Daniel L. Shawler

Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer.

PURPOSE Belagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition. PATIENTS AND METHODS We performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.25, 2.5, or 5.0 x 10(7) cells/injection) of belagenpumatucel-L on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety, and anticancer activity were monitored. RESULTS Seventy-five patients (two stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients) received a total of 550 vaccinations. No significant adverse events were observed. A dose-related survival difference was demonstrated in patients who received > or = 2.5 x 10(7) cells/injection (P = .0069). Focusing on the 61 late-stage (IIIB and IV) assessable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% and 52%, respectively for the higher dose groups combined and 39% and 20%, respectively, for the low-dose group. Immune function was explored in the 61 advanced-stage (IIIB and IV) patients. Increased cytokine production (at week 12 compared with patients with progressive disease) was observed among clinical responders (interferon gamma, P = .006; interleukin [IL] -6, P = .004; IL-4, P = .007), who also displayed an elevated antibody-mediated response to vaccine HLAs (P = .014). Furthermore, positive enzyme-linked immunospot reactions to belagenpumatucel-L showed a correlation trend (P = .086) with clinical responsiveness in patients achieving stable disease or better. CONCLUSION Belagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

Therapy of chronic lymphocytic leukemia and cutaneous T-cell lymphoma with T101 monoclonal antibody.

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.

Comparison of gene therapy with interleukin-2 gene modified fibroblasts and tumor cells in the murine CT-26 model of colorectal carcinoma.

We compared the efficacy of gene therapy mediated by interleukin-2 (IL-2) gene-modified tumor cells to gene therapy mediated by IL-2 transduced fibroblasts in the CT-26 model of murine colorectal carcinoma. We transduced CT-26 tumor cells and BALB/c 3T3 fibroblasts with three different retroviral vectors using three different promoters for the human IL-2 gene: DC/TKIL-2 (thymidine kinase promoter), LXSN-iIL2 (long terminal repeat promoter), and LNCX-iIL2 (cytomegalovirus promoter). These transductions resulted in CT-26 and 3T3 subclones that secreted different amounts of IL-2. Immunization of animals with either CT-26/IL-2 cells or with fibroblast/IL-2 cells mixed with CT-26 induced similar levels of immunity that protected 62-82% of animals against a subsequent tumor challenge with parental CT-26. However, mice developed tumors at the site of inoculation in 46% of the animals immunized with CT-26/IL-2 cells. In a separate experiment, CT-26/IL-2 cells were exposed to 6,000 cGy of gamma irradiation to prevent tumor growth at the site of inoculation. Although the CT-26/IL-2 cells continued to secrete IL-2 after irradiation, they were no longer effective at inducing antitumor immunity. In contrast, both irradiated and nonirradiated fibroblast/IL-2 cells, mixed with irradiated CT-26, were equally effective at inducing antitumor immunity. These data suggest that in the CT-26 model, fibroblast-mediated IL-2 gene therapy has advantages for the induction of antitumor immunity and abrogation of tumorigenic potential at the site of inoculation compared with tumor cell-mediated IL-2 gene therapy.

Antigenic and immunologic characterization of an allogeneic colon carcinoma vaccine

We report the immunological characterization of three colon carcinoma cell lines, COLO 205, SW620 and SW403, which we selected to combine with cytokine‐secreting fibroblasts for the development of an allogeneic tumour cell vaccine. The cell lines expressed HLA‐A2 as well as shared tumour‐associated antigens (TAAs) representative of colon carcinomas: CEA, Ep‐CAM, MUC1, HER2/neu and MAGE antigens. They did not secrete high levels of the immunosuppressive factors TGF‐β, IL‐10 or prostaglandins. The lines presented TAAs in a manner recognized by immune effector cells, which was demonstrated by the lysis of SW620 by HLA‐A2‐restricted anti‐p53 cytotoxic T lymphocytes (CTL). COLO 205 and SW620 were genetically modified to express the co‐stimulatory molecule CD80 (B7.1), which increased the ability of the cells to stimulate CTL in vitro. CTL clones derived from HLA‐A2+ peripheral blood mononuclear cells stimulated with the CD80‐expressing lines lysed the stimulator cell and an HLA‐A2+ colon cancer cell line, but did not lyse an isogeneic fibroblast line or an HLA‐A2− colon cancer cell line. CTL clones derived from colon carcinoma patients immunized with an allogeneic vaccine containing these lines demonstrated killing of autologous tumour cells, the vaccine cell lines and other HLA‐A2+ colon cancer cell lines, but not fibroblasts isogeneic to certain of the target cell lines. Our studies demonstrate that these colon carcinoma cell lines express shared TAAs that can induce CTLs which recognize and lyse other colon carcinoma cells, and support the continued clinical evaluation of the CD80 gene modified allogeneic colon cell/cytokine‐secreting fibroblast carcinoma vaccine.

The stability of breast cancer progenitor cells during cryopreservation: Maintenance of proliferation, self-renewal, and senescence characteristics.

Cancer stem cells are believed to be the driving force behind tumor progression and development. Despite extensive studies on the effects of cryopreservation on embryonic and hematopoietic stem cells there is only limited data that directly deals with in the cryopreservation of cancer stem cells. In this study, we looked at the effect of cryopreservation on breast cancer progenitor cells known as mammospheres, which are derived from the MCF7 breast carcinoma cell line. We focused on the effect of cryopreservation on the cell biology and function of tumor-initiating cells using a standard method of cryopreservation with 15% dimethyl sulfoxide (Me(2)SO). Cell proliferation and survival was analyzed by alamarBlue solution on cryopreserved cells stored for 1-12 weeks and also by the expression of Ki-67. To assess self-renewal, single cells were harvested by limiting dilution procedure and wells were scored once a week. In order to investigate senescence, the activity of beta-galactosidase was detected by histochemical staining. Our results indicate that cryopreservation of breast cancer initiating cells will not reduce the ability of the cells to proliferate following cryopreservation storage for up to 12 months. Similarly, self-renewal, a unique property of stem cells, was shown to be maintained during cryopreservation. In contrast, cryopreservation of the mammospheres significantly increases the rate of senescence-mediated pathways. These data suggest that although cryopreservation of tumor-initiating cells is feasible but further studies are necessary to achieve a trustable repository of tumor-initiating cells and the design of new therapeutic measures to specifically target these cells.

Construction and characterization of retroviral vectors for interleukin-2 gene therapy.

Summary Several investigators have employed interleukin-2 (IL-2) gene transfer to enhance the immunogenicity of tumor cell vaccines. We describe in this report the construction and characterization of retroviral vectors for IL-2 gene therapy. Human IL-2 cDNA with a chimeric rat preproinsulin/IL-2 DNA leader sequence was sub-cloned into the pLXSN (long terminal repeat promoter) and pLNCX (cytomegalovirus [CMV] promoter) vectors to generate the plasmids pLXSN-iIL2 and pLNCX-iIL2, respectively. Human IL-2 cDNA with a chimeric human tissue factor/IL-2 DNA leader sequence was utilized to construct the vector pLXSN-tIL2. The levels of IL-2 secreted by transduced tumor cells and fibroblasts were evaluated by enzyme-linked immunosorbent assay (ELISA) of culture supernatants and compared with those of normal peripheral blood mononuclear cells (PBMC) activated in vitro with calcium ionophore and phorbol 12-myristate 13-acetate. The highest levels of IL-2 secreted by transduced tumor cells (760 units/106 cells/24 h), adult fibroblasts (625 units/106 cells/24 h), and embryonic fibroblasts (3,975 units/106 cells/24 h) were 150− to 1,000-fold higher than that secreted by the activated PBMC (4 units/106 cells/24 h). Similar levels of IL-2 were expressed by human fibroblasts transduced with pLXSN vectors employing the preproinsulin (pLXSN-iIL2) or tissue factor (pLXSN-tIL2) leader sequences (range in IL-2 units/106 cells/24 h pLXSN-iIL2 = 375–625 vs. pLXSN-tIL2 = 90–440). Because IL-2-transduced cells for clinical applications are generally irradiated to prevent cellular proliferation, we evaluated the effects of radiation on IL-2 production. Radiation doses between 1,500 and 10,000 cGy resulted in gradual decreases in IL-2 secretion by transduced cells. The range of the decrease in IL-2 secretion was 7–11% by day 7, 0–29% by day 14, and 25–50% by day 35. For clinical applications, stable production of the vector in high concentrations is an important consideration. The retroviral vector pLXSN-tIL2 produced the highest viral titer and was chosen for further characterization. Southern blot analysis of SacI-digested genomic DNA from the LXSN-tIL2 producer cell line and SacI-digested pLXSN-tIL2 plasmid DNA revealed the expected 3.2-kbp fragment, suggesting the absence of transgene rearrangement and the suitability of this vector as a candidate for clinical applications.

GENE THERAPY APPROACHES TO ENHANCE ANTITUMOR IMMUNITY

Publisher Summary This chapter summarizes the advances in antitumor immunity enhancement and concentrates on several types of genetic manipulations that have been explored to enhance the efficacy of cancer immunotherapies. A number of cytokines, produced in purified form by recombinant DNA methodology, have been evaluated for their antitumor effects. In several clinical trials, cytokines and related immunomodulators have produced objective tumor responses in some patients afflicted with a variety of neoplasms. The development of antitumor immune responses as a consequence of cytokine gene transfer in the treatment of cancer has been demonstrated in several animal tumor models. A number of approaches have been developed to circumvent the need to establish cell lines or cultures ex vivo for immunogene therapy. Direct tumor injection of cytokine or allogeneic MHC gene vectors has been successful in animal models and this strategy is being evaluated in phase I clinical trials. Additional targets for immunogene therapy approaches include mutated oncogenes, tumor suppressor genes, and viral antigens. These potential tumor-associated antigens (TAAs) have been suggested as appealing targets for immunotherapy as they are not expressed by normal tissues. The chapter also provides a summary of immunogene therapy clinical protocols submitted to regulatory agencies worldwide. The results of these trials should provide insights regarding that of the immunogene therapy approaches are most appropriate for further clinical evaluation.

Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

SummaryWe have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.

Tissue distribution and serum kinetics of T101 monoclonal antibody during passive anti-cancer therapy

We have administered fifty-six 24 hr infusions of the anti-human T-cell monoclonal antibody T101 to 10 patients with cutaneous T-cell lymphoma (CTCL) and 6 patients with chronic lymphocytic leukemia (CLL) in doses of 10, 50, 100, 150, and 500 mg. The larger doses of T101 resulted in higher, more persistent serum T101 concentrations, and CTCL patients generally developed higher serum T101 levels than CLL patients given equivalent doses. The presence of host anti-mIgG antibodies prior to infusion was associated with decreased serum concentrations of T101. Treatments that demonstrated measurable serum T101 levels were also associated with in vivo T101 binding and cytodestruction of circulating target cells. Immunofluorescence analysis of bone marrow and lymph node biopsies in CLL, and skin biopsies in CTCL, suggested that T101 had reached extravascular tumor sites. Infusion of 111In-conjugated T101 showed uptake in the liver, spleen, lymph nodes, and (in CTCL) skin infiltrates. Our data demonstrate the tissue distribution of T101 and suggest that immunoconjugates of T101 with toxins, drugs, or radioisotopes may result in better therapeutic responses.

The use of monoclonal antibodies and flow cytometry to detect peripheral blood and bone marrow involvement of a diffuse, poorly differentiated lymphoma

Using monoclonal antibodies and flow cytometry, we were able to characterize the phenotype of a diffuse, poorly differentiated lymphoma and to isolate subpopulations of cells from the blood and bone marrow that expressed the malignant phenotype even though the patient exhibited no absolute lymphocytosis. Because the circulating clone reacted with the anti-T cell monoclonal antibody T101, we initiated serotherapy with T101 as part of a phase I study. A 10 mg infusion of T101 resulted in the rapid clearance of normal T cells from circulation, but the clone showed evidence of modulation and was not cleared. Twenty-four hours following infusion, all cell populations had returned to pre-treatment levels. Our study suggests that, by using monoclonal antibodies and flow cytometry, blood and bone marrow involvement of a lymphoma can be demonstrated in patients without absolute lymphocytosis, a finding which may influence the staging and treatment of the disease.