The In Vivo Immunomodulatory Effects of Recombinant Interferon gamma Plus Recombinant Tumor Necrosis Factor-alfa By Walter J. Urba, William C. Kopp, Jeffrey W. Clark, John W. Smith II, Ronald G. Steis, Christoph Huber, David Coggin, and Dan L. Longo We conducted a phase I study in which an intramuscular injection of interferon gamma (IFNy) at 10, 50, or 100 pIg/m2 was followed 5 minutes later by an intramuscular injection of 10, 50, or 100 pg/m' of tumor necrosis factor-alfa (TNFa) at another site every other day for 20 days (10 doses). The addition of TNFa to IFNy reduced both the magnitude and duration of IFNy-mediated effects on peripheral blood monocyte expression of Fc receptors (FcRs) and HLA-DR and production of hydrogen peroxide. This inhibition was related to the dose of TNFa. On the other hand, TNFa and IFNy appeared to have an additive stimulatory effect on the production of neopterin by monocytes. The highest serum levels of neopterin were detected in patients who received the highest doses of both IFNy, and TNFa. Thus, conflicting conclusions regarding the effect of the combination on immune activation are possible. If the activation of peripheral blood mono-

cytes is the appropriate surrogate measure of the immune enhancement of the combination, then the simultaneous administration of IFNy and TNFa is ineffective, and future attempts to exploit the potential additive or synergistic effects of this combination of cytokines in humans may need to explore sequential administration schemata. On the other hand, if serum neopterin levels are a more reliable index of immune activation, simultaneous administration of 100 pg/m' IFNy and 50 ILg/m' TNFa every other day (the maximally tolerated dose [MTD]) should be used in phase II testing. This dilemma points out the limitations of currently available methods of human immune assessment and the inadequacies in our capacity to gauge what particular immune measure or set of measures predict for in vivo antitumor effects. J Clin Oncol 9:1831-1839. This is a US government work. There are no restrictionson its use.

rTUMOR NECROSIS factor-alfa (TNFa) and interferon gamma (IFNy) can directly inhibit the in vitro growth of some tumor cell lines and exert antitumor effects in vivo in murine tumor models.' Unfortunately, except in rare instances, neither agent when used alone has demonstrated significant antitumor effects in patients with solid tumors.2-8 However, the presumed lack of efficacy of these agents is based on the limited number of tumor responses observed in phase I trials of both agents and, in the case of IFNy, in phase II trials using maximally tolerated doses (MTDs). Reports describing the synergistic antiproliferative properties of TNFa and IFNy against human malignant cell lines9"' led us to perform a clinical trial using both agents together.'2 Although a number of hypotheses have been proposed, the mechanism for their synergy is unknown. In addition to the additive or synergistic potential of their direct antiproliferative effects, synergy also may occur in their known immunologic effects. 3 IFNy can enhance natural-killer (NK) activity' and increase class II major histocompatibility complex (MHC) antigen expression on tumor cells.14 It is also a potent monocyte activator and augments

tumoricidal activity,' 5 hydrogen peroxide genera-

tion,16 antibody-dependent cellular cytotoxicity, receptors for the Fc portion of immunoglobulin G (IgG),' 7 and class II MHC (HLA-DR) antigen expression.'" TNFcx has been reported to affect

From the Clinical Immunology Services, Program Resources, Inc/DynCorp, and Biological Response Modifiers Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD; and Department of Haematology, JohannesGutenberg University, Mainz, FederalRepublic of Germany. Submitted October 1, 1990; acceptedApril 10, 1991. This project has been funded at least in part with federal funds from the Department of Health and Human Services undercontractno. NO1-CO-74102. The content of thispublication does not necessarily reflect the views or policies of the Departmentof Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsementby the United States Government. Address reprint requests to Walter J. Urba, MD, PhD, Clinical Immunology Services, Program Resources, Inci DynCorp, National Cancer Institute-Frederick Cancer Research and Development Center, PO Box B, Building 560, Room 11-62, Frederick,MD 21702-1201. This is a US government work. There are no restrictionson its use. 0732-183X/91/0910-0004$0.00/0

Journalof Clinical Oncology, Vol 9, No 10 (October), 1991: pp 1831-1839

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URBA ET AL

1832 neutrophils,19,20 endothelium, 21 class I MHC antigen expression, 22 and induction of colony-stimulating factors, 23 and to enhance both NK24 and macrophage tumoricidal activity. 25 Immunologically active doses of IFNy have been identified in patients with melanoma,13 but the results of extensive phase II testing of this dose in patients with metastatic disease have not been reported. Very little monitoring of immunologic effects was performed during the phase I trials of TNFca; therefore, data describing its in vivo immunomodulatory properties are not available. Since the in vivo antitumor properties of IFNy and TNFa may result from either direct antitumor effects, which might be expected to be optimal at the MTD, or indirect effects from activation of the immune system, which would be expected to be maximal at the optimal biologic dose (OBD), we performed both a careful analysis of toxicity and response to treatment,' 2 and an extensive evaluation of the effects of the combination on immune function. We chose immunomodulatory doses of IFN- 13 and combined them with various doses of TNFa. This report describes the effects of the combination of IFNy and TNFac on peripheral blood monocyte activation and function and on serum neopterin levels. MATERIALS AND METHODS PatientPopulation The details of patient selection and treatment are pre1 sented elsewhere. " All patients had a histologically confirmed diagnosis of cancer and were assessed and treated at the Biological Response Modifiers Program (BRMP), Frederick, MD. All patients voluntarily signed an informed consent for the protocol that had been approved by the institutional review boards of both the Frederick Cancer Research and Development Center and Clinical Oncology Program of the National Cancer Institute (NCI).

IFNy and TNFao Preparation Recombinant IFN-y (rIFNy) and recombinant TNFa (rTNFa) were prepared and provided by Genentech, Inc, South San Francisco, CA. Sterility, purity, pyrogenicity, and general safety met the standards of the Office of Biologics, Food and Drug Administration. Tests for endotoxin in the limulus assay routinely revealed less than 0.5 ng/mg protein. The specific activity of rIFNy was approximately 2 x 10' U/mg protein based on an antiviral bioassay with encephalomyocarditis virus and human A549 cells (standardized with National Institutes of Health [NIH] IFNy standard Gg23901350). The rTNFa was purified to more than 99% as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a specific activity of

approximately 4 x 10' U/mg of protein, as defined by the lysis of dactinomycin-treated mouse L929 cells.

Study Design Patients received intramuscular (IM) injections of both rTNFa and rIFNy into different sites within 5 minutes of each other every other day for 3 consecutive weeks, for a total of 10 treatments. Each patient received the same doses of each cytokine throughout treatment. Patients were assigned sequentially to different dose levels as described in Table 1. Three additional patients at the MTD received aspirin 650 mg orally four times per day starting 1 hour before therapy and continuing until 3 weeks of therapy were completed. The use of steroids or prostaglandin inhibitors was otherwise proscribed. Samples for immunologic monitoring (hydrogen peroxide [H 20 2] generation by monocytes, Fc receptor [FcR] number and HLA-DR expression on monocytes, and serum levels of neopterin) were obtained at baseline (two to three samples) and days 0, 1, 2, 3, 4, 15, 16, and 21. Therapy was begun on day 0. The day-21 samples were obtained 48 hours after the last treatment. As one of the primary objectives of this study was to evaluate the in vivo immunologic effects of fixed doses of rIFNy and rTNFa, dose reductions for subjective or objective toxicities were not permitted.

Cell Preparationand Cryopreservation To eliminate day-to-day variability in assay and staining procedures and instrumentation, all assays were performed on cryopreserved cells. At the completion of the treatment cycle, all samples from one individual were thawed and tested in a single assay. Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood by FicollHypaque density gradient centrifugation (LSM; Litton Bionetics, Rockville, MD). Cells were frozen using a CryoMed (Mt Clemens, MI) liquid nitrogen controlled-rate freezer. Immediately after freezing, the vials were transferred to the gaseous phase of a liquid nitrogen storage freezer until needed. Frozen cells were thawed rapidly by agitation in a 37'C water bath. Viability by trypan blue dye exclusion and total cell recovery was usually greater than 90%.

Measurement of HO, Release by Monocytes H 20, production by mononuclear phagocytes was detected by monitoring the horseradish peroxidase-catalyzed Table 1. Combination TNFa/IFNy Study Design Dose Level

rTNFc Dose (Ipg/m

2

)

rIFNy Dose

No. of

(pg/m')

Patients

1

10

10

3

2 3 4 5 6

50 100 10 50 100

10 10 50 50 50

3 3 3 3 3

7

10

100

3

8 9

50 100

100 100

6 + 3* 6

*Three patients were treated with aspirin at this dose.

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1833

IMMUNOMODULATORY EFFECTS OF IFNy + TNFa oxidation of fluorescent scopoletin." The results were calculated by microcomputer and expressed as nanomoles of H2 O2 produced per microgram of protein per hour. Our method has been described in detail previously."

Estimation of FcRs on Monocytes The number of receptor sites for the Fc portion of IgG on monocytes was estimated by comparing the immunofluorescence due to saturable binding of fluorescein isothiocyanate (FITC)-conjugated IgG (Copper Biomedical, Malvern, PA) to the U-937 myelomonocytic cell line with that due to patients' monocytes. The method was described originally by Guyre et al,17 and our methods for calculating FcR 3 number have been described elsewhere." 0

Measurement of HLA-DR Expression on Monocytes Enumeration of monocytes expressing HLA-DR and the level of expression of this class II MHC surface antigen were determined by flow cytometry. Analysis was performed using a Cytofluorograf 30H equipped with a 2150 computer (Ortho Diagnostics Systems, Inc, Westwood, MA). Monoclonal antibodies against HLA-DR were obtained from Becton-Dickinson and Co (Mountain View, CA). Nonviable cells were excluded from analysis by red fluorescence after staining with propidium iodide.

Measurement of Neopterin Serum levels of neopterin were measured using a radioimmunoassay kit (Neopterin-RIAcid, Henning Berlin GmBH, Berlin, Federal Republic of Germany). The manufacturer's instructions were followed in the performance of this assay. The normal value in our lab is less than 10 nmol/L.

StatisticalMethods Intra- and intergroup comparisons were made using the percentage difference between average on-treatment measurements and average baseline measurements over the average baseline measurement for each patient. The significance of changes from baseline (two to three samples per patient) for each immune parameter was assessed using the least squares mean test." Analysis of variance (ANOVA) followed by multiple comparison tests were performed using the General Linear Models procedure of Statistical Application System (SAS) software from the SAS Institute, Cary, NC. The statistical test results described are based on two-sided tests conducted at a .05 level of significance.

RESULTS H 20 2 Productionby Monocytes

We examined the combined effects of TNFa

and IFNy on monocyte H 20 2 production of peripheral blood monocytes. H 20 2 production by peripheral blood monocytes is enhanced 24 and 48 hours after IM or subcutaneous administration of rIFNyý at doses of 10, 100, or 250 pLg/m 2.13 Figure 1

compares H 20 2 production by patients' peripheral blood monocytes obtained on day 14 of treatment

10

50

100

0

10

50

100

ug TNF

Fig 1. H20 2 production by peripheral blood monocytes isolated from patients 24 hours after IM administered IFNy alone (0) or in combination with TNFa (0). Each 0 represents the mean of 10 patients receiving IFNy alone on day 14 of a previous study, and each C represents the mean on day 15 of three to nine patients receiving IFN-y and TNFa at the indicated doses. Data are presented as percentage of change compared with baseline H20 2 production plus or minus SE.

with IFNy alone on a previous study with H 20 2 generation from monocytes obtained on day 15 from patients receiving the same dose of IFNy in combination with TNFa on this study. In all cases, monocytes were obtained 24 hours after administration of the cytokine(s). The data are depicted as percentages of difference from baseline. The data for IFNy alone were obtained from patients treated with IFNy as adjuvant therapy for melanoma during an earlier trial and have been published previously.' 3 Since our previous trial did not in2 clude an IFNy dose of 50 Cpg/m , Fig 1 only includes data for patients receiving TNFca in combination with IFNy at 10 and 100 pig/m 2. Administration of IFNy alone, at either the 10 tpg/m 2 or 100 lpg/m 2 dose, resulted in a significant increase above baseline in monocyte H 20 2 production. Monocytes from patients receiving 10 ±pg/m 2 of TNFa in addition to IFNy at 10 [Lg/m 2 appeared to have a similar increase in H20 2 production, but monocytes from patients receiving the same dose of IFNy with 50 plg/m 2 or 100 ý±g/m 2 of TNFct had no increase in H ,O, 20 2 production. The increase in H 20 2 generation by peripheral blood monocytes that would have been expected after treatment with 100 pRg/m 2 IFNy was not observed when the same dose of IFNy was given in combination with TNFct at three different doses (Fig 1). When only IFNy dose was examined, regardless

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URBA ET AL

of TNFa dose, there was a significant increase above baseline at 24 hours for the 50 Ipg/m 2 dose (P = .011). The increase was lower than expected from our previous study and also dissipated more rapidly. After administration of IFNy alone, H 2 0 2 13 is increased significantly for 48 hours." Regardless of the dose of IFNy or TNFa, none of the 48-hour time points differed significantly from baseline. Table 2 lists the data from all patients 24 and 48 hours after their eighth treatment. Increased H 2 0 2 production was generally noted at 24 hours at all doses of IFNy when it was combined with the lowest dose (10 pLg) of TNFa. H 2 0 2 production was lower at the 50 and 100 pg/m 2 dose levels of TNFa than at the 10 pLg/m 2 level, which was the only level significantly elevated compared with baseline. Significant immunologic changes were observed only at 24 hours; by 48 hours there were no significant differences between any of the groups, and none of them were significantly different from baseline. FcR Expression by Monocytes Similar inhibitory effects of TNFa also were noted on IFNy-enhanced FcR expression on peripheral blood monocytes. When IFNy alone is used, FcR expression is enhanced at doses > 10 iLg/m 2 and remains enhanced for at least 48 hours."3 Figure 2 shows the percentage of change compared with baseline for all nine groups of patients on day 15, 24 hours after cytokine administration. For a given dose of TNFa, increasing doses of IFNy result in higher levels of FcR expression. Furthermore, at each dose of IFNy, increasing the dose of TNFa results in decreased FcR expression. Maximal FcR expression is noted at IFNy 100 Ig and TNFa 10 CIg, while minimal FcR expression is noted at IFNy 10 pg and TNFa 100 Ig. Using ANOVA, the IFNy dose of 50 pLg combined with TNFau at 10 I.g, and the IFNy dose of 100 ý.g with TNFa at 10 ý.g, 50 p.g, or 100 p.g

Table 2. Monocyte H20

2

TNF

Fig 2. FcR expression on monocytes 24 hours after IM IFNy administered in combination with TNFa. Each bar represents the mean of data from flow-cytometric analysis of monocytes from 3-9 patients on day 15. Data are presented as percentages of change compared with baseline FcR expression _+SE. The dose of each cytokine is listed in the margin, and the exact percentage of change is listed under each bar. (0) Indicates statistically significant difference from baseline.

were associated with significantly enhanced FcR expression compared with baseline. Higher doses of TNFa not only blunt the magnitude of the IFNy-induced increase in FcR expression, but also shorten the duration of this increase. In our previous trial using IFNy alone, enhanced FcR expression could be detected for 48 hours,13 whereas at 48 hours after combined TNFa/IFNy treatment, only the groups receiving IFNy at 100 tpg with the two lowest doses of TNFa (10 pg and 50 pLg) remained significantly different from baseline. Monocyte Cell-SurfaceMarkerAnalysis More than 90% of peripheral blood monocytes express HLA-DR antigens on their surfaces. IFNy treatment at 10 I.g/m 2 or 100 jig/m 2 has been

Production 24 and 48 Hours After Administration of TNFa/IFNy

IFNy 10 Ig + TNFe (pg/m')

Baseline Day 15* Day 16t

"i(

IFNy 50

Ag

IFN-y100 pg + TNFa (Lg/m')

+ TNFa (pg/m2)

10

50

100

10

50

100

10

50

100

93 167 84

110 98 145

104 64 131

157 293 147

164 279 213

138 147 277

147 184 258

201 241 240

195 185 135

NOTE. Data indicate nanomoles of H2 0 2 produced per microgram protein in 60 minutes. For details of the assay, see Methods. *Blood sample drawn 24 hours after the eighth treatment. tBlood sample drawn 48 hours after the eighth treatment.

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IMMUNOMODULATORY EFFECTS OF IFN-y + TNFa

1835

shown to increase monocyte HLA-DR expression.13 Concomitant treatment with TNFa appears to blunt the increase in HLA-DR expression seen after IFNy treatment. At the highest IFNy dose (100 jig), there was a significant increase in HLA-DR regardless of the dose of TNFat (Fig 3). At the intermediate dose of IFNy (50 jIg), enhanced HLA-DR expression was not observed at the highest dose of TNFa (100 RIg). The lowest dose of IFNy (10 jg), which previously has been shown to enhance HLA-DR expression, was unable to enhance expression of HLA-DR on peripheral blood monocytes even at the lowest dose of TNFa (10 Vig). In fact, HLA-DR expression decreased when TNFa at 50 jig or 100 jIg was combined with 10 ig/m' of IFNy. The stimulatory effects lasted longer than 24 hours only for patients receiving IFNy at 100 jig combined with TNFa at 10 jig or 50 pg doses. The percentage of monocytes bearing HLA-DR did not change; 90% to 95% of all cells with 900 scatter and forward light scatter properties of monocytes remained positive throughout treatment. However, there were changes in the percentage of cells expressing a different class II antigen (HLA-DQ). At baseline, 20% to 50% of patients' monocytes expressed HLA-DQ antigens. At the lowest dose of TNFa (10 jig), there was an IFNy--induced increase in the percentage of mono-

cytes bearing HLA-DQ antigens (data not shown). Similar to other IFNy-induced effects, when higher doses of TNFat were administered with IFNy, there was little change in the percentage of monocytes bearing HLA-DQ antigens. Neopterin Levels Neopterin is a pyrazino-pyrimidine compound derived from guanosine triphosphate (GTP) that is released from macrophages after stimulation with IFNy.2 IFNy treatment of patients with renal cell carcinoma is associated with dose-related increases in serum levels of neopterin.2 9 Sera were not available for neopterin measurement in patients treated with IFNy alone on our previous trial. In contrast to FcR and HLA-DR expression and H2,O, 02 production, IFNy-induced neopterin production appears to be increased by concomitant administration of TNFat, suggesting an additive effect. Figure 4 is almost the mirror image of FcR expression shown in Fig 2. Maximal neopterin production was noted when the highest doses of both agents were used (TNFa, 100 pg; IFNy, 100 jig); and minimal neopterin production was noted at the lowest doses of both agents (TNFat, 10 jig; IFNy, 10 jig). Aspirin Effects Once the MTD was determined, three additional patients were treated at the MTD (IFNy, 100 pig; TNFea, 50 jIg) with aspirin 650 mg given orally four times a day in an effort to decrease the toxicity. An analysis of H20,, production, FcR and HLA-DR expression, and neopterin production failed to detect any differences compared with patients treated at the same dose without aspirin (data not shown). DISCUSSION

I

TNF

Fig 3. HLA-DR expression on monocytes 24 hours after IM IFNy administered in combination with TNFa. Each bar represents the mean of data from flow-cytometric analysis of monocytes from 3-9 patients on day 15. Data are percentages of change in mean fluorescent channel compared with baseline plus or minus SE. The dose of each cytokine is listed in the margin, and the percentage of change is listed under each bar.

TNFa and IFNy may exert antitumor effects either directly by their antiproliferative effects or indirectly by their immunologic effects. Direct antitumor effects would be expected to be maximal at the MTD for both agents, so it is important to determine the MTD for this combination of biologic agents in the same way that would be performed for standard chemotherapeutic agents. The MTD for the combination of TNFa and IFNy in our study was 50 jig TNFa every other day and 100 jpg IFNy every other day for a 3-week treatment period.' 2 Other investigators also have deter-

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URBA ET AL

1836

TNF

Fig 4. Percentage of change from baseline in serum neopterin levels 24 hours after IM IFNy and TNFa administered at the indicated doses. Each bar represents the mean of data from 3-6 patients + SE. (0) Indicates significantly different from baseline.

mined the MTD of combined therapy with TNF and IFNy, but by different routes and schedules of administration 03"'31; their clinical data have been discussed in relation to ours in a separate report.12 For biologicals, data from animal models have demonstrated that there may be a wide disparity between the MTD and the optimal therapeutic dose.32 Therefore, the investigation of a biologic response modifier (BRM), or combination of BRMs, only at the MTD could overlook lower optimal immunomodulatory doses that might be more efficacious. Aulitzky et a129 recently demonstrated that treatment with immunologically active doses of IFN-y that are clearly below the MTD caused responses in 30% of patients with metastatic renal cell carcinoma. Responses were seen in patients with evidence of the most immunologic activation; in this case, increased serum levels of serum P32-microglobulin. These results support the notion that a complete evaluation of a BRM (or combination of BRMs) requires a determination of both the MTD and the OBD, followed by phase II efficacy testing of both dose levels if they differ. A major concern in the development of biologic agents is that one could draw a premature negative conclusion about an agent's efficacy before it had been tested adequately under the appropriate circumstances. Thus, we performed careful immunologic monitoring during our trial of combined

therapy with TNFa and IFNy to try to determine an optimal biologic dose in addition to an MTD. Our data show that despite the well-known salutary immunologic effects of TNFa and IFNy alone, coadministration of both agents every other day for 3 weeks led to unexpected results: significantly less immunomodulation in the PBMC than we would have expected based on our previous studies administering IFNy alone. This was true for class II MHC antigen and FcR expression on peripheral blood monocytes and for one measure of monocyte function, HO 2 2 production. Coadministration of TNFa and IFNy has been shown to enhance a variety of immunologic activities, including monocyte function in tumor-bearing mice. 33 TNFa and IFNy exhibited antitumor effects when given together to mice bearing B16-BL6 tumors at doses that would have been ineffective if either agent were given alone. The authors postulated that the additive effects of TNFa and IFNy were due to their combined immunomodulatory properties rather than direct cytostatic or cytotoxic effects 33; however, no immunologic monitoring was performed, so the proposed mechanism for improved efficacy is merely conjecture. Our study certainly does not support the notion that coadministration of TNFoa and IFNy results in additive or synergistic immune effects on peripheral blood monocytes. Although the mechanism for the apparent lack of additive or synergistic immunomodulation is unknown, one interpretation of our data would be that TNFa inhibits IFNy,'s effects on monocytes. This would not have been predicted based on preclinical animal experiments, but there were in vitro data indicating the potential for negative interactions between the two cytokines.3 4'35 Arend et a134 showed that li-

popolysaccharide, interleukin-1 beta, and TNFa were all capable of inhibiting the IFNy-induced FcR expression on human monocytes in vitro, although TNFoa was the least potent inhibitor. Leeuwenberg et a135 showed that TNFa could inhibit IFNy-induced class II MHC antigen expression on human umbilical vein endothelial cells in vitro. However, the timing of TNFa was important, as TNF administration before or simultaneous with IFNy administration resulted in inhibition, while TNF administration 24 hours later enhanced MHC antigen expression. Our study examined the effects of simultaneous administra-

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IMMUNOMODULATORY EFFECTS OF IFNy + TNFoa

1837

tion, a schedule that might not be optimal for enhancement of certain immunologic effects. Trivial explanations for our results would be that patients with metastatic disease, or patients with tumors other than melanoma, do not respond to IFNy with the same immunologic changes at the same doses as do patients with melanoma treated in the adjuvant setting. 13 These reservations are not likely to explain our findings, as data from a recently completed trial of our own document similar immunomodulatory effects of IFN-y in patients with metastatic melanoma (unpublished data, June 1991), and other authors have documented IFNy's immunomodulatory proper-

mechanism of the additive effects of TNFa and IFNy on neopterin levels is unexplained. An alternate interpretation of our data is that TNFa did not inhibit IFNy-induced immunologic changes at all, but that combination therapy activated monocytes very effectively and caused a shift of activated cells from the circulation to the vessel walls or other tissues (perhaps tumor). In this context, the decreased FcR and HLA-DR expression and reduced H 20 2 production would not be indicative of inhibition of IFNy effects, but would reflect the trafficking of activated monocytes out of the circulation and their replacement with newly generated monocytes unexposed or unresponsive to the IFNy and TNF. Cytokine administration is known to alter expression of adhesion molecules on monocytes, 42 and recent studies have shown transient monocytopenia in patients treated with TNFa or IFNy (W.E. Aulitzky, personal communication, January 1991). Although we did not observe changes in the number of peripheral blood monocytes in our patients, we looked at relatively late time points, and it is possible that redistribution of activated monocytes occurred in the first few hours after cytokine administration followed by repopulation with unactivated monocytes within 24 hours. This could also explain the additive effects of combined treatment on serum neopterin; activated monocytes sequestered by the endothelium or in peripheral tissues would still secrete their soluble products that could be measured in the serum. Therefore, the mechanism of TNFa's effects on IFNy-induced immunologic changes remains to be fully elucidated. In contrast with our previous trial of IFNy alone, we were unable to find doses of IFNy and TNFa that reproducibly enhanced the host's peripheral blood monocyte function. Our study documents both the importance of extensive immunologic testing before the phase II evaluation of biologic agents or their combinations, and the difficulty of interpreting the results. The determination of an MTD was straightforward, and appropriate doses for phase II testing can be selected.12 However, determining an unequivocal optimal immunomodulatory dose for phase II testing is not possible from these data. If one looks only at peripheral blood monocyte function, one would assume that TNFa inhibits IFNy-induced effects and that there is no effective dose to combine with IFNy-. If one examines neopterin levels as a

ties in other tumor types.2 ,936

In contrast to the inhibition of other IFNyenhanced functions, TNFat appeared to have doserelated additive effects with IFNy on neopterin induction. Neopterin is a low-molecular weight compound derived from GTP that is an intermediate product in the synthetic pathway of biopterin, itself an essential cofactor in neurotransmitter synthesis. 8 Elevated levels have been found in patients with acquired immune deficiency syndrome, during allograft rejection, in a variety of autoimmune disorders, and in patients undergoing 38 immunotherapy with a variety of cytokines.37' Although TNFac has not been shown to enhance neopterin production in vitro, patients receiving TNFat for treatment of malignant disease do have increased levels of neopterin in their serum.38 The elevated neopterin levels were felt to be due to secondary induction of IFNy in vivo. IFNy has been shown to increase the activity of the enzyme GTP cyclohydrolase I, which is important in the synthesis of biopterin.3 9 Monocytes activated with IFNy markedly increase the levels of this enzyme, but only show modest increases in enzymes further along the pathway toward the synthesis of tetrahydrobiopterin. 39 This results in a marked increase in neopterin. The functional significance of increased levels of neopterin is not entirely clear, but a recent report suggests that it may be an important cofactor in the generation of nitric oxide by macrophages. 40 Nitric oxides appear to be important in mediating the cytotoxic action of macrophages and also play a role in the toxicity of treatment with certain agents such as TNF41 or interleukin-2, because they have potent vasodilatory properties that can lead to hypotension. The

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URBA ET AL

1838 surrogate indicator of the state of activation of the body's monocytes, one would choose maximal doses of both agents for subsequent phase II studies. This study points out the difficulty in relying on peripheral blood findings to determine optimal biologic effects. If activated cells preferentially leave the circulation, examining peripheral blood cell-surface markers of activation and various cellular functions could be misleading. Selecting treatment strategies solely on this basis may not be optimal. This study also points out the need to consider potential negative interactions as well as additive or synergistic ones in the evaluation of biologic agents. Since many future trials will include combinations of cytokines, it will be important to measure the biologic (not just the clinical) effects of these agents when they are administered. Monitoring strategies should include mea-

surement of secreted soluble factors as measures of cellular activation as well as examination of cells in the peripheral blood. Ideally, methods to assess immune changes at the site of the tumor after treatment with cytokines will be developed so that biologic therapies can be tested rationally rather

than empirically. ACKNOWLEDGMENT We thank the nurses of the Biological Response Modifiers Program and the staffs of the Clinical Monitoring and Flow Cytometry Laboratories for their help in the performance of this study. We also thank Drs Steve Sherwin and Howard Jaffe from Genentech for their help during the developmental stages of the clinical trial and for generously providing both cytokines. We appreciate the excellent secretarial assistance provided by Judy Beacht, Donna Bialozor, and Terry Phillips, and the help in data analysis from Sharon Beck.

REFEREN ICES 1. Talmadge JE, Black PL, Tribble H, et al: Preclinical approaches to the treatment of metastatic disease: Therapeutic properties of rHuTNF, rHuIFN-y and rHuIL-2. Drugs Exp Clin Res 13:327-332, 1987 2. Bonnem EM, Oldham RK: Gamma-interferon: Physiology and speculation on its role in medicine. J Biol Response Mod 6:275-301, 1987 3. Rinehart JJ, Malspeis L, Young D, et al: Phase I/II trial of human recombinant interferon gamma in renal cell carcinoma. J Biol Response Mod 5:300-308, 1986 4. Edmondson JH, Long HJ, Creagan ET, et al: Phase II study of recombinant gamma-interferon in patients with advanced nonosseus sarcomas. Cancer Treat Rep 71:211213, 1987 5. Recombinant Human Interferon gamma (5-6810) Research Group on Renal Cell Carcinoma: Phase II study of recombinant human interferon gamma (5-6810) on renal cell carcinoma. Summary of two collaborative studies. Cancer 60:929-933, 1987 6. Newman HFV, Bleehen NM, Galazka A, et al: Small cell lung carcinoma. A phase II evaluation of r-interferon-y. Cancer 60:2938-2940, 1987 7. Creagan ET, Ahmann DL, Long HJ, et al: Phase II study of recombinant interferon-gamma in patients with disseminated malignant melanoma. Cancer Treat Rep 71: 843-844, 1987 8. Blick M, Sherwin SA, Rosenblum M, et al: Phase I study of recombinant tumor necrosis factor in cancer patients. Cancer Res 47:2986-2989, 1987 9. Williamson BD, Carswell EA, Rubin BY, et al: Human tumor necrosis factor produced by human B cell lines: Synergistic cytotoxic interaction with human interferon. Proc Natl Acad Sci USA 80:5397-5401, 1983 10. Williams TW, Bellanti JA: In vitro synergism between intetferons and human lymphotoxin: Enhancement of lymphotoxin-induced target cell killing. J inmunol 130: 518-520, 1983

11. Fransen L, van der Heyden J, Ruysschaert R, et al: Recombinant tumor necrosis factor: Its effect and its synergism with interferon-y on a variety of normal and transformed human cell lines. Eur J Cancer Clin Oncol 22:419-426, 1986 12. Smith JW II, Urba WJ, Clark JW, et al: Phase I evaluation of recombinant tumor necrosis factor given in combination with recombinant interferon gamma. J Immunother (in press) 13. Maluish AE, Urba WJ, Longo DL, et al: The determination of an immunologically active dose of interferongamma in patients with melanoma. J Clin Oncol 6:434-445, 1988 14. Balkwill FR, Stevens MH, Griffin DB, et al: Interferon gamma regulates HLA-DR expression on solid tumors in vivo. Eur J Cancer Clin Oncol 23:101-106, 1987 15. Kleinerman ES, Herberman RB: Tumoricidal activity of human monocytes: Evidence for cytolytic function distinct from that of NK cells. J Immunol 133:4-10, 1984 16. Nathan CG, Murray HM, Wiebe M: Identification of interferon-gamma as the lymphokine which activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 158:670-689, 1983 17. Guyre PM, Morganelli PM, Miller R: Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes. J Clin Invest 72:393-397, 1983 18. Basham TY, Merigan TC: Recombinant IFN--y increases HLA-DR synthesis and expression. J Immunol 130:1492-1494, 1983 19. Gamble JR, Harlan JM, Klebanoff SJ, et al: Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci USA 82:8667-8671, 1985 20. Larrick JW, Graham D, Toy K, et al: Recombinant tumor necrosis factor causes activation of human granulocytes. Blood 69:640-644, 1987

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21. Nawroth PP, Stern DM: Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 163:740-745, 1986 22. Collins T, Lapierre LA, Fiers W, et al: Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A, B antigens in vascular endothelial cells and dermal fibroblasts in vitro. Proc Natl Acad Sci USA 83:446-450. 1986 23. Koeffler HP, Gasson J, Ranyard J, et al: Recombinant human TNFa stimulates production of granulocyte colony-stimulating factor. Blood 70:55-59, 1987 24. Ostensen ME, Thiele DL, Lipsky PE: Tumor necrosis factor-a enhances cytolytic activity of human natural killer cells. J Immunol 138:4185-4188, 1987 25. Hori K, Ehrke J, Mace K, et al: Effect of human recombinant tumor necrosis factor (rH-TNF) on induction of macrophage tumoricidal activity. Proc Am Assoc Cancer Res 28:402A, 1987 (abstr) 26. De La Harpe J, Nathan CF: A semi-automated micro-assay for H202 release by human blood monocytes and mouse peritoneal macrophages. J Immunol Methods 78:323-336, 1985 27. Snedecor GW, Cochran WG: Statistical Methods. Ames, IA, Iowa State University, 1980 28. Huber C, Batchelor JR, Fuchs D, et al: Immune response-associated production of neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med 160:310-316, 1984 29. Aulitzky W, Gastl G, Aulitzky WE, et al: Successful treatment of metastatic renal cell carcinoma with a biologically active dose of recombinant interferon-gamma. J Clin Oncol 7:1875-1884, 1989 30. Abbruzzese JL, Levin B, Ajani JA: Phase I trial of recombinant human y-interferon and recombinant human tumor necrosis factor in patients with advanced gastrointestinal cancer. Cancer Res 49:4057-4061, 1989 31. Demetri GD, Spriggs DR, Sherman ML, et al: A phase I trial of recombinant human tumor necrosis factor and interferon-gamma: Effects of combination cytokine administration in vivo. J Clin Oncol 7:1545-1553, 1989 32. Talmadge JE, Adams J, Phillips H: Immunomodulatory effects in mice of polyinosinic-polycytidilic acid com-

plex with poly-L-lysine and carboxymethylcellulose. Cancer Res 45:1058-1065, 1985 33. Talmadge JE, Tribble HR, Pennington RW: Immunomodulatory and immunotherapeutic properties of recombinant y-interferon and recombinant tumor necrosis factor in mice. Cancer Res 47:2563-2570, 1987 34. Arend WP, Ammons JT, Kotzin BL: Lipopolysaccharide and interleukin-1 inhibit interferon-y-induced Fc receptor expression on human monocytes. J Immunol 139:18731879, 1987 35. Leeuwenberg JFM, Damme JV, Meager T, et al: Effects of tumor necrosis factor on the interferon-yinduced major histocompatibility complex class II antigen expression by human endothelial cells. Eur J Immunol 18:1469-1472, 1988 36. Crawford J, Ringenberg S, Propert K, et al: A dose response study of the biological activity of interferongamma (IFN-G) in patients with advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 8:867A, 1989 (abstr) 37. Fuchs D, Hausen A, Reibnegger G, et al: Neopterin as a marker for activated cell mediated immunity: Application in HIV infection. Immunol Today 9:150-155, 1988 38. Troppmair J, Nachbaur K, Herold M, et al: In-vitro and in-vivo studies on the induction of neopterin biosynthesis by cytokines, alloantigens and lipopolysaccharide (LPS). Clin Exp Immunol 174:392-397, 1988 39. Schoedon G, Troppmair J, Adolf G, et al: Interferony- enhances biosynthesis of pterins in peripheral blood mononuclear cells by induction of GTP-cyclohydrolase I activity. J Interferon Res 6:697-703, 1986 40. Kwon NS, Nathan CF, Stuehr DJ: Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J Biol Chem 264:20496-20501, 1989 41. Kilbourn RG, Gross SS, Jubran A, et al: N0 -methyl-Larginine inhibits tumor necrosis factor-induced hypotension: Implications for the involvement of nitric oxide. Proc Natl Acad Sci USA 87:3629-3632, 1990 42. Mentzer SJ, Faller DV, Burakoff SJ: Interferon gamma dependent induction of LFA 1 mediates homotypic adhesion of human monocytes. J Immunol 137:108-113, 1986

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