Previously we reported (

Li LH et al,
2006
,
Cancer Gene Therapy
13
:
215
–224
) rapid and efficient production of human CD40L+ (hCD40L) B-Chronic lymphocytic leukemia (B-CLL) tumor vaccine by electrotransfecting the cells with a DNA plasmid encoding hCD40L. The hCD40L-transfected B-CLL cells cryopreserved at 3 hrs post transfection showed cell viability ≥50%, and CD40L expression level ≥50% (N=10). Although costimulatory molecule upregulation was not detected at 3 hrs, we hypothesized the vaccine would upregulate costimulatory molecules in vivo, emulating levels seen in vitro among viable cells after 12–24h culture. The suboptimal immunologic and clinical results of our previous vaccine preparation reported last year (
Fratantoni JC et al,
2005
,
Blood
106
:
136a
) suggested that costimulatory molecule upregulation in vivo was insufficient. Simply increasing culture time of hCD40L-transfected B-CLL cells is limited by the resulting low cell viability caused by DNA uptake-mediated toxicity. In this study, we report a simple modified vaccine manufacturing method that yields a vaccine with good cell viability and expression of co-stimulatory molecules prior to injection. During vaccine production, a portion of the B-CLL cells were first transfected with pCMV-hCD40L via electroporation (provider cells) and then mixed with non-transfected autologous B-CLL cells (naïve recipients) followed by coculture for 12 to 24hrs. Our data show that hCD40L expression levels on the transfected provider cells and the ratio of provider cells to naïve recipient cells directly correlate with hCD40L molecule expression on the naïve recipient cells. The naïve recipient cells in the mixture maintained high cell viability, 80%–90%, when normalized by the input naïve cell number, while cell viability of the provider cells declined to 19 ± 9% at 1d and further down to 2 ± 1% at 7ds post transfection (n=4). The percentage of cells expressing hCD40L depended on the mixing ratio. When a 10:1 ratio (provider: naïve) was used, the hCD40L expression level in naïve cells was up to 80%. In order to make an hCD40L+ B-CLL vaccine with high cell viability, a 1:1 ratio was applied. The viability of the final tumor vaccine product including both provider and recipient cells was 56 ± 6%, while hCD40L was detected among 34%±12% of the cells at 12–24h post mixing (n=10). Expression of CD80, CD86 and CD54 in the mixed cells were increased by 16 ± 8, 10 ± 5 and 24 ± 17 folds respectively, when compared to those of the naïve B-CLL cells (3 patients). Furthermore, we examined the capacity of the vaccine product to present antigen using an allo MLR, and monitored IFN-g secretion and proliferation of CFSE-labeled allo PBL. Data from 3 CLL patients’ samples showed that vaccine prepared by the mixing process could induce 6.8 ± 0.01, 2.1 ± 0.35 and 2.5 ± 1 fold more allo PBL proliferation and ≥25 folds higher IFN-g production than the control B-CLL cells (p<0.05). In summary, we could produce viable functional hCD40L+ CLL tumor vaccine with upregulated costimulatory molecules using autologous B-CLL cells. The process can be scaled up to produce >2x1010 modified cancer cells. This simple, non-viral vaccine manufacturing process is practical and currently under evaluation in Phase I/II clinical study.

Disclosures: All authors are employees of Maxcyte, Inc; All employees receive stock options in MaxCyte as part of a standard compensation package.

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