Tatiana Seregina

Effective Treatment of Preexisting Melanoma with Whole Cell Vaccines Expressing α(1,3)-Galactosyl Epitopes

The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies recognizing alpha(1,3)-galactosyl epitopes (alphaGal) not present on human cells. We exploited this immune mechanism to create a whole cell cancer vaccine to treat melanoma tumors. B16 melanoma vaccines genetically engineered to express alphaGal epitopes (B16alphaGal) effectively treated preexisting s.c. and pulmonary alphaGal-negative melanoma (B16Null) tumors in the alpha(1,3)-galactosyltransferase knockout mouse model. T cells from mice vaccinated with B16alphaGal recognized B16Null melanoma cells measured by detection of intracellular tumor necrosis factor-alpha. We showed successful adoptive transfer of immunity to recipient mice bearing lung melanoma metastasis. Mice receiving lymphocytes from donors previously immunized with B16alphaGal had reduced pulmonary metastases. The transfer of lymphocytes from mice vaccinated with control vaccine had no effect in the pulmonary metastasis burden. This study unequivocally establishes for the first time efficacy in the treatment of preexisting melanoma tumors using whole cell vaccines expressing alphaGal epitopes. Vaccination with B16alphagal induced strong long-lasting cell-mediated antitumor immunity extended to B16Null. These data formed the basis for the testing of this therapeutic strategy in human clinical trials currently under way.

Herpes simplex thymidine kinase gene-transduced donor lymphocyte infusions

OBJECTIVE Donor lymphocytes mediate both a beneficial graft-vs-leukemia/lymphoma (GVL) effect as well as graft-vs-host disease (GVHD), the most dreaded complication of allogeneic hematopoietic stem cell transplantation (HSCT). Transduction of donor lymphocytes with a herpes simplex thymidine kinase (HSVtk) gene prior to infusion confers lethal sensitivity to the anti-herpes drug, ganciclovir (GCV). HSVtk-transduced donor lymphocyte infusions (DLI) have already been used and significant problems have limited the clinical experience to very few patients. To this end, we also report on a study of whether HSVtk-DLI induces GVHD/GVL and if infusion of GCV allows abrogation of GVHD by selective killing of donor lymphocytes. MATERIALS AND METHODS Nine patients with relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) were infused with HSVtk gene-modified donor lymphocytes. In brief, transgeneic lymphocytes were prepared by 3 days of activation, 1 day of transduction, 6 days of selection with G418, and 2 to 4 weeks of expansion. RESULTS From 5.0 to 199 x 10(6) CD3(+) DLI were infused. There were no toxicities and no correlation between CD3(+) cell dose and either GVHD or GVL was observed. Only one patient who had cutaneous T-cell lymphoma (CTCL) developed GVHD and that same patient is the only patient to have an anti-tumor response. The patient was infused with 23 x 10(6) CD4(+) and 9.7 x 10(6) CD8(+) HSVtk DLI. Following discontinuation of immune suppression and infusion of GCV, GVHD promptly resolved. Although the CTCL relapsed, it has been easily controlled with intermittent topical therapy. One patient with acute myelogenous leukemia (AML) had a remission inversion of undetermined significance. Two patients with AML, one patient with lymphoma, and four patients with chronic myelogenous leukemia (CML) did not respond. CONCLUSION HSVtk-DLI may provide an anti-tumor effect in vivo and may induce GVHD that is abrogated by GCV treatment. While technical aspects to improve response need to be perfected, HSVtk-DLI infusion to induce a transient GVL/GVHD may become an effective future therapy to minimize complications of allogeneic HSCT.

Rat Sodium Iodide Symporter for Radioiodide Therapy of Cancer

Design and development of new approaches for targeted radiotherapy of cancer and improvement of therapeutic index by more local radiation therapy are very important issues. Adenovirus-mediated delivery of the sodium iodide symporter (NIS) gene to cancer cells is a powerful technique to concentrate lethal radiation in tumor cells and eradicate tumors with increased therapeutic index. A replication-defective adenoviral vector expressing the rat NIS gene (Ad-rNIS) was used for in vitro gene delivery and into human prostate cancer xenografts to study antitumor effect. Robust function of the rat symporter was detected in DU145, T47D, and HCT-15 human cancer cell lines transduced with Ad-rNIS. All three cancer cell lines successfully transferred functionally active rat symporter to the plasma membrane, resulting in very high levels of iodine-125 accumulation. Three-dimensional multicellular tumor spheroids derived from DU145 human prostate cancer cells were transduced with Ad-rNIS and incubated with 131I for 24 hours. After treatment, spheroids rapidly decreased in size and disappeared within 10 days. In vivo data revealed an inhibition of tumor growth in athymic nude mice after intratumoral Ad-rNIS injection followed by 131I administration. Eighty-eight percent of experimental mice survived >30 days, whereas control groups had only 18% survival >30 days. This is the first report that demonstrates the rat NIS gene can effectively induce growth arrest of human tumor xenografts after in vivo adenoviral gene delivery and 131I administration. The data confirm our hypothesis that the rat NIS gene is an attractive suicide gene candidate for cancer treatment.

Effective Suppression of Class I Major Histocompatibility Complex Expression by the US11 or ICP47 Genes Can Be Limited by Cell Type or Interferon-γ Exposure

An impediment encountered in many viral-based gene therapy clinical trials has been the rapid destruction of the transgene by the hosts immune response. The processing and presentation of antigens through the class I major histocompatibility complex (MHC) pathway is the initial specific response to viral infection. Disruption of the class I MHC pathway by herpes simplex virus (HSV) or the human cytomegalovirus (HCMV) results in a decrease of the CD8(+) cytotoxic T lymphocyte (CTL) response and prolongs survival of infected cells in the host. Two viral immune suppression genes that interfere with the class I MHC presentation pathway, the HSV type I ICP47 gene and HCMV US11 gene, were cloned and each incorporated into a retroviral vector. HSV ICP47 and HCMV US11 transgenes were expressed in multiple cells lines and compared for their abilities to reduce antigen presentation on the cell surface by class I MHC. Retroviral supernatants were used to transduce human, canine, and rat cell lines. Fluorescence-activated cell sorter (FACS) analysis of US11- and ICP47-transduced cell lines demonstrated substantial reductions in class I MHC cell surface expression in most cell lines except in rodent cells where ICP47 is nonfunctional. The decrease in the level of class I MHC expression for ICP47 transduced cell lines ranged from 31-98% relative to negative controls. US11 decreased class I cell surface MHC by 67-96%. When both ICP47 and US11 are expressed in human cells, a further reduction of class I MHC was observed. Next, human A375 melanoma cells were tested to determine if the resulting reduction in cell surface class I MHC would reduce in vitro cytotoxicity by CTL. A375 cells expressing either ICP47 or US11 demonstrated a twofold to threefold reduction of specific lysis by primed CD8(+) CTL. These data clearly establish an ability to convey immune protection to human cells by viral genes. However, further analysis demonstrated that interferon (IFN)-gamma could reverse part or all of the downregulation of class I MHC induced by the ICP47 or US11 genes. The ICP47 and US11 genes, when expressed in target cells, decrease class I MHC presentation and as such might be used in strategies to create local immunosuppression against transgenes or allografts.

Adoptive Immunotherapy for Leukemia: Donor Lymphocytes Transduced with the Herpes Simplex Thymidine Kinase Gene

This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.

Transduction of hematopoietic stem cells with a retroviral vector expressing the neomycin phosphotransferase gene

Summary:Transduction of stem cells with a marking gene holds promise to determine if tissue repair or regeneration is derived from the adult hematopoietic stem cell and if relapse of an autoimmune disease should occur whether relapse arises from the stem cell compartment or from lymphocytes surviving the conditioning regimen. New safety concerns about gene-modified stem cell would entail new safety testing such as documentation of the insertional site prior to release.

Use of codon-modified, red-shifted variants of green fluorescent protein genes to study virus-mediated gene transfer.

Publisher Summary One central requirement of gene therapy is the need to analyze gene transfer efficiency into target cells either in vitro or in vivo. The most common methods of gene transfer employ modified viruses as delivery vectors. Viral vectors have achieved widespread use in phase I and II clinical trials. These initial trials indicate that a great deal of further effort is needed to optimize viral vectors. Continued improvements in the available viral vector classes are actively pursued, as well as the development of new types of viral vectors. This chapter reviews the examples of exploiting the green fluorescent protein (GFP) marking system to study viral vector gene transfer relevant to human gene therapy. Retroviral vectors can efficiently transducer rodent and human lymphocytes. Several groups now employ gene-modified lymphocytes as adoptive immunotherapy for patients who have failed allogeneic bone marrow transplantation. In addition to such ex vivo therapeutic approaches, a primary focus of more recent approaches for cancer has been the development of improved in vivo gene delivery vector systems. One such system is the herpes simplex vector amplicon system. Red-shifted, codon-modified GFP genes permit the rapid visualization of herpes vectors within a few hours after target cell transductions. The small gene size and remarkable ease of visualizing red-shifted GFP variants using standard fluorescein isothiocyanate (FITC) filter systems makes them ideal for studying virus-mediated gene transfer.

1133. A Phase I Study of Antitumor Vaccination Using Genetically Modified Tumor Cells Expressing |[alpha]|(1,3)Galactosyltransferase in Patients with Refractory or Recurrent Non-Small Cell Lung Cancer (NSCLC): Preliminary Results

Background: Despite new therapies lung cancer remains the leading cause of cancer death. In a phase I trial we examined the safety and feasibility of antitumor vaccination with three genetically altered human lung cancer cell lines (HyperAcute|[trade]| Lung Cancer Vaccine) engineered to express xenotransplantation antigens through retroviral transfer of the murine |[alpha]|(1,3)galactosyltrasferase gene in patients (Pts) with advanced NSCLC. Methods: Pts with stage IV, recurrent or refractory NSCLC, ECOG PS |[le]|2, |[le]|2 prior chemotherapies, adequate nutrition, bone marrow and organ function were eligible for the study. Cohorts of 3 Pts were scheduled to receive intrademal injections of 3 |[times]| |[Lgr]|6, 10 |[times]| 10|[Lgr]|6, 30 |[times]| 10|[Lgr]|6, or 100 |[times]| 10|[Lgr]|6 irradiated vaccine cells every 4-weeks |[times]| 4. Toxicity was assessed using standard criteria and response was determined by RECIST criteria. Immunological responses included anti-|[alpha]|Gal antibody titers, whole cell interferon-|[gamma]| ELISPOT and vaccine site skin biopsies. Results: To date, 9 Pts, 6 men and 3 women, median age 54 (range, 34-72 yrs.), median number of prior chemotherapies 1 (range, 1-2) were vaccinated with up to 30 |[times]| 10|[Lgr]|6 cells. Five Pts received 4/4 vaccinations, 1 Pt received 3/4 vaccinations and 1 Pt received 2/4 vaccinations, and 2 Pts. have received 1 vaccination. Adverse events (|[le]|grade 2) attributable to vaccination include injection site pain/discomfort, local skin reaction, fatigue, and hypertension. Other adverse events (|[le]|grade 2) include bradycardia, cough, diarrhea, dyspnea, headache, hyperglycemia, hyponatremia, nausea, pleural effusion, and vomiting. Four patients had stable disease |[ge]|16 weeks (range, 21-36+ weeks), three other Pts progressed and 2 Pts are not evaluable at this time. Skin biopsies 48-hrs after vaccination demonstrated vaccine cells in the dermis with infiltration of lymphocytes and eosinophils. Conclusions: Antitumor vaccination with genetically altered allogeneic human lung cells expressing |[alpha]|(1,3) galactosyltrasferase is safe and feasible.