Paul Aebersold

Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report.

Lymphocytes extracted from freshly resected melanomas can be expanded in vitro and can often mediate specific lysis of autologous tumor cells but not allogeneic tumor or autologous normal cells. We treated 20 patients with metastatic melanoma by means of adoptive transfer of these tumor-infiltrating lymphocytes and interleukin-2, after the patients had received a single intravenous dose of cyclophosphamide. Objective regression of the cancer was observed in 9 of 15 patients (60 percent) who had not previously been treated with interleukin-2 and in 2 of 5 patients (40 percent) in whom previous therapy with interleukin-2 had failed. Regression of cancer occurred in the lungs, liver, bone, skin, and subcutaneous sites and lasted from 2 to more than 13 months. Toxic effects of interleukin-2 occurred, although the treatment course was short (five days); these side effects were reversible. It appears that in patients with metastatic melanoma, this experimental treatment regimen can produce higher response rates than those achieved with interleukin-2 administered alone or with lymphokine-activated killer cells. It is too early to determine whether this new form of immunotherapy can improve survival, but further trials seem warranted.

Gene transfer into humans--immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction.

BACKGROUND AND METHODS Treatment with tumor-infiltrating lymphocytes (TIL) plus interleukin-2 can mediate the regression of metastatic melanoma in approximately half of patients. To optimize this treatment approach and define the in vivo distribution and survival of TIL, we used retroviral-mediated gene transduction to introduce the gene coding for resistance to neomycin into human TIL before their infusion into patients--thus using the new gene as a marker for the infused cells. RESULTS Five patients received the gene-modified TIL. All the patients tolerated the treatment well, and no side effects due to the gene transduction were noted. The presence and expression of the neomycin-resistance gene were demonstrated in TIL from all the patients with Southern blot analysis and enzymatic assay for the neomycin phosphotransferase coded by the bacterial gene. Cells from four of the five patients grew successfully in high concentrations of G418, a neomycin analogue otherwise toxic to eukaryotic cells. With polymerase-chain-reaction analysis, gene-modified cells were consistently found in the circulation of all five patients for three weeks and for as long as two months in two patients. Cells were recovered from tumor deposits as much as 64 days after cell administration. The procedure was safe according to all criteria, including the absence of infections virus in TIL and in the patients. CONCLUSIONS These studies demonstrate the feasibility and safety of using retroviral gene transduction for human gene therapy and have implications for the design of TIL with improved antitumor potency, as well as for the possible use of lymphocytes for the gene therapy of other diseases.

Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients.

We have administered 1039 courses of high-dose interleukin-2 (IL-2) to 652 cancer patients. Five hundred ninety-six patients had metastatic cancer that either had failed standard effective therapies or had disease for which no standard effective therapy existed, and 56 patients were treated in the absence of evaluable disease in the adjuvant setting. IL-2 was administered either alone (155 patients) or in conjunction with activated immune cells such as lymphokine activated killer (LAK) cells (214 patients) or tumor infiltrating lymphocytes (TIL) (66 patients), with other cytokines such as alpha interferon (a-IFN)(128 patients) or tumor necrosis factor (TNF)(38 patients), with monoclonal antibodies (32 patients), or with the chemotherapeutic agent cyclophosphamide (19 patients). Initial results with the treatment of high-dose IL-2 alone or in conjunction with LAK cells have indicated that objective regressions of cancer can be achieved in 20% to 35% of patients with selected advanced metastatic cancers. Although most responses have been seen in patients with metastatic renal cell cancer, melanoma, colorectal cancer, and non-Hodgkins lymphoma, many histologic types of cancer have not been treated in significant numbers. These regressions can be durable; of 18 patients achieving a complete response, ten have not experienced recurrence at intervals from 18 to 52 months. Although combinations of IL-2 with TNF do not appear to result in increased responses, there is a suggestion in our initial phase I studies that the combination of a-IFN and IL-2 is more effective than the administration of cytokine alone and this combination deserves further study. Similarly the adoptive transfer of TIL in conjunction with IL-2 also appears to be more effective than the use of IL-2 alone. The toxic side effects in patients treated with high-dose IL-2 are presented and include malaise, nausea and vomiting, hypotension, fluid retention, and organ dysfunction. Treatment-related deaths were seen in 1% of all treatment courses and in 1.5% of patients. These studies demonstrate that a purely immunologic manipulation can mediate the regression of advanced cancers in selected patients and may provide a base for the development of practical, effective biologic treatments for some cancer patients.

Culture of human tumor infiltrating lymphocytes in hollow fiber bioreactors

Human tumor infiltrating lymphocytes (TIL) from metastatic melanoma of six patients were grown using a new hollow fiber bioreactor system. After inoculating 0.35-10 X 10(8) TIL into the extra-fiber space (EFS), each Cellmax bioreactor was perfused with AIM-V medium, supplemented with rIL-2. The cells subsequently expanded 124-1170-fold to yield 1.5-5.4 X 10(10) TIL over a 14-32 day period. TIL were flushed from the EFS using 200 ml medium and possessed an average viability = 91%. The phenotype and the autologous tumor cell lytic capacity of these TIL were similar to those of TIL grown in the currently used gas-permeable culture bags. Tissue culture media use averaged 4.3 liters/10(10) TIL harvested. The TIL of one patient were re-expanded twice from cells remaining within the same bioreactor after harvest suggesting that one bioreactor cartridge could be used for repetitive, periodic studies. An estimated 80% decrease in technical time expended and in incubator space requirements were realized using this methodology. Cell culture on hollow fibers appears to be a useful method for producing large quantities of primary human lymphocytes for experimental, and perhaps, therapeutic needs.

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

SummaryStudies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneoR gene. The presence of theneoR gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneoR gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4+ and CD8+ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4+ and CD8+ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4+ or CD8+ cells. In other experiments highly purified CD4+ and CD8+ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneoR gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4+ and CD8+ gene-modified TIL.

A simplified automated procedure for generation of human lymphokine-activated killer cells for use in clinical trials

The administration of lymphokine-activated killer (LAK) cells along with interleukin-2 (IL-2) can mediate regression of tumors in selected patients. A closed automated system utilizing commercial blood cell separators has been developed for washing and Ficoll-Hypaque (FH) separation of lymphocytes, for lymphocyte culture in polyolefin bags, and for concentration of LAK cells out of culture prior to infusion. We now demonstrate that preparation of LAK cells can be simplified by elimination of the FH sedimentation step. Patient leukapheresis was performed using Fenwal CS-3000 blood cell separators, with a mean cellular yield per procedure of 54 X 10(9) WBC (95% lymphocytes), 184 X 10(9) RBC, and 306 X 10(9) platelets (n = 22). These cells were then washed in the same apheresis kit with a counter-current flow of saline, thereby eliminating 85% of platelets while retaining 88% of WBC. Aliquots of the washed cells were separated on FH gradients in 50 ml centrifuge tubes, and both FH-separated and washed-only cells were cultured at 3 X 10(6)/ml with 1500 U/ml IL-2 in polyolefin bags. Cytotoxicities of 22 preparations of LAK cells from 14 patients were evaluated in 4 h 51Cr release assays. Cells that were washed-only averaged 47, 35, and 9 lytic units/10(6) cells against K562, Daudi, and fresh tumor, while FH separated cells averaged 46, 33, and 6 lytic units/10(6) cells respectively. Cellular recoveries using the wash-only technique were 25% greater than when using FH sedimentation. Omission of FH separation saves time and expense in preparation and provides greater numbers of LAK cells for use in adoptive immunotherapy.