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Immunotherapy of cancer is an old concept and specific or non specific approaches have been developed for decades. These early trials have never demonstrated any benefit and have largely been abandoned. The advent of recombinant techniques allowing to produce large amounts of cytokines, the recent description of tumour antigens expressed by tumor cell surface, the rapid increase in immunological knowledge and the development of molecular biology have rekindled interest in immunological therapy of cancer. Extensive clinical experience using cytokines and more recently monoclonal antibodies is now available and allows us to report on immunotherapy-induced anemia.

This study shows that subcutaneous administration of increasing doses of IL-12, once a week, in 21 cancer patients increased the expression of cytokine genes (interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), IP-10, MIG, IL-10, IL-4) in peripheral blood mononuclear cells even at very low doses (30 ng/kg). Surprisingly, no circulating TNF-alpha or IL-4 could be detected in the plasma of patients treated with IL-12. However, a marked increase of soluble IL-4 receptor was demonstrated in the plasma of five of the six patients studied, which may represent an additional mechanism by which IL-12 inhibits the development of the Th2 response in vivo. A marked decline of IFN-gamma and IP10 induction was recorded after repeated cycles of IL-12. In contrast, in most patients IL-12 increased IL-10 expression with no subsequent decrease during the course of therapy, and even an earlier peak of IL-10 induction at the 6th cycle. In addition, a constant up-regulation of serum soluble IFN-gamma receptor levels was observed after each cycle of IL-12 treatment with a delayed peak compared with the IFN-gamma peak. The constant rise of IL-10 and soluble IFN-gamma receptor during IL-12 therapy may therefore contribute to the inhibition of IFN-gamma activity detected after repeated cycles of IL-12. Lastly, a marked heterogeneity of cytokine induction was observed from one patient to another, which appeared to be independent of the dose of IL-12 administered. These data may lead to a better understanding of the biological activity of IL-12 and the in vivo mechanisms of its regulation.

On the basis of compelling preclinical data in cats and dogs we initiated a clinical gene therapy study in nine patients with advanced solid tumors using xenogeneic fibroblasts secreting human IL-2 (Vero-IL-2 cells). Cohorts of three successive patients with tumors accessible to CT- or ultrasound-guided injection were treated repeatedly with 5 x 10(5), 5 x 10(6), or 5 x 10(7) Vero-IL-2 cells. Endpoints of the study were feasibility, toxicity, and clinical and biological effects of this novel approach to immunotherapy of cancer. Histopathological, immunological and molecular analyses were performed on biopsy specimens of tumors and blood samples from before, during and after treatment. Low levels of serum antibodies to Vero cells developed in 2/9 patients. Analysis of tumor biopsies showed increased expression of CD3 mRNA and enhanced tumor infiltration with varying lymphocyte subpopulations after treatment. In addition, monoclonal alterations of the TCR repertoire of blood and tumor lymphocytes were observed. Treatment was well tolerated and toxicity consisted of transient fever in one patient and short-lived, mild itching and erythema in two others. One patient with soft tissue sarcoma showed a more than 90% and more than 50% reduction of the volume of two distant, non-injected metastases, respectively, lasting for 22+ months. Four other patients showed stabilization of their disease for three to nine months, among whom was a patient with melanoma who developed marked vitiligo. We conclude that repeated injection of up to 5 x 10(7) Vero-IL-2 cells was safe and showed biological and clinical activity in heavily pretreated patients with advanced solid tumors. Further evaluation of intratumoral application of Vero-IL-2 seems warranted.

The recent discovery of the molecular structure of tumor antigens expressed by melanoma cells has contributed to the development of therapeutic strategies aimed at stimulating anti-tumor immunity. The first clinical trials in patients with cancer involved systemic administration of interleukin 2 alone or in combination with alpha-interferon or other chemotherapy agents. Objective clinical responses were observed in 15 to 25% of the patients with melanomas or renal cell cancer. In order to improve these encouraging early results, different clinical protocols are being evaluated with the goal of increasing intra-tumoral concentrations of the administered cytokines. Better optimization of methods for immunizing against specific tumor antigens naturally leads to the transition from non-specific stimulation of the human immune system to tumor antigen specific immunotherapy.

Cancer immunotherapy has been carried out since the early fifties and first involved nonspecific system (BCG, Corynebacterium parvum, levamisole...). More recently, the production of cytokines as interferons or interleukin 2, the introduction of monoclonal antibodies have allowed a new development to cancer immunotherapy. Nevertheless, these new approaches have to be considered as a step in the biological therapy of cancer.

Alterations of immunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL-2. Five-day infusions of rIL-2 at doses from 1 x 10(6) to 24 x 10(6) biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 x 10(6) U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analyzed at days 0, 8 and 11 of the rIL-2 courses. Following a leucopenia during rIL-2 infusion, a lymphocytosis was found in all patients except one. The lymphocytosis peaked at day 8 and was detected at doses of rIL-2 as low as 1 x 10(6) U/m2 per day, reaching a plateau at a dose of 16 x 10(6) U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL-2, some patients had predominant T cells (CD3+, NKH1(CD56)-), others had predominant natural killer (NK) cells (CD3-, NKH1 (CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL-2 was preceded by a course of 5 days infusion of 2 x 10(6) U/m2 per day recombinant interferon-gamma (rIFN-gamma). The infusion of rIFN-gamma prior to rIL-2 had no effect on the rIL-2-induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL-2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low-dose rIFN-gamma does not modify the immune stimulation by rIL-2.

Active immunotherapy of cancer is based on the hyperactivation of the immune system of tumor-bearing patients. Interleukin 2 (IL2) has proven to be the most potent immunostimulant used so far. Therapeutic activity of IL2 and IL2-activated lymphocytes has been reported in the treatment of certain tumors such as metastatic melanoma and renal carcinoma. Effectiveness is related to the dose of IL2 administered to the patients, as are important toxic effects. Other cytokines, such as interferon (IFN) and tumor necrosis factor (TNF), are used for their direct effect on malignant cells, particularly those of the lymphoid lineage. The development of these new immunotherapeutic approaches will depend on the quality of their scientific bases, the clinical follow-up of the toxic effects, and the social management of the costs.

A potential activity of interferons in the treatment of solid tumors has been suggested by in vitro experiments and, more recently, by phase I trials. Phase II trials have demonstrated a possible antitumoral activity in treating some solid tumors: malignant melanoma, renal cell carcinoma, ovarian adenocarcinoma, carcinoid tumor, superficial bladder cancer. Further trials are necessary to establish the precise role of interferons in treating solid tumors, when administered alone or in combination.