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.

We performed a phase I clinical trial in grade IV astrocytoma to assess the safety of a whole-cell vaccine comprising autologous tumor cells genetically modified by a transforming growth factor-b2 (TGF-b2) antisense vector. Blocking secretion of the immunosuppressive molecule TGF-b in this manner should inhibit one of the major mechanisms by which tumor cells evade immune surveillance and should lead to clinically effective antitumor immunity. Six patients with progressive WHO grade IV astrocytoma were enrolled in the trial. Patients received 2-7 subcutaneous injections of 5 Â 10 6 -2 Â 10 7 autologous tumor cells per injection. TGF-b2 secretion by the tumor cells used to vaccinate patients was inhibited by 53-98%. Treatment was well tolerated with only low-grade, transient treatment-related toxicities reported. Two patients had partial regressions and two had stable disease following therapy. The overall median survival was 68 weeks. Median survival of the responding patients was 78 weeks, compared to a historic value of 47 weeks for glioma patients treated conventionally. There were indications of humoral and cellular immunity induced by the vaccine. These findings support further clinical evaluation of vaccines comprised of TGF-b antisense-modified tumor cells.

In a previous dose escalation trial we demonstrated dose related survival correlation to Belagenpumatucel-L. In order to further evaluate safety and response at the previously defined optimal dose and schedule and to gain preliminary evidence on a hypothesis that the level of circulating tumor cells (CTCs) in blood may correlate with the overall survival of patients with stage IV NSCLC, we initiated a phase II trial. Patients received intradermal immunization of 2.5 Â 10 7 transfected allogeneic tumor cells (Belagenpumatucel-L, supplied by NovaRx) 1 Â every month for a total of 16 months. Circulating tumor cells (Veridex, Raritan, NJ) were measured every 4 weeks. Twenty-one advanced NSCLC patients were enrolled on this study. No significant toxic effect was observed. Overall survival was 562 days. The median survival was 660 days in patients having less than 2 CTCs at baseline compared to 150 days in patients with 2 or more CTCs (P ¼ 0.025). Phase II results of safety and response are consistent with prior experience following treatment with Belagenpumatucel-L and there is a suggestion that the number of circulating tumor cells at baseline appears to correlate with overall survival. A larger clinical trial is warranted to further explore this observation.

The findings accompanying the administration of 50 intravenous courses of monoclonal antibody to human T-cell (T101) in eight patients, four with chronic lymphocytic leukemia and four with cutaneous T-cell lymphoma are reported. Infusion rates of 0.7 to 1 mg/min were associated with unacceptable toxicity in the presence of circulating target cells, but slower rates were well-tolerated. Immunofluorescence techniques confirmed that circulating cells did bind the antibody in vivo and were subsequently removed from the circulation. Modulation of the antigen on target cells in the bone marrow and skin has important implications for the schedule of administration of such antibodies, and points out the possible limitation of effector cell-mediated cytotoxicity at the tissue level. Production of anti-mouse antibodies resulted in neutralization of therapy in two patients with cutaneous T-cell lymphoma, and suggests that whether such an anti-mouse response is produced may be secondary to the underlying immune status of the patient or the amount of mouse protein to which immunocompetent cells are exposed. The relative specificity and efficacy of monoclonal antibody therapy is encouraging, but the limited clinical benefit and problems of modulation and anti-mouse antibody production underscore the need for continued research into passive therapy and suggest that cytotoxic conjugates may be of more clinical value.

We have investigated in vitro the magnitude, nature, and regulation of spontaneous and mitogen-induced Ig secretion by splenic lymphocytes from several autoimmune murine strains (NZB, NZB X W, MRL/l BXSB) and appropriate, normal mice. All autoimmune strains had increased numbers of mature splenic B lymphocytes, which secreted and/or contained Ig, compared to age-matched normal strains. In NZB and NZB X W mice, the high frequency of mature B cells was apparent early in life, whereas in MRL/l and BXSB mice it was first noted shortly before the clinical onset of disease. Spleen cells from young autoimmune mice of all four strains secreted predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM secretors throughout the animals' lives. Approximately 15% of the total Ig-secreting cells in older NZB, NZB X W, and MRL mice were committed to secretion of anti-ssDNA antibodies. In both autoimmune and normal spleen cells, the B-cell population alone contained fewer secreting cells than the total lymphocyte population, indicating that T cells were required to achieve maximal levels of plaque-forming cells. Spleen cells of NZB and NZB X W mice had a greater response to lipopolysaccharide (LPS) than other autoimmune and normal strains. Responsiveness to LPS, as measured by the frequency of induced Ig-secreting cells, was considerably diminished with age and onset of disease in all autoimmune but not in normal strains. LPS-induced Ig secretion by B cells of autoimmune and normal mice was subject to regulation by splenic T cells. No significant differences were observed between concanavalin-A (Con A) stimulated spleen cells from young and older autoimmune mice and normal control strains in effectively suppressing spontaneous and LPS-induced Ig secretion. Moreover, B cells from autoimmune mice and from normal strains were equally receptive to Con A-induced suppressor signals. T cells from young and older NZB and BXSB mice added to a standard number of B cells from syngeneic young mice provided equal help in enhancing LPS-induced Ig secretion, and this help in turn was equivalent to that provided by T cells from normal mice of the same H-2 haplotype. The exception was the MRL/l strain; T cells from older animals provided considerably more help than T cells from young MRL/l or T cells from young and older H-2-compatible normal mice.

In young adulthood, MRL/Mp-lpr/lpr mice develop severe systemic lupus erythematosus (SLE)-like syndrome associated with massive T cell proliferation. The congenic MRL/Mp- mice lack the lpr gene and develop chronic SLE late in life. We have exchanged thymic transplants between these substrains so as to determine the role of the thymus in the development of early, severe SLE and of lymphoproliferation. The median survival times of unmanipulated lpr/lpr and mice were 160 and 510 d, respectively. The lpr/lpr and mice thymectomized when newborn and transplanted at 1 mo with the opposite type of thymus retained the diseases phenotype of their unmanipulated counterparts with 50% mortality at 186 and 498 d, respectively. In contrast, lpr/lpr mice thymectomized when newborn but not transplanted with thymus did not develop lymphoid hyperplasia and glomerulonephritis, and 100% of them were alive at 390 d. Serologically, the thymectomized but untransplanted lpr/lpr mice had significantly reduced levels of autoantibodies, whereas thymectomized and transplanted mice of either substrain were similar to unmanipulated controls. The results indicate that: (a) a thymus is essential for expression of lymphoproliferation and early SLE-like disease in the lpr/lpr phenotype; (b) the lpr/lpr disease is not a result of a unique hormonal or microenvironmental defect(s) of the thymus of this substrain because the genotype of the thymus is irrelevant for the development of T cell proliferation and early SLE; (c) differentiation of stem cells under the hormonal or microenvironmental influences of a thymus that possesses the lpr genotype does not lead to abnormal T cell differentiation or early autoimmunity; and (d) the lpr/lpr disease cannot be caused exclusively by an intrinsic B cell defect or environmental stimuli that cause B cell polyclonal activation.