Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Abstract

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

Site‐specific anti‐tumor immunity: Differences in DC function, TGF‐β production and numbers of intratumoral Foxp3+ Treg

“…TGF-b1 and TGF-b2 have pleiotropic functions (for review see [46]) and have been described to be expressed very strongly by glioma cells [5][6][7]. The importance of TGF-b in supporting glioma growth is attested by experiments blocking TGF-b production, which positively influence the survival of rats with established gliomas [47]. TGF-b regulates T-cell proliferation and T helper cell differentiation and it interferes with the generation of cytotoxic T cells [48], an effect which is central in tumor surveillance because CD8 1 T cells are necessary for efficient anti-tumor responses.…”

Medical Applications of Transforming Growth Factor-

“…83 In addition, since many tumors express increased amounts of TGF-β, agents that block TGF-β may be valuable in stimulating an immune response toward metastases. 84 Mechanisms to block TGF-β activity include soluble TβRII fragments, 85 decorin, 86 tranilast, 87 neutralizing antibodies, 88 threonine kinase inhibitors, 89 and RNA expression inhibitors such as anti-sense expression vectors or blocking oligonucleotides. 90 Impressive results have been obtained in animal models of fibrosis using TGF-β antagonists.…”

Phase II trial of Belagenpumatucel-L, a TGF-β2 antisense gene modified allogeneic tumor vaccine in advanced non small cell lung cancer (NSCLC) patients

“…Vaccine production and design of the antisense plasmid have been previously described. 14,17 Briefly, the vaccine consists of cells derived from 4 NSCLC cell lines [1 adenocarcinoma (SK-LU-1), 2 squamous cell carcinomas (NCI-H 520 and Rh 2), and 1 large cell carcinoma (NCI-H 460)] which were transfected via electroporation using a TGF-b2 antisense gene plasmid. Each cell line was required to have 435% knockdown of TGF-b2 following transfection.…”