent with G-CSF treatment. Spleen enlargement was modest. At first follow-up, all clinical and laboratory parameters had normalized. Leucocyte/lymphocyte counts and CD4/CD8 ratios were the same as during premobilization work-up and remained unchanged throughout. A single severe but likely unrelated adverse event, a case of papillary thyroid carcinoma, was reported. CONCLUSION: The studies add an observation time of almost 500 donor years to the growing body of evidence of the long-term safety of G-CSF for allogeneic donor stem cell mobilization.

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers.

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40 and +100 after transplantation. Median doses (and ranges) of infused NK- and T-cells per product were 1.21 (0.3-3.8) × 10(7)/kg and 0.03 (0.004-0.72) × 10(5)/kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHDgrade II, all receiving a total of 0.5 × 10(5) T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.

Oxygen measurements in platelet fluids - a new non-invasive method to detect bacterial contaminations in platelets.

BACKGROUND: The residual risk for bacterial contamination in blood components especially in platelets is one to two orders of magnitude higher than for transfusion relevant viral infections. The majority of all bacterial transmitted fatalities occurred at the end of platelet shelf life. Therefore, the maximum shelf life of platelet concentrates (PC) was reduced to 4 days after blood donation in Germany in 2008. METHODS: A new continuous non-invasive bacterial detection method was developed by O(2) measurements in the platelet fluids and tested with 10 transfusion relevant bacteria species. RESULTS: The bacterial concentration at the time point of a positive signal of PreSense O(2) ranged between 10(2) and 10(5) CFU mL(-1) . Harmful transfusion-transmitted bacterial infection would have probably been prevented by this novel technology. Only strict anaerobic bacteria strains like Clostridium perfringens were not detected within the study period of 72 h. CONCLUSIONS: The described non-invasive bacterial detection method represents a new approach to prevent transmission of bacterial infection in platelets. The method is characterised by the advantage that all investigations can be performed until right up to the time of transfusion, and therefore, reduce the risk for sample errors to a minimum.

Association of KIR2DL2 polymorphism rs2756923 with type 1 diabetes and preliminary evidence for lack of inhibition through HLA-C1 ligand binding.

Killer cell immunoglobulin-like receptors (KIRs) on chromosome 19q13.4 regulate the function of not only human natural killer (NK) cells but also T cells. An increase in activating KIR- human leucocyte antigen ligand pairs has been associated with an additional risk to develop type 1 diabetes (T1D). T1D families [n = 184 (552 individuals); n = 176 (528 subjects)], unrelated T1D patients (n = 380; n = 394) and healthy controls (n = 315; n = 401) from Germany and Belgium, respectively, were genotyped for the rs2756923 polymorphism within the KIR gene cluster haplotype B in exon 8 of the KIR2DL2 gene. We observed in both Germans and Belgians an overtransmission of the allele 'G' of the KIR2DL2-rs2756923 polymorphism (64.2% vs 35.8%, P = 3 x 10(-4) and 60.0% vs 40.0%, P = 0.02, respectively). In addition, this allele was more frequent in German patients than in healthy controls (78.4% vs 21.6%, P = 1 x 10(-3)). Preliminary results from a cytotoxicity assay suggest that inhibition of NK-cell cytotoxicity may be impaired in individuals carrying the rs2756923 G allele. These data suggest a potential role of the KIR2DL2-rs2756923 polymorphism in T1D in Germans and Belgians.

WT1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation.

The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.

CD8+ T-cell responses to tumor-associated antigens correlate with superior relapse-free survival after allo-SCT.

The GVL effect following allo-SCT is one of the most prominent examples showing the ability of the immune system to eliminate malignant hematological diseases. Tumor-associated Ags (TAA), for instance WT1 and proteinase-3, have been proposed as targets for T cells to establish a GVL effect. Here, we examined an additional TAA (MUC1) as a possible T-cell target of GVL-related immune responses. We have defined new peptide epitopes from the MUC1 Ag to broaden patients' screening and to expand the repertoire of immunologic monitoring as well as for therapeutic approaches in the future. Twenty-eight patients after allo-SCT have been screened for T-cell responses toward TAA (proteinase-3, WT1, MUC1). We could detect a significant relationship between relapse and the absence of a TAA-specific T-cell response, whereby only 2/13 (15%) patients with TAA-specific CTL relapsed, in contrast to 9/15 (60%) patients without TAA-specific CTL responses (P<0.05). In conclusion, CD8(+) T-cell responses directed to TAA might contribute to the GVL effect. These observations highlight both the importance and the potential of immunotherapeutic approaches after allo-SCT.

Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy.

Invasive aspergillosis is a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT. There is a growing body of evidence that T cells are important in the host defense against Aspergillus, and adoptively transferred anti-Aspergillus T-helper 1 (T(H)) 1 cells might reduce infectious mortality in hematopoietic transplant recipients. Here we present for the first time a simple and rapid method for the clinical-scale generation of functionally active anti-Aspergillus T cells according to good manufacturing practice conditions. A total of 1.1 x 10(9) WBCs derived from a leukapheresis product were incubated with Aspergillus antigens. Stimulated cells were selected by means of the IFN-gamma secretion assay and expanded. In three independent experiments, a median number of 2 x 10(7) CD3+CD4+cells (range, 0.9-3.2 x 10(7)) were obtained within 13 days. The cultured CD3+CD4+ cells exhibited almost exclusively a memory activated T-helper cell phenotype. Upon restimulation, the generated T cells produced IFN-gamma, but no IL-4 or IL-10, indicating a T(H)1-cell population. Additionally, the cells proliferated upon restimulation and showed reduced alloreactivity compared to unselected CD4+ cells. This method of generating is suitable for future prospective trials designed to evaluate the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis.

[Non-viral gene transfer results in therapeutic factor IX levels in haemophilia B mice]. / Nicht-viraler Gentransfer führt zu therapeutischen Faktor-IX-Spiegeln im Hämophilie-B-Mausmodell.

UNLABELLED: Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. METHODS: First, we constructed a strong liver-specific expression plasmid for human factor IX (FIX). Next, we tested the vector by injecting two doses under hydrodynamic conditions into the tail veins of FIX knockout mice. RESULTS: A single injection resulted in an increase in FIX expression over 100% of normal plasma levels. The FIX resulted fully functional. Further, no anti-FIX antibodies were observed and expression levels were vector dose dependent. CONCLUSION: The high expression obtained in small animals give hope for further development of non-viral gene transfer for the treatment of hemophilia B in humans.

Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter.

BACKGROUND: Lentiviral vectors have the capacity to transduce stably non-dividing, differentiated and undifferentiated cells of various tissues, including liver. To obtain high-level expression of transgenes, vectors often rely on viral promoters. However, recent data suggest that the supraphysiologic expression from ubiquitous viral promoters may not be beneficial and harbor the risk of oncogene activation. Therefore this study explored the lentiviral-mediated expression of human coagulation factor VIII (FVIII) driven by the physiologic FVIII gene promoter (FVIII-p), the liver-specific human alpha-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1alpha promoter (EF1alpha-p) and the viral CMV promoter. METHODS: Hepatic and non-hepatic cell lines were stably transduced with lentiviral vectors encoding FVIIIdelB and EGFP. To compare the different promoters, lentiviral vectors were cloned to drive FVIII expression from FVIII-p, EF1alpha-p, hAAT-p and CMV-p. RESULTS: As expected, the strong viral CMV-p and the ubiquitous EF1alpha-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/10(6) cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-alpha promoter 0.30 IU/mL/10(6) cells for 293T, 0.04 for HepG2, 2.75 for SK-Hep, 0.46 for Hepa1-6). While the hAAT-p resulted in low FVIII levels (0.10 IU/mL/10(6)cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/10(6)cells in Hepa1-6 and 0.44 in SK-Hep). DISCUSSION: These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.

Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.

BACKGROUND: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). METHODS: Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. RESULTS: Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. CONCLUSIONS: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .

Generation and characterization of human hematopoietic cell lines expressing factor VIII.

Considering the plasticity of hematopoietic stem cells (HSC), they would be ideal targets for gene therapy of hemophilia A by virtue of their progeny providing immediate access to the bloodstream. However, several attempts to show expression of recombinant factor VIII (rFVIII) by primary hematopoietic cells and cell lines have failed; this failure was attributed to the inability of HSC to secrete rFVIII. Here we describe the generation of stable, FVIII-secreting hematopoietic cell lines representing different blood-cell types using a bicistronic lentiviral vector encoding for a B-domain-deleted FVIII (FVIII Delta B) and enhanced green fluorescence protein (EGFP). Transduced cell lines with erythroid and/or megakaryocytic background, (K562-F8 and TF-1-F8) secrete high levels of FVIII in the order of 76.4 and 41.6 ng FVIII:C/ml, whereas moderate and low levels are observed in B lymphoblastoid Raji-F8 cells and the T leukemia line Jurkat-F8 which secrete 6.73 and 1.83 ng FVIII:C/ml, respectively. The capacity to secrete rFVIII appeared to depend on factors related to the cell lineage rather than on the transduction efficacy. Stimulation of transduced cells with the protein kinase C (PKC)-activator phorbol myristate acetate (PMA) resulted in a marked augmentation of rFVIII secretion and enhanced green fluorescent protein (EGFP). Incubation with 0.1 and 1 ng/ml PMA resulted in up to 2.7-fold (K562-F8, Raji-F8) and 1.8-fold (293T-F8) increased rFVIII secretion. The established cell lines should be helpful in further elucidating mechanisms that are able to improve FVIII secretion in hematopoietic cells on a post-translational level and suggest reanalysis of hematopoietic cells as target for gene therapy of hemophilia.

Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92.

For years activated natural killer (A-NK) cells have been explored with respect to their efficacy in anticancer therapy, but, except for some anectdotal reports, no clear clinical benefit has been shown. However, as the understanding about the interactions of NK cells and tumor cells advances, the use of A-NK cells might be revisited with more sophisticated approaches that pay tribute to mechanisms which allow tumor cells to escape immune surveillance. Here the highly cytotoxic NK cell line NK-92 seems to be an attractive alternative for use in adoptive immunotherapy, because it was shown to exhibit substantial antitumor activity against a wide range of malignancies in vitro as well as in xenografted SCID mice. NK-92 cells are characterized by an almost complete lack of killer cell immunglobulin-like receptors (KIRs) yet conserved ability to perforin and granzyme B-mediated cytolytic activity, which make them unique among the few established NK and T cell-like cell lines. NK-92 is the only natural killer cell line that has entered clinical trials. Here we discuss the current status of development of this cell line for adoptive immunotherapy (AIT) of malignancies and review our first clinical experience in patients with advanced cancer who have received repeated transfusions of irradiated NK-92 in a phase I/II trial. Also we discuss issues that address safety aspects of immunotherapy with clonal cell lines and describe further manipulations, which hold the potential of significantly improving the clinical outcome of AIT with NK-92.

Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy.

NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clinical use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by associated toxicity. Therefore, we describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92 CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that we have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clinical applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.

Sensitive determination of the RhD genotype in mixed samples using fluorescence-based polymerase chain reaction.

OBJECTIVES: Prenatal determination of the fetal RhD status in pregnancies of Rh-negative (Rh-neg) women by polymerase chain reaction (PCR) on DNA has become of increasing importance. Since determination of the RhD genotype in these cases is usually performed in samples containing both maternal Rh-neg and fetal Rh-neg or Rh-positive (Rh-pos) DNA, the sensitivity and specificity of PCR-based DNA detection are of crucial importance in the diagnosis of the fetal RhD status. METHODS: We developed a method for RhD typing using the PCR and a fluorescence-based detection method that allows us to determine RhD-pos DNA even if it is mixed with large amounts of Rh-neg DNA. RESULTS: Using this approach, Rh-pos DNA could be detected in dilutions with Rh-neg DNA of as high as 1 in 10,000 (Rh-pos/Rh-neg). Furthermore PCR amplification could also be carried out on DNA samples from persons with weak (Dweak) or partial (Dcat) RhD antigens. CONCLUSION: This method will be valuable in prenatal RhD typing after amniocentesis or after the isolation of fetal cells from maternal peripheral blood.