Transgenic human CD40 ligand (hCD40L) activates B-Chronic Lymphocytic Leukemia (B-CLL) cells by CD40 stimulation and may thereby enhance their capacity to present tumor antigens. Pre-clinical models show that co-expression of IL-2 further potentiates the immunogenicity of CD40L-expressing tumor cells. To discover whether these promising pre-clinical data could usefully be applied to patients with B-CLL, we used adenovectors to prepare hIL2- and hCD40L-expressing autologous tumor vaccines. Within these vaccines, a mean of 92% B-CLL cells were CD40L positive (versus <1% pre-treatment), and costimulatory molecules were also upregulated on the tumor cells (CD80 from 6% to 74% and CD86 from 16% to 73% positive cells). The mean secretion of IL-2 (measured by ELISA at 72 hours after transduction) was 1,780 pg/ml/10E6 cells (range = 175–400). To date, 8 patients have received between 5 and 8 subcutaneous injections in a modified Phase I design, in which the dose of IL-2 secreting cells was fixed at 2x10E7 per injection, while the dose of CD40L-expressing leukemia cells was escalated from 2x10E5 (level 1) to 2x10E7 (level 3) per injection. 12 additional patients will receive this final dose combination. Of those treated, two patients were in Rai stage 4; 2 in stage 3; 2 in stage 2; and 2 in stage 1. There were no adverse events from any injection in the patients. Injection-site biopsies revealed a modest infiltration of CD3+ cells, and the vaccine also had systemic immunomodulatory effects. Total T-cell numbers were significantly increased in only 2 patients, but an anti-B-CLL immune response was detected in 5/6 individuals currently analyzed. Using ELISPOT assays with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls, we found a rise in IFN-gamma spot-forming T-cells (<1/100,000 pre to >1/1500 post vaccination) and of Granzyme-B expressing T-cells (<1/100,000 pre to >1/2500 post). This reactivity is mediated at least in part by CD8-positive T cells with specificity to survivin, a known B-CLL associated tumor antigen. Although these immunologic effects have as yet been accompanied by only transient disease responses, our continuing accrual of additional patients who will receive immunomodulatory doses of vaccine should indicate whether the approach could contribute to the management of early or advanced B-CLL.

CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory T cells. We expressed both human CD40L and OX40L on B-Chronic Lymphocytic Leukemia (B-CLL) cells, by exploiting the phenomenon of molecular transfer from fibroblasts engineered to over-express both of these TNF-receptor superfamily members. We analyzed the effects of the modified B-CLL cells on the number, phenotype and cytotoxic function of autologous T cells in seven B-CLL patients. Transfer of CD40L and OX40L to B-CLL cells was observed in all patients (mean value from 1% pre to 76% post for CD40L; from 0.7% pre to 88% post for OX40L). Subsequent up-regulation of the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) was obtained after engagement of the endogenous CD40 receptor on B-CLL by the transferred CD40L molecules (mean value from 8% pre to 64% post for CD80; from 36% pre to 95% post for CD86). Co-culture of modified and unmodified B-CLL cells with autologous T cells revealed profound differences in the immune responses they induced. With unmodified B-CLL cells, or cells expressing either CD40L or OX40L individually, less than a 10-fold expansion of autologous T cells was observed, with a <100-fold expansion in tumor reactive T cells (measured by IFN-gamma Elispot with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls). By contrast, co-culture with B-CLL cells expressing both CD40L and OX40L induced a >40 fold expansion of autologous T cells - including both CD8+ T cells and CD4+ T cells with a Th1 pattern of cytokine release - and a >2500-fold increase in leukemia-reactive T cells. These expanded T cells were also directly cytotoxic to B-CLL targets, producing a mean 48% B-CLL killing at an E:T ratio of 10:1. A proportion of these tumor-reactive CD8+ T cells were specific for survivin, a B-CLL associated tumor antigen. Hence the combination of CD40L and OX40L expression by B-CLL cells allows generation of potent immune responses to B-CLL, which may be exploitable either by using active immunization with CD40L/OX40L-modified tumor cells or by adoptive immunotherapy with CD40L/OX40L generated tumor-reactive T cells.