Clinical trials utilising interleukin (IL)-2 with tumor-infiltrating lymphocytes (TIL) have demonstrated efficacy in the treatment of metastatic renal cell carcinoma (RCC). Several cytokine's, as well as growth factors have demonstrated modulatory effects upon the biological properties of TIL from RCC, suggesting a potentially important role for cytokines other than IL-2 in the development of active and tumor-specific TIL. IL-12 was recently characterized as a natural-killer-cell-stimulatory factor or cytotoxic-T-cell-maturation factor. These properties of IL-12 prompted us to investigate the impact of this cytokine upon the activation of TIL from human RCC. In an attempt to enhance the in vitro growth and activity of renal TIL, we have grown eight renal TIL cultures in varying concentrations of IL-2 (8, 40, 80, 400 U/ml) and IL-12 (200 U/ml). In addition, IL-12 (200 U/ml) was added to TIL cultures pre-activated with IL-2 (400 U/ml). Growth, cell expansion, and the ability of TIL to release certain cytokines upon tumor stimulation were determined. Proliferation assays, phenotypic analysis, and cytotoxicity assays were performed at an early and a late culture stage. IL-12, alone and when added to suboptimal concentrations of IL-2, failed to induce TIL growth. While the addition of IL-12 to optimal doses of IL-2 suppressed TIL culture expansion, sequential culture exposure first to IL-2 and then to IL-2 + IL-12 increased the number of cells expressing CD3(+)/CD56(+) and these cultures demonstrated enhanced in vitro lysis of autologous tumor. IL-12 clearly demonstrated a sequence-dependent impact of the biological behaviour of TIL from RCC. The optimal use of IL-12 in the in vitro expansion of renal TIL may result in cells with an enhanced specific anti-tumor effect.

Combination therapy with systemically administered interleukin-2 (IL-2) and tumor infiltrating lymphocytes (TIL) demonstrates significant clinical activity in some patients with metastatic renal cell carcinoma (RCC). The objective of this study was to identify predictors of therapeutic response in patients with IL-2- and TIL-based immunotherapy. We characterized and compared immunologic properties of tumors, TILs, peripheral blood lymphocytes (PBLs) and sera of responding (R, n = 8) with nonresponding patients (NR, n = 9). Before undergoing nephrectomy, responding patients exhibited a higher percentage of circulating natural killer (NK) cells (CD56+ CD3-) (43 +/- 20%) as compared with nonresponders (18 +/- 16%) (p < 0.01). After nephrectomy, the CD56+ CD3-/CD56- CD3+ ratio in responding patients (pre: 2.60 +/- 2.24; post: 0.28 +/- 0.19; p < 0.05) significantly decreased and was similar to that of patients not responding to therapy (0.42 +/- 0.36). Sera from patients responding to immunotherapy, obtained before and after completion of therapy, contained natural killer (NK)-enhancing factor(s) that significantly enhanced the proliferation (3.2 x 10(3) +/- 25%/ 3.6 x 10(3) +/- 13% counts/min) and cytotoxicity [17.6 +/- 4.0/18.0 +/- 1.9 lytic units (LU)] of fresh PBLs as compared with normal serum (1.8 x 10(3) +/- 8% counts/min; 13.4 +/- 2.5 LU) or sera from nonresponders (1.6 x 10(3) +/- 25%/1.5 x 10(3) +/- 20% counts/min; 8.3 +/- 5.9/6.8 +/- 4.8 LU). In contrast to noncultured tumor suspension, IL-2 cultivation induced TIL growth, cytotoxicity, and multicytokine synthesis, and a complete clearance of tumor cells. No significant differences were observed between responders and nonresponders in the in vitro characteristics of tumor/TIL, which include the degree of intratumoral lymphocytic infiltrate, TIL expansion, specific lysis of autologous tumor, phenotype, expansion time, quantity of TIL infused, cytokine release, and degree of tumor aggressiveness. We conclude that clinical response to TIL and IL-2-based immunotherapy is associated with patients' baseline natural immune status. The percentage of circulating NK cells and the presence of serum NK-cell-enhancing factors may serve as potential predictors of response in patients with advanced RCC. The in vitro study of RCC-TIL suggests that activated TIL may provide a synergistic effect to that of administered IL-2 on activation of cellular immune response in situ, rendering a tumor eradication, while the clinical outcome is largely dependent on the pretreatment immune status of patient.

The approach to disseminate renal cell carcinoma (RCC) has evolved significantly in recent years, largely owing to the progress of biologic therapy development. With increasing knowledge of biologic therapy come several dilemmas, including the choice of cytokine therapy, the appropriate mode of delivery, the selection of candidates for given therapeutic options, and the role of cytoreductive nephrectomy in immunotherapy protocols. This article reviews the historical development of immunotherapy and the lessons learned from previous experience and offers a logical approach to the patient with disseminated RCC.

Two distinct types of soluble tumor necrosis factor alpha receptors (sTNFRs), which are felt to represent proteolytic cleavage products of the extracellular domains of membrane-bound TNFRs of molecular mass, 55 and 75 kDa, are found in the serum and urine of humans. We have measured the serum concentrations of these receptors in eight patients with metastatic renal cell carcinoma during treatment with interleukin-2 (IL-2)-based immunotherapy. The mean pretreatment concentration of sTNFR-55 kDa (p < 0.05) but not sTNFR-75 kDa was significantly elevated prior to the onset of immunotherapy. In one patient the concentrations of both sTNFRs decreased dramatically following removal of the primary tumor. There were significant increases in the concentrations of both sTNFRs in patients treated with IL-2-based therapy that included in vivo primed tumor infiltrating lymphocytes (pTILs); some of these patients eventually achieved a complete response to therapy. These data demonstrate that sTNFR-55 kDa is elevated in patients with metastatic renal cell carcinoma prior to therapy and that IL-2-based therapy that included pTIL cells results in a further increase in sTNFRs.

Murine models demonstrate therapeutic synergy for the combination of interleukin-2, interferon-alpha and tumor-infiltrating lymphocytes. We treated 11 patients with metastatic renal cell carcinoma with a novel regimen consisting of in vivo primed tumor-infiltrating lymphocytes, interferon-alpha and interleukin-2. Patients received interferon-alpha before radical nephrectomy; in vivo primed tumor-infiltrating lymphocytes were isolated and expanded in vitro. Low dose continuous infusion interleukin-2 at a dose of 2 x 10(6) units per m.2 per day was administered for 96 hours during each treatment week and interferon-alpha was administered as a subcutaneous injection at a dose of 6 x 10(6) units per m.2 per day on days 1 and 4 of the interleukin-2 infusion. No therapy was given during the last 3 days of a treatment week. One course of therapy consisted of 3 weeks of therapy followed by 3 weeks of rest. Patients were treated until maximal response, disease progression or dose limiting toxicity. A maximum of 6 courses of therapy were administered. Eleven patients underwent interferon-alpha priming and subsequent radical nephrectomy. In vivo primed tumor-infiltrating lymphocytes were successfully expanded in all 11 patients with an expansion index of greater than 170. In vivo primed tumor-infiltrating lymphocytes maintained their lytic activity for greater than 5 to 8 weeks in culture as demonstrated in the 4-hour 51chromium release assay. Ten patients underwent multimodality biological therapy and 3 (30%, 95% confidence interval 6 to 65%) have achieved complete response (2 clinical and 1 surgical) with durations of 24+, 23+ and 5+ months. Patients with stable disease received no additional therapy. No deaths and no grade 4 toxicities occurred. Immunotherapy using a combination of interferon-alpha primed tumor-infiltrating lymphocytes, low dose continuous infusion interleukin-2 and interferon-alpha can induce significant and durable antitumor responses in some patients with advanced renal cell carcinoma.