Laurence Zitvogel

Bone Marrow‐Derived Dendritic Cells Serve as Potent Adjuvants for Peptide‐Based Antitumor Vaccines

Dendritic cells (DCs) are considered the most effective antigen‐presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM‐CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM‐CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte‐recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5‐14 tumors with peptide‐pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor‐mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide‐pulsed DCs.

IL-12-Engineered Dendritic Cells Serve as Effective Tumor Vaccine Adjuvants in Vivoa

The recent identification of tumor-associated antigens (TAA) and TAA-derived peptides presented by MHC molecules to T cells has provided the tools to design and test clinical vaccines for treating human malignancies, such as melanoma. While the most effective adjuvant for use in vaccine formulation remains unclear, autologous dendritic cells (DC) appear to be good candidate adjuvants. We have previously shown that syngeneic bone marrow-derived DC when pulsed ex vivo with relevant TAA-derived peptides can effectively vaccinate mice against a subsequent challenge with tumor or can effectively treat animals bearing established tumors. In this report, we have engineered murine interleukin-12 (mIL-12), a potent stimulator of cell-mediated immunity, into murine DC using retroviral-mediated or plasmid-based transfection procedures. Transfectants produced up to 25 ng rIL-12/10(6) cells/48 hours. These engineered cells are capable of promoting enhanced anti-tumor, antigen-specific CTL responses compared to nontransduced DC.

Interleukin-7 (IL-7) in colorectal cancer: IL-7 is produced by tissues from colorectal cancer and promotes preferential expansion of tumour infiltrating lymphocytes.

Despite increasing survival rates for patients with colorectal cancer, additional treatment options are required, including active or passive immunotherapy for patients with metastatic disease. Freshly harvested colorectal cancer specimens and in vitro cultured colorectal cancer cell lines were examined for IL–7 protein secretion in order to examine the potential role of this cytokine in the interaction between tumour cells and the host immune system. Freshly harvested colorectal cancer specimens (21/21), or normal adjacent mucosa (3/3), as well as long‐term established colorectal cancer cell lines (3/4) exhibited IL‐7 mRNA expression as detected by RT‐PCR and confirmed by Southern Blot analysis. Freshly harvested colorectal cancer tissue (16/18), or long‐term established colorectal cancer cell lines (2/4) secreted in vitro IL‐7 as detected by ELISA. In contrast, breast, pancreatic, or lung cancer cell lines, as well as several haematopoietic cancer cells lines, tested negative for IL‐7 mRNA and protein. The authors tested different cytokines (IL‐1β, IL‐2, IL‐7, or a combination of IL‐1β/IL‐7) in vitro for the ability to expand tumour ‐ infiltrating T lymphocytes (TIL) from individual patients (n=9) with colorectal cancer. TIL populations were tested at day 14 after in vitro propagation for phenotypic analysis by FACS and for reactivity directed against NK and LAK sensitive target cells and autologous cancer cells as measured by cytotoxicity and cytokine release. TIL obtained from colorectal cancer lesions can be efficiently expanded in the presence of IL‐7, some (3/9) of which appear to exhibit autologous tumour recognition as measured by cytolytic effector functions and by detection of IFNγ and TNFα release. Detection of IL‐7 mRNA expression in colorectal cancer, in normal mucosa adjacent to tumour, as well as the ability of colorectal cancer tissue to secrete IL‐7, raises new questions about the biology of the host / tumour interactions in colorectal cancer.

Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis.

In the past several years, s igdcant advances have been made in the development of gene therapy for the treatment of certain human diseases such as severe combined immunodeficiency’ and cystic f ibr~s is .~ .~ Given these recent successes in gene therapy, researchers are now trying gene transfer as a therapy modality for a wide variety of genetic and acquired diseases. To this end, both viral and nonviral delivery systems have been developed, including retroviruses, adenoviruses, adenoassociated viruses, herpes simplex viruses, picornaviruses, and liposomes, some of which have been approved for use in clinical mals. Of these viral vector systems, retroviral vectors currently represent the best characterized vector and the most utilized delivery system for human gene therapy applications. This communication will discuss the use of retroviral vectors for gene therapy of neoplastic diseases, focusing on the use of a novel heterodimeric cytokine, interleukin (IL,)-12, for immunotherapy of cancer. In addition, the application of retroviral vectors for gene therapy for Gaucher disease, a well-characterized autosomal recessive lysosomal storage disease, and for an acquired autoimmune disease, rheumatoid arthritis, will also be discussed.

Murine Models of Cancer Cytokine Gene Therapy Using Interleukin‐12a

Cytokine gene therapy, in particular IL-12 gene therapy, is one of the more novel and promising approaches in cancer therapy based on significant preclinical data derived mainly from murine tumor models. IL-12 is a heterodimeric cytokine that requires the simultaneous expression of both the p35 and p40 chains from the same cell for production of biologically active IL-12. A variety of biological functions have been attributed to IL-12 including the induction of IFN-gamma production and the promotion of predominantly Th1-type immune responses to antigens. Our previous studies using systemic administration of recombinant murine IL-12 have demonstrated profound antitumor efficacy against all tumors tested with the concomitant long-lived specific antitumor immunity in some cases. To determine whether the local secretion of IL-12 achieved by gene transduction has significant antitumor effects, fibroblast cell lines or murine tumor cell lines were transduced with expression plasmids or the retroviral vector TFG-mIL-12-Neo and inoculated intradermally (i.d.). Our first study using IL-12-transfected NIH3T3 cells admixed with the murine melanoma, BL-6, showed that local IL-12 expression suppresses tumor growth and promotes the acquisition of specific antitumor immunity. Subsequent studies showed that IL-12 gene therapy is also effective in treating established day 3 tumors. CD4+ and CD8+ T cells, as well as NK cells, appear to play important roles in the observed antitumor effects resulting from IL-12 paracrine secretion. Administration of neutralizing antibody specific for IFN-gamma also abrogated some of the IL-12-associated antitumor effects. Finally, this IL-12 gene therapy strategy to elicit an antitumor immune response was more effective when used in combination with the transduction of tumor cells with B7.1. Based on these promising results, a clinical protocol for the treating patients with cancer using genetically engineered fibroblasts to express IL-12 has been initiated at our institution.

Interleukin-12 Gene Therapy Prevents Establishment of SCC VII Squamous Cell Carcinomas, Inhibits Tumor Growth, and Elicits Long-term Antitumor Immunity in Syngeneic C3H Mice†

Interleukin‐12 (IL‐12) is an immunostimulatory agent with very promising antitumor activity. Using a retroviral expression vector, the authors have successfully transduced the genes encoding the two subunits of murine IL‐12 to the squamous cell carcinoma cell line, SCC VII. Once IL‐12 gene transcription and protein production were successfully verified, IL‐12 expression was found to inhibit the establishment of SCC VII tumors in syngeneic C3H/HeJ mice inoculated with 1 × 106 SCC VII/IL‐12 viable tumor cells. Mice immunized in this manner and rechallenged with parent SCC VII were capable of rejecting tumor up to 40% of the time. Treatment of established SCC VII tumors with irradiated IL‐12‐producing tumors cells led to significant tumor regression in a high percentage of animals.