Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells.

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.

Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes.

It has previously been shown that bone marrow-generated dendritic cells (DC) are potent stimulators in allogeneic mixed leukocyte reactions and are capable of activating naive CD4+ T cells in situ in an antigen-specific manner. In this study we have investigated whether bone marrow-generated DC are capable of inducing antigen-specific CD8+ T cell responses in vivo. Initial attempts to induce specific cytotoxic T lymphocyte (CTL) responses in mice injected with bone marrow-generated DC pulsed with ovalbumin (OVA) peptide were frustrated by the presence of high levels of nonspecific lytic activity, which obscured, though not completely, the presence of Ag-specific CTL. Using conditions that effectively differentiate between antigen-specific and nonspecific lytic activity, we have shown that bone marrow-generated DC pulsed with OVA peptide are potent inducers of OVA-specific CTL responses in vivo, compared with splenocytes or RMA-S cells pulsed with OVA peptide, or compared with immunization with free OVA peptide mixed with adjuvant. Antibody-mediated depletion experiments have shown that the cytotoxic effector cells consist primarily of CD8+ cells, and that induction of CTL in vivo is dependent on CD4+ as well as on CD8+ T cells. These results provide the basis for exploring the role of bone marrow-generated DC in major histocompatibility class I-restricted immune responses, and they provide the rationale for using bone marrow-generated DC in CTL-mediated immunotherapy of cancer and infectious diseases.

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.

Regression of bladder tumors in mice treated with interleukin 2 gene-modified tumor cells.

This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN-gamma genes were introduced and expressed in MBT-2 cells. The tumor-forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2-secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice.

Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors.

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene-modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow-generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.

Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

Gene expression in mice after high efficiency retroviral-mediated gene transfer.

A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression.

An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

Gene therapy for infectious diseases: the AIDS model.

Genetic manipulation of somatic cells may be of therapeutic value in a variety of infectious diseases, particularly in human immunodeficiency virus (HIV) infection. Stable insertion of custom-designed 'resistance genes' into cells susceptible to HIV could reduce the viral burden in infected individuals and potentially retard the characteristic progressive immune dysfunction. Alternatively, ectopic expression of genes that encode viral antigens might induce potent antiviral immune responses and form the basis for novel prophylactic and therapeutic vaccines. While laboratory studies have proved that the approach works in principle, preclinical and clinical studies will be necessary to evaluate the therapeutic benefit of such gene-based therapies.

Role of intron-contained sequences in formation of moloney murine leukemia virus env mRNA.

Formation of the Moloney murine leukemia virus envelope mRNA involves the removal of a 5,185-base pair-long intron. Deletion analysis of two Moloney murine leukemia virus-derived expression vectors revealed the existence of two short regions within the viral intron which are required for the efficient formation of the spliced RNA species. One region was present upstream from the 3' splice junction, extended at least 85 nucleotides beyond the splice site, and was not more than 165 nucleotides long. As yeast polymerase II introns, the Moloney murine leukemia virus intron contains the sequence 5'-TACTAAC-3' 15 nucleotides upstream from the 3' splice site. A second region located in the middle of the intron, within a 560-nucleotide-long sequence, was also essential for formation of the spliced RNA species. The efficient splicing of the env mRNA in the absence of expression of viral genes raises the possibility that similar mechanisms are used to remove introns of (some) cellular genes.

Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.

Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA.

Dendritic cells (DC) generated from the peripheral blood mononuclear cells of healthy individuals or from cancer patients transfected with carcinoembryonic antigen (CEA) mRNA stimulate a potent CD8+ cytotoxic T lymphocyte (CTL) response in vitro. DCs are effectively sensitized with RNA in the absence of reagents commonly used to facilitate mammalian cell transfection. RNA encoding a chimeric CEA/LAMP-1 lysosomal targeting signal enhances the induction of CEA-specific CD4+ T cells, providing a strategy to induce T-help that may be necessary to generate and/or maintain an optimal CD8+ CTL response in vivo. CEA RNA-transfected DCs also serve as effective targets in cytotoxicity assays, thus providing a general method for inducing, as well as measuring, CEA-specific CTL responses across a broad spectrum of HLA haplotypes.

Expression of functional epidermal growth factor receptors in a human hematopoietic cell line.

To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.

Retroviral vector-mediated gamma-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity.

Retroviral vectors were used to introduce the gamma-interferon (IFN-gamma) gene into CMS-5 cells, a weakly immunogenic tumor of BALB/c origin. After selection in G418-containing medium, colonies were isolated, cloned, and expanded to cell lines. IFN-gamma secretion was assessed using a bioassay and enzyme-linked immunosorbent assay, and high (25 units/ml) and low (5 units/ml) IFN-gamma producers were isolated. Tumor growth was followed after intradermal injection, and spleen cells were isolated at different time points. IFN-gamma secretion by tumor cells abrogated their tumorigenicity and induced a persistent and specific antitumor immunity. In contrast to the normally observed cellular immunosuppression in unmodified CMS-5 tumor-bearing mice, IFN-gamma-producing tumors induced a long lasting state of T-cell immunity, as judged by rejection of a CMS-5 tumor challenge and persistence of specific cytotoxic activity in the spleen cell population. High levels of tumor-specific cytotoxic activity could also be detected if IFN-gamma-secreting tumor cells, but not unmodified CMS-5 cells, were used as targets at a time point when immunosuppression was usually seen. These studies highlight the potential advantages of localized IFN-gamma secretion to induce potent antitumor immune responses.

Retroviral vector-mediated lymphokine gene transfer into human renal cancer cells.

Effective vaccination against cancer, either for prophylaxis or therapy, has been an elusive goal for years. Cytokine gene therapy offers a novel approach to generate immunogenic tumor cell vaccines. To examine the feasibility of cytokine gene transfer into human renal cancer (RC) cells, we introduced the cDNAs for human interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) into various RC cell lines with retroviral vectors. Using the NIH3T3 amplification assay, no replication competent retroviral particles were detectable in cell culture supernatants taken from gene-modified RC cell lines. Efficient expression of both lymphokines was achieved. Depending on the cell line and the vector construct used, lymphokine gene-modified human RC cell lines released 4 to 29 units/10(6) cells of IL-2, or up to 10 units/10(6) cells of IFN-gamma within 48 h. Fluorescence-activated cell sorter analysis of SK-RC-29 cells releasing IFN-gamma showed increased expression of major histocompatibility complex class I antigen, beta 2-microglobulin, and ICAM-1, as well as induction of major histocompatibility complex class II antigen expression [human leukocyte antigen(HLA)-DR, -DP], but no changes in these cell surface markers were observed with SK-RC-29 cells releasing IL-2. Following in vitro gamma-irradiation with 5,000 or 10,000 rad, growth of lymphokine gene-modified RC cells was abrogated, but their capability to release lymphokine and express lymphokine-induced antigenic determinants, such as HLA-DR, was retained. Tumor formation by the human RC cell line SK-RC-29 in BALB/c nude mice was not affected by IFN-gamma secretion, but was inhibited by in vivo release of IL-2 from s.c. injected tumor cells. These studies demonstrate the feasibility of retroviral mediated lymphokine-gene transfer into human RC cells and suggest a means for generating autologous or HLA-matched allogeneic tumor cell vaccines for the treatment of patients with renal cell carcinoma.

Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells.

Unique patient-specific tumor antigens may constitute the dominant antigens in the antitumor immune response. Hence, vaccination with the patient's own repertoire of tumor antigens may offer a superior strategy to elicit protective immunity. We have shown previously that dendritic cells transfected with mRNA isolated from tumor cells stimulate potent CTL responses and engender protective immunity in tumor-bearing mice. In the current study, we demonstrate that tumor mRNA, isolated from murine tumor cell lines or from primary human tumor cells microdissected from frozen tissue sections, can be amplified without loss of function. This study provides the foundations for an effective and broadly applicable treatment that does not require the characterization of the relevant antigenic profile in each patient and will not be limited by tumor tissue availability for antigen preparation.

Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells.

Dendritic cells (DCs), matured by CD40-ligand (CD40L), undergo marked changes in their ability to process and present antigen, resulting in augmented lymphocyte stimulatory activity. We demonstrate that the form of the tumor antigen (peptide or genetic material) used to load the DCs dictates the required sequence of antigen loading and maturation for antitumor immunotherapy. Optimal stimulation of carcinoembryonic antigen (CEA)-specific CTLs by peptide-loaded DCs occurs when DCs from cancer patients are matured with CD40L before exposure to CEA peptide, whereas optimal stimulation by RNA-transfected DCs occurs when the DCs are loaded with CEA RNA before maturation with CD40L.

Immunotherapy of prostate cancer in the Dunning rat model: use of cytokine gene modified tumor vaccines.

Adenocarcinoma of the prostate is the most common cancer in men. The majority of cancers are discovered once they have already metastasized, and there is no effective therapy for prostatic cancer at this stage. The use of cytokine-secreting tumor cell preparations as therapeutic vaccines for the treatment of advanced prostate cancer was investigated in the Dunning rat R3327-MatLyLu prostatic tumor model. IL-2 secreting, irradiated, tumor cell preparations were capable of curing animals with s.c. established tumors, and induced immunological memory that protected animals from subsequent tumor challenge. Immunotherapy was less effective when tumors were induced orthotopically, but nevertheless led to improved outcome, significantly delaying, and occasionally preventing, recurrence of tumors after resection of the cancerous prostate. Granulocyte-macrophage colony stimulating factor secreting tumor cell preparations were less effective, and interferon-gamma secreting cells had only a marginal effect. Induction of a potent immune response in tumor bearing animals against the nonimmunogenic MatLyLu tumor supports the view that active immunotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.

RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-gamma.

Two aspects of cytokine therapy of intracerebral tumors are considered in this study: modulation of tumor growth in vivo and central nervous system toxicity. Coimplantation of RG-2 glioma cells and retroviral vector producer cell lines was performed to provide a local source of interleukin-2 (IL-2) or IFN-gamma within the tumor and coinitiate an antitumor immune response. We demonstrated that local intratumoral production of IL-2 and IFN-gamma generates a cell-mediated antitumor response in vivo. This response was manifest as a diffuse infiltration of monocytes/macrophages, CD4+ and CD8+ T cells, and activation of microglial OX42+ cells in intracerebral RG2 tumors. The cell-mediated antitumor immune response resulted in the early suppression of intracranial and subcutaneous tumor growth, but the effect was not sustained and there were no tumor regressions. The absence of increased survival of animals with intracranial tumors is explained in part by the severe central nervous system toxicity caused by local production of IL-2 and IFN-gamma. Central nervous system toxicity induced blood-brain barrier disruption, vasogenic brain edema, and dislocation of the brain midline structures, as observed by dynamic magnetic resonance imaging and direct measurements of tissue water content. The clinical application of IL-2 and IFN-gamma gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with intracerebral production of these cytokines.