Smita K. Nair

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 patients 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.

Immunotherapy of cancer with dendritic-cell-based vaccines

Abstract 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.

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 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.

Regression of tumors in mice vaccinated with professional antigen-presenting cells pulsed with tumor extracts

Vaccination with tumor extracts circumvents the need to identify specific tumor rejection antigens and extends the use of active immunotherapy to the vast majority of cancers, in which specific tumor antigens have not yet been identified. In this study we examined the efficacy of tumor vaccines comprised of unfractionated tumor material presented by professional antigen‐presenting cells (APC): dendritic cells (DC) or macrophages (Mø). To enhance the relevance of these studies for human patients we used 2 poorly immunogenic murine tumor models and evaluated and effectiveness of the vaccination protocols in tumor‐bearing animals. APC (in particular DC) pulsed with unfractionated extracts from these “poorly immunogenic” tumors were highly effective in eliciting tumor‐specific cytotoxic T lymphocytes. A measurable CTL response could be detected after even a single immunization with tumor extract‐pulsed DC. DC or Mø pulsed with tumor extract were also effective vaccines in tumor‐bearing animals. In the murine bladder tumor (MBT‐2) model a modest extension of survival and 45% cure rate was seen in the animal groups immunized with DC or Mø pulsed with MBT‐2 tumor extract. DC or Mø pulsed with B16/F10.9 tumor extract were also remarkably effective in the B16 melanoma lung metastasis model, as shown by the observation that treatment with APC caused a significant reduction in lung metastases. Cumulatively, the CTL and immunotherapy data from the two murine tumor systems suggest that APC (in particular DC) pulsed with unfractionated cell extracts as a source of tumor antigen may be equally or more effective than genetically modified tumor vaccines. Int. J. Cancer 70:706–715, 1997.

Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

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+ CD80lowCD83lowCD14low. 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. Int. J. Cancer 82:121–124, 1999.

Induction of Tumor-Specific Cytotoxic T Lymphocytes in Cancer Patients by Autologous Tumor RNA-Transfected Dendritic Cells

ObjectiveTo demonstrate the feasibility of inducing tumor antigen-specific immune responses in patients with metastatic cancer using total tumor RNA-loaded dendritic cells (DCs). Summary Background DataThe authors have shown that DCs transfected with mRNA encoding defined tumor antigens induce tumor antigen-specific T-cell responses in vitro and in vivo. There may be significant advantages to inducing immune responses against the entire repertoire of antigens expressed by a patient’s autologous tumor. MethodsRNA was extracted from a metastatic colon cancer and used to load autologous DCs. The DCs were coincubated with autologous T cells and the cytolytic activity of the T cells was assessed by the ability to lyse the autologous tumor cells. RNA was then extracted from a metastatic lung cancer and used to load autologous DCs, followed by four injections of the DC vaccine given every 4 weeks. Tumor antigen-specific cytotoxic T lymphocyte activity was then evaluated by testing peripheral blood mononuclear cells for their ability to lyse an antigen-expressing target. ResultsDCs transfected with the total RNA content of autologous tumor cells stimulated antigen-specific T-cell responses that are capable of recognizing and lysing autologous, primary tumor cells in vitro. Tumor-specific immune responses were induced in a patient with a carcinoembryonic antigen-expressing adenocarcinoma after immunization with autologous DCs transfected with total tumor RNA. ConclusionsDCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of tumor antigens of surgically resected malignancies.

Injection of Immature Dendritic Cells into Adjuvant-Treated Skin Obviates the Need for Ex Vivo Maturation

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. 111Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.

RNA-transfected dendritic cells in cancer immunotherapy.

The field of cancer immunotherapy has been recently invigorated by the discovery that vaccination with dendritic cells (DCs) pulsed with tumor antigens is a potent strategy to elicit protective immunity in tumor-bearing animals. The recognition that the cellular arm of the immune response is best equipped to recognize tumor cells as foreign and to lead to their eradication has shifted the emphasis in vaccine development. Vaccines that induce cellular responses, especially by the CD8+ cytotoxic T lymphocyte (CTL) arm of the immune system, are now favored over those that activate humoral immunity. At the same time, DCs have emerged as the most potent antigen-presenting cells (APCs) for eliciting antitumor CTLs. DCs can be generated from cancer patients by culturing adherent PBMCs from the patients for 5–7 days in the presence of cytokines (1). The major research effort in many labs involves the choice of tumor antigen with which to load these DCs. The issues being addressed are, first, the composition of the antigen, whether a defined tumor antigen or an unfractionated mixture of tumor-derived antigens; and second, the form in which the antigen should be presented, whether as a polypeptide or a nucleic acid. Here, we focus on the use of RNA-transfected DCs in cancer immunotherapy, reviewing current data on the transfection of DCs with RNAs encoding either defined or unfractionated tumor antigens. We also consider the advantages and disadvantages of employing RNA transfection in loading DCs with tumor antigens and the merits of RNA transfection in situations where a low level of transiently expressed protein is sufficient to allow antigen presentation.