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.

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.

Patients with metastatic melanoma undergoing therapy with cyclophosphamide (CPM), tumor-infiltrating lymphocytes (TIL), and interleukin-2 (IL-2) were studied for the ability of their 111In-labeled TIL or peripheral blood lymphocytes (PBL) to localize in sites of tumor using gamma camera imaging and biopsies. Nineteen infusions of radiolabeled TIL were given to 18 patients, while five patients received radiolabeled autologous PBL during TIL therapy. Clear tumor localization was seen on 13 of 18 nuclear scan series performed on 111In-TIL recipients, while tumor was imaged in only one of four scan sequences on patients given 111In-PBL. Nineteen paired biopsies of tumor and normal skin were completed on 10 patients receiving 111In-TIL, while eight biopsies were done on three PBL patients receiving 111In-PBL. The mean percentage of total injectate activity localizing per gram of tumor tissue was 0.0049% in the TIL group and 0.0010% in the PBL group (P2 = .0004). The mean of the tumor to normal skin ratios of the 111In-TIL group was three times that for 111In-PBL (P2 = .0072). One patient was studied by nuclear scanning on three consecutive treatment courses of CPM, TIL, and IL-2. He initially demonstrated clear tumor localization by 111In-TIL at several sites, then faint localization with 111In-PBL at a single site, and subsequently positive tumor imaging on repeat 111In-TIL infusion at multiple sites. These results confirm and expand our initial data demonstrating that human TIL transferred with CPM pretreatment and followed by IL-2 preferentially localize to tumor sites and indicate that this localization is greater for TIL than PBL.

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).

Pyramidal inversion about nitrogen has been studied in saturated, cyclic amines containing from four to seven members.Only the nitrogen atom in each ring carries a substituent (chlorine or methyl). The a protons, when decoupled from the ß protons, in all cases change from a singlet to an AB quartet at lower temperatures. Complete line-shape analyses are reported for nitrogen inversion in V-methylazetidine (CHC1F2, £a = 12.5 kcal/mol; log A = 15.3), V-methylpyrrolidine (CHC1F2, Ex = 10.2; log A = 15.0), V-chloropyrrolidine (CFCL, ¿a = 13.8; log A = 16.4), and Á-chlorohomopiperidine (CHC12F, £" = 11.2; log A = 15.3); coalescencetemperature analyses are given for nitrogen inversion in V-chloroazetidine (CHC1F2, AG* (-20) = 13.4 kcal/mol) and (V-methylhomopiperidine (CHC1F2, AG*(-125) = 6.8). The barrier for V-chlorohomopiperidine is found to be relatively insensitive to changes in solvent. The methyl-substituted fiveand seven-membered rings offer the first unambiguous dynamic nmr examples of nitrogen inversion unhindered by ring strain or electronegative substituents. The observed rate processes in the six-membered rings, V-methylpiperidine (reported previously) and V-chloropiperidine (CH2C12, E,x = 17.0 kcal/mol; log A = 15.5), are thought to be ring reversal rather than nitrogen inversion. Unimolecular, pyramidal inversion about nitrogen has been the subject of an increasing number of investigations.(I) 2The rate of nitrogen inversion has frequently proved to be amenable to measurement by the convenient dynamic nuclear magnetic resonance (dnmr) method. The earliest such experiments3 relied on systems such as aziridines with ring strain to raise the barrier to the more accessible range above 10 kcal/mol. Acyclic systems and rings of larger size were avoided because it was thought that the rates would be too rapid for the method. Bottini and Roberts4 reported that A-alkyl-substituted azetidines, pyrrolidines, piperidines, and morpholines, in contrast to the aziridines, showed temperature-independent nmr spectra down to -71°. We report in the present paper that nitrogen inversion rates may be measured in these saturated nitrogen heterocycles by means of dnmr methods by operation at higher magnetic fields and lower temperatures.