adoptive transfer of tumor cytotoxic macrophages …transfusion service, amsterdam, the...

(CANCER RESEARCH 50, 7450-7456. December 1. 1990]

Adoptive Transfer of Tumor Cytotoxic Macrophages Generated in Vitro fromCirculating Blood Monocytes: A New Approach to Cancer Immunotherapy1

Reinhard Andreesen,2 Carmen Scheibenbogen, Wolfram Brugger, Stefan Krause, Hans-Gerd Meerpohl,

Hans-Georg Leser, Hans Engler, and Georg W. LÃ¶hrMedizinische Klink l und Frauenklinik der Albert-Ludwigs-Universitat Freiburg im Breisgau. Freiburg, West Germany

ABSTRACT

Cells of the macrophage lineage are considered to be of specialimportance in the defense of the host against tumor development andspread. Immunotherapeutic strategies to stimulate macrophage (MAC)tumor cytotoxicity make use of activating compounds such as -y-interferon

which are given systemically. However, there are several lines of evidencethat in malignant disease the generation of cytotoxic effector M Us isimpaired. Both defective cell maturation and loss of responsiveness toactivation are described. Here, a first clinical phase I trial of adoptiveimmunotherapy in cancer patients using autologous MACs generated invitro from blood monocytes (MOs) is reported. Mononuclear cells wereisolated by cytapheresis and density centrifugation and cultured in hydro-phobic Teflon bags for 7 days with 2% autologous serum and recombinanthuman 7-interferon being present for the last 18 h. Cytotoxic Moderimi MACs were then purified by countercurrent elutriation andreinfused into the patient. A total of 72 therapies have been performedwith patients being treated i.v. (n = 8) and i.p. (n = 7). In vitro generatedMACs proved to be mature as judged by the expression of maturation-associated surface molecules (MAX antigens, CD16, CD51, CD71), werecytotoxic to U937 tumor cells, and were efficient secretory cells. Celldose escalation was performed in the first patients beginning with 10"

MACs to finally infuse the total number of cells recovered from onesingle cycle of isolation and culture. MAC yield varied from 1 to 17 xIO8representing 13-79% of MOs initially seeded. Adoptive MAC transfer was well tolerated. Side effects observed were low-grade fever(<38.5Â°C),induction of the coagulation cascade, and abdominal discom

fort after i.p. application. The procoagulant activity of MAC autograftswas cell dose dependent and demonstrated by the detection of circulatingfibrin monomers and thrombin-antithrombin complexes. Biological responses observed included elevated serum neopterin levels and the appearance of interleukin-6 in sera and ascitic fluids. Indication of a possibletherapeutic effect was only observed in i.p.-treated patients and consistedof disappearance of malignant ascites in 2 of 7 patients.

INTRODUCTION

In the search for new strategies to combat cancer, modulationof the immune response of the host to the malignant cell hasreceived increasing attention during the last decade. Variousbiological response modifiers such as interferons and interleu-kins have entered phase II clinical trials ( 1). Yet, only Â¡nselectedcancers could therapeutic efficacy be demonstrated so far (2).Because experimental evidence indicates that in malignant disease cytotoxic cell generation may be impaired in situ (3-9),concepts have been developed to build up a tumor cytotoxiccell potential ex vivo that can be reinfused into the autologoushost (10). The first clinical results of adoptive immunotherapyusing lymphoid effector cells (lymphokine activated killer cellsand/or tumor-infiltrating lymphocytes) have been promising

Received 3/28/90; accepted 8/30/90.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.

1Supported by Bundesminister fÃ¼rForschung und Technologie und DeutscheForschungsgemeinschaft.

2To whom requests for reprints should be addressed, at Medizinische Klinikl, Hugstetter Str. 55, D-7800 Freiburg, West Germany.

but were achieved only at the prize of high, although reversible,toxicity (11, 12).

Within the host defense system against the development andspread of malignant tumors, cells of the macrophage lineageare considered to be of special importance (13, 14). In animalmodels, ex vivo activated MACs1 have demonstrated their therapeutic efficacy when injected into tumor-bearing mice (15-17). However, attempts to make use of MACs for adoptiveimmunotherapy in humans are very limited and have beenhampered by the lack of technology to generate large numbersof mature competent effector cells. Because we could show thatwith maturation of MOs in vitro the functional competence interms of cytotoxicity (18), secretory repertoire (19), and theability to respond to appropriate stimulation develops (19, 20),a rationalized approach would require mature effector cells.Wereport here the first clinical trial of the in vitro generation andreinfusion of autologous tumor cytotoxic MACs activated byIFN-7 in tumor patients. Adoptive transfer of MACs was donei.v. and i.p. It is shown that large-scale technology to growcytotoxic MACs from circulating blood MOs of cancer patientsis feasible and that their reinfusion into the donor patient iswell tolerated. Low-grade fever and induction of the coagulationcascade were observed as therapy-related side effects. This exvivo modulation of the mononuclear phagocyte system resultedin a biological response of the host as documented by increasedlevels of neopterin, IL-6, and C-reactive protein.

MATERIALS AND METHODS

Patient Population

Fifteen patients with advanced metastasized cancer in whom standard therapy had failed or did not exist were treated according to aprotocol that had been approved by the local ethical committee onhuman experimentation. Four patients had non-small cell carcinomaof the lung, four had ovarian carcinoma, two had malignant melanoma,two stomach cancer, one carcinoma of the Fallopian tubes, one hadsquamous cell carcinoma of the tongue, and one had renal cell carcinoma. All but one patient had measurable disease either by physicalexamination, radiographie studies, or serum parameters. For comparison, leukapheresis was also performed on nine healthy volunteers.Signed informed consent was obtained from all patients prior to entryinto the study as well as from volunteers prior to leukapheresis.

Clinical Parameters Monitored

Complete blood cell counts and hepatic and renal function studieswere performed. Initial assessment and poststudy evaluation also included electrocardiogram and echocardiogram. In addition to routineanalysis of coagulation parameters (prothrombin, partial thromboplas-tin time, fibrinogen), fibrin monomers were measured using a semi-quantitative test (FM test, Boehringer Mannheim GmbH, Mannheim,

3The abbreviations used are: MAC, macrophage; MO, monocyte; IL, interleu-kin; TAT, thrombin-antithrombin III complexes; GM-CSF, granulocyte-macro-phage colony stimulating factor; TNF-n, tumor necrosis factor-Â«;LPS, lipopoly-saccharides; FRG. Federal Republic of Germany; ELISA, enzyme-linked immu-nosorbent assay; CEA, carcinoembryonic antigen; B2M, ft-microglobulin.

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ADOPTIVE IMMUNOTHERAPY USING MAC AS EFFECTOR CELLS

FRG) prior to MAC infusion and 15 min, l h, and 4 h, as well as 1and 7 days, after infusion. In selected patients, TAT were measured byELISA (Enzygnost TAT, Behringwerke AG, Marburg, FRG; normalrange, 1-4.1 ^g/liter).

Leukapheresis, Cell Isolation, and Culture

See Fig. 1 for a schematic description. Large numbers of mononu-clear cells were obtained by 2-h leukapheresis with a continous-flowcell separator (CS3000; Baxter, Munich, FRG). Between 5 and 7 litersof whole blood was processed at a flow rate between 30 and 60 ml/min. Acid-citrate-dextrose was used as the anticoagulant. Mononuclearcells were further separated from RBCs and contaminating granulocytesby Ficoll-Hypaque gradient centrifugation (IBM 2997). The separatedcells were washed 3 times with phosphate-buffered saline and thenresuspended in RPMI 1640 supplemented with amino acids, polyvitam-ins, pyruvate, 2 x IO"5 M 2-mercaptoethanol, penicillin, and strepto

mycin, as well as 2% autologous noninactivated serum. Cells wereseeded into rectangular bags made of hydrophobic Teflon (Biofolie 25;Heraeus GmbH, Biberach, FRG) at 5 x 106/itil and incubated at 37Â°C

for 7 days (21). Eighteen h prior to harvest recombinant human IFN-7(Dr. Karl Thomae GmbH, Biberach, FRG) was added to give a finalconcentration of 200 lU/ml. On day 7 of culture, cells were harvested,pelleted, and resuspended in Hanks' balanced salt solution with 2%

human albumin. They were loaded onto an elm rial ion system (BeckmanJ6-ME, JE-5-0 rotor with standard elutriation chambers of either 15-or 30-ml volume, respectively) at a flow rate of 12 ml/min (67 ml forthe large volume chamber) and a constant speed of 2500 rpm. Lymphocytes and thrombocytes were eluted from the mixed cell populationat 15 ml/min (72 ml/min for the large volume chamber) and 20 ml/min (90 ml/min for the large volume chamber), respectively. Monocyte-derived MACs remaining in the elutriation chamber were collected at30 ml/min (132 ml/min for the large volume chamber, and rotor stop).They were resuspended in clinical grade 5% human albumin solutionsfor i.v. or i.p. infusion.

Administration of MACs

The MO-derived MACs were administered into either large peripheral or central veins over a period of 10-20 min using an infusion setfor blood products with a llQ-pm filter (Baxter, Munich, FRG) or i.p.using an implanted Port-a-Cath system. This was followed by theinfusion of an additional 250 ml of physiological saline to allow betterdistribution of the cells. If ascites was present, it was drained as

ELUTRIATION

completely as possible prior to cell infusion. In one patient with ascites(patient 10) i.p. infusion of cells was done by sterile needle puncture.

Functional and Phenotypic Analysis of Autografted MO

MO-derived MACs were analyzed for tumor cytotoxicity with apostlabeling assay using U937 promonocytic leukemia cells as targets(20).

Analysis of MAC Secretory Products. Macrophages were washedthree times in RPMI 1640 and seeded at 5 x 105/ml supplemented

RPMI 1640 without or with 1 jig/ml Salmonella abortus equi LPS (giftfrom Dr. C. Galanos, Max Planck Institut, Freiburg, FRG) or 200 IU/ml recombinant human IFN-7 (Thomae, Biberach, FRG), respectively.After 48 h incubation the supernatants were collected, filtered (0.2 ^m),and stored frozen at â€”20Â°C.The ELISA technique was applied to

measure the amount of fibronectin (own development), a2-macroglob-ulin (own development), TNF-a (provided by Thomae, Biberach, FRG),and GM-CSF (courtesy of Dr. G. Zenke, Sandoz, Basel, Switzerland).Neopterin was measured using a commercially available radioimmuno-assay (IBL, Hamburg, FRG) and IL-6 was measured in a bioassay usingthe IL-6-dependent cell line B9 (courtesy of Dr. L. Aarden, Amsterdam,The Netherlands).

Immunoperoxidase Staining (22). Cells were attached to adhesionslides and prefixed in 0.05% glutaraldehyde on ice. They were incubatedwith the following mouse monoclonal antibodies: anti-CD 14 (My4)(Coulter, Krefeld, FRG), MAX. 1, MAX.3, MAX.21 (HLA-DR) (allown laboratory; Ref. 19), anti-CD71 (OKT9, anti-transferrin receptor;Ortho-Diagnostics, Neckargmund, FRG), anti-CD51 (13C2; Dr. Hor-ton, Imperial Cancer Research Fund Laboratories, London, England),anti-CDI6 CLB FcRIII (Dr. P. Tetteroo, Netherlands Red Cross BloodTransfusion Service, Amsterdam, The Netherlands), anti-CDw32FcRII (CIKM5), and anti-CD64 FcRI (10.1; Dr. N. Hogg, ImperialCancer Research Fund, London, England). A four-layer peroxidase-antiperoxidase technique was applied, followed by postfixation withOsO4 as described elsewhere (19).

Assessment of a Biological Response in Patients Receiving MO Auto-grafts

Blood samples were collected as indicated and assayed for TNF-Â«(Biermann, Bad Nauheim, FRG; sensitivity, >10 pg/ml) and B2M(Elias, Freiburg, FRG; normal value, <3 /Â¿g/ml)by ELISA, neopterinby radioimmunoassay (IBL, Hamburg, FRG; normal value, <2.5 ng/ml), lysozyme with photometric assays (normal range, 3-9 ng/ml), andIL-6 with a bioassay as mentioned above (normal value, <4 units/ml).

Evaluation of Tumor Response

Quantitative changes of tumor size were assessed at 4-week intervalsby clinical examination, chest roentgenograms, abdominal sonography,and computed tomography. Stable disease was defined as <25% changein the size of mÃ©tastases.Progressive disease was defined as an increaseof more than 25% in the size of measurable lesions and/or the appearance of new lesions.

CYTAPHERESIS RESULTS

F/H SEPARATION

18h. IFN-r

MAC MOTEFLON CULTURE

Fig. I. Schematic description of adoptive immunotherapy using cytotoxicmacrophages derived from peripheral blood monocytes.

In this pilot phase I study, autologous in vitro generatedtumor cytotoxic MACs were reinfused into 15 patients withadvanced metastatic cancer (Table 1): i.v. through a peripheralor central venous catheter in 8 patients with systemic mÃ©tastasesof solid tumors and i.p. in 7 patients with metastatic spreadrestricted to the peritoneal cavity, either through implantedPort-a-Cath systems or through needle puncture if ascites waspresent. Patients were treated with 1-9 cycles of adoptiveimmunotherapy using IFN-7-activated MACs at 2-weekly intervals for systemic and weekly intervals, respectively, for i.p.administration. At the beginning of the study, patients includedin the i.v. protocol received escalating cell doses at each dose

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Table 1 Characteristics of patients receiving MAC adoptive immunotherapy

No. ofpatientsSex(M/F)Age

range(yr)TumorhistologyLung

(non-smallcell)MelanomaSquamous

cellcarcinomaRenalcellcarcinomaOvarian

cancerCancerof FallopiantubesStomach

cancerPrevioustherapySurgeryChemotherapyRadiotherapyNonei.v.85/328-6942114221i.p.72/531-514127503

Table 2 Cytapheresis products of cancer patients and normal volunteers

Total cellscollected"Blood

processed(liters)WBCs(xlO*/ml)RBCs(XIOVfil)Platelets

(xlOV^DGranulocytes(%)Mononuclear

cells(%)Totalmononuclear cells(xlO')Totalmonocytes (xlO9)Patients*

(n =53)5.42

Â±0.9r36.1Â±13.93.31

Â±0.982790Â±127015.9Â±

18.884.0Â±18.96.03Â±2.501.67Â±1.18Normal

volunteers*(Â»=9)4.9027.71.9518563.696.40.3613.20.707204.17.13.96

Â±2.040.45Â±0.62

Â°Cells were collected by eytapheresis into a total volume of 200 ml and counted

in a Contraves cell counter.* Data were obtained from 9 healthy donors (one cytapheresis/donor) and 12

cancer patients (with a total of 53 cytapheresis procedures).' Mean Â±SD.

level: 10" (dose level I), 2 x 10" MACs (dose level II), 4x10"

MACs (dose level III), the maximal number of MACs obtainedthrough 1 isolation/culture cycle (dose level IV), and the maximal number given twice on alternate days (dose level V) werereinfused. Similarly, a dose escalation was performed for thei.p. protocol, starting with 2 x IO8 MACs but omitting dose

level V. Dose escalation for both the i.p. and i.v. administrationwas done intraindividually, i.e., patients were treated at thehigher dose level if no major side effects were observed.

Ex Vivo Generation of Cytotoxic Macrophages. Leukapheresiswas performed 7 days prior to reinfusion of cells. As shown inTable 2 cytapheresis products from cancer patients differedfrom those obtained from healthy blood donors. In general,more mononuclear cells and particular more MOs were recovered. In addition, more granulocytes, platelets, and RBCscontaminated the cytapheresis product. Mononuclear cells werecultured for 7 days with IFN--y added 18 h prior to cell harvest.

Subsequent to harvesting, cells were purified from lymphocytesby countercurrent elutriation. The scheme of cell isolation,culture, and adoptive transfer is depicted in Fig. 1. Dependingon the individual patient but also varying from each separation/culture cycle, between 1 and 17x10" MACs could be recovered

from the cultures representing an average yield between 13 and79% (64.6 Â±32.8%) of MO initially seeded (50 Â±13% for cellsfrom normal donor). Since purity of elutriated MACs (as judgedby morphology and expression of CD 14) was low and unsatisfactory with the use of the small volume elutriation chamberwe changed to the large volume chamber which resulted in amean MAC purity of 93% (95.7 Â±5% for cells from normaldonors). These MACs expressed the CD14 and HLA-DR antigens as well as the FcRI (CD64) and FcRII (CDw32). Theyproved to be mature by the expression of the maturation-associated antigens MAX.l, M AX.3, CD71 (transferrin receptor), CD51 (a chain of vitronectin receptor), and CD 16 (Fc-

RIII). See Table 3 for details. Generated MACs were active astumor cytotoxic effector cells; with U937 cells as targets ateffectorrtarget ratios of 1:1, 5:1, and 10:1, ex vivo generatedMACs used for adoptive transfer therapy induced 70 Â±27, 81Â±15, and 91 Â±10% growth inhibition, respectively (n = 18;see also Ref. 20). They secreted high concentrations of neop-terin suggestive of mature MACs (23). Upon further stimulation with LPS, IL-6, TNF-a, and GM-CSF are produced also.In addition, Â«2-macroglobulin and fibronectin which are notproduced in circulating blood MOs (20) are secreted in largeamounts, further proving the unimpaired maturation of MACs.A detailed description of the phenotypic and functional analysisof adoptively transferred MO-derived MACs is given in Table4.

MAC Adoptive Transfer. Table 5 describes the the individualtherapies done with the generated MACs in terms of purity andtotal cells infused. MACs were reinfused over 10-20 min eitheri.v. or Â¡.p.,the latter being followed by i.p. infusion of 500 mlof physiological saline. If present, ascites was drained completely prior to cell infusion.

Clinical and Biological Responses. Clinical side effects wereusually mild (Table 6): after i.v. infusion only low-grade fever(<38Â°C)with a maximum at about 2-6 h was observed in 4 of

8 patients. Patients receiving cells i.p. also had low-grade feverwith a maximum around 8-12 h. malaise and fatigue (4 of 7patients), and often slight peritoneal irritation (3 of 7 patients).These clinical side effects did not seem to correlate with thenumber of MACs being reinfused, e.g., fever occurred in 2 of 4patients treated at dose level I (10*), 1 of 5 patients treated atdose levels II and III (2 x 10* and 4 x 10"), and none of 3patients treated with >5 x 10" MACs. From the laboratory

Table 3 Phenotype analysis of MO-derived MAC from cancer patients andhealthy donors

' positive MAC"

Antibody Defined molecule Patients* Normal volunteers*

My4MAX.21MAX.lMAX.3OKT913C2anti-CD

16CIKM510.1CD14HLA-DRgp64gp65CD7

1, transferrinreceptorCD51,vitronectinreceptorCDI6,

FcRIIICDw32,FCRIICD64,

FcRI90.7

Â±6.5r89.2

Â±10.867.9Â±24.765.0

Â±24.167.422.662.923.464.321.957.415.385.6

10.383.9

Â±13.179.3Â±18.587.5Â±13.676.5Â±23.467.5

19.656.522.948.315.668.024.177.0

18.7'' Antigen expression on single cells was evaluated by immunoperoxidasc

staining on adhesion slides. Data arc given as percentage of positive MACexpressing the antigen indicated.

* Data were derived from tested MACs in 12 cancer patients (total of 43 MAC

autografts analy/ed) and from 16 healthy volunteers.' Mean Â±SD.

Table 4 Secretory activity of MAC used for adoptive transferMACs (5 x 10'/ml) were seeded in scrum-free RPMI 1640 medium without

or with.V. abortus equi LPS ( 1jjg/ml)or IFN--y (200 ID/ml) for 48 h. Supernatantswere tested as described in "Materials and Methods."

Fibronectin(ng/ml)(t;-Macroglobulin

(ng/ml)Neopterin

(ng/ml)TNF-a

(ng/ml)lnterleukin-6

(units/ml)GM-CSF

(ng/ml)Control422

Â±324Â°(35-859)229

Â±188(10-578)3.3

Â±2.2(0.6-9.8)0.04

Â±0.036(0.04-0.15)<3<0.14Â°

Mean Â±SD of 35 experiments: ranges, in+

LPS424

Â±291(43-579)152

+126(23-430)4.9

Â±2.8(1.0-8.9)18.38

Â±16.3(8.27-51.16)303

Â±169(120-650)2.37

Â±2.04(0.16-7.26)parentheses.+lFN-r369

Â±299(20-658)155Â±

121(21-406)5.1

Â±2.7(0.7-9.3)0.073

Â±0.1(0.01-0.17)<3<0.14

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Table 5 Characteristics of macrophages reinfused

PatientNo.123456789101112131415No.oftherapies6311465727477310MACs/infusion(XIO")2.8(1.0-5.1)Â°2.3(1.0-4.0)2.02.04.65

(2.4-6.9)7.6(4.0-17.0)2.5(1.1-4.2)7.7(1.1-11.3)3.0

(2.0-4.0)4.5(2.0-6.2)2.6(1.0-4.0)5.9

(2.0-9.9)5.3(2.4-8.4)6.8(5.3-8.5)5.9

(4.4-7.6)Total

MACs(X10")16.97.02.02.018.645.812.554.26.031.510.435.737.227.354.2Purity(mean%)69*59*71*50Â»78Â»76*79Â»9593919089939995

" Mean MACs of total therapies performed; range, in parentheses.* F lui rial Â¡ondone with small volume (15 ml) chamber.

parameters tested, the only abnormal finding was the detectionof circulating fibrin monomers together with a slight drop infibrinogen levels shortly after MAC transfusion which was moreoften seen after i.v. infusion. Fibrin monomers suggested induction of the coagulation cascade most likely induced by theinfused MAC expressing tissue factor on their cell surfaces.This could be quantitated in two patients by measuring TAT.As can be seen from Fig. 2 TAT levels corresponded to fibrinmonomers and in this patient seemed to correlate with thenumber of MACs infused. Neither clinical nor laboratory parameters characteristic of disseminated intravascular coagulation were observed, because there were no thromboemboliccomplications, and clotting times and platelet counts remainedunchanged (not shown). The infusion of ex vivo manipulatedMACs also did not induce autoimmune reactivity because an-tinuclear antibodies remained negative 1 month after therapy.Due to RBC loss during repeated leukapheresis, transfusionswere needed in 4 of 15 patients.

Neopterin, B2M, lysozyme, TNF-a, and IL-6 were measuredin serum and/or ascites to monitor functional activity of auto-

grafted MACs. Elevated levels of serum neopterin at days 1and 3 posttherapy could be demonstrated in 5 of 8 patients

receiving cells i.v. and 2 of 7 patients receiving cells i.p. (seeFig. 3 for 2 representative i.v. patients), whereas B2M did notchange significantly. Three of 7 i.p. patients had detectable IL-6 in serum (>4-60 units/ml) already 4 h after therapy whichreturned to below detection limits (<4 units/ml) within 1 week.In contrast, IL-6 in the ascites fluid of 1 patient (patient 10)with peritoneal carcinomatosis secondary to stomach cancercontinued to increase following each successive therapy cycle(Fig. 4). This was accompanied by an increase in circulating C-reactive protein. In patients treated i.v., serum IL-6 could onlybe detected in 2 patients. In none of the patients could TNF-a

be detected in the serum and no increase in circulating lysozymewas seen.

All patients had progressive disease prior to inclusion in theimmunotherapy protocol. No objective tumor responses interms of regression of measurable tumor sites were observed.However, 3 patients (patients 9, 12, and 13) receiving MACsi.p. showed stable disease during therapy for 4 weeks, 8 weeks,and 6 months after end of therapy, respectively. Two patientscontinued to progress rapidly during therapy, developing ileus-like symptoms, and had to be withdrawn from the study. In 1(patient 10) with massive ascites production prior to therapy,ascites disappeared completely paralleled by normalization ofmarkedly elevated CEA in ascites fluid (Fig. 5). Ascites did notrecur for 15 weeks off-study. Furthermore, 3 patients had amarked decrease in elevated serum CA-125. In another i.p.patient, ascites production could be stopped too, with a markeddecline of CA-125 concentration both in the ascites fluid andin serum (Table 6). Here, recurrence of ascites was not seen for11 weeks.

DISCUSSION

We report a clinical phase I study of adoptive immunotherapyof cancer using monocyte-derived MACs as effector cells. Thefollowing may be concluded from this study which included 72therapies in 15 patients: (a) large numbers of tumor cytotoxicmacrophages can be generated from circulating blood mono-cytes, (b) their adoptive transfer is possible without major side

Table 6 Clinical and biological response to MAC adoptive immunotherapy

ClinicalresponseRoute

of ad-Patientministration1

i.v.2i.v.3i.v.4i.v.5i.v.6i.v.7i.v.8i.v.9

i.p.10i.p.11

i.p.12i.p.13i.p.14i.p.15

i.p.Side

effectsFever''NDFeverFeverNDFeverNDNDFever/AD

Fever/ADNDFever/ADNDFeverNDTumor

site"PDPDNE'NE'PDPDPDPDSDDisappearance

ofascites'PDSDSDDisappearance

ofascile/NE"Biochemical

marker*NDNDNDNDCEAfNDCEAfNDCA-125J

NDCEA(A)UCA-125iCA-125

iNDCA-125

UCA-125(A)UNDBiological

responseNP'

IL-6+â€”

â€”++

++â€”

â€”++

+++-(A)

+(A)â€”â€”+â€”â€”â€”--(A)

-I-(A)++

" Tumor response was evaluated 4 weeks poststudy.* In serum or ascites.' NP, neopterin; PD, progressive disease; SD, stable disease; NE. not Ã©valuable;AD, abdominal discomfort; ND, not done; A, ascites; ] or J, >30% increase or

decrease; [[, >90% of pretreatment value; â€”,no change; +, increase after MAC infusion.d Temperature elevation >0.8"C.' Withdrawn from study after one therapy because of deteriorating performance status.'Ascites was the only clinical parameter assessible.' Minimal nodular metastasis.

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lig/t

120

E 100aoI 80

1 5Â°c| 40

f 20 -

0

111,1, MAC (IO81

123456

Therapy Cycles

Fig. 2. Induction of fibrin monomers (FM) and TAT in sera from patientstreated with autologous macrophages. At weekly intervals, patient 8 received theindicated numbers of macrophages. TAT and FM were measured before (blackcolumn) and 4 h (diagonal hatched column) and 18 h (vertical hatched column)after therapy, respectively.

ng/ml

upon reinfusion remains unclear. Furthermore, the experimental evidence for an antitumor function of MACs also indicatesthat this function may be severely impaired in patients withmalignancies (3-9, 26), a defect that could be demonstrated toimply both loss of responsiveness to activating signals (27) and,probably even more important, failure to differentiate to matureeffector cells (28, 29). In addition, in response to tumor site-specific signals infiltrating blood MOs might be induced todifferentiate into a subpopulation of tumor-associated MACswhich actually promote tumor growth (30) by releasing growthfactors (31), inducing neoangiogenesis (32) and lending theirmigratory capacity to tumor cells through somatic hybridization(33). Thus, terminal differentiation of MACs in vitro whichproduces cells of end stage mature, stable phenotype (22) to usseems to be an important first step in the design of a MAC-based adoptive immunotherapy modality. In comparison withblood MOs, monocyte-derived MACs exhibit more spontaneous tumor cytotoxicity (18), are highly responsive to activation by IFN-7 (20), express all three types of Fc receptorsnecessary for antibody-dependent cellular cytotoxicity (20, 34),and, upon stimulation by LPS, secrete a distinct pattern ofmonokines which are highly cytotoxic to cancer cells through10-fold increased TNF-a levels (19) but might be less harmfulto the recipient through low levels of IL-1 (19), IL-6 (19), and

mg/dl

7 U 21 28 35

Day of Study

Fig. 3. Circulating neopterin in sera of two patients treated with increasingnumbers of autologous macrophages. MO-derived, IFN--x-activated MACs weregenerated as described in "Materials and Methods" and infused via peripheral

venous catheters. Therapy cycles were repeated every 2 weeks with increasing celldoses. Pat., patient.

effects, (c) a biological response is elicited in the autologousrecipient, and (d) preliminary evidence might suggest a possibletherapeutic efficacy of this novel treatment modality.

The rational to develop this therapeutic approach is derivedfrom the assumption that the macrophage system plays anessential role in the surveillance against the occurrence andspread of malignant tumors (13, 14). Adoptive immunotherapyusing in vitro activated MACs has been done successfully inmurine tumor models (15-17). In humans, only preliminaryinformation is available concerning a study using freshly isolated circulating blood monocytes (24). Those circulating bloodMOs, however, are relatively short-lived immature precursorcells which have to undergo further maturation and differentiation in order to become competent immune effector cells, aprocess that is initiated upon emigration of the cells from thecapillary bed into tissues and body cavities (25). Thus, even ifblood MOs are successfully activated ex vivo their fate in vivo

4567

Weeks of Study

Fig. 4. Induction of IL-6 in ascites fluid and acute phase response observed ina patient receiving repealed i.p. infusions of autologous macrophages. Ascites wasdrained completely prior to cell infusion (arrows) and analyzed for IL-6 activity( ). When clinical ascites disappeared, cell therapy was stopped but ultrasound-guided aspiration was still possible to recover some fluid for furtheranalysis. C-reactive protein ( ) was measured in sera taken on indicated days.

ng/mg albumin liter

1 2 173456

Weeks of Study

Fig. 5. Disappearance of malignant ascites and concomitant decrease in itsCEA content during repeated i.p. infusions of autologous macrophages. Macrophage adoptive therapy was done weekly with ascites drained completely prior totherapy. The amount of ascites fluid is given ( ) as well as the CEA valuescorrected for albumin content ( ).

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platelet-activating factor (35). Macrophages were taken fortherapy at day 7 in culture because at this stage the response toIFN-7 as measured by neopterin release (23), secretion ofmacrophage colony-stimulating factor (19), and induction ofprocoagulant activity was demonstrated to be maximal (36).

Monocyte-derived MACs can be induced to release an additional multitude of signals capable of interacting with other cellsystems and regulatory circuits of the normal physiology of thebody (37). The biological response seen in our patients withlow-grade fever, rise in circulating and peritoneal cavity IL-6,

and the induction of the coagulation cascade may have beenexpected. Yet, it is not clear whether this is due directly tocytokines produced from the MAC autografi or by secondaryendogenous signals. Neopterin secretion and expression of pro-coagulant activity is the only MAC activity stimulated ex vivoby IFN-7 alone. Thus, elevated serum neopterin and detectionof fibrin monomers and thrombin-antithrombin III complexesare considered to be direct, cell dose-related effects of theinfused cells (Fig. 3). The induction of thromboplastin-factorVII complexes on MAC cell surfaces by IFN-7 'Â«vitro is mostprominent on monocyte-derived MACs on day 7 in culture (36)and the documented procoagulatory effects of MAC autograftsin vivo may need special attention in the further developmentof this type of adoptive immunotherapy.

Based on current concepts, IFN-7 might be the best ex vivostimulus for MAC adoptive immunotherapy. It rather selectively augments those biological activities wanted for antitumoreffects: cell-mediated cytotoxicity (20), oxidative burst formation (38), induction of Fc receptors (39), and HLA class IIantigens (40). However, for other activities such as secretion ofTNF-a, IL-1, and IL-6, IFN-7 Â¡sa potent priming signal whichsynergizes with bacterial products (19). It needs to be determined in further studies whether the use of endotoxin as anadditional ex vivo stimulus might produce MACs of highereffectiveness or of intolerable toxicity.

An important issue that needs to be addressed in phase IItrials will be the appropriate dose and timing of repeatedtherapies. It may well be that, as it was reported recently forthe treatment of metastatic renal cell carcinoma with IFN-7(41), a biologically active dose much below the maximal tolerable dose will be more therapeutic. Animal data are also suggestive of this assumption (15).

Furthermore, in this ongoing phase II trial attention shouldbe paid to whether, depending on the recipient's infectious

status, i.e., of circulating or tissue-localized endotoxin concentrations, the biological response to adoptively transferred, IFN-7-activated MACs might differ. Twice, MACs were reinfusedinto febrile patients and elicited a pronounced biological response.

A possible therapeutic response of the tumor could only beseen with i.p. treatment. Here, the accumulation of effectorcells at tumor sites is possible and could be documented bylabeling studies. The tumor response consisted of disappearanceof ascites and a substantial decrease in tumor-associated biochemical markers (3 of 7 patients). Until now, labeling studiesdemonstrated that i.v. infused MACs are captured in the lungsfor 1-3 h and then distributed to the liver and spleen.4 For i.v.

immunotherapy, strategies need to be developed to target theeffector cells to sites of tumor growth, e.g., by the induction of

' R. Andreesen, S. Kopf, B. Hennemann, C. Scheibenbogen, and C. SchÃ¼-

micher, manuscript in preparation.' R. Andreesen, C. Scheibenbogen. W. Brugger. and S. Krause, unpublished

observation.

chemoattractant signals in tumor sites or by sequential application of monoclonal antibodies against tumor antigens.

A trial using locoregional adoptive MAC therapy by theregional perfusion through tumor-supplying arteries is in prog

ress.In conclusion, it seems possible to generate large quantities

of tumor cytotoxic MACs in vitro and to reinfuse these cellsinto the autologous host without major side effects. Furtherstudies will focus on the following: (a) improvement of MACyield by pretreatment of patients with growth factors or bydefining optimal culture conditions through the addition ofhematopoietins and other differentiation-inducing factors suchas 1,25-OH vitamin D} (42), (b) further enhancement of thefunctional competence of ex vivo generated MACs by additionof stimuli other than IFN-7, sucn as LPS and macrophagecolony-stimulating factor (43), and (c) combination therapywith monoclonal antibodies directed against tumor-associated

antigens.Since MAC autografts can be safely stored in liquid nitrogen,5

this treatment modality might not only apply for use in malignancy. MACs are potent microbicidal cells and secrete a numberof hematopoietic factors (37). Their adoptive transfer may beused as a prophylactic or therapeutic measure in the management of certain infectious diseases or as supportive care duringthe reconstitution after bone marrow ablative chemotherapy.In the setting of bone marrow transplantation, MAC autograftsmay be of special benefit to cure minimal residual disease(analogous to graft-verms-leukemia effect), to accelerate hematopoietic recovery, and to prevent opportunistic infections.

ACKNOWLEDGMENTS

The authors wish to thank Annegret Rehm, Birgit Bieber, and UseLiebhardt for their excellent technical assistance and R. Mertelsmannfor helpful discussion and critical review of the manuscript.

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1990;50:7450-7456. Cancer Res Reinhard Andreesen, Carmen Scheibenbogen, Wolfram Brugger, et al. Cancer Immunotherapy

from Circulating Blood Monocytes: A New Approach toin VitroAdoptive Transfer of Tumor Cytotoxic Macrophages Generated

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