Reducing toxicity of 4-1BB costimulation: targeting 4-1BB ligands to the tumor stroma with bi-specific aptamer conjugates.

Systemic administration of immune modulatory antibodies to cancer patients is associated with autoimmune pathologies. We have developed a clinically feasible and broadly applicable approach to limit immune stimulation to disseminated tumor lesions using a bi-specific agonistic 4-1BB oligonucleotide aptamer targeted to a broadly expressed stromal product (e.g., VEGF or osteopontin). The stroma-targeted aptamer conjugates engendered potent antitumor immunity against unrelated tumors and exhibited a superior therapeutic index compared to non-targeted agonistic 4-1BB antibody.

Vaccination against Foxp3(+) regulatory T cells aggravates atherosclerosis.

OBJECTIVE: Regulatory T cells are crucial for immune homeostasis and an impaired regulatory T cell function results in many pathological conditions. Regulatory T cells have already been described to be protective in atherosclerosis. However the exact contribution of Foxp3-expressing natural regulatory T cells in atherosclerosis has not been elucidated yet. METHODS AND RESULTS: In this study we vaccinated LDL receptor deficient mice with dendritic cells which are transfected with Foxp3 encoding mRNA and studied the effect on initial atherosclerosis. Vaccination against Foxp3 resulted in a reduction of Foxp3(+) regulatory T cells in several organs and in an increase in initial atherosclerotic lesion formation. Furthermore we observed an increase in plaque cellularity and increased T cell proliferation in the Foxp3 vaccinated mice. CONCLUSION: We further establish the protective role of Tregs in atherosclerosis. The results illustrate the important role for Foxp3-expressing regulatory T cells in atherosclerosis, thereby providing a potential opportunity for therapeutic intervention against this disease.

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA.

Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.

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.

Induction of cytotoxic T lymphocytes with dendritic cells transfected with human papillomavirus E6 and E7 RNA: implications for cervical cancer immunotherapy.

Human papillomavirus (HPV) infection is associated with cervical cancer. The high-risk HPV E6 and E7 oncoproteins are constitutively expressed in most cervical carcinoma cells, and are, therefore, attractive antigens for cytotoxic T-lymphocyte (CTL)-mediated immunotherapy. The objective of this study was to evaluate the use of dendritic cells (DCs) transfected with RNA encoding the E6 and E7 protein for cervical cancer immunotherapy. The authors have shown that DCs transfected with RNA-encoding antigen stimulate potent antigen-specific CTL responses in vitro and in vivo. In this study, they tried to determine whether DCs transfected with E6 and E7 RNA stimulate primary, antigen-specific CTL responses in vitro. The results show that DCs pulsed with E6 or E7 RNA stimulate antigen-specific CTL responses that recognize and lyse DCs transfected with E6 and E7 RNA and human cervical carcinoma cells expressing the E6 and E7 products, and the lysis was comparable to that achieved with E6 and E7 peptide-pulsed DCs. Dendritic cells cotransfected with both E6 and E7 RNA stimulate CTLs that are more effective at lysing human cervical cancer cells. This study provides a rationale for the development of cervical carcinoma immunotherapy using DCs transfected with HPV E6 and E7 RNA.

Effectiveness of combined interleukin 2 and B7.1 vaccination strategy is dependent on the sequence and order: a liposome-mediated gene therapy treatment for bladder cancer.

We have developed a novel liposome-mediated immunogene therapy using interleukin 2 (IL-2) and B7.1 in a murine bladder cancer model. A carcinogen-induced murine bladder cancer cell line, MBT-2, was transfected with cationic liposome 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleolylphosphatidylethanolamine and IL-2 plasmid. The optimized transfection condition generated IL-2 levels of 245-305 ng/10(6) cells/24 h, 100-fold higher than the levels seen with retrovirus transfection. Ninety percent of the peak level of IL-2 production was maintained for up to 11 days after transfection. Animal studies were conducted in C3H/HeJ female mice with 2 x 10(4) MBT-2 cells implanted orthotopically on day 0. Multiple vaccination schedules were performed with i.p. injection of 5 x 10(6) IL-2 and/or B7.1 gene-modified cell preparations. The greatest impact on survival was observed with the day 5, 10, and 15 regimen. Control animals receiving retrovirally gene-modified MBT-2/IL-2 cell preparations had a median survival of 29 days. Animals receiving the IL-2 liposomally gene-modified cell preparation alone had a median survival of 46 days. Seventy-five percent of animals receiving IL-2 followed by B7.1 gene-modified tumor vaccines were the only group to show complete tumor-free survival at day 60. All of these surviving animals rejected the parental MBT-2 tumor rechallenge and survived at day 120 with a high CTL response. In conclusion, liposome-mediated transfection demonstrates a clear advantage as compared with the retroviral system in the MBT-2 model. Multi-agent as opposed to single-agent cytokine gene-modified tumor vaccines were beneficial. These "targeted" sequential vaccinations using IL-2 followed by B7.1 gene-modified tumor cells significantly increased a systemic immune response that translated into increased survival.

Phenotypic and functional maturation of dendritic cells mediated by heparan sulfate.

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.

Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro.

Although immunological tolerance to self Ags represents an important mechanism to prevent normal tissue injury, there is growing evidence that tolerance to tumor Ags, which often represent normal peripherally expressed proteins, is not absolute and can be effectively reverted. Prostate-specific Ag (PSA) is a self Ag expressed by both normal and malignant prostatic epithelium, and therefore offers a unique opportunity to examine the ability of self Ags to serve as specific CTL targets. In this study, we investigated the efficacy of autologous dendritic cells (DC) transfected with mRNA encoding PSA to stimulate CTL against PSA Ags in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL responses among many cancer patients independent of their HLA repertoire. In this study, we show that PSA mRNA-transfected DC were capable of stimulating primary CTL responses against PSA Ags in vitro. The PSA-specific CTL did not cross-react with kallikrein Ags, a protein, which shares significant homology with PSA, suggesting that harmful autoimmune toxicity may not represent a significant problem with this approach. PSA RNA-transfected DC generated from male or female healthy volunteers or from cancer patients were equally effective in stimulating PSA-specific CTL in vitro, implying that neither natural tolerance to PSA Ags nor tumor-mediated T cell anergy may represent major barriers for CTL generation against the self Ag PSA. This study provides a preclinical rationale for using PSA RNA-transfected DC in active or adoptive immunization protocols.

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.

How tumors escape immune destruction and what we can do about it.

There is strong circumstantial evidence that tumor progression in cancer patients is controlled by the immune system. As will be detailed below, this conclusion is based on observations that tumor progression is often associated with secretion of immune suppressive factors and/or downregulation of MHC class I antigen presentation functions. The inference is that tumors must have elaborated strategies to circumvent an apparently effective immune response. Importantly, a tumor-specific immune response cannot be detected in most individuals. While this failure is in part technical, it also suggests that the magnitude of the immune responses to which tumors have to respond is low. This raises the concern, which is the underlying theme of this commentary, that a more robust immune response elicited by deliberate vaccination will exacerbate the rate of immune escape and nullify the potential benefits of immune-based therapies.

Induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic malignancies expressing CEA.

The application of dendritic cells (DC) to the active immunotherapy of cancer currently relies on the generation of potent DC capable of presenting tumor antigens such as carcinoembryonic antigen (CEA). It is unknown whether the T cells of patients with advanced malignancies can be reliably stimulated against tumor antigens by their autologous DC. In this study, starting with the peripheral blood mononuclear cells (PBMC) of patients with metastatic malignancies expressing CEA, autologous DCs were generated in vitro in serum-free media supplemented with GM-CSF and IL-4. The DCs from HLA A2 positive patients were loaded with the CEA peptide CAP-1 and the DCs from HLA A2 negative patients were depleted of bystander lymphocytes and loaded with mRNA encoding CEA. The DC preparations were tested to determine their phenotype and were used to stimulate autologous PBMC twice, separated by 10-14 days. The stimulated cells were then tested for their ability to lyse CEA-expressing target cells. We successfully generated an adequate number of DC for a clinical trial from all patients. The harvested DC preparations contained 49% DC and 87% DC if depleted of bystander lymphocytes. Phenotypic analysis showed the typical pattern of CD11c+ CD40+ CD86+ HLA-DR+ CD80(low) CD83(low) CD14(low). All preparations but one were able to stimulate CEA-specific cytotoxic T-lymphocyte (CTL) activity, suggesting that the majority of patients are not anergic to CEA and possess functional DC. The CTL activity was similar for the CEA peptide and CEA RNA-loaded DC.

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.

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.

Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells.

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mphi) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/ME progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.

High level accumulation of an aberrantly spliced human DHFR RNA species.

Cells transduced with either of two human DHFR minigenes express an RNA product which is considerably shorter than what would be predicted from the size of an unspliced transcript expressed from its DNA template. RNA blotting analysis has shown that this short transcript accumulated to exceedingly high levels which were comparable to the levels reached by the highly abundant endogenous actin mRNA, or MoMLV RNA expressed in chronically infected cells. RNA blotting, RNase mapping, primer extension, RT-PCR, and sequencing have shown that this highly expressed transcript, termed TBN, is a spliced RNA product which utilizes cryptic splice signals present in the normally spliced DHFR mRNA. Subcloning experiments have demonstrated that all the information required for the generation and high level accumulation of the TBN transcripts is encoded in the 1.6 kb DHFR DNA minigene. TBN transcripts were generated with comparable efficiency from DNA templates containing either the human ADA or the early SV40 promoters. Since neither the ADA nor the SV40 promoter are considered to be particularly "strong" promoters, this observation argues that initiation of transcription is not the rate limiting step in determining the amount of the TBN transcripts which accumulate in the cell. Insertion of a foreign sequence into the DHFR DNA minigene led to the expression and high level accumulation of a chimeric transcript, suggesting that the unusual properties of this expression system may be used for high level expression of foreign sequences. These observations offer new insights into the mechanisms which control the accumulation of translatable mRNA in the cell, and have potentially important implications for experiments involving optimization of gene expression for gene therapy applications.

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.

Immunotherapy of cancer with dendritic-cell-based vaccines.

Animal studies have shown that vaccination with genetically modified tumor cells or with dendritic cells (DC) pulsed with tumor antigens are potent strategies to elicit protective immunity in tumor-bearing animals, more potent than "conventional" strategies that have been tested in clinical settings with limited success. While both vaccination strategies are forms of cell therapy requiring complex and costly ex vivo manipulations of the patient's cells, current protocols using dendritic cells are considerably simpler and would be more widely available. Vaccination with defined tumor antigens presented by DC has obvious appeal. However, in view of the expected emergence of antigen-loss variants as well as natural immunovariation, effective vaccine formulations must contain mixtures of commonly, if not universally, expressed tumor antigens. When, or even if, such common tumor antigens will be identified cannot be, predicted, however. Thus, for the foreseeable future, vaccination with total-tumor-derived material as source of tumor antigens may be preferable to using defined tumor antigens. Vaccination with undefined tumor-derived antigens will be limited, however, by the availability of sufficient tumor tissue for antigen preparation. Because the mRNA content of single cells can be amplified, tumor mRNA, or corresponding cDNA libraries, offer an unlimited source of tumor antigens. DC transfected with tumor RNA were shown to engender potent antitumor immunity in animal studies. Thus, immunotherapy using autologous DC loaded with unfractionated tumor-derived antigens in the form of RNA emerges as a potentially powerful and broadly useful vaccination strategy for cancer patients.