Phase I clinical trial with IL-2-transfected xenogeneic cells administered in subcutaneous metastatic tumours: clinical and immunological findings.

Various studies have emphasized an immunodepression state observed at the tumour site. To reverse this defect and based upon animal studies, we initiated a phase I clinical trial of gene therapy in which various doses of xenogeneic monkey fibroblasts (Vero cells) genetically engineered to produce human IL-2 were administered intratumorally in 8 patients with metastatic solid tumours. No severe adverse effect was observed in the 8 patients analysed during this clinical trial even in the highest dose (5 yen 107 cells) group. This absence of toxicity seems to be associated with rapid elimination of Vero-IL-2 cells from the organism. Indeed, exogenous IL-2 mRNA could no longer be detected in the peripheral whole blood 48 hours after Vero-IL-2 cell administration. In addition, we did not find any expression of exogenous IL-2 mRNA in post-therapeutic lesions removed 29 days after the start of therapy. A major finding of this trial concerns the two histological responses of two treated subcutaneous nodules not associated with an apparent clinical response. The relationship between local treatment and tumour regression was supported by replacement of tumour cells by inflammatory cells in regressing lesions and marked induction of T and natural killer cell derived cytokines (IL-2, IL-4, IFNg ...) in post-therapeutic lesions analysed 28 days after the start of Vero-IL-2 administration. Gene therapy using xenogeneic cells as vehicle may therefore present certain advantages over other vectors, such as its complete absence of toxicity. Furthermore, the in vivo biological effect of immunostimulatory genes, i.e IL-2-, may be potentiated by the xenogeneic rejection reaction.

Interleukin 17, a T-cell-derived cytokine, promotes tumorigenicity of human cervical tumors in nude mice.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

Major histocompatibility complex class I presentation of exogenous soluble tumor antigen fused to the B-fragment of Shiga toxin.

Targeting exogenous antigen into the MHC class I-restricted presentation pathway is a prerequisite for the induction of cytotoxic T lymphocytes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections and tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin derived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins carrying an active or inactive endoplasmic reticulum retrieval signal (the C-terminal peptides KDEL or KDELGL, respectively) could be presented by peripheral blood mononuclear cells in an MHC class I-restricted manner to Mage 1-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In further analysis, we showed that treatment with brefeldin A or paraformaldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracellular processing of the antigen. Immunofluorescence analysis also revealed that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct antigen into the exogenous class I-restricted pathway makes it an attractive non-living and non-toxic vaccine vector.

Characterization of soluble gp130 released by melanoma cell lines: A polyvalent antagonist of cytokines from the interleukin 6 family.

gp130 acts as a common transducing signal chain for all receptors belonging to the interleukin (IL)-6 receptor family. The IL-6-related cytokines [IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin] often modulate tumor phenotype and control the proliferation of many tumor cell lines. We demonstrate that melanoma cell lines release, in vitro and in vivo (when transplanted in nude mice), soluble gp130 (sgp130), a potential antagonist of cytokines from the IL-6 family. Biochemical analysis revealed that sgp130 derived from melanoma patients' sera or from culture supernatants of melanoma cell lines is a Mr 104,000 protein that resolved after deglycosylation as a Mr 58,000 protein. PCR and Northern blot analysis only identified one gp130 membrane mRNA, suggesting that the soluble form of gp130 is generated by proteolytic cleavage. OSM reproducibly increases sgp130 released by melanoma cell lines, whereas leukemia inhibitory factor stimulates the production of sgp130 in only one of three cell lines tested. This tumor-derived sgp130 is functional because it binds in solution to the IL-6-soluble IL-6 receptor (gp80) complex. Recombinant sgp130 inhibits the growth inhibitory activity of the IL-6-soluble IL-6 receptor complex and OSM on some melanoma cell lines. Therefore, this soluble gp130 represents a natural antagonist of cytokines from the IL-6 family.

Variable expression of CD3-zeta chain in tumor-infiltrating lymphocytes (TIL) derived from renal-cell carcinoma: relationship with TIL phenotype and function.

It has been reported that in mice and in humans, tumor-infiltrating lymphocytes (TIL) may exhibit a defect in CD3-zeta-chain expression. Therefore, the level of CD3-zeta was analyzed in TIL derived from patients with renal-cell carcinoma, and its correlation with TIL phenotype and function was assessed. We identified 4 out of 13 tumor-infiltrating lymphocytes derived from renal-cell carcinoma, with a significant decrease in CD3-zeta-chain expression as compared with control peripheral-blood lymphocytes. This defect was never found after culturing TIL with IL2. In one case, the low expression of zeta chain observed in TIL on day 0 was reversed when TIL were cultured with IL2. The zeta-chain level did not seem to predict the growth of TIL in response to IL2. All the TIL, irrespective of the level of zeta-chain expression, exhibited lower proliferative response when stimulated with PHA or anti-CD3 MAb in comparison with normal peripheral-blood mononuclear cells. Nevertheless, in this limited series of patients, a correlation was observed between the level of zeta-chain expression and T-cell infiltration (p = 0.02). After TIL stimulation with PHA or anti-CD3, in contrast to IL2 or IFN-gamma production, a trend towards a relationship between TNF alpha induction and the level of zeta-chain expression was observed.

Serum interleukin 6 and C-reactive protein levels correlate with resistance to IL-2 therapy and poor survival in melanoma patients.

Interleukin 6 and C-reactive protein (CRP) were determined prior to IL-2 therapy in sera from metastatic melanoma patients. Patients with elevated serum IL-6 (> 20 pg ml-1) and/or CRP (> 10 mg l-1) levels were associated with resistance to IL-2 therapy. A correlation between high serum IL-6 levels and a shorter median survival was also observed.

Phenotypic and functional analysis of tumour-infiltrating lymphocytes from patients with renal cell carcinoma.

Twenty renal cell carcinomas were cultured in the presence of 200 IU/ml of recombinant interleukin-2; tumour-infiltrating lymphocytes (TIL) developed in 70% of cases; the phenotypic profile was heterogeneous in 11 TIL tested on day 30: 4 were mostly CD8 positive, 4 mostly CD4 and 3 showed CD4-CD8-CD56 mixed phenotypes. The cytotoxic activity was also heterogeneous, and no TIL developed a tumour-specific cytotoxic activity. The phenotypic profile and cytotoxic activity were also tested after thawing, and this study demonstrated that TIL can be frozen at the time of the nephrectomy, then thawed and cultured for the purposes of therapeutic trials when metastases appear. The differences between TIL derived from renal cell carcinoma and TI1 derived from melanoma are discussed.

[CD4 TIL (Tumor Infiltrating Lymphocytes) induce complete response in patients treated with IL-2 (Interleukin-2). Preliminary study]. / Les TIL (Tumor Infiltrating Lymphocytes) CD4 induisent des résponses complètes chez des patients traités par IL-2 (Interleukin-2). Etude préliminaire.

This study analyzed clinical response induced by TIL in patients with renal cell carcinoma previously treated by interleukin-2. Six patients (4 men, 2 women, mean age 44.3 years) with measurable metastatic localizations have been treated by TIL reinjection (0.02 to 7.6 x 10(10) cells). TIL phenotype was a combination of CD4 and CD8 in 3 patients, predominantly CD4 in two patients and predominantly CD8 in one patient. Previous treatment by interleukin-2 induced one partial response, 2 stabilizations of the disease and 3 tumoral progressions. TIL led to an amelioration for 4 patients: 2 were in complete response, 2 were stabilized and 2 had tumoral progression and decreased. This study shows that CD4 TIL may improve an initially response induced by IL-2 therapy.