Cytotherapy, 2013; 15: 1563e1570

Treatment of patients with advanced cancer with the natural killer cellline NK-92

TORSTEN TONN1,2,�, DIRK SCHWABE3,�, HANS G. KLINGEMANN4, SVEN BECKER1,2,RUTH ESSER3,7, ULRIKE KOEHL3,7, MEINOLF SUTTORP6, ERHARD SEIFRIED1,2,OLIVER G. OTTMANN5 & GESINE BUG5

1Institute for Transfusion Medicine and Immunohematology, Goethe University Hospital, Frankfurt am Main,Germany, 2German Red Cross Blood Donation Service, Baden-Württemberg-Hessen, Germany, 3Pediatric Hematologyand Oncology, University Hospital, Frankfurt am Main, Germany, 4Tufts University Medical School, Boston,Massachusetts, USA, 5Department of Medicine, Hematology/Oncology, Goethe University Hospital, Frankfurt amMain, Germany, 6Division of Paediatric Haematology and Oncology, Children’s Hospital, University Hospital CarlGustav Carus, Technical University, Dresden, Germany, and 7Institute of cellular Therapeutics, IFB-Tx, HannoverMedical School, Hannover, Germany

AbstractBackground aims. Natural killer (NK) cells, either naive or genetically engineered, are increasingly considered for cellulartherapy of patients with malignancies. When using NK cells from peripheral blood, the number of expanded NK cells can behighly variable and the need for NK cell enrichment can make the process expensive. The NK-92 cell line (CD56þ/CD3�)that was isolated from a patient with lymphoma has predictable high cytotoxic activity and can be expanded under goodmanufacturing practice conditions in recombinant interleukin-2. Methods. Fifteen patients (age, 9e71 years) with advanced,treatment-resistant malignancies, either solid tumors/sarcomas (n ¼ 13) or leukemia/lymphoma (n ¼ 2), received two in-fusions of NK-92 cells, given 48 h apart. Three cohorts of patients were treated with escalating doses of NK-92 cells (n ¼ 7at 1 � 109, n ¼ 6 at 3 � 109 and n ¼ 2 at 1 � 1010 cells/m2). Results. No infusion-related or long-term side effects wereobserved. The dose of 1010 cells/m2 was considered the maximum expandable cell dose with the use of an established culturebag system. Three fourths of patients with lung cancer had some anti-tumor response. Only one patient of seven haddevelopment of human leukocyte antigen antibodies. The persistence of NK-92 cells (male origin) in the circulation wasconfirmed by Y chromosomeespecific polymerase chain reaction in two female patients. Conclusions. Infusions of NK-92cells up to 1010 cells/m2 were well tolerated. Despite the allogeneic nature of NK-92, development of human leukocyteantigen antibodies in these patients with cancer appears to be rare. The cells can persist in the recipient’s circulation for atleast 48 h. Some encouraging responses were seen in patients with advanced lung cancer.

Key Words: cellular immunotherapy, phase I clinical trial, cancer, natural killer cell, NK-92

Introduction

Chemotherapy is not curative for most cancersbecause it only affects dividing cells, allowing the truecancer stem cells with slow or minimal turnover toremain unaffected. Cancer cells also develop resis-tance to drugs, which ultimately contributes to dis-ease recurrence. Although new drug developmentshold some promise, thus far the survival benefit fornon-hematological cancers has been marginal, largelybecause the malignant clone rapidly adjusts to thepathway with which the drug interferes.

�These authors contributed equally to this work.Correspondence: Dr Torsten Tonn, German Red Cross Blood Donation ServiceFaculty Carl Gustav CaruseUniversity of Technology Dresden, Blasewitzer Stras

(Received 16 March 2013; accepted 29 June 2013)

ISSN 1465-3249 Copyright � 2013, International Society for Cellular Therapy. Phttp://dx.doi.org/10.1016/j.jcyt.2013.06.017

Cell-based therapies involving cytotoxic cells arebelieved to attack and kill malignant stem cellsthrough alternative mechanisms that include DNAdegradation through the perforin/granzyme pathwayand release of immuno-stimulatory (ie, interferon[IFN]-g) or cytotoxic cytokines (ie, tumor necrosisfactor [TNF]-a) (1). These pathways are cytolytic totarget cells independent of cell cycle status and celldivisions and are not cross-reactive with other anti-cancer treatments. The cells of the immune system—

largely T lymphocytes and natural killer (NK) cells—are now recognized as being able to provide control

North-East, Center for Regenerative Therapies Dresden (CRTD), Medicalse 68/70, D-01307 Dresden, Germany. E-mail: ttonn@blutspende.de

ublished by Elsevier Inc. All rights reserved.

1564 T. Tonn et al.

of cancer cell growth. Patients in whom leukemiarelapses after an allogeneic hematopoietic stem celltransplant (HSCT) can achieve a remission whendonor lymphocytes are infused (2,3). Furthermore,when comparing the incidence of relapse after syn-geneic and allogeneic transplant, there is an approx-imately 30% higher recurrence rate after syngeneictransplant because of the identical genetic make-upof the transplanted cells that do not evoke a donor-derived cellular immune response (4).

More recently, NK cells have received someattention because they recognize tumor cells through adifferent pathway than T-lymphocytes. Generally, theactivity of NK cells is blocked by killer cell immuno-globulin receptors (KIR) when they encounter cellswith the cognate (“self”) major histocompatibilitycomplex (MHC) pattern (5,6). Conversely, they canbecome activated when they encounter non-self. Thiswas first observed in the context of MHC haplotype-mismatched HSCT when T-lymphocytes wereremoved from the donor marrow (7,8). This obser-vation was recently confirmed in a large, retrospectiveanalysis with patient/donor samples from the NationalMarrow Donor Program (9). Transplantation ofdonor/recipient KIR/ligand mismatches provided asurvival advantage because of a lower relapse rate.In the non-transplant setting, infusion of allogeneic,CD3-depleted mononuclear cells enriched forCD56þ NK cells could induce remission in some25% of patients with acute myeloid leukemia (10).Allogeneic MHC-mismatched NK cells can also begiven to patients as part of a sibling HSCT (11) as wellas early after an autologous HSCT without concern ofgraft-versus-host disease or marrow suppression (12),provided that T cells are removed.

Several laboratories are actively working onisolating and expanding clinical scale numbers ofNK cells for treatment of patients with cancer (13).Since infusions of autologous NK cells, even withconcomitant administration of cytokines such asinterleukin (IL)-2, are clinically ineffective (14), thefocus has shifted to allogeneic NK cells from a relatedor unrelated donor. Because the majority of peripherallymphocytes obtained by apheresis are T-cells, theymust be removed to prevent graft-versus-host diseasein the recipient, which adds another step of manipu-lation and expenses to the expansion process (13).There is also substantial donor-dependent variabilityin terms of NK cell yield (12,13,15e17). For thesereasons, a continuously growing NK cell line, pro-viding predictable numbers of highly cytotoxic NKcells on expansion, is attractive for cellular therapy.

The activated NK cell line NK-92 is highlycytotoxic against a broad spectrum of malignant cells(18e22); the cells lack all currently known inhibitoryKIR receptors, except for KIR2DL4, which is

expressed at low levels, while expressing the fullspectrum of activating receptors (22). The expansionkinetics of NK-92 have been delineated, and pre-dictable numbers of activated NK-92 cells can bereproducibly obtained after culture in IL-2 (23). Aclinical phase I trial with three infusions given everyother day has been reported in patients withadvanced renal cell cancer and melanoma (24), andpreliminary results from this trial have also beenpresented (25). The infusions of NK-92 cells werewell tolerated, and some of the patients with end-stage cancer showed some tumor responses.

In the present report, we show results from a phaseI trial with NK-92 cells that were administered to agroup of patients with predominantly end-stage solidtumors and with a different infusion schedule thanpreviously reported (24). This study also tested for thepresence of human leukocyte antigen (HLA) anti-bodies and persistence ofNK-92 cells in the circulationof selected patients. Infusions of NK-92 cells were welltolerated, even after re-treatment at a later time, withsome patients with advanced chemotherapy-resistantlung cancer having disease responses.

Methods

Patients

Eligibility criteria included patients with malignantdiseases who were resistant to standard therapy, had aKarnofsky performance status of >50% and adequateorgan function, defined as a total bilirubin <2.0mg/dL and creatinine <2.0 mg/dL. The study wasapproved by the Ethics Committee at the University ofFrankfurt/Germany according to the Declaration ofHelsinki and was registered with the Paul-Ehrlich-Institute (Langen, Germany). All patients signedinformed consent before entering the study.

Study design

To be eligible for the study, patients were confirmedto have a negative cross-match with NK-92 cells. Acommercial lymphocytotoxicity assay (One LambdaInc, Canoga Park, CA, USA) was used. NK-92 cellswere seeded at a density of 2.000 NK-92 cells andincubated with the patient’s serum according to themanufacturer’s instruction in the presence of rabbitcomplement. To detect dead cells, Fluoroquenchwas added. The reaction was considered negativeif the number of dead cells was not increasedcompared with the negative control (NK-92 withanti-HLA antibody negative control serum). For thisphase I study, patients were treated at three differentdose levels with two infusions of NK-92 cells 48 hapart at the following doses: 1 � 109 cells/m2 (n ¼ 7)

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NK-92 treatment of cancer 1565

3 � 109 cells/m2 (n ¼ 6) and 1 � 1010 cells/m2

(n ¼ 2). No lymphocyte-depleting chemotherapy wasgiven before the NK cell infusion. The protocol waslater amended to allow repeated NK-92 infusions inpatients who had a presumed clinical benefit and didnot have HLA antibodies against NK-92 cells.

0

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0 2 4 7 9 120 144 168 192 216 240 264 288 312 (hrs)

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Figure 1. Culture expansion of NK-92 cells in VueLife culturebags. NK-92 cells were seeded at an initial concentration of 2e5 �104 cells/mL in X-Vivo 10 media supplemented with 1000 IU/mLIL-2 and 5% heat-inactivated human AB plasma. The expansionwas performed as batch culture to determine the doubling time ofNK-92 cells (32 h) and the time of harvest for a given cell dose. Anadditional 1000 IU/mL IL-2 was added to the cultures the daybefore the harvest. On the basis of these results, a time point forharvest was chosen that allowed a cell density of approximately 5 �105 to 1 � 106 NK-92 cells/mL (0.5e1 � 109/L).

Expansion of NK-92 cells

Amaster cell bank ofNK-92 cells had been establishedpreviously, with a total cell number of 5 � 107 NK-92kept in a 1-mL vial in 10% dimethyl sulfoxide, 40%heat-inactivated human plasma (German Red CrossBloodDonation Service, Baden-Würtemberg-Hessen,Germany) and 50% X-Vivo 10 medium (Lonza,Belgium). NK-92 cells were sterile and free of myco-plasma and human or animal—including porcine—pathogen viruses (Analysis Biomedical Test GmbH,Cologne, Germany). For the generation of individualcell dosages, a vial of the master cell bank of NK-92cells was thawed and expanded in the presence of 1000IU human recombinant IL-2 (Proleukin, Novartis,Basel, Switzerland) until cell growth was established.The VuelLife culture bags (Cellgenix TechnologyGmbH, Freiburg, Germany) were prefilled with 3 L ofX-Vivo 10 medium with 1000 IU IL-2 and 5% heat-inactivated human plasma. NK-92 cells were added ata concentration of 2e5 � 104 cells/mL. Samples forsterility testing were collected from each bag at the dayof culture initiation. The expansion was performed asbatch culture with a doubling time of 32 h (Figure 1).The day before harvest, an additional 1000 IU/mL ofProleukin was added to the culture bags. Harvest ofcells was performed in a closed system under class Aconditions. Briefly, the total volume of up to 20 L wastransferred to 500-mL transfusion bags (Fresenius,Bad Homburg, Germany) and centrifuged for 20min at 2000 rpm. The supernatant was subsequentlytransferred to empty transfusion bags that were con-nected to the transfusion bags before centrifugationwiththe use of a sterile connection device (T-SCD,Terumo,Eschborn, Germany). This process was repeated untilall cell pellets were pooled into one 500-mL blood bag.Finally the NK-92 cells were spun down again andresuspended in 300 mL phosphate-buffered saline/eth-ylenediamine tetra-acetic acid buffer (Miltenyi Biotech,Cologne, Germany). Cell numbers were assessed withthe use of a Sysmex XT 1800 automated cell analyzer.

Cytokine production by NK-92 into the culturesupernatant was determined after 24-h culture of thecells in X-Vivo 10 supplemented with 1000 IU/mLIL-2. A commercial cytokine bead array (BectonDickinson, Heidelberg, Germany) was used tomeasure the following cytokines: IFN-g, TNF-a,IL-1b, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-12p70.

Before release, cell viability was determined bymeans of Trypan blue exclusion. Additionally, thepotency was assessed in a flow cytometric cytotoxicityassay (PKH-Assay) against K562. A further aliquotwas taken for sterility testing (BactAlert, Biomerieux,Nürtingen, Germany). NK-92 cell preparations werereleased if they fulfilled the following criteria: negativesterility at the time of batch culture initiation, viability>80% and cytotoxicity >50% at effector-to-targetratio of 10:1 against K562. Cells were irradiated with10 Gy before intravenous infusion.

Infusion of NK-92

Before NK-92 infusion, patients were started onhydration with normal saline at 250 mL/h for 2 h.Patients were pre-medicated with methylpredniso-lone 2 mg/kg and an anti-histamine. The secondscheduled infusion of NK-92 cells 48 h later wasgiven only if the first infusion was well tolerated andwithout adverse events grade >2.

Post-infusion monitoring

Regular blood work (complete blood cell count, elec-trolytes, creatinine and liver function tests) was per-formed weekly for 4 weeks after the first infusion.Peripheral blood samples from two female patientsreceiving treatment were obtained 24 and 48 h afterNK-92 infusion and analyzed for the presence ofthe XY-positive NK-92 cells with the use of a Y chro-mosomeespecific polymerase chain reaction (PCR).Briefly, 1 mg of genomic DNA extracted from periph-eral blood was used in a single PCR reaction, and all

1566 T. Tonn et al.

experiments were performed in duplicate. Primers Y1Forward (50-TCCAC TTTAT TCCAG GCCTGTCC-30, position 3511-3533 inYH10) andY2Reverse(50-TTGAA TGGAA TGGGA ACGAA TGG-30,position 78-100 inYHl10), spanning the EcoRI re-striction site of the tandem repeat target sequencepHY10, were synthesized by a commercial supplier(Biometra, Göttingen, Germany). To determine thelower threshold of sensitivity for detection of maleDNA, mixtures of decreasing proportions of maleDNA within excess female DNA were prepared,keeping the total amount of DNA constant. With theuse of NK-92 cells as the male DNA source, thethresholdof detectionwas5e10 cells in106 female cells(0,001% ¼ þ, 0.01% ¼ þþ and 0.1% ¼ þþþ)(Figure 2).

To document any immune response againstNK-92 cells, HLA antibodies were determined bymicrocytotoxicity cross-match (One Lambda Flow-Pro, Canoga Park, CA, USA) in seven patients at 1and weeks after the last NK-92 infusion.

Serum levels of the following cytokines weremeasured at 0, 1, 4, 20, 24, 48 and 72 h after the firstand second infusion using a commercially availablecytokine bead array (BectonDickinson): IFN-g, TNF-a, IL-1b, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-12p70.

Patients had response assessment 28 days aftercompleting NK-92 infusions. Responses were eval-uated as complete response, partial response, stabledisease and progressive disease, on the basis ofResponse Evaluation Criteria in Solid Tumors(RECIST) (26). Patients were not regularly scannedafter 4 weeks but received imaging studies whenrecurrence was suspected.

Results

Patients

Fifteen patients with end-stage, chemotherapy-resistant cancer were enrolled in the study between1999 and 2006. All patients had failed conventional

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Figure 2. Persistence of NK-92 cells in the blood circulation: PCRfor Y chromosomeepositive NK-92 cells in two female recipients.The threshold of detection was 5e10 cells in 106 female cells(0.001% ¼ þ, 0.01% ¼ þþ and 0.1% ¼ þþþ).

prior therapies for recurrent disease and had nofurther treatment options (Table I). Median age ofthe study population was 50 years (range, 9e71years). Patient characteristics are listed in Table I.

Toxicities

All infusions were well tolerated by all patients, evenat the highest dose level, with the exception of onepatient. The second transfusion in patient 2 wasdiscontinued after he had lower back pain thatresponded to morphine. The back pain was probablyrelated to abdominal distension caused by the fluidbolus given before and during the NK-92 infusion.The patient had extensive intra- and retro-peritonealtumor formation with partial obstruction of the ure-ter. He did not receive the second scheduled infu-sion. No effect on marrow function was observed inany patient with stable blood cell counts over the first4 weeks after NK-92 treatment. Likewise, nochanges in renal and hepatic functions were noted.One patient (patient 14) with lung cancer who hadstable disease after NK-92 infusions received fiveadditional infusions over a period of 6 months,beginning 4 months after initial treatment.

Clinical outcomes

Three patients with advanced lung cancer had clin-ically significant responses to NK-92: two patients(patients 7 and 13) had mixed responses and onepatient (patient 14) had stable disease for approxi-mately 2 years (Table I). Of the two mixed responses,one occurred in a woman (patient 7) with small-celllung cancer (SCLC) who had a supraclavicularlymph node metastasis. After treatment with twoinfusions of 3 � 109 NK-92 cells/m2, the lymph nodecould no longer be detected. Patient 13 had SCLCwith multiple metastases in the liver and a 1-inch,round-shaped, single metastasis in the lung justbehind the sternum. After two transfusions of 3 �109 NK-92 cells/m2, the lung metastasis had almostcompletely disappeared, as confirmed by radiog-raphy performed 14 days after NK-92 treatment. Noresponse was observed for liver metastases.

Patient 14, with non-SCLC, had multiple lungmetastases resistant to conventional chemotherapy.She was the first patient to receive the 1010 NK-92cells/m2 dose level. The two infusions were welltolerated, and the patient believed that her pain hadbeen relieved by the treatment. The disease wasstable, and 4 months later she received four addi-tional infusions of NK-92 (5 � 109e1010 NK-92cells/m2) over a period of 6 months, again withoutany side effects. According to imaging studies, thedisease remained stable until she had development of

Table I. Patient characteristics and outcomes.

Patient DiseaseSex/age(years) Prior therapy

Metastaticsites

NK-92 starting celldose (109/m2)

NK-92 total celldose (109)

Response to NKcell therapy

OS indays

1 PNET F/22 Surgery,Chemo,HSCT

L, LN, B,kidney

1 2.3 PD 44

2 Sarcoma(soft tissue)

M/18 Surgery, Rad,Chemo

L, LN, B,soft tissue

0.85 2.4 PD 262

3 Rhadomyosarcoma M/16 Surgery, Rad,Chemo

L, LN, B,soft tissue

1 4.0 PD 42

4 Osteosarcoma F/16 Surgery,Chemo

L 1 2.6 PD 99

5 CLLetransformed M/64 Surgery, Rad,Chemo

N/a 1 2.0 PD 163

6 Adrenal carcinoma M/64 Surgery, Rad,Chemo

Soft tissue 1 2.0 PD 801

7 SCLC F/50 Surgery, Rad,Chemo

L, LN 1 3.6 MR 388

8 Sarcoma (softtissue)

F/17 Chemo L, LN, B 3 9.4 PD 13

9 Medulloblastoma F/25 Surgery, Rad,Chemo

CNS 3 8.6 PD 593

10 Medulloblastoma F/9 Surgery, Rad,Chemo

None 3 6.6 PD 19

11 Colorectal cancer F/66 Surgery, Rad,Chemo

L, LN, liver 3 12 PD 310

12 SCLC F/56 Chemo L 3 5.8 PD 21813 SCLC M/64 Chemo Pancreas 3 10 MR 24214 NSCLC F/69 Surgery, Rad,

ChemoL 10 42.4 SD 707

15 B-NHL M/71 Rad, Chemo LN, BM softtissue

10 18 PD 296

B, bone; BM, bone marrow; L, lung; LN, lymph node; PD, progressive disease; SD, stable disease; MR, mixed response; PNET, primitiveneuroectodermal tumor; CLL, chronic lymphocytic leukemia; NSCLC, non-SCLC; NHL, non-Hodgkin lymphoma; Rad, radiation; OS,overall survival.

NK-92 treatment of cancer 1567

SCLC and died on day 707 after the first NK-92infusion.

Correlative studies

Only one of seven patients had an antibody responsedirected against the HLA antigens expressed by NK-92 at the time of testing 1 and 4 weeks after NK-92infusions. However, this patient also had receivedblood transfusions after NK-92 infusion with po-tential transmission of antibodies. To determine howlong NK-92 cells would persist in the blood circu-lation after infusion, the presence of male NK-92cells in two female recipients was tested by Y chro-mosomeespecific PCR 24 and 48 h after infusion.Both recipients had NK-92 cells present in the cir-culation (Figure 2).

NK-92 cells are known to express genes for andsecrete a large number of cytokines (22). With theuse of the same culture conditions as for the clinicalscale expansion, the concentration of cytokines in theculture supernatant was measured at 24 h (Figure 3).Significant amounts of INF-g and IL-10 were presentin these cultures with lower concentrations of IL-6

and IL-8. Elevated concentrations of IL-10 andINF-g were also detected in the serum of two patientsin whom we obtained levels after NK-92 infusion,with a maximum peak at 2 h after the infusion. Noneof the other cytokines showed detectable serum levels(results not shown).

Discussion

This phase I trial in patients with advanced, pre-dominantly solid organ cancers confirmed earlierresults that the infusion of NK-92 cells is safe andwell tolerated even at higher dose levels (24).Compared with the previous trial, though, this pro-tocol had only two infusions but had an additionaldose level of NK-92 (1010/m2), essentially twice thenumber of infused cells reported in the earlier trial.Although only two patients received the highestcell dose, one patient (patient 14) received five addi-tional infusions over a 6-month period, which shetolerated well. Infusional side effects occurred in onlyone patient (abdominal pain), who had ureter ob-struction before infusing NK-92 caused by a large

Figure 3. Cytokine concentration in the culture supernatant ofNK-92 seeded at 5 � 105 cells/mL. Cells were cultured for 24 h inX-Vivo 10 medium with 1000 IU/mL of IL-2. The concentrationof different cytokines was determined with a commercial beadarray. Data points represent results from one representativeexperiment.

1568 T. Tonn et al.

intra-abdominal tumor mass. The pre-NK-92 hy-dration probably caused abdominal distension. Itis possible that more infusional side effects wereprevented by methylprednisone as part of the pre-medication. Because there is the potential that ste-roids mitigate any anti-tumor effect of NK-92 andside effects appear to be manageable without ste-roids, as shown in the study by Arai et al. (24), futurestudies with NK-92 will not use steroids as part ofthe pre-medication.

Because phase I data are not powered to assessdisease response, only limited conclusions withrespect to efficacy should be drawn from these data.At the higher NK-92 dose level, 75% of patients withlung cancer either had a mixed response or stabledisease for some time. Considering the expectedrapid progression of lung cancer, stable disease isconsidered to be a positive therapeutic effect (27). Abeneficial effect in lung cancer could be expectedbecause murine studies with NK-92 cells have shownthat they migrate to and reside in the lung within thefirst hours after intravenous administration, wherethey stay for several hours before they circulate todistant organs (Klingemann H., et al., unpublishedobservation).

It remains to be determined whether the observedanti-tumor effect in patients with lung cancer iscaused by cellular cytotoxicity (perforin/granzyme-mediated) or caused by cytokine release in the lung(or a combination of both), because NK-92 cellsproduce significant levels of IFN-g and IL-10. Thisprobably would be a localized cytokine effect,because we were unable to demonstrate serum levelsof either cytokine. Serum concentration measure-ment of cytokines, however, can be inconclusivebecause of the rapid dilution of the cytokines inserum and the ex vivo degradation of the protein ifthe sample is not rapidly processed.

Because of their allogeneic nature, NK-92 cellscan be expected to evoke a humoral immuneresponse against the HLA antigens they express. Wewere able to analyze seven patients at 1 and 4 weeksafter the infusion for HLA antibodies. One patienttested positive at 4 weeks. Because he had receivedblood transfusions in the meantime, this result maynot be conclusive.

Miller et al. (10) have shown in patients withacute leukemia who received infusions of MHChaplotype-mismatched blood NK cells that diseaseresponses occurred only in patients who had in vivoclonal expansion of NK cells. We were able here toshow in two patients the persistence of NK-92 cellsfor at least 48 h after infusion. NK-92 cells are irra-diated before administration and are not expected toproliferate in vivo. It is unclear if these cells still hadsome cytotoxicity and whether this could have beenmaintained with concomitant treatment of IL-2.Future phase II studies will try to address this issue.It is known, however, that NK-92, like blood NKcells, is capable of multiple repeated encounters withmalignant cells before they become apoptotic (28)(also Wels W., unpublished observation). As withblood NK cells, homing studies in humans have notbeen performed, but murine studies have shownlocalization of NK-92 to tumor sites (29).

To further optimize the efficacy of NK-92 celltreatment, additional strategies may be considered:Murine models have shown that depletion of regu-latory T cells before cell therapy enhances theanti-tumor efficacy of transferred donor cells (30).Lymphodepletion has also been shown to augmentthe efficacy of cell therapy by increasing exposure tocytokines (IL-7, IL-12 and IL-15) and eliminatingcompeting elements of the immune system (“cyto-kine sink”) (31). Clinical trials with immune cells(mostly T cells) have made use of this strategy bycombining fludarabine and cyclophosphamide. Suchtreatment also provides some immunosuppression toprevent the rejection of allogeneic cells (10,32).

NK-92 cells do not express any currently knownKIR receptors, except for KIR2DL4 at a low levelthat has activating and inhibitory functions (22). Thisimportant and unique feature may account for thehigh cytolytic activity of NK-92 against a broad rangeof malignancies (19e21). Moreover, NK-92 cellscan be genetically engineered to express specific re-ceptors for targeted tumor cell recognition. To date,NK-92 cells have been armed with chimeric antigenreceptors that recognize specific ligands on targetcells such as Her-2 (33), CD19 (34,35), CD20 (36),CD38 (37) GD2 (38), EpCAM (39) and EBNA(40). In addition to chimeric antigen receptors, cy-tokines have been transfected into NK-92 that couldmaintain its in vivo cytotoxicity (41). Another way to

NK-92 treatment of cancer 1569

optimize NK-92 activity and to override any inhibi-tory tumor signals is the combination with mono-clonal antibodies that predominantly utilizeantibody-dependent, cell-mediated cytotoxicity.Because the original NK-92 cell line lacks expressionof the CD16 (FcgRIIIA) receptor, it has been engi-neered to express a high-affinity Fc receptor (42).

In summary, this study confirms earlier obser-vations (24) that the continuously growing NK-92cell line (i) has no significant adverse effects oninfusion into patients with cancer, even at thehighest 1010/m2 cell dose, (ii) has a low propensityto evoke a humoral immune response, (iii) persistsin patients after infusions for at least 48 h and (iv)appears to have some anti-tumor effect in patientswith lung cancer.

Acknowledgments

This study was supported by a grant (no. 00/02) fromthe Jose Carreras Research Foundation and the HeldHecker funds of the Johann Wolfgang Goethe Uni-versity Clinics Frankfurt/Main. We thank C. Seidl,MD, for technical help (cross-matching) and G.Suck, PhD, for comments on the manuscript.

Disclosure of interests: HK is co-founder andchairman of Conkwest Inc (Del Mar, CA). The otherauthors declare no competing financial interests.

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