Rapid and efficient nonviral gene delivery of CD154 to primary chronic ...

Cancer Gene Therapy (2006) 13, 215–224 All rights reserved 0929-1903/06 $30.00

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ORIGINAL ARTICLE

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells LH Li1, E Biagi2, C Allen1, R Shivakumar1, JM Weiss1, S Feller1, E Yvon2, JC Fratantoni1 and LN Liu1 1

MaxCyte, Inc., Gaithersburg, Maryland, USA and 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporationbased gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64715%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in longterm cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system. Cancer Gene Therapy (2006) 13, 215–224. doi:10.1038/sj.cgt.7700883; published online 5 August 2005 Keywords: B-CLL cells; electroporation; CD154; tumor vaccine

Introduction

One of the major goals of cancer immunotherapy is to increase the immunogenicity of tumor cells. Patients with chronic lymphocytic leukemia (CLL) have circulating malignant B cells in the peripheral blood (B-CLL cells).1–3 Despite the strong expression of major histocompatibility complex I (MHC I) and class II (MHC II) molecules, the neoplastic B-CLL cells generally lack the surface expression of costimulatory molecules; thus, they are generally ineffective stimulator cells in mixed lymphocyte reactions.3–9 CD154 (CD40L) belongs to the tumor necrosis factor superfamily10,11 and is normally expressed transiently by activated CD4 T-helper lymphocytes.12,13 It interacts with the receptor CD40 usually expressed by B cells, dendritic cells (DC) and other antigenpresenting cells (APC) to proliferate, differentiate, upregulate costimulatory molecules, such as CD80 and CD86, and adhesion molecules (e.g. ICAM-I) to Correspondence: Dr LN Liu, MaxCyte, Inc., 22 Firstfield Rd., Suite 250, Gaithersburg, MD 20878, USA. E-mail: [email protected] Received 25 March 2005; revised 26 May 2005; accepted 27 May 2005; published online 5 August 2005

increase antigen presentation.4,13–15 CD154/CD40 interaction plays an essential role in the regulation of both humoral16 and cellular immune responses.17,18 Patients with B-CLL have a similar immunodeficiency as those observed in patients with inherited CD154 deficiency that their CD4 T cells fail to express CD154 upon ligation of CD3.19,20 To help overcome the immunological defect found in B-CLL cells, Kipps’s group used an adenovirus-based vector to forcedly express CD154 ectopically in B-CLL cells and demonstrated that this induces an autologous immune recognition of the B-CLL cells in vitro and in patients.3,5,19,21,22 However, transduction by recombinant adenovirus requires an extremely high MOI (multiplicity of infection, virus particles per cell) because B-CLL cells and many other cancer cells lack the essential Coxsackie and Adenovirus Receptor (CAR).21 Previous phase I clinical study results showed that 2000 adenovirus particles were needed for transduction of one B-CLL cell.22 Furthermore, the mouse CD40L (mCD40L) gene was used for the trial because the adenovector encoding human CD154 did not transduce patient cells.22 To increase B-CLL cell susceptibility by adenovector, Takahashi et al.7 showed some improvement by stimulating the B-CLL cells via MRC-5 feeder cells prior to recombinant adenovirus transduction. Biagi et al.6 further modified

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the procedure and demonstrated that CD154 could translocate from adenoviral vector-transduced MRC-5 cells to B-CLL cells, by a mechanism of bystander intercellular transfer. Several other viral vectors have been reported to transduce B-CLL cells in preclinical studies. Vectors based on recombinant adeno-associated virus (rAAV) mediated sufficient transgene expression in primary B-CLL cells;23–25 however, a feeder layer expressing mCD40L (HeLa/SF) was needed. Thus, it was not clear whether the detected CD40L was due to the direct transgene expression in the B-CLL cells or translocation from the HeLa-CD40L feeder cells. In addition to the complicated procedures that involve coculturing and removal of the feeder cells, the costs associated with banking of the master CD40L-expressing cell line and the production of the viral vector present great challenges for the treatment of large numbers of patients. While there are recent reports of helper virus-free Epstein–Barr virus (EBV)-based vector25,26 and Herpes simplex virus (HSV)based vectors25,27 (especially HSV amplicons) for B-CLL transduction, the safety and efficacy of these vectors require further exploration. Electroporation has been established as an efficient method of loading a wide range of cell types with a variety of biomolecules, including genetic constructs.28–31 Numerous studies have shown that electroporation is capable of mediating transgene expression and cellular uptake of various molecules in vitro,32,33 ex vivo34,35 and in vivo.36–38 Since it is a physical approach, electroporation-mediated ex vivo delivery of plasmid DNA avoids limitations related to viral vectors, such as a requirement for specific cell surface receptors. Nonviral gene therapy strategies also may be safer than viral strategies. Conventional, bench-top electroporation systems are already widely used in biomedical research laboratories. However, for clinical applications, the limited processing volume and open cuvettes used in these systems are not optimal. We have developed an electroporation-based nonviral, gene delivery system (MaxCyte GT) that can be scaled up to a wide range of volumes, configured as a sterile and closed system, and designed for clinical gene/cell therapy.35,39,40 Here, we report efficient transgene delivery to human primary B-CLL cells. Transgene CD154 expression could be detected as early as 3 h post electroporation, and the CD154-transfected B-CLL cells maintained transgene expression after long-term cryopreservation. Furthermore, electroporation-mediated transgene expression was functional in that the forced CD154 expression in B-CLL cells induced the upregulation of costimulatory CD80/CD86 expression, and it restored B-CLL cell immunogenic function. Moreover, the cell processing protocol was scaled up to process clinically relevant volumes (up to 5 108 cells), and its consistency among various CLL patients was demonstrated at the clinical site under the current Good Manufacturing Practice (cGMP) regulations. This novel approach is feasible for utilizing ex vivo genetically modified autologous primary leukemia cells as a cancer vaccine for the treatment of patients.

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Materials and methods

B-CLL cells After informed consent, peripheral blood was collected from B-CLL patients at Washington Cancer Institute (Washington, DC) or at Baylor College of Medicine (Houston, TX). B-CLL cells were isolated by standard Ficoll Paque gradient separation followed by cryopreservation. A small fraction of the cells was saved and characterized by flow cytometry. FACS analysis of CLL patients’ peripheral blood mononuclear cells (PBMC) revealed that 490% of the cell population was CD5, CD19 and CD20 positive. Antibodies and reagents Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Mab) specific for IgG1, CD154 and MHC II (HLA-DR), phycoerythrin (PE)-conjugated Mab specific to hCD54 (ICAM-1), hCD80 (B7-1) and hCD86 (B7-2), Cytochrome-conjugated Mab specific to CD5, CD19 and CD20 were purchased from BD Pharmogen (San Diego, CA). FITC-labeled dextran (500 kDa) was obtained from Sigma (St Louis, MO). FITC-conjugated AVD-FMK was purchased from Promega (Madison, WI). Plasmids Full-length cDNA encoding for CD154 was amplified from a human leukocyte cDNA library (Clontech, San Jose, CA) by PCR amplification using primers engineered with a NheI restriction enzyme digestion site at the 50 end, and a NotI digestion site at the 30 end. After enzyme digestion and gel purification the PCR fragment was subcloned into the same restriction sites on pDsRed-N1 (Clontech, San Jose, CA) backbone replacing the DsRed transgene. The transgene expression is regulated by the CMV promoter. PCR-amplified CD154 was examined by DNA sequencing to confirm nucleotide sequence with the one in public domain (PubMed). The pCD154 plasmid was manufactured by Althea Technologies (San Diego, CA) following the current Good Manufacturing Practice (cGMP) guideline that the entire construct was sequenced three times to exclude the possibility of any mutated nucleotides. The plasmid was first tested at MaxCyte and were used later for patients’ samples at Baylor College of Medicine. Electroporation-based nonviral gene delivery Cryopreserved B-CLL cells were thawed following standard procedure and incubated in 371C prewarmed complete culture medium (RPMI-1640 supplemented with 10% FBS; 2 mM L-glutamine) for 30 min. The B-CLL cells were then washed one time and resuspended at a cell concentration ranging from 1 108 to 5 108 cells/ml in electroporation (EP) buffer (Hyclone). DNA plasmid at a final concentration of 0.44 mg/ml or FITC-dextran at final concentration of 0.5 mg/ml was added to the cells. The cell mixture was then transferred to a clinical-grade (CL-1) processing chamber by a syringe. The B-CLL cells were electroporated by attaching the processing chamber


onto the MaxCyte GT electronic module and initiating the software-controlled cell loading process (details of the process protocol are filed with FDA Master File #BBMF10702). After pulsing, the processing chamber was detached from the processor. The processed B-CLL cells were retrieved by a sterile syringe and then transferred to a clean tube followed by incubation at 371C for 20 min. The transfected B-CLL cells were cultured in complete media at 371C in a CO2 incubator for 3 h prior to cryopreservation.

Flow cytometric analysis Transgene CD154 expression, the presence of other cell surface markers and cell viability were routinely analyzed by flow cytometric analysis. The cells at various time points post transfection were harvested by centrifugation and washed with PBS one time. The cells were then immunostained with specific, fluorescence-conjugated Mab for 20 min at 41C followed by a PBS wash. The labeled cells were examined using a FACSCalibur (BD, San Jose, CA) with proper gating using isotype Mab immunostained cells as a control. Gating was set at p0.5% of the control cells to be fluorescent positive cells. The cell viability was analyzed by incubating the cells with propidium iodide (PI) (2 mg/ml; Roche Diagnostics, Indianapolis, IN) prior to flow cytometric analysis and calculated by PI exclusion. Transactivation assay Primary B-CLL cells were first loaded with FITCDextran (500 kDa) using the same MaxCyte GT protocol that was employed for transfection. After washing with PBS three times, FITC-Dextran incorporated cells were cocultured with an equal amount of autologous CD154transfected B-CLL cells for 24 h. The total cell mixture was then immunostained with PE-conjugated anti-CD86 Mab. FACS analysis was performed to evaluate the expression of CD86 in FITC-dextran incorporated B-CLL cells indicated by FITC and PE double positive cell. Mixed lymphocyte reaction HLA-mismatched lymphocytes were obtained from leukapheresis products by Ficoll Paque gradient isolation and further purified by removal of the attached monocytes in T175 flasks. In a 96-well plate, 4 105 allogeneic lymphocytes were mixed with 2 105 hIL2-transfected B-CLL cells and 4 105 CD154-transfected B-CLL cells or control untransfected B-CLL cells. After 40–48 h coculturing, the culture media were removed and analyzed using a commercially available ELISA kit (R&D Systems) for IFN-g production. Statistical analysis Unpaired Student’s t-test (Microsoft Excel) with two tails was used to determine the significance of results. Data were presented as mean7standard deviation. Statistical significance was determined as Po0.05.

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Results

Efficient delivery of marker gene and CD154 to primary B-CLL cells We used a standard DNA plasmid carrying a full-length cDNA encoding the eGFP marker gene to optimize our transient transfection procedure for B-CLL cells. Numerous experiments were performed to test various cell handling procedures, electroporation parameters, DNA and cell concentration and other factors. The optimized procedure was documented in FDA Master File #BBMF10702 and is described in the Materials and methods. Figure 1a shows transient transfection results using eGFP plasmids. Transgene eGFP expression could be observed within a few hours post transfection. eGFP-transfected B-CLL cells (lower panel) reveal strong eGFP expression, while the cells maintain good cell morphology at 5 h post transfection. When the eGFP-transfected B-CLL cells were analyzed by flow cytometry, 52% of the processed cells expressed the eGFP marker gene. After we optimized transfections using the eGFP marker gene, we next examined transient transfection of B-CLL cells using the same plasmid backbone as the one described above, but carrying the full-length cDNA encoding for human CD154 (hCD40L). Figure 1b shows FACS analysis of the control (left panel) and CD154transfected (right panel) B-CLL cells at 3 h post transfection. CD154 expression was detected among 56% of the transfected cells, which were CD5 and CD19 positive. Consistent nonviral gene delivery to primary B-CLL cells Consistent and efficient gene delivery to primary B-CLL cells is essential for CLL clinical immunotherapy. We intended to develop a rapid process procedure that can be effectively applied to individual patient samples. We followed the above protocol and processed a total of seven CLL patients’ samples. All patients’ cells were cryopreserved after they were isolated from peripheral blood. On the day of processing, the cryopreserved BCLL cells were thawed and cultured according to the procedures described in the Materials and methods. The transfected cells were then cultured at 371C for 3 h allowing transgene expression, and then they were cryopreserved and stored in liquid nitrogen. Figure 2 shows the cell viability (Figure 2a) and CD154 transgene expression (Figure 2b) of these transfected cells. Cells from 3 donors were analyzed within 10 min of thawing at MaxCyte (Gaithersburg, MD), whereas the other four donors’ cells were g-irradiated (30 Gy) immediately after thawing and analyzed at Baylor College of Medicine (Houston, TX). Although viability of the CD154-transfected B-CLL cells and the control, untransfected cells was 68719% and 8376% respectively when analyzed within 10 min after thawing, the viable transfected cell population declined after 24 h in culture at a rate greater than the controls, regardless of whether the cells were girradiated or not (Figure 2a). As shown in Figure 2b, the expression of CD154 among the viable cells was 69716% immediately after thawing and culturing in vitro, and this expression level could be maintained up to 48 h (66720%

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Cell Viability (%)

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100 90 80 70 60 50 40 30 20 10 0 0

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Time Post Thawing (hours) Figure 2 Long-term culture of cryopreserved CD154-transfected B-CLL cells. The CD154-transfected B-CLL cells from seven donors were cryopreserved at 3 h post transfection and stored in liquid nitrogen for various times (up to 1 week). Control cells were B-CLL cells that were mock transfected with no DNA. The cells were either cultured directly or g-irradiated immediately after thawing. The cells were analyzed for cell viability by PI exclusion (a) and CD154 expression (b) either immediately,40 or 24 and 48 h after thawing by flow cytometric analysis.

Figure 1 Efficient transgene expression in primary B-CLL cells via electroporation-based, nonviral gene delivery system. (a) Human primary B-CLL cells were transfected with a standard DNA plasmid carrying full-length cDNA encoding for eGFP. Images were taken 5 h post transfection. Upper panel: Control B-CLL cells; bottom panel: eGFP-transfected B-CLL cells; left panel: bright field; right panel: fluorescence field. (b) Human primary B-CLL cells were transfected with a standard DNA plasmid carrying full-length cDNA encoding CD154. The CD154-transfected B-CLL cells were immunostained with FITC-conjugated Mab against CD154, Cychrome-labeled Mab against CD5 (middle panel) and PE-labeled Mab against CD19 (bottom panel). Left panel: Control B-CLL cells; right panel: CD154transfected B-CLL cells. The upper panel illustrates typical cell scatter gating in R1 of control and DNA plasmid-transfected B-CLL cells at 3 h post transfection.

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at 48 h). Furthermore, we found that the g-irradiation had no effect upon the overall CD154 expression. It was not clear what caused the viability of the CD154transfected B-CLL cells to decline at a higher rate than that of the control cells. The same phenomenon was observed in eGFP-transfected B-CLL cells (data not shown), suggesting that the effect was not induced by the transgene itself. As the transfected cells were frozen at 3 h post transfection, the high cell viability upon thawing (82723% when normalized by the viability of the control, untransfected cells) indicates that electroporation per se causes a low level of physical damage to the cells. The lower cell viability of the long-term cultured (424 h) but not the short-term cultured CD154-transfected cells might be due to the DNA-uptake-mediated apoptosis, as observed in other cell types.41–44 The CD154-transfected B-CLL cells were immunostained with FITC-conjugated VAD-FMK (FITC-VAD-FMK) 48 h after thawing as the upregulation of caspases have been reported to be markers of apoptosis.45,46 VAD-FMK irreversibly binds to activated caspases.47 FACS analysis of the CD154transfected B-CLL cells reveals that there were two cell populations (Figure 3). The cells in the Gate R2, representing approximately 60–70% of the whole cell


Cell Viability (%) CD154 Expression (% among viable cells)

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Figure 3 DNA uptake induces apoptosis in long-term cultured BCLL cells. The CD154-transfected B-CLL cells were collected after 48 h culture in vitro and immunostained with FITC-conjugated AVDFMK and PI prior to FACS analysis. (a) represents the forward/side scattering plot used for cell gating. (b) represents the cells without FITC-AVD-FMK incubation. (c) and (d) are the gated cells, with FITC-AVD-FMK incubation, in regions R1 and R2, respectively. The right shift in fluorescence intensity shown in (d) indicates that cells in gate R2 were apoptotic.

population, were mainly apoptotic and nonviable as they were predominantly FITC-VAD-FMK positive (Figure 3), and they were also PI positive (data not shown). In contrast, the cells in the Gate R1 (higher forward scatter) were FITC-VAD-FMK negative, nonapoptotic and viable (PI negative, data not shown).

Cryopreserved CD154-transfected B-CLL cells are stable Cell stability is essential for a cell-based therapeutic product. We next investigated whether CD154-transfected B-CLL cells were stable after long-term storage in liquid nitrogen. FACS analysis showed no significant changes in cell viability or CD154 expression of the cryopreserved, CD154-transfected cells after 5 months storage in liquid nitrogen (Figure 4). The transfected cells from one of these donors were stored up to 8 months, and there was no significant alteration on the cell viability or CD154 expression level. These results highlight the feasibility of using cryopreserved, transfected patients’ primary cells in the clinic. CD154 upregulate immunoaccessory molecules in transfected B-CLL cells Forced expression of CD154 in B-CLL cells has been shown to induce the upregulation of accessory surface markers such as CD80/CD86. To test whether the CD154 delivered by transient transfection is functional, the CD154-transfected B-CLL cells were cultured in vitro for 48 h post thawing followed by immunostaining with Mab specific for CD154, HLA-DR, CD86, CD80 and CD54. As described in Figure 3, the viable transfected cells gated in R1 were analyzed by FACS for costimulatory protein expression. Figure 5a shows a typical example of forced expression of CD154 (darker lines)

100.0

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Figure 4 Stability of cryopreserved, CD154-transfected B-CLL cells. The CD154-transfected B-CLL cells were cryopreserved at 3 h post transfection and stored in liquid nitrogen up to 5 months (n ¼ 4). One donor’s cells were cryopreserved up to 8 months (n ¼ 1). The cryopreserved cells were thawed at the indicated time points and analyzed for the CD154 expression and cell viability by flow cytometry.

leading to increased expression of HLA-DR, CD86, CD80 and CD54. Figure 5b and c summarizes the upregulation of HLA-DR, CD80, CD86 and CD54 molecules in CD154-transfected cells from three B-CLL donors’ cells. Although the percentage of HLA-DR-, CD86- and CD54-positive cells did not increase significantly after CD154 transfection (Figure 5b), the expression level of HLA-DR, CD86 and CD54 increased dramatically as indicated by the higher mean fluorescence intensity of fluorophore-conjugated Mab (Figure 5c, P-value o0.04, 0.05 and 0.03 as compared to the control cells, respectively). Furthermore, both the percentage of CD80 þ cells and the expression level of CD80 were significantly higher in the CD154-transfected cells (57727%) than in the control cells (4.372.5%, Po0.04). Hence, CD154 was functional, and caused the upregulation of immunoaccessory molecules in the transfected B-CLL cells.

CD154-transfected B-CLL cells induced allogeneic immune responses B-CLL cells are poorly immunogenic and fail to trigger allogeneic T-cell responses.3–9.Forced expression of CD154 in B-CLL cells can rescue their immunogenic function, and hIL-2 synergistically enhances this action.4,5,7,19,21,27 To further demonstrate that the CD154transfected B-CLL cells are functional, the control, mocktransfected and CD154-transfected B-CLL cells were mixed with allogeneic lymphocytes for 48 h together with hIL-2-transfected B-CLL cells (approximately 1 ng/ml hIL-2 expressed). Culture media were then analyzed for IFN-g production as an index of T-cell stimulation. As shown in Figure 6, significantly more IFN-g (Po0.001) was detected in the medium obtained from the wells with

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Figure 5 Upregulation of accessory molecules in CD154-transfected B-CLL cells. Primary B-CLL cells were transfected with CD154 DNA plasmid and immunostained with FITC-conjugated Mab against CD154, HLA-DR, CD86, CD80 and CD54 (ICAM-1) and analyzed by FACS 48 h post transfection. Only the cells gated in R1 as described in Figure 3 were analyzed. (a) The right shift of CD154-transfected B-CLL cells with fluorophore-conjugated specific Mab indicates an upregulation of HLA-DR, CD80, CD86 and CD54 expression. (b) The percentage of positive cells for each surface molecule is graphed. Open bars represent mock-transfected control cells, whereas dark bars represent the CD154transfected B-CLL cells. Results were graphed as the means7standard deviations (n ¼ 3). (c) The mean fluorescence intensities of each accessory molecule are graphed (n ¼ 3).

CD154-transfected cells than from the ones with control B-CLL cells. Neither GFP nor hIL-2-transfected B-CLL cell lymphocytes triggered any significant IFN-g production when mixed with the allogeneic control cells (data not shown). These data demonstrate that the restoration of the allogeneic response of the B-CLL cells was due to the expression of CD154. Consistent with previous reports,7 we found that IFN-g production was enhanced with the presence of either recombinant hIL-2- or hIL-2-transfected cells. Specifically, with one donor’s transfected cells, we found that the kinetics of IFN-g production were significantly reduced (only 63713 pg/ml IFN-g) and delayed when we omitted recombinant hIL-2- or hIL-2transfected cells. Thus, we routinely included the hIL-2transfected B-CLL cells or recombinant hIL-2 in the

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mixed allogeneic lymphocyte reaction with CD154transfected B-CLL cells.

Upregulation of immunoaccessory gene in control cells by CD154-transfected B-CLL cells CD40L expressed by MRC-5 feeder cells can transactivate B-CLL cells and enhance the expression of costimulatory molecules.6 To examine whether transactivity occurs in the CD154-transfected B-CLL cells, control B-CLL cells were first color labeled by electroporating them with FITC-conjugated dextran (500 kDa). Figure 7a reveals that FITC-dextran was incorporated into greater than 95% of the control B-CLL cells (upper panel). The FITClabeled cells were then cocultured with CD154-transfected B-CLL cells from the same donor for 24 h followed by


Relative IFN-γ production (pg/mL)

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Donor Number Figure 6 CD154-transfected B-CLL cells elicited IFN-g secretion in mixed lymphocyte reaction. Cryopreserved CD154-transfected BCLL cells from four donors were thawed following the procedure as described in Materials and methods. In a well on a 96-well plate, 4e5 allogeneic lymphocytes and 2e5 hIL2-transfected B-CLL cells were mixed with either 4e5 CD154-transfected B-CLL cells (black bar) or control B-CLL cells (empty bar) and cocultured for 40–48 h. The conditioned culture media were analyzed for IFN-g production by a commercially available ELISA kit. The standard deviation was given from four repeated experiments with a P-value at Po0.001.

immunostaining with PE-conjugated Mab against CD86. Analysis of the FITC-positive cell population showed that the expression level of CD86 increased significantly on the control cells after coculturing them with the CD154transfected B-CLL cells (Figure 7a, bottom panel). The mean intensity of CD86 expression increased by more than four-fold, and this result was repeated with cells from two different donors (Figure 7b). These data illustrate that CD154-transfected B-CLL cells could upregulate CD86 expression on control cells by a bystander effect.

Discussion

CD154 is known to play a pivotal role in the regulation of both humoral and cellular immune responses by upregulating various costimulatory molecules’ expression in APC. Forced expression of CD154 in various cancer cells has been shown to enhance cancer cell immunogenicity;12 however, direct gene delivery of CD154 to primary cancer cells including B-CLL is difficult. Although the adenovector has been widely used for transgene delivery because it can efficiently transduce a variety of human cells, many cancer cells including B-CLL are refractory to the adenovector transduction due to the lack of the adenoviral receptor – CAR.6,21,22,48 While many groups have modified adenoviral transduction procedures by increasing MOI,21,22 utilizing feeder cells6,7 or using rAAV-based vector,23–25 they are not optimal for B-CLL or many other cancer immunotherapies because of the manufacturing complexities. Establishing and maintaining a master cell bank of CD40L-expressing feeder cells is labor and time consuming, which makes it impractical to use the ex vivo CD154 genetically modified autologous B-CLL cells, or any other tumor cells for immunotherapy

Figure 7 Upregulation of CD86 of control B-CLL cells by CD154transfected B-CLL cells. Efficient loading of fluorescence-labeled macromolecules into B-CLL cells. Primary B-CLL cells were electroporated with 0.5 mg/ml FITC-conjugated dextran (500 kDa MW) and analyzed by FACS 24 h post processing. Greater than 95% of the B-CLL cells incorporated with FITC-dextran (upper panel). (a) typical upregulation of CD86 on FITC-labeled, control B-CLL cells. FITC-labeled, control B-CLL cells were cocultured with an equal amount of either control or CD154-transfected B-CLL cells for 24 h followed by FACS analysis, as described in Materials and methods. FITC-positive cell population was selected for examination. ControlAb and control þ Ab represent the samples that the control cells were either not stained, or stained with PE-CD86, respectively; mixed þ Mab represents the sample where control cells were cocultured with CD154-transfected cells and then stained with PECD86. Right shift of CD86 indicates the upregulation of CD86 (lower panel). (b) The mean fluorescence intensity of CD86 on B-CLL cells from three separate experiments. Two donor samples were studied.

to treat large numbers of patients. Several other viral vectors, including EBV- and Herpes simplex virus (HSV)based vectors (especially HSV amplicons), may hold promise for direct gene delivery to B-CLL cells,25–27 but safety in the clinical setting remains to be established. On the other hand, electroporation, a physical procedure, can rapidly introduce transgenes into both dividing and nondividing cells instantly when the cell/DNA

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mixture is applied to an electric field. It does not require specific cell surface receptors, the major obstacle for many viral-based gene delivery systems. Furthermore, compared to systems employing viral vectors or chemical/ lipid, electroporation-mediated gene transfer is time- and labor-efficient. We demonstrated here, with a standard, simple DNA plasmid, that electroporation-mediated direct transfection of pCMV-CD154 consistently resulted in efficient and rapid expression of CD154 in the primary B-CLL cells. The whole manufacturing procedure from thawing cryopreserved patient cells to freezing the vaccine product takes approximately 5 h. Electroporated CD154-transfected B-CLL cells behaved differently from AdCD154-transduced cells after 48 h in culture.7 As revealed in Figure 2a, the cell viability of the CD154-transfected B-CLL cells declined faster than that of the control cells. Although X70% viable cells were observed both at 3 h post transfection and after cryopreservation upon thawing, only 20% viable cells remained at 48 h post thawing. The lower cell viability after longterm culturing in vitro was due to apoptosis, since cellular caspases were elevated in the 48 h cultured, transfected cells. While further studies are needed to investigate the mechanism, a similar phenotype has been reported in other cell types41–44 as a result of incorporation of doublestranded DNA plasmids. We observed that FITCdextran-electroporated B-CLL cells maintained the same viability as the control, nonmodified cells (Figure 7), as did mRNA- and siRNA-electroporated B-CLL cells (our unpublished data); therefore, electroporation per se does not contribute to the lower cell viability of long-term cultured, transfected cells. Our procedures were developed so that a short period of culturing, for example, 3 h, is sufficient prior to cryopreservation. After sterility and other release tests, the processed B-CLL cells will be g-irradiated, frozen in aliquots and then thawed immediately prior to administration to patients. By following this procedure, in vitro culture-caused apoptosis is limited. It was reported previously that apoptotic bodies were favorable for DC presenting tumor antigens.48–52 Therefore, apoptosis of the CD154-tranfected cells after administration in vivo may even be favorable for immunotherapy. Human clinical studies are underway to determine the effectiveness of CD154-transfected B-CLL cells in producing in vivo immune responses. This cell process protocol was transferred to the Center for Gene and Cell Therapy (CAGT), Baylor College of Medicine, where a clinical, closed cell processing chamber was used. Following cGMP guidelines, to date, six B-CLL patients’ cell samples were processed in a scale ranging from 1 108 to 5 108 cells for each transfection. At 3 h post transfection, prior to cryopreservation, the viability of the processed B-CLL cells was 8274%, and the CD154 expression reached 64715%. By the end of the process, greater than six cancer vaccine doses (2 107 cells/vial) were frozen for each patient. Depending on the starting cell number, the total number of modified cells ranged from 1 108 to 5 108. For some patients, 10 doses were cryopreserved. The whole process from thawing patients’

Cancer Gene Therapy

B-CLL cells to cryopreservation of the vaccines took approximately 5 h, which was significantly shorter than the process using a viral vector (424 h). We showed that forced expression of CD154 mediated by electroporation was functional and was able to upregulate costimulatory molecules and restore B-CLL cell immunogenicity in a mismatched MLR. Furthermore, we demonstrated that the cryopreserved, CD154-transfected primary B-CLL cells maintained transgene expression and high cell viability after 8-month storage in liquid nitrogen, suggesting that the transfected cells are stable and that it is feasible to give patients multiple dosages of their own cancer cell-based vaccines. Longer term studies of the stability of the genetically modified cancer cells based vaccines are ongoing. Using the same technology, we have demonstrated efficient loading of bioactive molecules including cDNA plasmid, in vitro transcribed mRNA, siRNA and proteins into hard-to-transfect human primary cells, such as B cells, T cells, DC, CD34 þ cells, myoblast, fibroblast, endothelial progenitor cells, HUVEC and mesenchymal stem cells.40 Our process does not need extensive washes, which are required for the viral vector procedures to remove viral components prior to administration to patients, suggesting that this rapid nonviral cell loading technology is cost effective and more suitable for ex vivo cell/gene manipulation under the cGMP guidelines. In summary, we have developed a rapid, clinical-grade, electroporation-based nonviral gene delivery system that can efficiently and consistently transfect primary B-CLL cells. The CD154-transfected B-CLL cells can be cryopreserved, and maintain their immunomodulator function upon thawing. Vaccination strategies for patients with BCLL using autologous tumor cells manipulated ex vivo may be simplified by this rapid and efficient nonviral gene delivery system. Acknowledgements

We thank Dr Malcolm K Brenner for comments on the manuscript, and Nicholas Chopas, Sarah Wang, Nat Forgotson and Sergey Dzekunov for instrumentation support.

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