Belagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor p (TGF-P3). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-P8 expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-j3 expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) MATERIALS AND METHODS Construction of Vectors and Genetic Modification of 9LCells. TGF-132 antisense vector. To generate the TGF-,3 antisense vector, a DNA fragment containing bases 1-870 of simian TGF-,32 cDNA (15) was ligated in reverse orientation in the HindIII-XhoI sites of the pCEP-4 vector (Invitrogen) (Fig. 1). Expression of the antisense molecule in pCEP-4 is driven by the cytomegalovirus promoter of the vector. The pCEP-4 vector also contains the hygromycin resistance gene driven by the herpes simplex virus thymidine kinase promoter, the Epstein-Barr virus origin of replication, and the gene for the Epstein-Barr virus nuclear-associated antigen protein 1. Genetic modification of 9L cells with the pCEP-4/TGF-/3 antisense vector or an empty pCEP-4 control vector was performed by electroporation using a BTX (San Diego) electroporator (16). Pools of clones were selected with hygromycin at 200 ,ug/ml (Sigma). TGF-3 secretion was measured by the previously described TF-1 cell bioassay (17). The growth of TF-1 cells (from Mire-Sluis, ref. 17) is inhibited in a dosedependent manner by . To confirm the specificity of TF-1 growth inhibition by TGF-13, the conditioned 9L supernatants were incubated with neutralizing concentrations of turkey anti-TGF-f3 antiserum. Normal (nonimmune) turkey sera was used as a negative control. Standard curves generated with known concentrations of purified TGF-13 (Sigma) served as a positive control and permitted quantification of TGF-3 levels in the test samples. Antisense inhibition of TGF-,3 has been maintained for >1 yr and has been confirmed by the TF-1 bioassay.IL-2 vector. The construction of LNCX/IL-2 retroviral has been described (18). Virus-containing supernatant from PA317/LNCX/IL-2 packaging cell line was used to transduce the 9L and TGF-/32 antisense-modified 9L cell cultures as described (18). The levels of interleukin 2 (IL-2) in tissue culture supernatants of IL-2-transduced cells were measured by previously described ELISA, and the IL-2 biological activity was confirmed as described (18

Although the overall trial did not meet its survival endpoint, improved survival for belagenpumatucel-L is suggested in patients who were randomised within 12weeks of completion of chemotherapy and in those who had received prior radiation. Further studies of belagenpumatucel-L in NSCLC are warranted.