B-chronic lymphocytic leukemia (B-CLL) cells transfected with CD40 ligand (CD40L) demonstrate up-regulation of co-stimulatory and adhesion molecules and enhanced antigen-presenting capacity. Preclinical models show that co-expression of IL-2 further potentiates the immune response elicited by human CD40L (hCD40L)-expressing cells. However, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems (

Weirda, et al, Blood 96:2917–2924, 2000
;
Biagi, et al, Blood 104: abstract 768, 2004
;
Bergwelt-Baildon, Ann Oncol 15: 853–857, 2004
). We report here use of a non-viral, clinical-grade, electroporation-based gene delivery system, plus standard cDNA plasmids encoding for hCD40L and human IL-2 (hIL-2), to efficiently express hCD40L and hIL-2 in B-CLL cells. Vaccines for clinical study prepared using B-CLL cells from 10 patients - 3/10 lots manufactured into qualification lots and 7/10 lots were infused into patients. Flow cytometric (FACS) analysis of B-CLL samples showed unmodified B-CLL cells had undetectable (< 1%) expression of hCD40L. Three hours post transfection, mean cell viability was >85% (trypan blue). FACS analysis of hCD40L-transfected cells showed that 54 ± 18% (mean ± SD) of viable cells expressed hCD40L. For cells loaded with hIL-2, mean expression of hIL-2 was 10,100 ± 4100 pg IL-2/106 cells, secreted in culture medium following 18 hrs culture post transfection. Costimulatory molecules (CD80, CD86), MHC-II molecule (HLA-DR) and adhesion molecule (ICAM-I) in hCD40L-transfected B-CLL cells were assayed by FACS during manufacture of 3 qualification lots. Significant increase in level of cell surface expression was detected for all molecules in hCD40L-loaded B-CLL cells. Geometric mean fluorescence intensity (GMFI) for expression of CD80, CD86, HLA-DR and ICAM-1 was 193±77, 1007±586, 2563±862 and 3205±1576, respectively, 18 hrs post transfection, vs 19+1, 54+19, 870+400, and 103+43, respectively, in unmodified B-CLL cells. To demonstrate transfected B-CLL cells are functional, mock transfected and hCD40L-transfected B-CLL cells were mixed with allogeneic lymphocytes for 48 hrs together with hIL-2-transfected B-CLL cells. T cell activation was assessed by measurement of IFN-γ secretion in medium: 400 - 4000 pg/mL in T cell cultures stimulated with hCD40L-transfected B-CLL cells, vs 50 - 250 pg/mL in control (mock transfected) cultures (p < 0.001). The final vaccine product was cryopreserved; stability of hCD40L and hIL-2 was demonstrated after 8 months in storage. Prior to administration, the cryopreserved final product was thawed, washed and irradiated (3000 cG). Patients received subcutaneous injections of vaccine q 1 week X3, then q 2 weeks X3. Patients were grouped into 2 dose cohorts and received 2x107 hIL-2-loaded cells mixed with 2x105 (Low Dose) or 2x107 (High Dose) hCD40L-loaded cells. There were no adverse events from any injection. An anti-B-CLL response was seen in some patients. White blood cell counts and ALC were stable over 24 week follow up and 1 patient demonstrated a transient decrease (>50% surface area) in local adenopathy. This approach has successfully produced a biologically active cellular product and we believe it can be used for development of a practical cell-based therapeutic.

Topics:
adverse event, aldesleukin, antigens, autologous tumor cells, bodily secretions, cd40 ligand, cd80 antigens, cell adhesion molecules, congo, cryopreservation

Author notes

Corresponding author

2005, The American Society of Hematology
2005
Sign in via your Institution