[CANCER RESEARCH 47, 2020-2027. April 15, 1987]

Direct Antiproliferative Effects of Recombinant Human Interferon-«B/D Hybridson Human Tumor Cell Lines1

Isaiah J. Fidler,2Ruediger Heicappell, Ikuo Saiki, Markus G. Grutter, Michel A. Horisberger, and Jakob Nuesch

Department of Cell Biology [1. J. F.. R. H., I. SJ, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030, andPharmaceuticals Division ¡M.G. G., M. A. H., J. N.J, Ciba Geigy Ld., Basel CH 4002 Switzerland

ABSTRACT

The purpose of these studies was to examine the antiproliferativeproperties of 16 recombinant human IFN-a B/D hybrids against varioushuman tumor lines of different histológica! origin and to determinewhether any of the hybrid molecules possessed immunomodulating activity that could activate antitumor properties in peripheral blood monocytesof normal donors.

Hybrids with the B domain at the Ml? terminal end exhibited higheractivity for antiviral activity and a higher level of direct antitumorantiproliferative activities as compared with hybrids with the D domainat the Ml; terminal end. The positive hybrids were directly cytostatic tomelanoma, glioblastoma, renal carcinoma, colon carcinoma, and prostaticcarcinoma cells. Tumor cell sensitivity to IFN-a hybrids was independentof sensitivity to ll-'V-y or to Adriamycin. The growth of a normal cell

line (human embryo fibroblast) was unaffected by IFN-a hybrids but wascompletely arrested by Adriamycin. Some of the IFN-a hybrids werealso cytostatic to mouse melanoma, lung carcinoma, and fibrosarcomacell lines, albeit at lower levels than they were to human cells.

The incubation of monocytes with IFN-a hybrids with the B domainat the Ml. terminal end was also associated with marked antitumorcytotoxicity. Kinetic studies, however, indicated that this activity wasattributable to IFN-a carried on monocytes and acting directly on tumorcells. We conclude that recombinant human IFN-a B/D hybrids possesspotent direct antiproliferative activity against a large variety of humantumor lines.

INTRODUCTION

The IFNs3 are a large group of inducible proteins with potent

antiviral, antitumor, and immunomodulating activities (1, 2).The IFN are classified according to antigenic specificities into3 broad classes: the leukocyte derived IFN-a; fibroblast-derivedIFN-/8; and T-lymphocyte-produced IFN-7. Native, partiallypurified, and recombinant IFN-a have been used clinically andfound to have antitumor activities against several tumors (1,3-5). Two major mechanisms have been proposed to account forthe antitumor effects of IFN-a: (a) direct antiproliferative effects on tumor cells or induction of a differentiation phenotypein the tumor cells (6-11); and (Z»)activation of nonspecific hostdefense mechanisms, such as natural killer cell (6, 12, 13) ormacrophage tumoricidal properties (13, 14).

Recent progress in recombinant DNA technology has allowedIFN-a to be expressed and produced in microorganisms. IFN-a subtypes have been shown to express different antiviral andantiproliferative activities (11, 15-18). The multiple biologicalactivities or effects of the IFN-a subtypes have prompted investigations into the determinants on the protein that are respon-

Received 8/25/86; revised 1/14/87; accepted 1/16/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported in part by funds from Ciba Geigy A.C., Basel, Switzerland.1To whom requests for reprints should be addressed, at Department of Cell

Biology, The University of Texas, M. D. Anderson Hospital and Tumor Instituteat Houston, 6723 Bertner Avenue (173), Houston, TX 77030.

3The abbreviations used are: IFN, interferon; ADM, Adriamycin; HBSS,Hanks' balanced salt solution; PBS, phosphate-buffered saline (20 mM NaH2PO«-80 mM K2HPO.-0.15 mM NaCl, pH 7.2); RFU, relative fluorescence units; PMSF,phenyl-methyl-sulfanyl-fluoride.

sible for such activities. To better define the sites on the IFNmolecule that mediate these biological effects, Meister et al.(19) have constructed hybrid molecules between recombinantlymphoblastoid IFNs, Ly IFN-D (a 1) and Ly IFN-B (a 8). Thecombination of various parts of 2 different interferon subtypesresults in the formation of new molecules with biological activity similar to one or both parental molecules or, more likely,with a different spectrum from that of either parental molecule.

The availability of a large number of new and unique IFN-ahybrid molecules prompted us to examine whether any of thesemolecules possessed antitumor activities. We also wished todetermine whether the antiproliferative activity is direct or canalso involve the activation of macrophages, the antitumor cytostatic and cytolytic activities of which are well established(20).

MATERIALS AND METHODS

Interferon Hybrid Construction. B/D hybrid interferons were constructed as outlined previously (19). Briefly, the coding sequences ofthe parent expression vectors were cleaved at 3 common restrictionsites to generate fragments comprising the coding sequences for theamino acids 1-60, 61-92, 93-150, and 151-166, respectively. Thesefragments were used to generate the hybrid interferon genes uponligation of the appropriate DNA segments. B/D hybrid interferonconstructions were transferred to the yeast PH05 promoter and transformed into Saccharomyces cerevisiae GRF18 on a high-copy-numbervector [yeast 2 urn vector p3DB207 (19)).

Interferon Preparation. S. cerevisiae strains harboring one of the 14indicated B/D hybrid interferon expression plasmids were grown in 30-liter fermenters. After fermentation, cells were harvested and resus-pended in 0.1 M Tris-HCl (pH 8.0)-5 mM EDTA-0.5 M NaCl-20 /¿Mphenylmethylsulfonyl fluoride at 10 ml of buffer/1 g of cell pellet. Afterthe suspension was cooled to 5-1 (!"(.'. it was passed through a Dyno

Mill (type KDL Pilot 0.6 liter) provided with polyurethane agitatordisks and 500 nil glass beads 0.5-0.75 mm in diameter. The suspensioncontaining the broken cells was clarified by centrifugation (Sonali GSArotor, 12,000 rpm at 4°Cfor 20 min). Polyethyleneamine (Polymin P,

pH 8.0) was added to the IFN-containing solution to a final concentration of 0.25% (w/v). The solution was stirred for l h and then centri-fuged. The IFN hybrids were further purified by affinity chromatogra-phy using a monoclonal antibody designated SA-144 (22), essentiallyas described previously (23).

The active IFN fractions were dialyzed against PBS. The concentration of the purified proteins was determined by the method of Bradford(24) using bovine serum albumin as a reference. The purity of the B,D, and hybrid IFN preparations was tested by sodium dodecyl sulfatopolyacrylamide gel electrophoresis (19) and estimated to exceed 90%.The IFN-a hybrids were free of endotoxins, as determined by theI,¡mullísamebocyte lysate assay (<0.1 ng/ml) (Cape Cod Associates,Woods Hole, MA).

Other Reagents. Recombinant human IFN-7 (specific activity, 2 xIO6 un its/nig) was obtained from Ciba Geigy Ltd. ADM, which was

obtained from the National Cancer Institute, was dissolved in 0.9%NaCl solution to 1 mg/ml and stored at —20°Cuntil used. Ca2+- andMg2+-free HBSS, RPMI 1640 medium, and Eagle's minimal essential

medium were purchased from M. A. Bioproducts, Walkersville, MD.A stock solution of hydroethidine was prepared by dissolving 7 mg ofhydroethidine in 1 ml of /V.TV-dimethylacetamide (Polysciences, Inc.,

2020

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

Warrington, PA). The working solution (28 /ig/ml) was prepared bystirring 40 /<I of stock solution into 10 ml of PBS containing ( V ' andMg2*. The resultant solution was filtered through a 0.22-jiin filter.

Cell Cultures—Targets. The human cell lines used were A375 melanoma parental and metastatic variant, HT-29 colon carcinoma andNataseli glioblastoma, SN12C and SN12L1 renal cell carcinomas, PC-3 prostatic carcinoma parental and PC-3M metastatic variant, andnontumorigenic human embryo lung fibroblasts (20).

The mouse cell lines used were B16F10 melanoma (25); 3LL lungcarcinoma (26), and UV-2237 fibrosarcoma parental line and UV-2237ADM resistant variant line (27). All cell lines were cultured in vitro in75-cm2 flasks (Falcon Plastics, Oxnard, CA) with Eagle's minimal

essential medium supplemented with 10% fetal bovine serum, vitamins,sodium pyruvate, nonessential amino acids, and L-glutamine (M. A.Bioproducts). The cultures were incubated at 37°Cin a humidified

atmosphere containing 5% CO2-95% air. All cultures were free ofpathogenic mouse viruses and of Mycoplasma.

Characterization of IFN-a Hybrids for Antiviral Activity. The specificactivity of recombinant IFNs was determined in an antiviral assay thatmeasured the plaque reduction of vesicular stomatitis virus. Confluentcultures of human embryonic foreskin diploid cells (Flow 7000) andprimary calf kidney cells were incubated with serial dilutions of IFNfor 18 h before infection with the virus. The procedures for plaqueassay and cell cultivation have been described in detail previously (28).Each assay included a laboratory standard calibrated against the Leukocyte Human Reference Interferon G-023-901-527. The specific activities on human and bovine cells were expressed in international unitsper mg of protein.

In Vitro Assay for Direct Tumor Cell Growth Inhibition of IFN-aHybrids and Other Agents. Tumor cells were seeded into wells of 96-well Microtest II tissue culture plates (Falcon Plastics) at a density ofIO4cells/well of 38 mm2. (Previous studies established that this is the

optimal seeding concentration that avoids full confluency for up to 4days). One day later, the cultures were washed and incubated for 3 dayswith various amounts of test materials. The monolayer cells were thenwashed 5 times with PBS (Dynawasher II 96-channel washer/aspirator;Dynatech Laboratories, Alexandria, VA) and incubated with PBS containing 28 ¿ig/mlof hydroethidine for 30 min at room temperature.During this incubation period, the plates were covered with aluminumfoil to prevent exposure to light. After 5 washes with PBS, the fluorescence-labeled cells were lysed with 1% Triton X-100. The number of

labeled cells was determined by the relative fluorescence monitored bya Microfluor Reader (Dynatech Laboratories, Springifeld, VA)equipped with a 250-nm excitation filter and a 400-nm high-passemission filter. The results were expressed as RFU.

The percentage of cytostasis activity was calculated by the formula

[(A - B) -!-A]x 100

where . I is the RFU in tumor cells incubated with control medium andBis the RFU in tumor cells incubated with test materials. The statisticalsignificance of the results was determined with the 2-tailed Student's t

test.Isolation and Culture of Human Peripheral Blood Monocytes. Mono-

nuclear cells were obtained from incidental samples derived from collections of platelet concentrates using an IBM 2997 blood cell separator(29). Monocytes were isolated from the mononuclear cell sample bycentrifugal elutriation using a JE-6B Beckman elutriation rotor, asdescribed previously in detail (30, 31). Monocytes with greater than97% viability were washed twice with HBSS, resuspended in RPMI1640 supplemented with 5% human AB serum to a concentration of 5x 10s monocytes/ml, and plated into 96-well Microtest II plates (FlowLaboratories, McLean, VA) at a concentration of 1 x 10* cells/well.After a 2-h incubation, the nonadherent cells were removed by vigorouswashing with medium. At this point, the purity of monocytes was morethan 99%, as assessed by examining cell morphology, phagocytosis,and nonspecific esterase staining.

In Vitro Incubation of Monocytes by IFN-a Hybrids and Other TestAgents. Monocytes were incubated in endotoxin-free medium alone inmedium containing the various IFN-a hybrids, or in IFN--y for 18-24

h at 37°C.The monocyte cultures were then rinsed thoroughly with

media before tumor-target cells were added.Monocyte-mediated Cytolysis-Cytotoxicity. Cytotoxicity was assessed

by measuring the release of radioactivity, as described previously (32).Target cells in their exponential growth phase were incubated in theappropriate medium which contained [125I]iododeoxyuridine (0.3 ¿iCi/

ml; specific activity, more than 2000 ci/mmol; New England Nuclear,Boston, MA) for 24 h. The cells were then washed twice to removeunbound radioiodine, harvested by a 1-min trypsinization (0.25 Difco

trypsin:0.02% EDTA), and washed. The labeled cells were resuspendedin medium, and 1 x IO4 cells were plated into culture wells to obtain

an initial target:effector cell ratio of 1:10. Radiolabeled cells were platedalone as a control group. After 16 h, the cultures were washed to removethe nonadherent target cells, refed with fresh medium, and then culturedfor an additional 2 days. At this time, the cultures were washed twicewith HBSS, and the adherent viable cells were lysed with 0.1 ml of 0.5N NaOH. The radioactivity of the lysate was measured in a gammacounter, and the percentage of specific cytotoxic activity of the monocytes was calculated as

l(A-B)+A}x 100

where A is cpm in cultures of control monocytes and target cells and Bis cpm in cultures of monocytes pretreated with test agents and targetcells.

Monocyte-mediated Cytostasis-Cytotoxicity. Tumor cells were seededinto wells containing monocytes preincubated in endotoxin-free medium (control) or with dilutions of IFN-a or IFN-f. Seventy-two hlater, we determined the number of cells in the wells after they wereincubated with hydroethidine, as described above. Previous studies (33,34) have established the accuracy of this assay in monitoring cytotox-icity because it produces results identical to those obtained by visualtechnique (cell count) and by release of radioactive label from targetcell DNA. The percentage of cytostasis activity was calculated by theformula

[(A-B) +A]x 100

where A is the RFU in wells containing target cells and control monocytes and B is the RFU in wells containing target cells and monocytespreincubated with IFN-a or IFN--y.

Control Studies: Kinetics of IFN-a Interaction with Monocytes forCytostasis Activity. Tumor cells or adherent monocytes were incubatedwith 100 or 1000 units/ml of BBBB, BBDD (positive) or DDDD,DDBB (negative) IFN-a hybrids for periods ranging from 2-24 h before

adding tumor cells. By the end of a 24 h incubation period, the tumorcells or the monocytes were washed 3 times, and 1 x IO4 A375 cells

were added to each well of monocytes. Three days later, cell growthwas assessed and the percentage of cytostasis was calculated as describedearlier.

In the final set of experiments, treatment groups for the directcytostasis assay were medium for 24 h (control), IFN-a for 24 h(positive), medium for 8 h followed by IFN-a for 16 h, medium for 20h followed by IFN-a for 4 h, medium for 22 h followed by IFN-a for 2h, and medium for 23.5 h followed by IFN-a for 0.5 h. The treatmentgroups in the monocyte assays were monocytes incubated in mediumfor 24 h (control), IFN-a for 24 h (positive), IFN-a for 20 h followedby medium for 4 h, IFN-a for 16 h followed by medium for 6 h, IFN-

a for 2 h followed by medium for 22 h, medium for 22 h followed byIFN-a for 2 h, and medium for 23.5 h, followed by IFN-a for 0.5 h. In

the direct antiproliferative assay, the target cells were washed and refedwith medium, and cytostasis was determined 72 h thereafter, as described above. For the monocyte assays, the monocyte cultures werewashed thoroughly, and 1 x IO4 [I25l]iodoxuridine-labeled A375 cellswere used. The percentage of cytotoxicity (monocytes) - cytostasis(direct assay) was calculated as described above.

Statistical Analysis. The statistical significance of differences betweentest groups was analyzed by the Student's t test (2 tailed).

2021

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

RESULTS

Antiviral Activities of IFN-a B/D Hybrids. Before embarkingon studies detailing the antiproliferative activity of the variousIFN-a hybrid molecules, we examined their antiviral effects(against vesicular stomatitis virus) on human embryonic foreskin diploid cells. Table 1 lists the hybrids in their decreasingorder of activity on human diploid cells. We also list the specificactivity on bovine cells because these units approximate theprotein concentration.

The antiviral potency of the hybrids (on human cells) isdistributed within a broad range. The IFN-a B hybrids (BBBB,BBB-, BB- -, B—) exhibited the highest potency. The lowestpotency (IO3 less than IFN-a B) was exhibited by the IFN-a Dhybrids (DDDD, ODD-, DD- -, D- - -).

Direct Antiproliferative Effects of IFN-a B/D Hybrids againstHuman A375 Melanoma Cells. In the first set of antiproliferative experiments, A375 melanoma cells were cultured in medium (control) and in medium containing 1, 10, 100, or 1000units/ml of the various IFN-« B/D hybrids. Seventy-two hlater, the number of viable tumor cells was determined byincorporation of the vital fluorescence dye hydroethidine. Thisexperiment was repeated 4 times with nearly identical results.The data from a typical experiment, shown in Fig. 1, clearlydemonstrate that the IFN-a hybrids containing the B domainat the Ml.- terminal end produced significant cytostasis againstthe A375 melanoma cells (P < 0.001). In contrast, the majorityof the IFN-a hybrids with the D domain at the NH2 terminalend were far less effective in mediating tumor cytostasis.

Direct Antiproliferative Effect of IFN-a B/D Hybrids againstVarious Human Tumor Lines. In the next set of experiments,we examined the spectrum of antiproliferative activity of theIFN-a B/D hybrids against several human tumor lines of different histological origin. Specifically, we examined the effectsof IFN-a DDDD and DDBB (negative) and BBBB and BBDD(positive) hybrids. In these studies, we also examined the antiproliferative activities of recombinant human IFN--y. The an-

titumor effects of ADM served as a positive control.The data are summarized in Figs. 2 and 3. IFN-a BBBB and

BBDD hybrids produced significant cytostasis (albeit at different levels) against the A375 melanoma metastatic variant,Natasch glioblastoma cell line, and 2 human prostatic carcinoma cell lines (P < 0.001) (Fig. 2). Two lines recently derivedfrom a human renal cell carcinoma (SN12C and SN12L1), andthe HT-29 colon carcinoma cells were also sensitive to cytostasis by the BBBB and BBDD IFN-a hybrids (P < 0.005).

Table 1 Specific antiviral activities of recombinant human interferon-a B/Dhybrids on human and bovine cells

Specific activity (IU x 10*/mg) on

Interferon hybridmoleculeBBBBBBDDBBDBBDBBBDBDBDDDBDDBBBBDDBDDDBDBDDDBDDDDDBBDDBBBDDBBDDBDHuman

diploidcells2.521.71.10.50.50.40.30.150.10.050.040.010.0090.0040.001Bovine

diploidcells2.52.50.50.81.30.70.70.51.70.80.61.40.50.50.71.6

100

£ 80

tf V>

^ S 60

i i££ 4C¿S£ <r

LUÕ" 20

A375 MELANOMA

"2§Units/ml

IFN HYBRID DDDD DDDB DDBB DBBB DBOD OBBD DDBD DBDB

100

«80CJ-y.LUiliit

3Jg60ce"•

z 40affiuSES|°-

200•[|

1iI1iirit.1.1"2§1 "2§§"2^P "2?§ "s§§"s§§"s§§"s?§

IFN HYBRID BBBB BBBD BBDD BDDD BDBB BDDB BBDB BDBDFig. 1. Antiproliferative effects of recombinant human IFN-a B/D hybrids

against the A375 human melanoma cells. Ten thousand melanoma cells wereplated into culture wells and incubated with the various IFN-a hybrids at theindicated doses for 72 h. The percentage of growth inhibition was calculated bycomparing triplicate cultures with test materials with triplicate control cultures.The results are from one of 4 experiments. Variation from the mean did notexceed 10%. Values exceeding 40% cytostasis were highly significant (/' f. 0.001).

Neither IFN-a nor IFN-7 were cytostatic to a normal humanfibroblast line derived from fetal embryo lung (Fig. 3).

The susceptibility of the human cell lines to the cytostaticeffects of IFN-a was independent of their susceptibility to thecytostatic effects of IFN-7 or ADM. For example, ADM inhibited the growth of the normal embryo lung fibroblasts, but thegrowth of these cells was unaffected by any of the IFN-ahybrids.

Antiproliferative Effects of Recombinant Human IFN-a B/DHybrids on Murine Tumor Cells. In the next set of studies, weexamined the direct antiproliferative effects of recombinanthuman IFN-a B/D hybrids, recombinant mouse IFN-7, andADM against several mouse tumor cell lines (B16 melanoma,3LL carcinoma, and UV-2237 fibrosarcoma, ADM sensitiveand resistant). The data are shown in Fig. 4. In general, theantiproliferative effects of the recombinant human IFN-a hybrids were not as remarkable as compared with the resultsobtained with the various human cell lines. Once again, sensitivity or resistance to the chemotherapeutic agent ADM wasdissociated from sensitivity to the direct cytostatic effects ofIFN-a. We base this conclusion on data showing that ADM-resistant UV-2237 fibrosarcoma cells were as sensitive to highdoses of the IFN-a (BBDD) hybrid as were parental UV-2237fibrosarcoma cells sensitive to ADM (P < 0.01).

Interaction of IFN-a B/D Hybrids with Peripheral BloodMonocytes: Tumor Cell Cytostasis. Peripheral blood monocytes

2022

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

Fig. 2. Antiproliferative effects of recombinant human IFN-a B/D hybrids, recombinant human 11N -

and Adríamycinagainst the human cell lines A375melanoma metastatic variant, Natusch glioblastoma,PC-3 prostatic carcinoma, and PC-3 prostatic carcinoma metastatic variant. Experimental conditions andcalculations of data were identical to those describedin the legend for Fig. 1. Results are from one of 3experiments. U, units.

Fig. 3. Antiproliferative effects of recombinant human IFN-a B/D hybrids, recombinant human IFN-%and Adriamycin against the human cell lines SN12Crenal cell carcinoma, SN12L1 renal cell carcinomametastatic variant, HT-29 colon carcinoma, and normal embryo lung fibroblasts. Experimental conditionsand calculations of data were identical to those described in the legend for Fig. I. Results are from oneof 3 experiments. (7, units.

100io£<*

80itsSÌ60P*""SS

40=;SOCJ|S

ai5n-100A375

Melanoma Metastatic Variant¿¿--.-,!!

iÜÕ2

80ElUJ

!_Se

60Iz

*0—.LUO

UIf

20Z«Cn-Glioblastoma--_-II1-s?l

-28| -28| -sap -58| ss-sg ~-?s ~ss| •*§!~-§|"28|is-ssg

S S g -8_ggìsi si si ¿-Êtï|i9=¿5 93 95 5= J f fj5i I £ S£-100GOS

w80SÌ

60p££

400ÃŒJOu

Su£ "•200-100PC-3

Parent¿_---,1"w80£gLU

,_.

- , 11S"^5

«ou££I 20n-eo

co oeSCO o 'O1 al ^ ? ^"^*""ESt

!± it«PC-3

MetastaticVariant_---•¡

;-sap-sSj -sas, -sg| -ss| ss-ss ' -SB1.\\[I|-*S¡ -S8| -28| -Sg||S-aSaeoma

c aeocoa .cSCDOOO "ô aaocoa u —— en = o — en — >~= »--5 aWfflSIsWffl'B >-? i" "p

aEmEaEcoE.>E E E aEaoEaEcoE^E E fi95 9= 9= ^5 Õ5. S g1 9=^5^5.95£5|?

£ £ £ £ S itti«100§

¡280!>e

wstS"¡i

«ÜI*20nptoo«SN12C

RanalCellCarcinoma u coggu?C/9__-Ml

,_^S

60„li!»—J,5<->oc

SS40SS§E£E

P"- 20npSN12

li RanalCellCarcinoma__-ÕI"

-S8| -28| -=8§ -S8| -S8| S™»$ 'S8| "S8| "S8| -S8| -S8|BS'Sgooacoa

.= ococoo.ga— E 9 S <V 3 V >-? ^- "c a-ECD~a=oa= x-Ç »-•£

aEcoEoEcoE^E E E oEoDEaEa3EzÊ EE^=¿= ^=¿sÕ2 =^¿s¿a¿s¿s£3S£100cÄ5s2

80U.CO14*

(_ë|

60¡B55

«OCJa.

uj120<

Ap100HT-29

ColonCarcinoma--û

..„O

O,109¡g

»UJ

5Sa

60S

"|S40OU|ff

20n-Normal

Ftooblasts---,1|

-S8| -28| -2S| -S8| SS-ag " 'S?i 'si| -SS| -=1| -«i|S3""lOOCOO

cO0O3Oco CD en TÎ _ ers oa m en'Sgì si st si i- i lï gìst gìsi ^1ri¿593 ¿5 9= Õ5 |? ¿5 ¿5 ¿5 ¿5 £= |?

were incubated for 18-24 h with the 16 IFN-a B/D hybrids, was repeated 3 times with nearly identical results, promptingAfter washing the monolayers, A375 melanoma cells were us to examine whether IFN-a treatment of blood monocytes

added and 3 days later, we determined the extent of cell growth, also activated the cells for antiproliferative properties. TheseThe data shown in Fig. 5 suggest that IFN-a hybrids with the data are shown in Fig. 6. Once again, incubating the monocytesB domain at the NH2 terminal end activated monocytes to with the IFN-a hybrids with the B domain at the NH2 terminal

become cytotoxic to the A375 melanoma cells. The experiment end produced significant cytostasis of the A375 cells.2023

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

100CJ

CO80tiSÌ

60r;h-i"ÕÕ

£40ocjceocOL.

LÌ-"*)fipò.20Z«t

nBIG

FIO MelanomaÉ-_-

i,i,1

„1,l

100

l 60

20

3LL Carcinoma

-sg| -eg| -sg| -sg| -sg| ss-a

Fig. 4. Antiproliferative effects of recombinant human IFN-a B/D hybrids, recombinant mouse IFN--y,and Adriamycin against the mouse tumor linesB16FIO melanoma, 3LL carcinoma, UV-2237 fibrosarcoma, and UV-2237 fibrosarcoma resistant to Adriamycin. Experimental conditions and calculations ofdata were identical to those described in the legend forFig. 1. Results are from one of 3 experiments. U, units.

SE Q E e E -ÎE~. _ •-__ « ^ * ^ill

W80604020n_

UV-2237Patent--,.l

.

100

i¡ç/280

¡S

!jf 60

u «O: cc\* 20

0

a ,E m E Q E !

£ £~ £~ ;

UV-2237AOMR

sg| -sg| ss-g

¡I

£ gil il<?3 örsz~ g-

The remarkable similarity among the 3 assays (direct anti-proliferative effects, monocyte-mediated cytostasis, and monocyte-mediated cytotoxicity) prompted us to examine the possibility that the antitumor effects in all 3 assays were attributableto the direct effects of the IFN-a molecules on the target cells.

Control Studies for the Effects of IFN-a B/D Hybrids onMonocytes-Tumor Interaction. Tumoricidal activation of mon-ocytes by lymphokines requires that the lymphokine first bindto a monocyte surface receptor. Once internalized, the lymphokine must act on intracellular sites to begin the process ofactivation, which may require 6-18 h for completion (20). Weexamined the specific kinetics of the interaction of IFN-a withmonocytes to generate cytostasis against A375 cells. Nearlyidentical results were obtained in both the direct antiprolifera-tive assay (IFN-a and A375 cells) and the "monocyte-mediated"

cytostasis-cytotoxicity assay. The incubation of either the tumorcells or monocytes for 24 h with 100 or 1000 units/ml of IFN-a BBBB or BBDD hybrids produced significant cytostasis bythe end of a 72-h incubation period. Direct treatment of tumorcells or of monocytes for 2 h with IFN-a hybrids followed by a22-h incubation with medium (before tumor cells were addedto the monocyte monolayer) was not associated with significantantiproliferative effects. In contrast, cytostasis was significant(P < 0.001) when the A375 cells (direct) or monocytes (indirect)were first incubated with medium for 22 h followed by a 2-hincubation with IFN-a (before A375 cells were added to mon

ocytes).In the final set of experiments, we expanded the kinetics

study to determine the minimal interaction time of IFN-a withtarget cells (Fig. 7) or with monocytes (Fig. 8) necessary togenerate cytostasis or cytotoxicity against A375 melanomacells. The data in Fig. 7 clearly indicate that the sequence oftumor cell treatment with IFN-a and medium did not alter theresults. At any treatment combination, the BBBB and BBDDIFN-a hybrids produced significant cytostasis of A375 cells.

00 = >.:«P-§ z .

l CO COI = CO = O —i E co E o E

-28§ "S8§ Sg-'ag

9 . •§-l'I Il-- |-

Moreover, even a 30-min incubation of A375 cells with IFN-aBBBB and BBDD hybrids was sufficient to produce significanttumor cell cytostasis (P < 0.001). The activity of these IFN-ahybrids was dose and time dependent, i.e., 1000 units/ml produced higher levels of cytostasis than did 100 units/ml. A 24-or 8-h incubation period produced higher levels of cytostasis ascompared with a 2-h or 30-min exposure of tumor cells to IFN-a hybrids.

The data obtained with the monocyte cytotoxicity-cytolysisassay were different. As shown in Fig. 8, the timing of IFN-atreatment was crucial to the outcome. If the monocytes weretreated with IFN-a for even 16 h and then incubated in mediumfor 8 h, no antitumor activity was found. For example, treatment of monocytes for only 2 h or even 30 min was associatedwith significant tumor cytotoxicity provided that the treatmentoccurred just before the tumor cells were added. These datadiffer from those obtained from the direct interaction of IIVa with target cells (Fig. 7). Moreover, because lymphokineactivation of monocytes requires at least a 16-h period (35, 36),the data in Fig. 8 suggest that cytotoxicity against the A375cells was directly mediated by the IFN-a hybrids and not viathe pathway of IFN-a activation of macrophage antitumorproperties.

DISCUSSION

The major purpose of our studies was to determine thebiological activities of a new group of IFN-a hybrids. B/Dhybrids were constructed after cleaving the parent molecules togenerate fragments comprising the coding sequences for aminoacids 1-60, 61-69, 93-150, and 151-166 (17). The 14 newhybrids and the 2 parental molecules (BBBB, DDDD) wereexpressed in 5. cerevisiae. We examined these 16 differentmolecules for their ability to neutralize vesicular stomatitis virusinfection (Table 1) and for direct antiproliferative activities

2024

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

100 r

Fig. 7. Minimal interaction time of recombinant IFN-a B/D hybrids with A375 melanoma cells required to bring about growthinhibition. Tumor cells were incubated withthe indicated IFN-a hybrids and medium forthe specified sequence and duration. The percentage of cytostasis was determined 72 h later.Results are from one of 3 experiments. Alldeterminations were calculated from triplicatecultures. Variation from the mean did not exceed 10%. All values larger than 35% werehighly significant (/' £0.001).

id 52u- CO

80

I o 60

i«i z 40

20

Units/ml

IFN Hybrid

Sequence ofTreatment

i iIFN-a 24 hours Medium8 hours

IFN-a 16 hours

§§?| §| §§

Medium20 hoursIFN-a 4 hours

§1 Õ1 i§ i§

Medium22 hoursIFN-a 2 hours

§§Ü S§ 8g

I I I I

Medium 23.5 hoursIFN-a 0.5 hour

Fig. 8. Minimal interaction time of recombinant IFN-a B/D hybrid with human bloodmonocytes necessary to subsequently producegrowth inhibition of A37S melanoma cells.Monocytes were incubated with the indicatedIFN-a hybrids and medium for the specifiedsequence and duration. The cultures werewashed, and A37S tumor cells were added.Growth inhibition was determined 72 h later.Results are from one of 3 experiments. Alldeterminations were calculated from triplicatecultures. Variation from the mean did not exceed 10%. All values larger than 35% werehighly significant (P < 0.001).

Recombinant human IFN-a A/D hybrids have been shownto cross species barriers and to activate mouse immune cells todestroy mouse tumor targets (38). For these reasons, we examined the antiproliferative effects of the IFN-a B/D hybridson 4 different mouse tumor lines. Although antiproliferativeactivity was detected, the level of activity was far lower thanthat observed for recombinant mouse IFN-7 or for ADM. Here,again, the antiproliferative activity of the IFNs was shown tobe independent of the antiproliferative activity of ADM. Webase this conclusion on the study showing that the growth ofthe ADM-resistant mouse tumor line UV-2237 (27) was unaffected by ADM (even at the dose of 500 ng/ml), yet 40%cytostasis of these cells was achieved with either the recombinant IFN-T or IFN-a BBDD hybrid.

Several reports have indicated that IFN-a (A/D) moleculescan activate human monocytes /'// vitro to lyse human tumor

cells in vitro (13, 14). Because IFNs can indeed function in vivothrough modulation of host immunity (1, 2), we wished toexamine this possibility in detail. Indeed, the initial studies (3independent experiments) suggested that IFN-a BBBB, BBB-,BB- -, and B— hybrids activate monocytes to become tumorcytostatic and cytolytic (Figs. 5 and 6). However, subsequentkinetic studies (Figs. 7 and 8) suggested that such was not thecase. Kinetic studies of macrophage activation by lymphokinesor synthetic immunomodulators suggest that these moleculesmust first bind to the cell surface to become internalized intothe cytoplasm of the cells, where the "activation" process occurs(35, 36). These events require a lag time of at least 8-16 h innonprimed monocytes (36). In the experiments shown in Fig.8, it is clear that when IFN-a is incubated with monocytes foreven 30 min before sensitive A375 melanoma cells are added,high levels of tumor cytostasis occur. These are not the expected

100oLU<

on—OU"S

60S

20UJ0Units

mlIFN

HybridSequence

ofTreatment-_..if

§| S|iililiIFN-a

24 hoursig

i¡ i|!|liliIFN-a

20 hours

Medium 4 hoursif

§| §|ifliliIFN

a 16 hoursMedium8 hoursii

fi SSIIliliIFN-a

2 hours

Medium 22 hours;|if

ig ififliliMedium

22 hoursIFN a 2 hoursSÌ

S| i|ifi

i iiMedium23.5 hours

IFN-a 0.5 hour

kinetics for a macrophage-activating agent, e.g., IFN--y (34).The data we show in Figs. 7 and 8 suggest that the IFN-amolecules can rapidly bind to the surface of monocytes or tumorcells. Once bound onto cells, the molecules can be released toaffect the growth of the sensitive tumor target cell. Thus, themonocyte can be considered to be a carrier for IFN-a, and theanti-proliferative effects shown in Figs. 5, 6, and 8 are notattributable to the activation of antitumor properties in macrophages but to "carry-over" effects. This issue can also be

resolved by using radiolabeled IFN-a, and these studies are nowunderway.

In conclusion, we have demonstrated that members of a newclass of IFN-a hybrids, which consists of fragments from the Band D subtypes, have significant antiviral and antitumor activities. Because the biological activities of IFN molecules willdepend on their interaction with target cells, the availability ofthe IFN-a B/D hybrids and their broad spectrum of antitumoractivity encourages continued studies of their effects under invivo conditions.

ACKNOWLEDGMENTS

We thank Lola Lopez for her expert assistance in preparing thismanuscript.

REFERENCES

1. Borden, E. C., and Fall, L. A. Interferon: biochemical, cell growth, inhibitory,and immunological effects. Prog. Hematol., 350: 1-41, 1981.

2. Langer, .1.. and Pestka, S. Structure of interferons. Pharmacol. Ther., 27:371-401, 1985.

3. Gutterman, J. U., Fein, S., Quesada, J., Horning, S. J., Levine, J. F.,Alexanian, R . Bernshardt, I .. Kramer, M , Spiegel, II.. Colburn, W., Trown,P., Merigan, T., and Dziewanowski, Z. Recombinant leukocyte A interferon:

2026

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

IFN-a B/D HYBRIDS

pharmacokinetics, single-dose tolerance, and biologic effects in cancer patients. Ann. Intern. Med.. 96: 549-556, 1982.

4. Quesada. J. R., Reuben, .1.. Manning. J. T., Hersh. E. M., and Gutlerman.J. U. Alpha interferon for induction of remission in hairy-cell leukemia. N.Engl. J. Med., 310: 15-18, 1984.

5. Taylor-Papadimitriou, J., and Balkwill, F. R. Implications for clinical applications of new developments in ¡nterferonresearch. Biochim. Biophys. Acta,695:49-67, 1985.

6. Gast, L. G., Maith, C., Leiter, E., Gattringer, C., Mayer. I., Daxenbichler,G., Flener, R., and Huber, C. Effects of human recombinant «2arg-interferonand y-interferon on human breast cancer cell lines: dissociation of antipro-liferative activity and induction of HLA-DR antigen expression. Cancer Res.,«.•2957-2961.1985.

7. Blalock, J. E.. Geordiades, J. A., Langford. M. P., and Johnson, H. M.Purified human interferon has more potent anticellular activity than fibro-blast or leukocyte interferon. Cell. Immunol., 49: 390-394. 1980.

8. Borden, E. C., Hogan, T. F., and Voelkel, J. G. Comparative antiproliferativeactivity in vitro of natural interferons a and .. for diploid and transformedhuman cells. Cancer Res., 42:4948-4953, 1982.

9. Schiller, J. H., Groveman, D. S., Schmid, S. M., Willson, J. K. V., Cum-mings, K. B., and Borden, E. C. Synergistic antiproliferative effects of humanrecombinant ..54 or ßserinterferon on human cell lines of various histo-genesis. Cancer Res.. 46:483-488, 1986.

10. Rubin, B. Y., and Gupta, S. L. Differential efficacies of human type I andtype II interferons as antiviral and antiproliferative agents. Proc. Nati. Acad.Sci. USA, 77:5928-5933, 1980.

11. Greiner, J. W., Fisher, P. B., Pestka, S., and Schlom, J. Differential effectsof recombinant human leukocyte interferons on cell surface antigen expression. Cancer Res., 46:4984-4990. 1986.

12. Talmadge. J. E., Herberman, R. B., Chirigos. M. A., Maluish, A. E.. Schneider, M. A., Adams, J. S., Phillips, H., Thurman, G. B., Varesio, L., Long,C. W., Oldham. R. K . and Wiltrout, R. H. Hyporesponsiveness to augmentation of murine natural killer cell activity in different anatomical compartments by multiple injections of various immunomodulators including recombinant interferons and interleukin 2. J. Immunol., 135: 2483-2488, 1985.

13. Jett, J. R.. Mantovani, A., and Herberman, R. B. Augmentation of humanmonocyte-mediated cytolysis by interferon. Cell Immunol., 54: 425-434,1980.

14. Utsugi, T., and Soné,S. Comparative analysis of the priming effect of humanimri ti-i-im -.. a and .1on synergism with muramyl dipeptide analog for anti-tumor expression of human blood monocytes. J. Immunol., 136:1117-1122,1986.

15. Streuli, M., Hall, A., Boll, W., Stewart II. W. E., Nagata, S., and Weissman.C. Target cell specificity of two species of human interferon-a produced inEscherichia coli and of hybrid molecules derived from them. Proc. Nati.Acad. Sci. USA. 78: 2848-2852. 1981.

16. Week. P. K., Apperson, S., Stebbing, N., Gray, P. W., Leung, D., Shepard,M. H., and Goeddel, D. V. Antiviral activities of hybrids of two major humanleukocyte interferons. Nucleic Acids Res., 9:6153-6166. 1981.

17. Rehberg, E.. Kelder, B., Hoal, E. G., and Pestka, S. Speci lie molecularactivities of recombinant and hybrid leukocyte interferons. J. Biol. Chem..257: 11497-11502, 1982.

18. Sen, G. C., Herz, R., Davatelis, V., and Pestka, S. Antiviral and proteininducing activities of recombinant human leukocyte interferons and theirhybrids. J. Virol., 50: 445-450, 1984.

19. Meister, A., Uze, G., Mogensen, K. E., Gresser, I., Tovey, M. G., Grutter.M., and Meyer, F. Biologic activities and receptor binding of two human

recombinant interferons and their hybrids. J. Gen. Virol., 67: 1633-1643,1986.

20. Fidler. I. J. Macrophages and metastasis: a biological approach to cancertherapy. Cancer Res., 45:4714-4726, 1985.

21. Hinnen, A., Hicks. T. B.. and Fink, G. R. Transformation in yeast. Proc.Nati. Acad. Sci. USA. 75: 1929-1933. 1978.

22. Alkan, S. S., Weideli. H. J., and Schuerch, A. R. Monoclonal antibodiesagainst human leukocyte interferons for a definition of subclasses and theiraffinity purification. In: H. Peelers (éd.).Protides of Biological Fluids, pp.495-498, New York: Pergamon Press, 1983.

23. Staehelin, T., Hobbs, D. S., Kung, H.-F., Lai, C.-Y., and Pestka, S. Purification and characterization of recombinant human leukocyte interferon(IFLrA) with monoclonal antibodies. J. Biol. Chem. 256: 9750-9754, 1981.

24. Bradford. M. M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248-254, 1976.

25. Fidler, I. J. Selection of successive tumor lines for metastasis. Nat. NewBiol., 242:148-149, 1963.

26. Talmadge. J. E., and Fidler, I. J. Enhanced metastatic potential of tumorcells harvested from spontaneous métastasesof heterogeneous murine tumors. J. Nati. Cancer Inst.. 69: 975-980, 1982.

27. Giavazzi, R., Scholar, E., and Hart, I. R. Isolation and preliminary characterization of an Adriamycin-resistant murine fibrosarcoma cell line. CancerRes., «.-2216-2222, 1983.

28. Horisberger, M. A., and de Staritzky, K. Sensitivity of influenza A viruses tohuman IFNs in human diploid cells. FEBS Lett., 29: 207-210, 1985.

29. Hester. J. R., Kellogg, R. M., Mulzet, A. P., Kurger, V. R., McCredie, K. B..and Freireich, E. J. Principles of blood separation and component extractionin a disposable continuous-flow single-stage channel. Blood. 54: 254-262,1979.

30. Lopez-Berestein, G., Mehta, K., Mehta, R., Juliano, R. L., and Hersh, E. M.The activation of human monocytes by liposome-encapsulated muramyldipeptide analogues. J. Immunol.. 130: 1500-1504. 1983.

31. Turpin, J., Hester, J. P., Hersh, E. M., and Lopez-Berestein, G. Centrifugalelutriation as a method for isolation of large numbers of functionally intacthuman peripheral blood monocytes. J. Clin. Apheresis., 3:11-18, 1986.

32. Kleinerman, E. S., Schroit, A. J., Fogler, W. E., and Fidler, I. J. Tumoricidalactivity of human monocytes activated in vitro by free and liposome-encapsulated human lymphokines. J. Clin. Invest., 72: 1-12, 1983.

33. Bucana. C., Saiki, I., and Nayar, R. Uptake and accumulation of the vitaldye, hydroethidine, in normal and neoplastic cells. J. Histochem. Cytochem.,3*1109-1115, 1986.

34. Saiki, I., Bucana, C., Tsao, J. Y., and Fidler, I. J. A quantitative fluorescentmicroassay for identification of antiproliferative compounds. J. Nati. CancerInst., 77:1235-1240.1986.

35. Kleinerman, E. S., Fogler, W. E., and Fidler. I. J. Intracellular activation ofhuman and rodent macrophages by human lymphokines encapsulated inliposomes. J. Leukocyte Biol., 37:571-584. 1985.

36. Saiki, L, Soné,S.. Fogler, W. E.. Kleinerman, E. S., Lopez-Berestein. G.,and Fidler, I. J. Synergism between human recombinant gamma-interferonand muramyl dipeptide encapsulated in liposomes for activation of antitumorproperties in human blood monocytes. Cancer Res., 45:6188-6192, 1985.

37. Balkwill, F. R., Goldstein, L.. and Stebbing, N. Differential action of sixhuman interferons against two human carcinomas growing in nude mice. Int.J. Cancer, 35:613-617, 1985.

38. Nishimura, J., Nitsui, K., Ishikawa, T.. Tanaka, Y., Yamamoto, R., Suhara.Y., and Ishitsuka. H. Antitumor and antimetastatic activities of humanrecombinant interferon alpha A/D. Clin. Exp. Métastases.3:295-304,1985.

2027

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

1987;47:2020-2027. Cancer Res   Isaiah J. Fidler, Ruediger Heicappell, Ikuo Saiki, et al.  

B/D Hybrids on Human Tumor Cell LinesαInterferon-Direct Antiproliferative Effects of Recombinant Human

  Updated version

  http://cancerres.aacrjournals.org/content/47/8/2020

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/47/8/2020To request permission to re-use all or part of this article, use this link

Research. on August 14, 2021. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from