A Practical Approach for Achieving Clinical Immunotherapy of CLL with hCD40L- and hIL-2-Expressing Autologous Tumor Cells.
Authors: Fratantoni Joseph C. , Li Linhong , Liu Linda N. , Peshwa Madhusudan V. , Brenner Malcolm , Yvon Eric , Carrum George , Biagi Ettore 
Published: October 2019

Abstract
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, Blood96:2917–2924, 2000; Biagi, et al, Blood104: abstract 768, 2004; Bergwelt-Baildon, Ann Oncol15: 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.