Adoptive immunotherapy for stage IV renal cell carcinoma: A novel protocol utilizing periodate and...

CLINICAL STUDIES

Adoptive Immunotherapy for Stage IV Renal Cell Carcinoma: A Novel Protocol Utilizing Periodate and Interleukin-Z-Activated Autologous Leukocytes and Continuous Infusions of Low-Dose Interleukin-2

JOHN WANG, M.D., Ph.D. ALEXANDER WALLE, M.D., Ph.D. BRUCE GORDON, M.D. ABRAHAM NOVOGRODSKY, M.D., MANIKKAM SUTHANTHIRAN, M.D. ALBERT L. RUBIN, M.D. HARVEY MORRISON, M.D. RICHARD T. SILVER, M.D. KURT H. STENZEL, M.D.

New York, New York

Ph.D.

In a pilot study involving 13 patients with advanced stage IV renal cell carcinoma, anti-tumor effects and toxicity of a novel form of adoptive immunotherapy were determined. The protocol utilizes infusions of autologous mononuclear leukocytes treated with the oxidizing mitogen sodium periodate (lO;i) and cultured in medium containing human recombinant interleukin-2 (IL-2), and continuous infusions of low-dose IL-2 (mean f SD dose = 39.5 f 8.8 X lo3 U/kg/24 hours). Leukocytes (5 to 10 X log) were removed by leukapheresis three times per week, mononuclear cells were separated, activated with 104 and cultured in medium containing IL-2 (500 U/ml) for 48 to 72 hours. The cells were re-infused following the next leukapheresis procedure. IL-2 was administered five days per week. Treatment was continued for two three-week cycles. An increase in peripheral blood mononuclear cells bearing the natural killer cell (NK) surface marker, Leu 11, an increase in NK- and antibody-dependent cell-mediated cytotoxicity, and a slight increase in spontaneous cytotoxicity for non-NK targets were noted. Regresskms (more than 50 percent decrease in tumor mass) of pulmonary, liver, bone, or soft tissue metastases were induced in six patients. Severe fluid retention did not develop in any patient and no patient required treatment in the intensive care unit. Five of the patients who showed a response have experienced a relapse at 5.2 f 1 .O (mean f SD) months. These observations indicate that lOa/ IL-2-activated killer cells plus continuous infusions of low-dose IL-2 can result in regression of metastatic renal cell carcinoma.

From the Rogosin Institute, and the Departments of Medicine, Surgery, and Biochemistry, Cornell University Medical College and the New York Hospital, New York, New York. This work was supported in part by Public Health Service Re- search Grant RR00047 from the Division of Re- search Resources, National Institutes of Health, and by grants from the Elsa U. Pardee Foundation and the HRC Foundation. Requests for reprints should be addressed to Dr. Kurt H. Stenzel, Ro- gosin Institute, 429 East 71st Street, New York, New York 10021. Manuscript submitted August 18, 1987, and accepted September 24, 1987.

An attractive form of immunotherapy for cancer is adoptive transfer of autologous peripheral blood mononuclear leukocytes that have been activated to exhibit cytotoxicity for tumor targets. Recentiy, preliminary studies were reported on the use of autologous lymphokine-activated killer (LAK) cells and systemic administration of interleukin-2 (IL-2) in patients with advanced cancer [I]. LAK cells are cytotoxic for fresh tumor cells in vitro [2] and, either alone [3] or in combination with systemic IL-2 [4], inhibit pulmonary metastases in mouse tumor models. Objective evidence of tumor regression was noted in 11 of 25 patients in the first clinical study reported [I]. Subsequent studies by Rosenberg et al [5] and West et al [6] have confirmed the observation that tumor

1016 December 1967 The American Journal of Medicine Volume 83

REGRESSION OF RENAL CELL CARCINOMA-WANG ET AL

regression can be observed in some patients using LAK cells and IL-2. Rosenberg et al [5] noted a response in 13 of 36 patients with renal cell carcinoma and West et al [6] recorded such a response in three of six patients. Time to relapse was relatively short (six to IO months). Unfor-tu- nately, this therapy was associated with considerable toxicity, including severe fluid retention and, in some cases, pulmonary edema, requiring intubation and intensive care.

Cytotoxicity for tumor targets can also be generated by treating peripheral blood mononuclear ceils with the oxidizing mitogen sodium periodate (103) [7]. This agent induces cell activation by generating aldehyde groups on cell-surface glycoproteins [8]. The aldehyde modification is accomplished in a brief period (15 to 30 minutes) and 102 can then be completely removed. We found that IO;i induced IL-2 receptors on peripheral blood mononuclear cells and that IO;i and IL-2 are synergistic in promoting the acquisition of major histocompatibility complex unrestrict- ed cytotoxicity. Augmentation of cytotoxic activity was found at concentrations of IL-2 that alone resulted in minimal generation of cytotoxic cells [7]. Preclinical studies indicated that IO;i-treated cells inhibit growth of a human renal cancer cell line in nude mice and of a pulmonary metastasizing melanoma in a mouse tumor model. These findings suggested that clinical anti-tumor effects might be achieved using a combination of 102 and IL-2 to activate killer cells along with systemic administration of low-dose IL-2.

We report preliminary results of a phase II clinical study to determine the efficacy and toxicity of this method of adoptive immunotherapy for the treatment of metastatic renal cancer. Our data suggest that regression of metastatic renal cell carcinoma can be achieved using lOa/lL- 2-activated killer cells and continuous infusion of low- dose IL-2.

PATIENTS AND METHODS

Clinical Protocol. Thirteen patients with stage IV renal ceil carcinoma were included in this study. Criteria for inclusion include documented stage IV renal cell carcinoma with measurable disease, age 21 to 70, and a performance status of (Eastern Cooperative Oncology Group) 0 to 1. We did not insist on prior nephrectomy, although all but two patients (Patients 3 and 7) had undergone nephrectomy. Two patients (Patients 6 and 11) who had had solitary cerebral metastases removed were also included in the study. All patients were hospitalized for each treatment cycle in the Clinical Research Center at the New York Hospital/Cornell Medical Center. Signed informed consent was obtained from all patients before entry into the study. Leukocytes (5 to 10 X 1 Og) were removed by leukapheresis three times per week (Monday, Wednesday, and Friday). Peripheral blood mononuclear cells were isolated by Ficoll- Hypaque density gradient centrifugation and treated with 104 (2mM) at O°C for 30 minutes [9]. The cells were then cultured at a density of 1.5 X 1 06/mi In 500iml disposable

SCHEMA OF ADOPTIVE IMMUNOTH&APY PROTOCOL 1

Day of Treatment I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 I8 I9 , I 1 1 I I

IL2 IL2 IL2

Figure 1. Schema of adoptive immunotherapy protocol. Arrows indicate when leukapheresis and cell re-infusion were done. On Day 1, only leukapheresis was done and on Day 19, only cell re-infusion was done.

culture flasks in RPMI 1640 medium containing 2.5 percent autologous serum and 500 U/ml of human recombinant IL-2 [IO]. The recombinant IL-2 used in this study was provided by the Cetus Corp., Emeryville, California. The cells were harvested after 48 to 72 hours by centrifugation and washed extensively in lactated Ringer’s solution for re-infusion. A sample of cells was sent for overnight microbial culture the day prior to harvest and for long-term culture on the day of harvest. Pyrogen assays were done by the Limulus lysate method. Cells were re-infused following the next leukapheresis procedure. Recombinant IL-2 was diluted in 500 ml of 5 percent dextrose and water and given as a continuous infusion for five days per week at a dose of 10,000 to 125,000 U/kg/24 hours (mean f SD dose = 39.5 f 8.6 X IO3 U/kg/24 hours) beginning the day of the first leukapheresis. We decreased the maximum dose to 50,000 U/kg/24 hours as the study progressed. This treatment was continued for three weeks. Figure 1 is a scheme of our treatment protocol. Following a three-week rest period, another three-week treatment cycle was given. Evaluation of responses,.including chest radiographs and appropriate com- puterized tomographic (CT) scans, was done prior to and at intervals following completion of the treatment protocol. Laboratory Studies. Cytotoxicity assays were done as have been previously described [l I]. In brief, target cells (K-562, used as a natural killer-sensitive target [NK activity], or SK-RC-29, a human renal carcinoma cell line, used as an NK-insensitive target) are labeled with 300 uCi of sodium 51-chromate for one hour in complete medium (5 percent human AB serum in RPMI 1640 with L-glutamine). They are then washed twice with phosphate-buffered saline and resuspended in complete medium at 1 X IO5 cells per ml. Effector cells, prepared as just described, are washed and mixed with target cells at various effector to target ratios and incubated in round-bottom microtiter plates for three hours at 37OC in a 5 percent carbon dioxide incuba- tor. The culture supernatants are harvested and counted in a liquid scintillation counter. Maximum isotope release (MR) is produced by incubation of the targets with Triton X. Spontaneous release (SR) is measured by incubating the targets with medium alone. The percentage of cell lysis is calculated by: ER - SR/MR - SR, X 100, where ER is the release mediated by the experimental effector cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) was performed in accordance with the guidelines provided by the standardized procedures of the National Institutes of Health [ 121. Spontaneous blastogenesis, an assay that determines spontaneous incorporation of 3H-thymidine dur-

December 1987 The American Journal of Medicine Volume 83 1017

REGRESSK~N 0F RENAL CELL OAROIN~~~A---WANG ET AL

TABLE I Response of Patients with Metastatic Renal Cell Carcinoma after Treatment with IOz/lL-2-Activated Leukocytes and Continuous Infusions of IL-2

Site of Initial Prior Time to Sites of Current Patient Age Sex Metastatic Disease Nephrectomy Response Relapse* Relapse Status

1 57 M PHL, Lu, 60 Yes s 8 Bra, Lu, f30 Died 2 65 M Lu Yes PR 6 Lu, Bo, skin Died 3 51 M Lu, Bo No P - - Died 4 57 F Lu Yes PR 4 Bra, Lu, skin Alivet 5 62 M Lu, Bo, St Yes PR 6 Bra, Lu, Bo Died 6 63 F Lu, Bra Yes P - - Alive 7 46 F Bo No MR 4 Bo Died 8 42 F Lu, skin Yes PR 4 Lu, Bo, skin Alive+ 9 57 M Lu Yes P - - Alive

10 38 F Lu, Li Yes P - - Died 11 61 M Lu, Bra, Li Yes PR 6 Bo Alive? 12 65 M Lu, Ivc Yes P - - Alive 13 60 M Lu, Bo, adrenal, kidney Yes PR No recurrence at 6 mos. Alive

Lu = lung; Li = liver; Bo = bone; Bra = brain; PHL = perihilar lymph node; ST = subcutaneous tissue; Ivc = inferior vena cava; PR = partial response (greater than 50 percent decrease in two-dimensional measurements of metastatic lesions); MR = mixed response; S = stabilization (no increase in size of lesions); P = continued progression. * Months after initial treatment. + These patients are being re-treated.

5 June 1986

31 March 1986

ing an 18-hour incubation, was done as described [ 11,131. Cell responses of the peripheral blood mononuclear cells to two mitogens, concanavalin A and phytohemagglutinin (PtjA), were determined as previously reported [ 11,13]. Cell-surface antigens were determined using the following markers: monoclonal antibodies Leu 11 and anti-IL-2 recep- tor antibody (both from Becton-Dickinson; Mountain View, California), and anti-T3, T4, T8, and Tl 1 (Coulter Immunolo- gy, Hialeah, Florida). Labeled cells were analyzed by direct

23 June 1986

29 May 1986

Figure 2. Chest CTscans of Patients 4 (fop) and 2 (bottom) prior to initiation of therapy and after therapy The follow-up studies were done 18 days after initiation of the experimental protocol in Patient 4 and 59 days after initiation of the prote col in Patient 2.

immunofluorescence and measured in an EPICS C flow cytometer (Coulter). All mononuclear cells identified by 90 degree and forward angle light scatter were included in the flow cytometric evaluation of fluorescent cells. All blood samples were split in two series, in which one was stained with the specific antibodies and the other one with the appropriate isotypic controls. Positive cells were defined as cells that carried a fluorescence signal above that of isoty- pit controls.



30 June 1986

26 August 1986 Figure 3. Pelvic CT scan of Patient 5, prior to and 57 days after initiation of the experimental protocol. There is a decrease in the soft tissue mass surrounding the right iliac region and re-calcification of areas of /ytic bone destruo tion.

RESULTS

Clinical Response. Thirteen patients have thus far com- pleted the study protocol (Table I). There were eight men and five women, and their ages ranged from 38 to 65 years (mean f SD = 55.7 f 8.5). The sites of metastatic involvement shown in Table I indicate that all patients had advanced disease.

Six patients (46 percent) had a partial response, defined as a decrease of more than 50 percent in bidimen- sional measurements of metastatic lesions. Among the seven patients in whom we consider treatment to have failed, five had progressive disease. One patient, however, had no additional progression of disease for eight months (stable), and one patient had a mixed response, with both healing and progression of different bone le-

sions. Responses were seen in the lung (Figure 2), bone (Figure 3), liver (Figure 4), skin, lymph node, adrenal, and kidney.

The number of cells and amount of IL-2 administered are shown in Table II. All but two of the patients who sustained any type of response (including partial response, mixed response, and sustained) received a cu- mulative dose of IL-2 exceeding 1.2 X IO6 U/kg and all but one of the patients who did not show a response received 0.9 X 1 O6 U/kg or less. The large variation in IL- 2 administered resulted from our attempt to minimize toxicity by varying the dose of IL-2 depending upon the toxic effects experienced by each individual patient. The

3 February 1987

31 March 1987 Fjgure 4. CT scan of liver and spleen of Patient 11. The hepatic lesion could no longer be detected in the liver. Note the marked hepatosplenomegaly. This was seen in all the patients.



TABLE II Number of Activated Killer Cells and Amount of IL-2 Administered

Cells Administered (x log) Total Mean/infusion

IL-2 Administered (U X 103/kg) Total Mean124 hours

Responders: * 1 115 3.0 1,284 58 2 80 2.7 1,217 40 4 21 1.8 868 36 5 30 2.0 1,265 42

7t 35 2.0 1,222 40 8 61 3.8 1,201 41

11 14 0.9 1,348 48 13 76 4.7 1,023 38

Non-Responders: 3t 41 2.6 1,435 48 6 34 2.3 904 35 9 43 2.4 900 33

10 28 1.9 860 26 12 55 3.5 814 28

l For this analysis, the two non-responders who had a mixed response (MR) or stabilization (S) are included. 7 These patients did not have prior nephrectomy.

I2345678 Leukapheresis

No.

Figure 5. Number of activated cells re-infused (mean f SEM) 48 to 72 hours following leukaphereses 1 to 8, doring the first cycle of treatment. Data during the second cycle were essentially the same.

number of cells received did not bear any apparent rela- tionship to response.

Of the six patients who initially had a partial response, five have experienced a relapse at 5.2 f 1.0 (mean f SD) months and one continues in remission since April 1987. Recurrences were seen in areas where patients had previous disease, as well as in the brain. Cerebral metastases subsequently developed in three of the responding patients. Immunologic Alterations. The yield of activated mononuclear cells gradually decreased during the first week of IL-2 infusions and leukaphereses (Figure 5) as repot-ted by others [6]. The pattern during the second cycle of treatment was essentially the same (data not shown). During the second and third weeks, a rebound occurred, and the number of cells obtained was substantially greater than the baseline level (leukapheresis I), prior to the patients’ receiving 11-2.

The distribution of lymphocyte subsets during the course of treatment revealed a progressive increase in cells bearing the Leu 11 marker, characteristic of NK cells (Figure 6). NK activity increased even more dramati- cally (Figure 7). Both Leu 1 l-positive cells and NK activity remained elevated during the three-week interval between treatment cycles. There were no significant changes in Ti 1, T3, T4, or T8 cells during treatment. ADCC activity increased moderately (Figure 7).

The potency of IOJ/lL-2-activated killer cells for a non- NK-sensitive target increased during the first cycle of treatment and continued to increase during the second treatment cycle (Figure 6). Spontaneous cytotoxicity for this target also increased slightly during treatment.



A B C D Figure 6. Distribution of selected cell surface markers on peripheral blood mononuclear cells of study patients prior to therapy (A), during the first cycle of therapy (B), prior to the second cycle of therapy (C), and during the second cycle of therapy (0). Data are expressed as mean f SEM.

There were no significant differences in the immunologic studies done between the patients who had a response and those that did not. There was a trend, however, toward higher spontaneous cytotoxicity for the renal cell carcinoma cell line (SK-RC-29) in the responding patients compared with the non-responding patients. Be- cause of the small numbers, the differences are not significant. Toxicity. Several serious side effects of this treatment were encountered, but none required treatment in the intensive care unit (Table III). Fever developed in all patients (maximum temperature elevation was 40.0°C), and chills that lasted 60 to 90 minutes developed after cell infusions. A low-grade fever often persisted during IL-2 infusions (38 to 39’C). All patients experienced malaise, muscle aches, and joint pains. Mild nausea and diarrhea were also common. Severe fluid retention did not develop in any patient, although mild dyspnea developed in some patients shortly after infusion of cells. The mean increase

180-

160-

140-

120-

‘Y-7+- 80 1 f 60 1

A B C D

‘lgure 7. ADCC (0) and NK( 0) activity (percent of simul- taneous normal controls) of study patients. A, B, C, and D are as indicated in Figure 6. Data are expressed as mean f SEM.

60 1 IO,-/IL2

50- activated cells -

\/

I

40- 1

30-

20 _ /

I Spontaneous killing

‘O /</S r I I I A B C D

Figure 6. Spontaneous (0) and activated (/OJ/ILZ) (0) cytotoxicity of peripheral blood mononuclear cells of study patients for the renal cell cancer line, SK-IX? 29. Activated killing is shown at an effector to target ratio of 3O:l and spontaneous killing is shown at 100: 1. A, 6, C, and D are as indicated in Figure 6. Data are expressed as mean f SEM.



TABLE Ill Toxicity and Side Effects of Treatment with IO~/lL-2-Activated Leukocytes and Continuous Infusions of IL-2 (n = 13)

Toxicity Number* Percent

Malaise 13 100 Fever 13 100 Chills 13 100 Nausea 13 100 Anemia 13 100 Eosinophilia 13 100 Hepatosplenomegaly 13 100 Weight gain 12 92 Hypotension 11 85 Dyspnea 10 77 Vomiting 8 62 Diarrhea 8 62 Creatinine increase to >2.0 mg/dl 6 46 Pruritus 3 23 Rash 2 15 Nasal congestion 2 15 Bilirubin increase to >2.0 mg/dl 2 15

* Number of patients experiencing at least one episode of toxic effect.

in body weight during the course of treatment was 2.2 f 2.1 percent (mean f SD, range 0 to 6.9 percent). Asymptomatic hypotension developed in most patients and was controlled by increasing fluid intake or by de-. creasing the rate of infusion of IL-2. Increases in serum creatinine levels (greater than 2 mg/dl) were noted in six patients. The most seriously affected was Patient 1 in whom transient acute renal failure developed with a creatinine level that peaked at 17.0 mg/dl (baseline creatinine: 1.8 mg/dl). This was associated with a marked eosinophilia (35 percent), eosinophiluria, and white blood cell casts. Renal function rapidly improved with steroid therapy and cessation of IL-2 infusions. Clinically, the renal failure closely resembled an acute allergic interstitial ne- phritis. It occurred early in the study at doses of IL-2 up to 125,000 U/kg/24 hours. The mean pretreatment serum creatinine level in the remaining 12 patients was 1 .O f 0.29 mg/dl (range: 0.7 to 1.5 mg/dl). The mean peak creatinine level during therapy was 1.75 f 0.54 mg/dl (range: 0.8 to 2.7 mg/dl). All patients recovered from the transient renal dysfunction after completion of treatment. Some degree of hepatosplenomegaly developed in all patients during the course of treatment. A transient increase in serum bilirubin levels occurred in two patients (highest values were 3.9 and 3.4 mg/dl, respectively). Both levels returned to baseline after IL-2 dosages were reduced.

COMMENTS

This phase II study was undertaken to determine whether regression of metastatic renal cell carcinoma could be achieved using low-dose IL-2 and cells that were sensi-

tized to the effects of IL-2 by treatment with IO,. We previously treated five patients with metastatic renal cancer with oxidizing mitogen-activated mononuclear leukocytes alone. None of these patients had an objective tumor response. Little toxicity was encountered, save for fever and chills that began 30 to 60 minutes following cell infusion and lasted for about one hour. Based on our in vitro studies indicating synergy between IO;i and IL-2 in generating activated killer cells [7] and the data of Rosen- berg et al, both experimental [4] and clinical [ 1,5], that the combination of activated killer cells and systemic IL-2 was more effective than cells alone, we developed the current protocol. This phase II study was undertaken to determine whether regression of metastatic renal cell carcinoma could be achieved using low-dose IL-2 and cells that were sensitized to the effects of IL-2 by treatment with 102. Of the 13 patients we treated, six had a response, characterized by a significant partial regression of metastatic lesions.

Side effects were significant. The patient in whom acute renal failure developed received the highest IL-2 dose. Since decreasing the maximum dose to 50,000 U/ kg/24 hours, only mild degrees of renal insufficiency have occurred. A major toxic effect of IL-2 appears to be a dose-related increase in vascular permeability [ 141. The current protocol minimizes this toxicity while at the same time inducing tumor regression. This observation sug- gests that anti-tumor effects and the toxic effect of increased vascular permeability might be dissociated.

The current study does not differentiate between effects of IL-2 alone and IL-2 plus IOJIL-2-activated killer cells. Cells alone appear to be ineffective in our experi- ence and in that of Rosenberg et al [I]. In the latter study, IL-2 alone did not induce tumor regression. Thus, it appears that in renal cell carcinoma, a combination of activated cells and IL-2 is required, although controlled studies should be done to test this hypothesis.

Five of the six patients who showed a partial response to therapy subsequently experienced a relapse. Other studies have also documented frequent and relatively rapid relapse following cessation of adoptive immunotherapy [5]. As an approach to this problem, we are evaluat- ing monthly treatments with IL-2 and IOa/IL-2-activated cells as a maintenance treatment for patients who show an initial response. Cerebral metastases were noted in three of our patients following treatment. Approaches to this problem might include local treatment, reported for malignant glioma [ 151, combinations of other biologic response modifiers, and/or prophylactic brain irradiation,

Adoptive immunotherapy with activated killer cells is a highly labor-intensive procedure requiring specialized fa- cilities for cell culture, quality control, and immunologic monitoring. An interesting facet of the use of IOj-treated cells is that this activating agent needs only 15 to 30



minutes of exposure to cells to generate aldehyde groups on cell-surface sialyl residues. This modification is then sufficient to generate activated killer cells in vitro with no further exposure to the oxidizing agent. If this phenomenon proceeds in vivo as well, it raises the possibility of on- line treatment and re-infusion of cells, coupled with continuous infusions of 11-2. Such a procedure would greatly simplify current technology and make adoptive immunotherapy available to many more patients.

Whether adoptive immunotherapy as practiced today will extend patient survival or not requires further study. Our study indicates, however, that 104/lL-2-activated killer cells plus continuous infusions of low-dose IL-2 can

result in initial regression of metastatic renal cell carcinoma in lung, liver, bone, soft tissue, skin, lymph node, adrenal, and kidney.

ACKNOWLEDGMENT

We are deeply indebted to Drs. Stuart Saal and Neil Bander for their help, advice, and encouragement, to Rachel Schwartz and Mila Lagman for their superb techni- cal assistance, to Bonita Dennis and Karen Lorigan for their expert nursing and data-gathering skills, and to the nursing staff of the Clinical Research Center for their superb patient care. We also thank Mrs. Yinking Lai and Ms. Mary Stenzel for typing this manuscript.

REFERENCES

1. Rosenberg SA, Lotze MT, Muul LM, et al: Observations on the systemic administration of autologous lymphokine- activated killer cells and recombinant interleukin 2 to patients with metastatic cancer. N Engl J Med 1985; 313: 1485-1492.

2. Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA: Lym- phokine-activated killer cell phenomenon: lysis of natural killer-resistant fresh solid tumor cells by interleukin-2- activated autologous human peripheral blood lymphocytes. J Exp Med 1982; 155: 1823-1841.

3. Mazumder A, Rosenberg SA: Successful immunotherapy of natural killer-resistant pulmonary melanoma metastases by the intravenous adoptive transfer of syngeneic lymphocytes activated in vitro by interleukin 2. J Exp Med 1984; 159: 495-507.

4. Mule JJ, Shu S, Schwartz SL, Rosenberg SA: Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2. Science 1984; 225: 1487-1489.

5. Rosenberg SA, Lotze MT, Muul LM, et al: A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 1987; 316: 889-897.

6. West WH, Tauer KW, Yannelli JR, et al: Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J Med 1987; 316: 898-905.

7. Wang J, Suthanthiran M, Walle A, et al: Anti-tumor proper- ties of lymphocytes activated by the oxidizing mitogens. J lmmunol 1986; 136: 4735-4739.

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Novogrodsky A, Katchalski E: Membrane site modified on induction of the transformation of lymphocytes by periodate. Proc Natl Acad Sci USA 1972; 69: 3207-3210.

Novogrodsky A, Stenzel KH, Rubin AL: Stimulation of human peripheral blood lymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin: differ- ential effects of adherent cells. J lmmunol 1977; 118: 852-857.

Wang A, Lu SD, Mark D: Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 1984; 244: 1431- 1433.

Suthanthiran M, Williams PS, Solomon SD, Rubin AL, Sten- zel KH: Induction of cytolytic activity by anti-T3 monoclonal antibody. J Clin Invest 1984; 74: 2263-2271.

Roman0 P: Cytolytic assays for soluble antigens. In: Rose NR, Friedman H, Fahey JL, ed. Manual of clinical laboratory immunology. Washington: American Society for Micro- biology, 1986; 72-77.

Suthanthiran M, Garovoy MR: Immunologic monitoring of the renal transplant recipient. Urol Clin North Am 1983; 10: 315-325.

Rosenstein M, Ettinghausen S, Rosenberg SA: Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin-2. J lmmunol 1986; 137: 1735-1742.

Jacobs SK, Wilson DJ, Kornblith PL, Grimm EA: Interleukin-2 or autologous lymphokine-activated killer cell treatment of malignant glioma: phase I trial. Cancer Res 1986; 46: 2102-2104.


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