JOURNAL OF HEMATOTHERAPY & STEM CELL RESEARCH 10:535–544 (2001)Mary Ann Liebert, Inc.

Research Report

Cellular Immunotherapy of Malignancies Using the ClonalNatural Killer Cell Line NK-92

TORSTEN TONN,1,3 SVEN BECKER,1 RUTH ESSER,2 DIRK SCHWABE,2 and ERHARD SEIFRIED1

ABSTRACT

For years activated natural killer (A-NK) cells have been explored with respect to their efficacy inanticancer therapy, but, except for some anectdotal reports, no clear clinical benefit has been shown.However, as the understanding about the interactions of NK cells and tumor cells advances, the useof A-NK cells might be revisited with more sophisticated approaches that pay tribute to mechanismswhich allow tumor cells to escape immune surveillance. Here the highly cytotoxic NK cell line NK-92 seems to be an attractive alternative for use in adoptive immunotherapy, because it was shownto exhibit substantial antitumor activity against a wide range of malignancies in vitro as well as inxenografted SCID mice. NK-92 cells are characterized by an almost complete lack of killer cell im-munglobulin-like receptors (KIRs) yet conserved ability to perforin and granzyme B-mediated cy-tolytic activity, which make them unique among the few established NK and T cell-like cell lines.NK-92 is the only natural killer cell line that has entered clinical trials. Here we discuss the currentstatus of development of this cell line for adoptive immunotherapy (AIT) of malignancies and re-view our first clinical experience in patients with advanced cancer who have received repeated trans-fusions of irradiated NK-92 in a phase I/II trial. Also we discuss issues that address safety aspectsof immunotherapy with clonal cell lines and describe further manipulations, which hold the poten-tial of significantly improving the clinical outcome of AIT with NK-92.

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POTENTIAL OF NATURAL KILLERCELLS IN CELLULAR THERAPY

NATURAL KILLER (NK) CELLS are a subgroup of lym-phocytes that play an essential role in the cellular-

based immune defense of virus-infected and -transfectedcells. NK cells do not rearrange their immune receptorgenes, and the cytotoxicity toward tumor and virally in-fected cells is not major histocompatibility complex(MHC)-restricted (1,2). In addition, a sensitization of NKcells is not prerequisite for cytotoxic activity, which, onthe other hand, is enhanced upon activation of NK cellswith a variety of cytokines (3,4). These activated NK

cells, typically expanded from the peripheral blood andactivated by interleukin-2 (IL-2) are sometimes also re-ferred to as lymphokine-activated killer cells (LAK) cells,although the majority (.90%) of IL-2-activated bloodmononuclear cells represent polyclonal T cells. Recentlythe term activated natural killer cells (A-NK) has beenintroduced, due to the fact that adherence and activationis necessary to expand highly lytic NK cells and that anincreasing number of different cytokines are reported toactivate NK cells (5). In patients with malignant disor-ders, NK cell function has been shown to be impaired interms of a reduced in vitro proliferative response and re-duced cytotoxic activity (6; reviewed in ref. 7). Although

1Institute for Immunhematology and Transfusionmedicine, Red Cross Blood Donor Service Hessia, Frankfurt/Main, Germany.2Pediatric Hematology and Oncology, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.3Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Franfurt am Main, Germany.

dysfunctions in NK cell populations might allow the oc-currence and metastasis of tumor cells in these patients(7), there is also evidence that tumor cells themselvesmay have developed mechanisms to escape NK cell im-munosurveillance, e.g., by up-regulation of classical(HLA-A,B,C) and nonclassical (HLA-G,E) human leuko-cyte antigens (HLA) (8,9). These HLA alleles are able toinhibit NK cell function upon ligation with certain killercell immunglobulin-like receptors (KIRs) on effectorcells, such as NK cells (10).

Numerous studies have been conducted, and are stillongoing, aimed at improving the antitumor effect of NKcells by either endogenous activation of the patients’ ownNK cells through administration of cytokines (11–14) orthrough adoptive use of ex vivo-expanded autologous(15–17) or donor derived (18,19) A-NK or LAK cells, ora combination thereof (15,16,20). The safety and efficacyof adoptive A-NK cell therapy has been examined in solid(16–18,20) tumors as well as in hematological malig-nancies (15,21) where NK cells are used in combinationwith high-dose chemotherapy to promote the graft-ver-sus-leukemia effect without inducing the potentiallylethal graft-versus-host reaction that is largely mediatedby T cells. Although the systemic application of IL-2 hasproved to be less well tolerated in doses necessary toachieve relevant concentrations at the site of the tumor(20), the repeated intravenous transfusion of up to 1011

A-NK cells in some trials was well tolerated and did notlead to adverse reactions (18). However, only a few pa-tients could benefit from these treatments thus far (7).

There are limitations as to the efficacy of the reinfu-sion of ex vivo-expanded autologous and allogeneic A-NK cells: (1) NK cells from patients that suffer from ma-lignant diseases often show an impaired NK cell function,which usually cannot be fully reconstituted through exvivo expansion and cytokine activation (5,6,22). (2) Thepotential of expanding of A-NK is limited and poorlystandardized between different clinical trials, as are theresulting phenotypes of A-NK cell populations with re-gard to their therapeutic potential and the risk for adversereactions. (3) A-NK cell populations usually consist ofcells derived from NK cells with a CD32,CD561 phe-notype and are either positive or negative for CD8 andCD16 (7). Expansion with anti-CD3 monoclonal anti-body (mAb) and interferon (IFN) favors the generationof T cell-derived CD31,CD561, alpha-, beta- T cell re-ceptor (TCR-a/b)-positive killer cells (23–25). Thesecells were termed cytokine-induced killer (CIK) cells, todistinguish them from CD32 and apparently less cyto-toxic A-NK cells (26). Although the culture conditionsand cytokine supplementation favor the generation of cer-tain killer cell populations, interdonor related variations,which can hardly be exogenously influenced, lead to theexpansion of distinct A-NK types after long-term culture(27). In addition, the clinical benefit of individual A-NK

subpopulations seems to be influenced by the duration ofthe culture period. Short-term (5 days) cultures of murineadherent NK cells retain their capacity to infiltrate murineB16 pulmonary metastasis; however, this ability wasgradually lost when the ex vivo culture was extended upto 20 days, a culture period usually needed to expand suf-ficient numbers of A-NK for adoptive immunotherapy(AIT) (28).

These drawbacks have prompted several investigatorsto explore the properties of established NK- (29) or Tcell-derived (30) cytotoxic cell lines with regard to theiruse in cellular therapy. The T cell-derived TALL-104 cellline was the first cytotoxic cell line to be developed andclinical applied for AIT of malignancies. This MHC non-restricted T cell line was shown to induce regression oftransplantable human hematopoietic and nonhematopoi-etic malignancies in severe combined immunodeficient(SCID) mice (30,31), as well as in immunocompetentmice with leukemia (32). Recently, the tolerability of re-peated infusions of high doses of TALL-104 cells in pa-tients with advanced cancer could be shown in a phase Itrial (33).

Only few NK cell lines could be established from pa-tients with large granular lymphoma (reviewed in ref. 34),and, although some of them were shown to have main-tained cytotoxicity, they differ considerably with regardto the in vitro tumoricidal properties (34,35). The mostpotent and suitable NK cell line with regard to clinicaluse in cancer and viral infections seems to be the lineNK-92, which was established and suggested to be usedin AIT by H.-G. Klingemann et al. (36). Besides TALL-104, NK-92 cells are the only other cell line, and the onlytrue NK cell line, that continuously has been developedfor AIT in cancer and has entered clinical trials. In con-trast to other established NK cell lines and the T cell lineTALL-104, NK-92 cells were shown to have superior cy-totoxicity to a wide range of tumor cells in vitro and inxenografted human leukemia and malignant melanomaSCID mouse models, respectively. The mechanisms thatallow NK-92 cells to recognize and eradicate malignantand viral infected cells highly effectively without affect-ing nonmalignant allogeneic cells are not well under-stood. However, this superior cytotoxicity is likely me-diated by the lack of KIR expression on NK-92 cells.

CHARACTERISTICS OF NK-92 CELLS

NK-92 cells were established in 1992 from the pe-ripheral blood lymphocytes of a patient with large gran-ular lymphoma (LGL) (36,37). NK-92 cells are similarto A-NK cells with respect to surface receptor expressionand functional characteristics (Table 1), with the excep-tion of the Fc-receptor (CD16), which usually mediatesantibody-dependent cytotoxicity (ADCC) and is absent

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on NK-92 cells (36). However, in comparison with A-NK cells, NK-92 cells display a much higher cytolyticactivity against a significantly broader spectrum of tumortargets (29,35,38), which might at least in part be due tothe lack of inhibiting NK cell receptors (NKRs) on NK-92 cells. NK-92 cells were shown to lack KIR receptorsof the p58 complex (39), which through binding to HLAantigens on target cells generally inhibit NK cells suchthat no target cell killing occurs (40). In fact, analysis ofthe expression of 13 currently known KIRs by allele-spe-cific RT-PCR revealed the lack of KIR expression, de-spite KIR2DL4 receptor and the CD94/NKG2A complex,both of which are common to all NK cells (10), indicat-ing the clonal derivation of NK-92 cells from an earlyundifferentiated NK cell progenitor (M. Uhrberg, per-sonal communication). Transfection of the p58 gene intoNK-92 cells was shown to silence the cytotoxicity againsttarget cells, expressing the matching HLA allele on theirsurface (39). Although malignant cells are effectivelykilled, there is no toxicity against nonmalignant allo-geneic cells. Moreover, the proliferative potential of he-matopoietic cells is not impaired after cocultivation withNK-92 cells (29). NK-92 cells can be continuously grownand easily expanded in tissue culture, with doubling timesof 24–36 h. The growth of NK-92 cells is IL-2 depen-dent and cannot be promoted by other cytokines, such asIL-7 or IL-12. With IL-2 deprivation cytotoxicity de-clines after 24 h; however, substantial cytotoxicity canbe maintained for up to 48 h in vitro (29). Although IL-2 seems to be most crucial for proliferation and cytotoxicactivity, co-culture of NK-92 with other cytokines, suchas IL-18 and IL-12, was shown to increase cytotoxicityand broaden the target range (29,41).

PRECLINICAL STUDIES ON THEEFFICACY OF NK-92

Since their establishment in 1992, NK-92 cells havebeen continuously developed for the use of AIT of can-cers. Screening of the cytotoxic activity of NK-92 cellsagainst cell lines derived from leukemia, lymphoma(29,35), and solid tumors, such as malignant melanoma(38), prostate cancer, and breast cancer (Klingemann,personal communication), has confirmed the superiorkilling activity of NK-92 cells compared to blood-derivedLAK cells and, in case of leukemia, also to other NK celllike lines, such as YT (35), respectively.

However, since the sensitivity of established cell linesto NK-92 cells may possibly not predict its effect on pri-mary malignant cells, NK-92 cells have also been testedagainst primary leukemic blasts. In testing 45 primaryleukemias for their sensitivity to NK-92 cells mediatedkilling, O’Reilly et al. were able to show that about halfof newly diagnosed and relapsed acute myelogenous leu-

kemia (AML), T-cell adult lymphocytic leukemia (T-ALL), B-lineage-ALL, and chronic myelogenous leuke-mia (CML) cells were sensitive to lysis by NK-92 cells.In vitro NK-92 cells could effectively kill a variety ofleukemic cells isolated from either the peripheral bloodor bone marrow from patients with AML, T-ALL, CML,and B-lineage ALL. These experiments also confirmedthe superior cytotoxic effect of NK-92 cells when com-pared to LAK cells, and to the NK cell line YT and theT cell-derived cell lint TALL-104, respectively. This wasnot only true in terms of tumor cell lysis at different ef-fector-to-target ratios, but also with regard to the overallspectrum of leukemic cells killed in vitro. Of 46 patient-derived leukemic samples, 24 (52%) were either sensi-tive or highly sensitive to NK-92 mediated in vitro cy-totoxicity. Leukemia blasts derived from 6 out of 12(50%) patients with AML, 7 of 7 (100%) T-ALLs, and5 of 14 (35.7%) B-lineage-ALLs were either sensitive orhighly sensitive to NK-92-mediated lysis. In addition, therepeated i.p. injection of NK-92 was able to prolong thesurvival of SCID mice, which had been xenografted withpatient-derived T-ALL or AML cell lines TA27 andMA26, respectively. Although the mice died from the ag-gressively growing tumors when no NK-92 or IL-2 alonewas given, single or repeated injections of NK-92 wereable to prolong survival significantly, and in case of thecombined application of NK-92 cells with rhIL-2 couldeven lead to cure in some mice (35). These studies dem-onstrate the efficacy of NK-92 cells against leukemia-ini-tiating cells (leukemia stem cells) and their efficacy invivo.

Hence, to eliminate disease in NK-92 cell-sensitivemalignancies, the prolonged treatment with fully IL-2-activated NK cells seems to be important. This ultimatelyled to the construction of two stable IL-2-secreting NK-92 clones, NK-92 CI and NK-92MI, which constitutivelysecrete IL-2 at low levels and moderate levels, respec-tively, thereby allowing NK-92 cells at the site of actionto be activated fully, without the toxicity that may be as-sociated with a systemic administration of IL-2 (42). Be-cause the transfection of these clones was carried out us-ing a nonviral gene transfer method such asparticle-mediated gene gunning, adoptive transfer ofthese cells can be considered relatively safe with regardto viral safety aspects, hence making them suitable forAIT.

In concordance with the tumorigenicity observed withhematopoietic malignancies, NK-92 cells and their IL-2transfectants have also been shown to be effective againstsolid tumors, such as malignant melanoma. NK-92, aswell as NK-92CI and NK-92MI have been tested for theirability to kill human melanoma cell lines efficiently invitro and in xenografted mice (38). Again, NK-92 cellsexhibited a much higher cytotoxicity to various mela-noma cell lines in vitro than did LAK cells and also

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showed significant cytotoxicity at very low effector-to-tar-get ratios (1:1), where no lysis could be observed with LAKcells at all (38). In addition, administration of NK-92 cellsand their variants in human melanoma-bearing To-SCIDmice was able to prolong survival significantly and reducetumor growth, when a single dose of effector cells was ad-ministered systemically or locally, respectively.

However, especially in mice inoculated with the veryaggressive melanoma line (WM1341), which led to thedeath of mice within 25 days, the administration of ef-fector cells turned out only to be effective when they wereadministered shortly before or simultaneously with thetumor cells. In case of the slightly less aggressive grow-ing metastatic melanoma cell line MEWO, administra-tion of NK-92 or NK-92MI was able to show significantbenefit, even when the cells were administered 24 h af-ter injection of the melanoma cells (38).

As indicated from the target cell profile of naturallyoccurring NK cells, NK-92 cells are also able to recog-nize and eradicate viral infected cells specifically, suchas Epstein-Barr virus (EBV). NK-92 cells effectively rec-ognize and eradicate B lymphocytes from normal donorsthat have been infected with EBV, whereas uninfected Bcells were not lysed by NK-92 (43).

Besides the development of NK-92 cells for adoptiveimmunotherapy, NK-92 cells may also have beneficialeffects in terms of a “biological purging agent” of pe-ripheral blood stem cell (PBSC) grafts (29,37). Contam-ination of autologous PBSCs with tumor cells may ulti-mately lead to relapse of the underlying disease. Purgingwith biological means, such as A-NK or NK-92 cells,might be of special interest in cases where tumor cellscannot be identified by specific cell-surface markers thatallow depletion of the tumor cells using, e.g., magneticseparation methods. In these cases, the co-cultivation ofcontaminated PBSC preparations with autologous NKcells or clonal NK cell lines, such as NK-92, may leadto depletion of tumor cells. In BCR/ABL-positive PBSCfrom CML patients, a significant reduction of the malig-nant clone could be obtained after purging for 4–8 h, asdocumented by PCR (37).

SAFETY ASPECTS OF NK-92

NK-92 cells are a clonal, IL-2-dependent, growing NKcell line, which upon adoptive infusion to immunocom-promised patients potentially harbors the risk to graft inthese patients and to induce secondary lymphoma. There-fore, to assess the risk of NK-92 cells to induce cancergrowth in recipients, repeated injections of nonirradiatedNK-92 cells were performed in immunocompromisedSCID mice, as well as in NK cell-deficient pfp-Rag-2mice. No NK-92 were seen to permanently engraft, de-spite the ongoing administration of recombinant IL-2

(35,38). In addition, no toxicity could be observed inthese experiments, as monitored by histopathologicalmonitoring of bone marrow, spleen, kidney, lung, andbrain (35).

With regard to safety concerns, these data underlinethe low tumorigenic risk of NK-92 cells in immuno-compromised patients. Nevertheless, this issue is beingaddressed in current open-phase I trials. However, tak-ing into account the lack of tumorigenic potential ofNK-92 cells in the murine system, in our point of viewNK-92 cells harbor a lower risk to induce secondarylymphoma in immunocompromised patients, whencompared to, e.g., the T cell-like cell line TALL-104,which was initially established as an independentlygrowing cell line from clones that had grafted in SCIDmice (44,45). However, for both cell lines, methodshave been developed to prevent effectively further pro-liferation and at the same time retain a maximum of cy-totoxic activity (29,33). For NK-92 cells, irradiationwith 500 cGy was shown to prevent further cell divi-sion, whereas the same time substantial cytotoxicity canbe retained up to irradiation doses of 1000 cGy for upto 48 h in vitro (42).

Because of the potential for infection of the cell linecould cause life-threatening infections in immunocom-promised patients, NK-92 cells have been tested by PCRand were shown to be negative for HIV, HCV, HSV1/2,and CMV (36). In addition, to prepare the clinical appli-cation of NK-92 cells in the United States and Europe,the cells have been tested by two independent GLP-cer-tified laboratories for human, porcine, or bovine patho-gens. It was shown that no viral particles are replicatedin NK-92 cells and that there are no bacterial, fungal, ormycoplasma contaminations in the established mastercell banks.

CLINICAL-GRADE EXPANSION OF NK-92

In preparation of the clinical trials, work has focusedon the large-scale expansion of NK-92 under conditionsthat follow good manufacturing practices (GMP). In con-trast to autologous or allogeneic A-NK cells, which aredifficult and laborious to generate in high purity ex vivo,NK-92 cells can be easily expanded to large numbers andare readily available on demand in standardized qualityfor adoptive therapy (Fig. 1). Starting from a master cellbank, which has been tested for infectious agents, indi-vidual samples of NK-92 cells can be thawed and ex-panded in FDA-approved therapeutical-grade media, sup-plemented with 500 IE IL-2 and 5% heat-inactivatedquarantine tested fresh frozen plasma (FFP). Under theseconditions, NK-92 can be grown in batch culture and un-der GMP conditions to cell densities of about 1 3 106/ml,without impairment of viability and cytotoxicity, adding

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to a volume of approximately 10 L for a cell dose of 1010

cells. The geometry of culture containers used does notseem to be critical, as comparable results have been ob-tained using stirrer flasks and Teflon-coated bags (46) orcell factory flasks (47). However, substantial improvementcould be obtained using a optimized cell culture mediumand a controlled stirred bioreactor. Under these conditions,NK-92 can be grown up to densities of 4 3 106/ml, whichreduces the cell culture volume for a cell dose of, e.g., 1010

NK-92 to 2.5 L (S. Schmidt, personal communication). Asmentioned above, the doubling time of NK-92 cells rangesbetween 24 and 36 h (Table 1), so that starting with 107

cells, 1010 cells can be obtained after approximately 11–16days. After harvest of NK-92, cells are washed once andresuspended in a volume of 200–300 ml of media and ir-radiated at 1000 cGy. Before transfusion, the quality of theexpanded NK-92 cells is verified with regard to their via-bility and lytic activity against the NK-sensitive referencecell line K562. Viability usually exceeds 95% and there areno limitations as to cytotoxicity, when compared to non-expanded and nonirradiated NK-92 cells.

PRELIMINARY RESULTS FROM OPENCLINICAL PHASE I/II TRIALS

On basis of the preclinical data described above, whichshow the highly cytotoxic activity of NK-92 cells against

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FIG. 1. Strategy of nondirected adoptive immunotherapy using NK-92 cells.

FIG. 2. NK-92 cells selectively adhere to and invade a spher-oid of the bladder cancer cell line RT-4.

a wide range of tumors (Fig. 2) and the lack of toxicityand tumorigenic potential in immunodeficient SCIDmice, NK-92 to our knowledge is the first NK cell linethat has entered clinical application (48). Currently, twophase I/II trials in children and adults, respectively, areopen in Europe and in United States, NK-92 cells haveachieved FDA approval to be tested in patients with ad-vanced malignant melanoma and renal cell carcinoma(H.-G. Klingemann, personal communication, 2001).

The most advanced clinical trial in Europe, which isinitiated and conducted by our group aims at analyzingthe safety and efficacy of two infusions of irradiated NK-92 in children and young adults, with advanced cancer.The open, single-armed phase I study consists of a doseescalation scheme, with 3 patients each receiving two

transfusions of 109, 3 3 109, and 5 3 109 NK-92/m2 bodysurface, 48 h apart, respectively. The patients’ inclusioncriteria for phase I study was advanced-stage cancer, ei-ther relapsed or therapy resistant after several lines of con-ventional therapy, so that there was only hope for cure us-ing experimental strategies. Thus far, 7 patients haveentered the clinical trial, all of which suffering from child-hood advanced cancer (4 sarcoma, 2 medulloblastoma, 1PNET) in terminal stage with tumor masses exceeding25% of the body weight in some cases (Table 2). All but2 patients tolerated the repeated transfusions of NK-92cells without any side effects. One patient, receiving 1 3

109 NK-92/m2 developed mild fever of 101.0°F about 6h after the transfusion that resolved quickly. In anotherpatient suffering from advanced-stage epitheloid sarcoma

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TABLE 1. FEATURES OF NK-92 CELLS

Criteria Characteristic

Morphology Large granular lymphocytes (LGLs), azurophilic granulesImmunophenotype Positive: CD56, CD2, CD7, C11a, CD28, CD45, CD54

Negative: CD1, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, HLA-DRTCR genes in germline configuration

Karyotype Numerical and structural alterationsPositive: KIR2DL4, CD94/NKG2a

NKR expression* Negative: (activating receptors) 2DS1, 2DS2, 2DS3, 2DS4, 3DS1Negative: (inhibitory receptors) 2DL1, 2DL2, 2DL3, 3DL1, 3DL2

Cytotoxicity Highly lytic to several cell lines and primary cells of: leukemia,lymphoma, malignant melanoma, prostate cancer, squamous cellcarcinoma, breast cancer, viral infected cells

Infection Negative: HIV1, HBV, HCV, CMV, porcine and bovine viruses. Lackof bacterial, fungal and mycoplasma contaminations

Growth characteristics Il-2 dependent (100–500 IU/ml), doubling time 24–36 h,maximal density 1–4 3 106/ml in optimized clinical-grade media

Radiosensitivity Growth inhibition .250cGY

This table is a synopsis of published data (29, 35, 36, 38, 42, 44, 46, 47).*M. Uhrberg, personal communication, 2001.

TABLE 2. PATIENTS INCLUDED IN THE PHASE I/II TRIAL IN CHILDREN

WITH ADVANCED CANCER AND NK-92 CELLS TRANSFUSED

Previous Sites of NK-92/ Total NK-92 cellsPatient Age Disease therapy metastasis dose (109/m2) transfused (109)

1 22 PNET S, R, PBSC L, LN, B, K 1 2.32 18 Soft tissue sarcoma S, R, Ch L, LN, B, ST 0.85a 2.43 16 Rhabdomyosarcoma S, R, Ch L, LN, B, ST 1 44 16 Osteosarcoma S, Ch L 1 2.65 17 Soft tissue sarcoma Ch L, LN, B 3 9.46 25 Medulloblastoma S, R, Ch CNS 3 8.67 9 Medulloblastoma S, R, Ch None 3 6.6

aSecond transfusion discontinued. For further details, see text.Abbreviations: B, bone; Ch, chemotherapy; CNS, central nervous system; K, kidney; L, lung; LN, lymph node; PBSC, pe-

ripheral blood stem cell transplantation; PNET, primitive neuroectodermal tumor; R, radiation; S, surgery; ST, soft tissue.

and receiving the same cell dose, a lower back pain de-veloped during the second infusion. The pain respondedto morphine and the patient remained asymptomatic. Thisreaction was felt to be due to the fluid bolus given concomitantly with the NK-92 infusion in this special pa-tient, who displayed massive intraabdominal and re-troperitoneal tumor formation with partial obstruction ofthe urether. However, because this patient did not com-plete the cell dose of two transfusions of 1 3 109/m2 (to-tal NK-92 cells transfused were 2.4 3 109), another pa-tient entered the clinical trial to complete the group ofpatients receiving two doses of 1 3 109 NK-92. Asidefrom these 2 patients, no other side effects could be ob-served in any of the patients treated. So far, we have suc-cessfully completed the second dose escalation and areabout to begin the third and highest dose escalation in thisphase I study with young adults. However, in analogy tothe phase I/II trial with children and young adults, the firstadult patient suffering from transformed B-cell ALL hasalso entered treatment with NK-92 cells in a study con-ducted by the hematology department at the universityhospital in Frankfurt (48). Taking this into account, a to-tal of 8 patients have received two doses of 1–3 3 109

NK-92 cells/m2 body surface without any significant sideeffects related to NK-92 toxicity. The highest total NK-92 cell dose transfused to one patient was 9.4 3 109 cells.Although the disease in this patient, who suffered fromalveolar soft tissue sarcoma, progressed, the patient isalive 69 weeks after NK-92 treatment and there has beenno sign of adverse reactions due to NK-92 treatment.

Because of the advanced nature of the cancers in allpatients treated so far, no conclusions as to the efficacyof treatment with NK-92 cells can be drawn. Six patientsdied of disease progression. The other patients were givenanother experimental treatment. In summary, these pre-liminary phase I observations in 8 patients support thenotion that intravenous administration of up to 3 3 109

irradiated NK-92 m2 cells is safe.

REJECTION OF NK-92 CELLS BY THERECIPIENTS IMMUNE SYSTEM—OPEN

QUESTIONS

Because NK-92 cells have so far only been tested inimmunocompromised mice, one of the main efforts inour studies is to analyze whether adverse reactions of therecipients’ immune system would possibly limit the ef-fect of NK-92 cells. Therefore, we monitored the recip-ients’ adverse reactions to the transfused NK-92 cells interms of HLA-antibody formation and a T cell response.However, in none of the patients treated so far could weobserve HLA-antibody formation against NK-92 cells asassessed by a microlymphocytotoxicity crossmatch of thepatients’ serum and NK-92 cells. Neither could we ob-

serve the induction of a T cell response, which was mon-itored by a mixed lymphocyte reaction of the patients’lymphocytes with irradiated NK-92 cells over the timecourse and the monitoring of an increase of the activa-tion marker CD69 on the patients’ CD4 and CD8 lym-phocytes, respectively. However, these preliminary datamight as well reflect the fact that the patients that wereincluded in the study thus far are widely immunocom-promised due to chemo- and radiation therapy. There-fore, in the next patients to be included in the study, wedecided to monitor further the immune status by analyz-ing the cellular and humoral immune response to com-mon recall antigens using a patch test.

Homing of the adoptively transfused NK-92 cells tothe site of the tumor could not be addressed in this study,but certainly this is one of the criteria that may influencethe therapeutic efficacy of NK-92 cells as anticancer ther-apy. To reach the site of metastasis, intravenously trans-ferred effector cells need to cross the endothelial celllayer and migrate into the tissue (reviewed in ref. 7).Monitoring of the kinetics of NK-92 cells in the periph-eral blood of the transfused patients revealed that a largeportion of NK-92 cells apparently leave the circulationwithin minutes of transfusion. However, although we arenot able to say if these cells were trapped in the liver orlung, a portion of the NK-92 cells reappear in the circu-lation 48–72 h after the second infusion, indicating a re-distribution of NK-92 cells within the recipient (M. Sut-torp, unpublished data).

SUMMARY AND OUTLOOK

NK-92 cells seem to be unique in their highly pre-served cytotoxicity against a wide range of tumors amongall currently established NK and T cell-derived clonal celllines (29,35). Moreover, as indicated by experiments inSCID mice, as well as the preliminary results of our phaseI study, repeated infusions of NK-92 are well toleratedand do not lead to toxicity or the development of sec-ondary lymphoma when adoptively transfused into pa-tients. The maximal tolerated dose, as well as the best-suited treatment schedule, i.e., number of infusions, withor without IL-2, remain to be elucidated for different dis-eases. On the basis of the current experience in our group,we would estimate that the maximal tolerable doses maybe substantially above the 5 3 109/m2, which will be themaximal dose transfused in this study. Compared to thecell doses, which showed efficacy in SCID mice, trans-fusions of about 5 3 108/kg should be targeted. More re-cent studies have added a dose of 1010/m2 to the dose es-calation scheme.

Although there is evidence from studies in mice usingeither adoptively transfused A-NK cells (49,50) or theTALL-104 cell line (51) that a fraction of the transfused

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cells are capable of localizing at the site of the tumor,our group is preparing a clinical trial with indium-labeledNK-92 cells to analyze further the homing of NK-92 cellsafter adoptive transfer.

Because NK-92 cells, like NK cells, do not require pre-sensitization and are able to kill tumor cells in a non-MHC-restricted manner, they are as an independently growingcell line best suited for GMP conforming and undirectedcellular therapy supplying effector cells that are readilyavailable upon demand. Based on the safety of the parentalNK-92 cells, they do as well hold the option to be furtherengineered to increase the efficacy and to allow, e.g., spe-cific retargeting of NK-92 cells to an increasing numberof tumor-specific antigens yet to be identified.

Retargeting of NK cells to tumor- or HIV-infected cellswith either bispecific antibodies (52,53) or chimeric anti-gen receptors (54,55) was shown to mediate specific ly-sis of target cells, carrying the corresponding antigen. Onthe basis of these observations, variants of NK-92 havebeen constructed that express an erbB2-specific chime-ric receptor complex (56). These variants have beenshown to recognize and lyse specifically a variety of tu-mor cell lines expressing the erbB2 growth factor recep-tor, as well as erbB2-positive primary breast cancer cellsthat were otherwise resistant to NK-mediated killing (W.Wels personal communication; see C. Uherek et al., thisissue). Meanwhile, additional chimeric variants havebeen constructed that either target the CD19 antigen orHIV epitopes to be used in AIT of B-ALL and HIV, re-spectively. Variants of NK-92 cells that are redirected tothe CD20 antigen expressed on B cell lymphoma are cur-rently under development.

Other concepts may include strategies to circumvent thedisadvantage of limited effector cells, as given with irradi-ated NK-92 cells. As shown with T cells, it may be desir-able to control NK-92 cells in vivo via the activation of sui-cide genes, such as the herpes simplex virus thymidinekinase (HSV-TK) suicide gene (57). This would ultimatelyallow the in vivo expansion of NK-92 and the subsequentganciclovir-induced elimination of NK-92 cells.

As with other cellular immunotherapies, the tumor bur-den seems to be critical for the clinical outcome of adop-tive therapy with NK-92 cells, and, as the phase I stud-ies have been completed successfully and the geneticmodification of NK-92 proceeds, AIT with NK-92 cellsshould be viewed as part of a comprehensive cancer treat-ment approach that may include conventional and ex-perimental therapies (58).

ACKNOWLEDGMENTS

This work was supported in part by grants of the“Stiftung Hämotherapie-Forschung (Bonn)” to TorstenTonn and the Held/Hecker foundation at the University

Hospital, Frankfurt. Ruth Esser holds a grant of the “Hilfefür krebskranke Kinder e.v. Frankfurt.” We thank W.Glienke, M. Suttorp, and M. Uhrberg for sharing unpub-lished data. We are indepted to H.G. Klingemann for ad-vice and support throughout the clinical trial.

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Address reprint requests to:Dr. Torsten Tonn

Chemotherapeutisches ForschungsinsitutGeorg-Speyer-Haus

Paul-Ehrlich Strasse 42-44D-60596 Frankfurt am Main, Germany

E-mail: ttonn@bsdhessen.de

Received April 8, 2001; accepted May 2, 2001.

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