autologous peripheral blood stem cell transplantation and ...€¦ · autologous peripheral blood...

Vol. 1, 607-614, June 1995 Clinical Cancer Research 607

Autologous Peripheral Blood Stem Cell Transplantation and

Adoptive Immunotherapy with Activated Natural Killer

Cells in the Immediate Posttransplant Period’

John Lister,2 Witold B. Rybka,

Albert D. Donnenberg,

Margarida deMagalhaes-Silverman,

Steven M. Pincus, Elana J. Bloom,

Elaine M. Elder, Edward D. Ball, and

Theresa L. Whiteside

Division of Hematology/Bone Marrow Transplantation, Department

of Medicine [J. L., W. B. R., A. D. D., M. d-S., S. M. P., E. J. B.,

E. D. B.], and Departments of Pathology and Otolaryngology

[E. M. E., T. L. W.], Pittsburgh Cancer Institute, University of

Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213-2582

ABSTRACT

Relapse after high-dose chemotherapy supported by pe-ripheral blood stem cell transplantation (HDC-PBSCT) is the

main cause of therapeutic failure in patients with lymphomaand breast cancer. Adoptive immunotherapy with activatednatural killer (A-NK) cells and interleukin 2 might eliminate

surviving residual tumor without adding to toxicity.Eleven patients with relapsed lymphoma and one with

metastatic breast cancer were entered on a pilot clinical trial

of HDC-PBSCT followed on day 2 after transplant by infu-sion of cultured autologous A-NK cells. Simultaneously, re-

combinant human interleukin 2 (rhIL-2) was initiated as a

4-day continuous i.v. infusion at 2 x 106 IU/m2/day, referredto as high-dose rhIL-2. Therapy with high-dose rhIL-2 wasfollowed by a 90-day continuous i.v. infusion at 3 x i05IU/m2/day, referred to as low-dose rhIL-2. All patients en-grafted and nine completed treatment. Posttransplant daysto a neutrophil count of 500/pJ and to a platelet count of50,000/p.l were similar to comparable patients treated withHDC-PBSCT alone. Generation of A-NK cells for therapywas feasible in all patients except the three patients with

Hodgkin’s disease, whose cells did not proliferate in culture.Overall toxicity associated with early posttransplant trans-fer of A-NK cells and interleukin 2 did not differ from thatobserved with peripheral blood stem cell transplantationalone in comparable patients. There was early amplificationof natural killer cell activity in the peripheral blood of fourpatients that appeared to result from the transfused A-NKcells. Adoptive transfer of A-NK cells and rhIL-2 during the

pancytopenic phase after HDC-PBSCT was feasible and well

tolerated, did not adversely affect engraftment, and resultedin amplified natural killer activity in the peripheral bloodduring the immediate posttransplantation period.

INTRODUCTION

The use of HDC3 and hemopoietic stem cell support has

become widely employed in the treatment of relapsed lym-

phoma and metastatic breast cancer (1-7). In chemotherapy-

sensitive lymphoma and metastatic breast cancer, HDC can

provide disease-free survival at S years in 40% and 15% of

remitting patients, respectively. Nevertheless, the majority of

completely remitting patients will relapse and die of their dis-

ease. Dose escalation of chemotherapy and radiation therapy

above conventional transplant doses has not improved disease-

free survival.

AlT with in vitro-activated autologous effector cells, either

LAK cells or tumor-infiltrating lymphocytes, in combination

with IL-2 has been shown to induce long-term responses in a

small proportion of patients with metastatic melanoma or renal

cell carcinoma (8, 9). These two types of cancer are generally

chemoresistant but seem to be among the most responsive to

biological therapy. Experience with the therapeutic use of cel-

lular effectors and IL-2 in lymphoma or breast cancer has been

limited, and only patients with advanced disease have been

treated (10). Nevertheless, a few responses have been seen (1 1),

indicating that AlT might be effective in these diseases. The

treatment protocol described here is an attempt to evaluate the

feasibility of AlT with A-NK cells and IL-2 in the setting of

HDC-PBSCT in patients with lymphoma or breast cancer at the

time of maximum chemotherapy effect.

A-NK cells are a subset of CD3�CD56�m peripheral blood

NK cells selected by adherence to plastic in the presence of

22 nM of IL-2 (12). A-NK cells can be expanded in culture with

IL-2 for 2 to 3 weeks (12). This subset of NK cells has been

shown recently to preferentially enter human tumor spheroids or

established human tumor xenografts in nude mice and to kill

NK-resistant tumor cells (13). In murine models of tumor me-

tastasis, systemically delivered A-NK cells have been shown to

preferentially localize to metastases (14). Our hypothesis has

Received 11/i 1/94; accepted 3/9/95.

1 This study was supported in part by the Alcoa Foundation (Pittsburgh,

PA), NIH/National Cancer Institute Grant 5U01-CA58271-02, and

Pathology Education and Research Foundation.

2 To whom requests for reprints should be addressed, at Division ofHematology/Bone Marrow Transplantation, University of Pittsburgh

Medical Center, 200 Lothrop Street, Pittsburgh, PA 15213-2582.

3 The abbreviations used are: HDC, high-dose chemotherapy; AlT,

adoptive immunotherapy; LAK. lymphokine-activated killer cells; IL-2,

interleukin 2; A-NK, activated natural killer; PBSCT, peripheral blood

stem cell transplantation; NK, natural killer; G-CSF, granulocyte-

colony-stimulating factor; PBSC, peripheral blood stem cell; rhIL-2,

recombinant human IL-2; HD, high dose; LD, low dose; MNC, mono-

nuclear cells; PE, phycoerythrin; ANC, absolute neutrophil count; CNS,

central nervous system; UPN, unique patient number; PR, partial re-

sponse(s); LU, lytic unit(s).

Research. on August 1, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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608 Stem Cell Transplantation and Immunotherapy

Table I Patient characteristics

UPN

Age at Transplant

(yr) Diagnosis

Time between diagnosis and

transplant (months) Prior therapy”

380 46 BRC” 56 CMF (6), ROTh, ADRIA (3)

386 60 FML 32 CHOP (10)

385 49 DSCCL 12 ProMACE-CYTABOM (6), CNOP (2)

430 49 FML 50 ProMACE-CYTABOM (2), DICE (3)

448 42 DLCL 12 CHOP (6), ProMACE (1)459 26 HD-NS 43 MitoxBVD (8), ROTE, MOPP/ABV

463 29 HD-NS 46 MOPP-ABVD (12)

465 27 HD-NS 32 MOPP (6)

469 56 FSCCL-DML(T) 61 CHOP (6)

475 40 DML 17 ProMACE-CYTABOM (4), ROTh

485 61 FSCCL-DLCL(T) 131 COAMP (6), ROTL

61 DLCL 51 COP-BLAM (6), CVP (5)

“BRC, breast cancer;FML, follicular mixed lymphoma; DLCL, diffuse largecell lymphoma; HD-NS, nodular sclerosing Hodgkin’s disease;

FSCCL-DML(T), transformation (FSCCL to DML); ROTL, limited field radiotherapy; ROTE, extensive field radiotherapy; DML, diffuse mixed

lymphoma; DSCCL, diffuse small cleaved cell lymphoma; FSCCL-DLCL(T), transformation (FSCCL to DLCL).

h Numbers in parentheses, cycles of chemotherapy given.

been that A-NK cells mediate significant antitumor effects when

administered systemically and home to tumor metastases grow-

ing as solid tissue. Thus, systemic infusion of A-NK cells

supported with IL-2 might eliminate residual tumor surviving

HDC in patients with advanced cancer and decrease the relapse

rate following HDC-PBSCT. As a first step in testing this

hypothesis, this study demonstrates the feasibility of generating

A-NK cells in patients with advanced lymphoma and of corn-

bining HDC-PBSCT with AlT using A-NK cells and IL-2

during the cytopenic phase immediately after the transplant.

MATERIALS AND METHODS

Patients. All patients gave informed consent for partici-

pation in the AlT protocol, which was approved by the Pitts-

burgh Cancer Institute and the Institutional Review Board of the

University of Pittsburgh. The patient characteristics are listed in

Table 1 . Six women and six men were entered on the study. The

mean age of the patients was 44 (range, 26-61) years. Eight

patients had lymphoma, three had Hodgkin’s disease, and one

had metastatic breast cancer. Compassionate approval was ob-

tamed for treatment of the breast cancer patient. These patients

received transplants between November 1992 and February

1994. All patients had failed at least one chemotherapy regimen.

Five of the 12 patients entered on this protocol were judged to

have chemotherapy-responsive disease, demonstrated as a par-

tial or complete response to ifosfamide chemotherapy on eval-

uation 4-8 weeks after its administration.

The patients participating in this study were compared to

42 patients who had received transplants consecutively, between

March 1992 and February 1993, with autologous PBSCT for

recurrent lymphoma or Hodgkin’s disease on another protocol

open at our institution. This protocol targeted the same patient

population, and all patients would have been eligible for trans-

plant on the AlT protocol. Reasons for not participating in the

AlT protocol were patient refusal, or a period of time, January

1993 to April 1993, when the AlT protocol was not open for

accrual, because it was under review by the Food and Drug

Administration. All of the 42 patients who received PBSCT

alone received G-CSF after transplant to accelerate leukocyte

recovery and to potentially reduce the length of hospital stay.

Patients on the AlT protocol did not receive G-CSF posttrans-

plant, so as not to mask any adverse effect of AlT on engraft-

ment or to add a confounding variable to the attribution of

toxicity posttransplant. The comparison targeted toxicity and

engraftment, looking for clinically relevant differences.

Treatment Protocol. The treatment schema for the lym-

phoma patients is shown in Fig. 1. Patients were admitted to

hospital and had a Hickman pheresis catheter placed in the

subclavian position under local anesthesia. Chemotherapy con-

sisting of ifosfamide and Mesna at 3 g/m2/day by continuous i.v.

infusion for 4 and S days, respectively, for the lymphorna

patients and cyclophosphamide and mesna at 5 g/m2 by contin-

uous i.v. infusion for 1 and 2 days, respectively, for the patient

with breast cancer was given. Following chemotherapy, G-CSF

(Neupogen; Arngen, Thousand Oaks, CA) at S p.gfkg/day s.c.

was administered. Daily blood counts were performed. On the

first week day, after ifosfamide-induced leukocyte nadir, with

WBC >1000/pA, leukapheresis for collection of PBSC was

started. A total of 7-9 X 108 MNC/kg were collected.

On the first Monday after PBSC collection, rhIL-2 (Chiron

Corp., Emeryville, CA) at 3 X i0� IU/m2/day by continuous i.v.

infusion was started, using a Pharmacia-Deltec CADD-1 pump

(Pharmacia Deltec, St. Paul, MN) and was continued for 6 days.

The following Monday, daily 4-h leukapheresis began and con-

tinued for 4 days (second leukapheresis series). The time from

the first day of leukapheresis for PBSCs (first leukapheresis)

and the beginning of leukapheresis for A-NK cells (second

leukapheresis) varied between 2 and 4 weeks. The WBC was

normal at the time of the second leukapheresis procedure. The

collections were processed by the Cellular Adoptive Immuno-

therapy Laboratory of the Pittsburgh Cancer Institute and were

cultured for the generation of A-NK cells as described below.

The patients were then admitted to the Bone Marrow

Transplant Unit to begin HDC. Supportive care included single

rooms with positive pressure high efficiency particular air fil-

tered air, low microbial content diets, prophylactic antibiotics

and acyclovir, blood product support, and i.v. antibiotics and

amphotericin as clinically indicated. The patients with lym-



Begin LD - CII - LL-2 day +6at 3 X 105 lU/rn2 IV CII daily

continue to day +96

Clinical Cancer Research 609

( High dose Ifosfamide I Mesas

I 3 gm/m2/d CII X 4 days

� MesnaCil

r� G-CSF

I to begin after chemotherapy and to continue� until end offirst leukapheresis J

Fig. I Flow diagram of the treatment plan for patients with lymphomaundergoing HDC-PBSCT followed by AlT with A-NK cells and IL-2.

cli, continuous iv. infusion; LD-Cll-IL-2, low-dose continuous iv.

infusion of IL-2.

phoma received busulfan (16 mg/kg) and cyclophosphamide

(120 mg/kg) as conditioning (15). The patient with breast cancer

received cyclophosphamide (1500 mg/m2/day), thiotepa (125

mg/m2/day), carboplatinum (200 mg/m2/day), and mesna (1500

mg/m2/day), all for 4 days (day-6 to day-3) by continuous i.v.

infusion (16).

Patients received the PBSCT product on day 0 and the

A-NK cells on day +2, both as a rapid i.v. infusion. On day +2,

coincident with the A-NK cell infusion, a continuous i.v. infu-

sion of rhIL-2 at 2 X 106 IU/m2/day (HD-rhIL-2) was started

and continued for 4 days. Thereafter, the dose of rhIL-2 was

reduced to 3 X i0� IU/m2/day (LD-rhIL-2), and IL-2 was

continued as outpatient therapy for 90 days.

Clinical evaluations of response were performed at 1 and 3

months posttransplantation and then every 3 months for 2 years.

Toxicity grading was performed daily during hospitalization and

then weekly during outpatient treatment.

PBSC Product. PBSCs were obtained daily by 4-h leu-

kapheresis (Cobe Spectra; Cobe BC!’, Inc., Lakewood, CO)

until the total collected MNC count was 7-9 X 108/kg. All

patients received 7 X 108 MNCIkg, with the excess portion

reserved for treatment of graft failure. A median number of

seven collections (range, 5-1 1) was performed per patient. Total

cell counts were determined on an automated cell counter

(Coulter ZM; Coulter, Hialeah, FL). The pheresis product (50%

v/v) was mixed with 10% DMSO (v/v, Cryoserv; Research

Industries Corporation, Salt Lake City, UT), and 20% autolo-

gous plasma (v/v) in 20% Medium 199 (Life Technologies, Inc.,

Grand Island, NY). The cell concentration was adjusted to not

exceed 2 X 10� cells/ml, and 60-ml aliquots were transferred to

bags (Cel Freeze Cryogenic Storage Containers; Chartermed,

Lakewood, NJ) for controlled rate freezing. The bags were

stored in liquid nitrogen and thawed in a 37#{176}Cwater bath at the

bedside immediately prior to infusion.

A-NK Cell Product. Following low-dose IL-2 priming

(see Fig. 1), leukapheresis was performed daily for 4 consecu-

tive days to collect cells for the generation of A-NK cells. MNC

were separated by Ficoll-Hypaque density gradient centrifuga-

tion and depleted of monocytes by treatment with 5 m�i phe-

nylalanine methyl ester (Terumo Medical Corp., Elkton, MD)

for 40 mm at room temperature (17). Monocyte depletion was

previously found to be necessary for optimal culture of A-NK

cells (12). The monocyte-depleted MNC were washed and re-

suspended at a concentration of 5 X 10” cells/ml in the complete

culture medium (RPMI 1640 from GIBCO, Grand Island, NY)

containing 6000 IU/ml rhIL-2 (Chiron Corp.), 10% pooled,

heated-inactivated human AB serum (v/v; Nabi, Miami, FL),

and 50 p.g/ml gentamicin (GIBCO). A-NK cells were captured

on the surface of plastic culture flasks during 4-5-h incubation

in the presence of 6000 lU/mi of IL-2 in an atmosphere of 5%

CO2 in air at 37#{176}C,as previously described (12). Nonadherent

MNC were removed by vigorous washing of the flasks with

warm culture medium containing 2% human AB serum (v/v).

Fresh complete culture medium containing 6000 lU/mI rhIL-2

was added back to the plastic culture flasks, and the number of

cells adherent to plastic determined using an inverted micro-

scope. A-NK cell cultures were supplemented with irradiated

allogeneic concanavalin A-preactivated MNC on day 1, as de-

scribed earlier (12), and cultured for 14-18 days. A-NK cell

cultures were monitored three times a week for growth and

viability of cells by hemacytometer counts performed with

trypan blue dye on aliquots of the culture. Cultures were main-

tamed at a cell density of 1-2 X 10” cells/mi by supplementa-

tion with complete culture medium containing rhIL-2 every 4

days. On the day of patient infusion (day +2), aliquots of the

A-NK cell product were sampled for flow cytometry, and as-

sessments of cytotoxic activity against K562 and Daudi cell

lines as well as bacterial and fungal culture were made. The

A-NK cell product was characterized by purity of the cells

(% CD3CD56�), fold expansion in culture based on the initial

and final cell counts (12), and cytotoxic activity against the

selected tumor cell targets (Table 2).

Flow Cytometry. MNC were isolated from the patients’

peripheral blood by centrifugation over Ficoll, washed, and

incubated with FITC- and PE-conjugated mAbs in preparation

for two-color flow cytometry. In some cases, peridinin chloro-

phyll protein-conjugated mAbs were also used for three-color

flow. Isotype controls used were IgG1-FITC, IgG2A-PE, and

IgG1-peridinin chlorophyll protein. Gates were set to include




Table 2 Proportions of NK cells a nd NK activity in the pheresis products used to start the A-NK cell cul

generated for therapy

tures and character istics of A-NK cells

Pheresis product A-NK cells

Fold proliferation’

Total no. ofcells infused”

CD3CD56�(%)“

Cytotoxicityvs. Daudi”UPN

CD3CD56�(%)(i

Cytotoxicityi.s. K562’�

380

386

385

430

448

459

463

465

469

475

485

501

2511

2

9

8

11

8

18

1 1

11

14

10

348

319

36

97

177

116

120

147

48

313

341

153

75

148

30

12

76

1

1

1

19

9

1

3

2.4 X i01#{176}

4.0 X iO’#{176}

1.6 x 10”

8.3 x 10�2.0 x iO’#{176}

1.2 X 10�

6.8 X 108

1.2 x i0�

4.6 X 10�

1.2 X iO’#{176}

oe1.0 X 10”

98

91

64

7080

95

69

97

87

88

95

95

3,363

6,174

3,631

23,435

5,388

14,411

2,621

5,389

14,437

8,712

5,398

13,733

a Determined by two-color flow cytometry.

I, Cytotoxicity expressed as LU24,/107 cells determined in 4-h 51Cr release assays.

( Total number of cells on day 14 of culture divided by number of initial plastic-adherent cells.

(I Number of therapeutic culture cells infused.

(� I . 1 X iO� cells were generated from this culture; however, these were not infused because the patient was taken off protocol on day -5 due

to the appearance of symptomatic CNS lymphoma.

CD45 � cells and to exclude debris. The mAb-labeled cells were

analyzed with a FACScan flow cytometer using Consort-32

software (Becton Dickinson, San Jose, CA). The mAbs used for

staining were specific for CD3, CD45, CD56 (Becton Dickin-

son), and CD16 (Medarex, West Lebanon, NH). When cell

numbers permitted, i04 gated events were analyzed (approxi-

mately 90% of samples).

Total numbers of CD34� and lineage negative

(CD34 ‘ ‘“‘ ) cells were determined in the daily PBSC product

by flow cytometry. An aliquot of the PBSC daily product was

stained with two anti-CD34, FITC-conjugated mAbs (8G12;

Becton Dickinson and Qbend-10; GenTrak, Plymouth Meeting,

PA) and four lineage specific PE-conjugated mAbs with spec-

ificity for CD3, CD11b, CD14, and CD19 (Becton Dickinson).

U937 cells were used to gate out autofluorescence, and debris

was excluded by reference to 6-p.m beads. The percentage of

each CD34� population in 20,000 events was used to quantify

the total count of each population in the PBSC product.

Cytotoxicity Assay. The 51Cr release assay used in our

laboratories has been described in detail previously (18).

Briefly, resting NK or A-NK cells were incubated with 51Cr-

labeled K562 or Daudi target cells for 4 h at E:T ratios ranging

from 6 to 50: 1 for resting NK cells and from 0.3 to 6:1 for A-NK

cells. Cytotoxicity was expressed as LU2�J107 effector cells,

where one LU20 is the number of cellular effectors required for

20% lysis of 1 X � target cells (18).

Statistics. The Wilcoxon rank sum test (two-tailed) was

used to compare engraftment parameters between the control

patients and the patients participating in the AlT protocol.

Patients from both groups who received <6 X 10” CD34�”

cells/kg and who had reached the target engraftment parameters

before 40 days after transplant were compared. Patients in both

groups who had received >6 X 106 CD34�” cells/kg all

engrafted rapidly and were excluded from the analysis.

RESULTS

Generation of A-NK Cells. A-NK cells obtained from

the peripheral blood of normal volunteers were previously found

to proliferate extensively (up to 5000-fold expansion during 14

days of culture) in our hands (12). However, it was uncertain

whether it would be feasible to generate these effector cells in

patients with lymphoma or metastatic breast cancer. We found

that in all patients, it was possible to generate highly purified

A-NK cells from leukapheresis products collected after 6-day

priming with LD-rhIL-2 (Table 2). In three patients with

Hodgkin’s disease, A-NK cells failed to expand in culture under

conditions used routinely in our laboratory (Table 2). The levels

of proliferation in 14-day cultures of A-NK cells obtained from

patients with NHL varied from 1- to 148-fold. Neither the

percentage of CD3CD56� cells nor levels of NK activity were

lower in the pheresis products of patients with HD, as compared

to the patients with lymphoma (Table 2). All three patients with

Hodgkin’s disease had a nodular sclerosing histology. Extent of

prior treatment, time interval between diagnosis and transplant,

and disease extent did not predict for the ability to generate

A-NK cells in this group of patients. Overall, in 6 of 12 patients,

it was possible to obtain > 1 X 10’#{176}A-NK cells for AlT. In 8

of 12 patients, cultured A-NK cells were >85% CD3CD56�

cells, and the A-NK cell culture with the lowest purity contained

64% CD3CD56� cells.

Engraftment. All patients engrafted and became trans-

fusion independent following HDC-PBSCF and AlT. The me-

dian day to reach an ANC of 500/pA was 14 (range, 1 1-19) days.

The median day to reach a platelet count of 50,000/pA was 13.5

(range, 9-200) days. The median time for granulocyte recovery

was significantly (P < 0.001) shortened in 42 patients treated

with G-CSF after HDC-PBSCT relative to 1 1 patients who

received AlT with A-NK cells posttransplant. This difference is

consistent with G-CSF administration, suggesting no adverse



BA

Liz.<

0

0

I I I

PBSC+A-NK #{149}

PBSC + G.CSF 0

S

15 5

S SS 0

0

0 �

10 5 00

0 0

0

5

I I I

0 40


20

10 20 30

Day to Platelet Count > 50,000/pi

Fig. 2 Scatterplot of day posttransplant to an ANC > 500/�i.l and a

platelet count > 50,000/pJ in patients undergoing PBSCT with AlT or

G-CSF posttransplant. All patients received <6 X 10” CD34�1��

cells/kg.

effect on neutrophil engraftment from AlT. Seven patients had

rapid, sustained platelet engraftment and achieved a platelet

count of 50,000/pA 1-3 days before their ANC reached 500/pA.

Five of these patients received less than 6 X 10” of CD341�_

cells/kg, and in comparison to control patients, whose grafts

contained similar numbers of stem cells, their platelet engraft-

ment was significantly more rapid (Fig. 2; P < 0.001). Our

experience has been that regardless of the type of transplant or

the source of stem cells, any patient receiving >1 X i0�

CD34�� cells/kg rapidly engrafted, achieving an ANC >

500/pA and a platelet count > 50,000/pA on or before day + 13.

The same was true for PBSCT, when >6 X 10” CD34�”�

cells/kg were transplanted. Therefore, we compared patients

who received <6 X 10” CD34�’”’ cells/kg and reached a

platelet count of 50,000/pA before 40 days in both the AlT and

G-CSF transplant groups. Those patients who received AlT had

significantly accelerated platelet production after transplantation

(Fig. 2).

In aggregate, these data indicate that AlT with A-NK cells

plus IL-2 did not adversely affect hematopoietic recovery, in a

clinically relevant fashion, when administered immediately after

PBSCT and may have accelerated platelet engraftment in a

subset of patients.

Toxicity. Therapy was discontinued in three patients

(UPN 485, 465, and 448 on days + 1, +5, and +25, respective-

ly). Reasons for discontinuation were: CNS lymphoma (UPN

448 and 485) and severe anxiety (UPN 465). Both patients with

CNS lymphoma were free of symptoms on entry into the study,

but developed signs of CNS involvement before A-NK cells and

HD-rhIL-2 were administered. One patient (UPN 448) received

intrathecal chemotherapy and radiation to the pons for CNS

lymphoma before HDC began. She continued on treatment until

day +25, when she requested to be withdrawn from the proto-

col, because of persistent quadraparesis. Four patients have

died; UPN 385 on day + 109 from interstitial pneumonitis, UPN

448 on day +72 and UPN 485 on day + 162 from CNS lym-

phoma, and UPN 469 on day + 171 from recurrent lymphoma.

Toxicity from the rhIL-2 and A-NK cells was limited to mild

hypotension and edema occurring from days +3 to +7. Two

patients received low-dose dopamine for hypotension and had

reduced urine output during this time. No patient experienced

renal failure, veno-occlusive disease, hemorrhagic cystitis, or

diffuse alveolar hemorrhage.

Immunological Modulation. The percentage of

CD3CD56� or CD3CD56�CD16� (NK) cells and NK ac-

tivity against K562 targets in the patients’ peripheral blood were

measured at various times during the treatment. Table 3 shows

the results of NK phenotyping and NK activity in four patients

for whom serial data were available before and in the early

posttransplant period. Of the four patients, three (UPN 380, 386,

and 475) had normal NK activity before therapy. During the

period between day +3 and +8, when the absolute leukocyte

count was <200/pA, variable but detectable cytotoxicity was

observed in the blood, which might be attributable to A-NK cell

infusion. Fig. 3 shows serial results in UPN 386, demonstrating

the initial peak of NK activity followed by the gradual rise

attributed to LD-rhIL-2 over 90 days posttransplant. In general,

the number of circulating NK cells after transplant did not

correlate with NK activity. As illustrated in Fig. 3, despite a

rapid fall in cytotoxicity from the peak on day +3, the number

of NK cells continued to increase in the peripheral blood.

Similar increases in the absolute number of circulating NK cells

were seen in other patients. Spontaneous cytolytic activity of

fresh peripheral blood mononuclear cells against Daudi targets

was detected in the peripheral blood of two patients (UPN 385

and 386) in the immediate posttransplant period, and this could

be interpreted as additional evidence in support of the presence

of circulating transferred A-NK cells.

Ifosfamide. Ifosfamide chemotherapy was used to test for

chemosensitivity of each lymphoma and for mobilization of

PBSCS. Five of 10 patients responded to ifosfamide (2 complete

responses, 3 PR, overall response rate of 50%). No patient exhib-

ited progressive disease on ifosfamide. One patient (UPN 485)

developed a Fanconi-like renal wasting syndrome that recovered

spontaneously. Outside of predictable pancytopenia, there were no

other side effects directly attributable to ifosfamide.

Busulfan/Cyclophosphamide. There was no mortality

attributable to the conditioning regimen, and toxicity was tran-

sient. The 3-month response evaluations showed that response

had improved in five patients, was unchanged in three, and had

worsened in two. Three of the five patients experienced im-

provement from a PR to complete response and two from stable

disease to PR. This conditioning regimen was well tolerated and

active in this group of patients.

DISCUSSION

This clinical trial demonstrates the feasibility of adding

AlT with autologous A-NK cells and IL-2 in the pancytopenic

period immediately after PBSCT. This is a rational combination

of therapies, because they differ in their mechanism of action,

mechanism of disease resistance, and toxicity. In our hands, AlT

was used in an effort to exert a further antitumor effect at the

time of minimal disease after HDC-PBSCT.

The choice of A-NK cells for AlT was based on our

preliminary data indicating that this subset of IL-2-activated



Table 3 NK activity and percentage of MNC with NK cell phenotype in the blood of patients treated with HDC-PBSCT and AlT

UPN (diagnosis)”Before therapy”

LU’

Days +3 to �8d Day +90�

LUC

% NKcellse LU�

% NKcellse

380 (BRC)

385 (DSCCL)

386 (FML)

475 (DML)

319

33

325

415

772

2641673

287

32

2

41

n/a

833

440883

1290

25

3

48

32

a Only the patients cited exhibited increased NK activity in the blood on days +3 to +8, the other patients had values between 0 and 33 LU (see

Table 1 for diagnosis codes).6 This measurement (LU) was obtained before any protocol therapy had started and at least 4 weeks past any prior therapy.

C NK activity was measured in the PBMC of patients using 4-h 51Cr release assays with K562 as targets and is expressed as LU2(,/107 effector

cells (LU). The normal range in our laboratory is 50-300 LU, with a median of 132 LU.d The absolute leukocyte count was <200/jil at the time of this measurement. No lytic activity is detectable in the absence of A-NK cell infusion.

e % NK cells was determined by flow cytometry in two (CD356�) or three-color (CD356� 16k) analysis. The normal range for our laboratoryis 6-21%, with a median of 12%.

1The absolute leukocyte count was normal at the time of this measurement.

400

0

350 B�C’

0�

C’

0

zC.)

C’

.�

0-I

0�

00

0.0

-20 0 20 40 60 80 100

Day relative to transplant

Fig. 3 Changes in NK activity (LU7�1/107 cells) and in the absolute number of NK cells/pA in the peripheral blood of UPN 386 between days -20and + 100 of therapy relative to transplantation (day 0).


1800

. 1600

� 1400

U

� 1200

� 1000

I: 800

.� 600

� 400

200

0

CDrCD56(�mCD16_ or dim NK cells has potent antitumor

activity in vitro and in vivo (12, 19) and is particularly effective

in targeting metastases (14, 20). A-NK cells have been shown to

be able to produce a variety of cytokines (20, 21), localize to

metastases (20), and rapidly eliminate established metastases in

animal tumor models (13, 14, 19). Activities of A-NK cells are

strictly dependent on the presence of IL-2 (10, 22). At 2 X 106

IU/m2/day (HD-rhIL-2), the serum concentration of IL-2 was

estimated to be sufficient to bind high affinity IL-2 receptor

(22). Thus, we reasoned that the infused A-NK cells, a propor-

tion of which expresses the high affinity IL-2 receptor (12),

were likely to be stimulated in vivo, at least during the initial 4

days after adoptive transfer.

We hypothesized that the timing of A-NK cell transfusion

might be critical for cure. Animal models of combined chemo-

therapy and AlT have shown that the smaller the time interval

between the two therapies, the greater the cure rate (23, 24). On

300

250

200

150

100

50

0

the basis of earlier observations, we planned to give the A-NK

cells and IL-2 as close to the chemotherapy as possible.

Our main concerns were the possible adverse effect of AlT

on engraftment and the added toxicity of AlT in the pancyto-

penic phase after HDC-PBSCT. Our preclinical studies showed

no suppression of colony formation in vitro by human A-NK

cells and that at the higher A-NK to progenitor ratios, there may

actually be enhancement of growth (25). Murphy et a!. (26, 27)

and Siefer et a!. (28) have shown accelerated platelet production

after marrow transplantation, in a murine model, that is depen-

dent upon infusion of murine NK cells. We believe that we may

have observed an analogous phenomenon in humans.

The administration of IL-2 at doses sufficiently high to

support the antitumor activity of A-NK cells was an essential

component of the therapy. To reduce IL-2-related toxicity, two

design principles were used in this trial. First, the doses of

rhIL-2 used were one to two orders of magnitude less than those




used previously for therapy. Second, during the pancytopenic

phase, induction of cytokine secretion from monocytes and

thromboxane A2 from neutrophils would be absent. These sec-

ondarily generated molecules are thought to mediate the toxic

effects of IL-2 (29), and their absence would be expected to

reduce IL-2-induced toxicity. Indeed, toxicity attributable to

IL-2 was tolerable in this trial. The subsequent 90-day infusion

with LD-rhIL-2 was used in the hope of sustaining or expanding

in vivo antitumor effector cells. It has been shown previously to

expand the number of circulating CD56� NK cells without

affecting the number of T lymphocytes in patients with cancer

and prime for LAK activity generated in vitro with higher

concentrations of IL-2 (30-32).

The NK and LAK activity observed in the blood of these

patients can be explained by the persistence in the circulation of

functional transfused A-NK cells. NK activity in the blood is

normally absent after transplant and returns no earlier than the

third week. In Fig. 3, the initial peak of activity observed at day

+3 coincides with the transfusion of the A-NK cells and their

support with HD-rhIL-2. The subsequent fall in activity can be

ascribed to the death of the A-NK cells, which may at least

partially be due to the withdrawal of HD-rhIL-2. The presence

of cells with NK phenotype but lack of activity suggests the

presence of a nonfunctional precursor that acquires NK activity

with maturation. The cells responsible for this activity are prob-

ably not of transfused A-NK cell origin.

The ability to consistently generate A-NK cells from the

peripheral blood of patients with advanced lymphoma or solid

tumors has also been of concern (33). To facilitate A-NK cell

generation, a 6-day-long infusion of LD-rhIL-2 before the series

of phereses for A-NK cell generation was included in the

protocol. The intent was to increase the yield of A-NK cells in

culture by priming their progenitors in vivo. Using this approach

and the improved culture conditions, i.e., short adherence time

and irradiated feeder cells, as described earlier (12), we have

been able to generate cell cultures which were highly enriched

in CD3CD56� cells in all patients with lymphoma. However,

A-NK cells obtained from the peripheral blood of the 3 patients

with Hodgkin’s disease showed poor in vitro growth. No spe-

cific cause for this poor A-NK cell generation in culture could

be discerned, and it did not appear to be related to the extent of

prior treatment these patients received or to the extent of their

disease. Both the number of NK cells in the peripheral circula-

tion and NK activity were normal at baseline in these three

patients. Hodgkin’s disease is known to be associated with a

profound cellular immune deficit (34), and it is intriguing to

speculate that the observed inability to generate A-NK cells

might be related to a low number or poor function of A-NK cell

precursors in the peripheral blood of these patients.

Using a combination of HDC-PBSCT and AlT with A-NK

cells and IL-2 had no clinically relevant adverse effects on

engraftment and toxicity, which were both comparable to those

seen with HDC-PBSCT alone. In a subset of patients treated

with AlT, platelet engraftment may have been accelerated. In

addition, early amplification of NK cell function in the cytope-

nic phase after transplantation, that could have its origin in the

transfused A-NK cells, was observed in some patients. Future

studies will attempt to optimize AlT with A-NK cells and to

dissect the contribution of each transfused cell population to the

immunological changes observed.

ACKNOWLEDGMENTS

We thank Dr. Ronald B. Herberman and Dr. A. Louie for critical

review of the treatment protocol.

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1995;1:607-614. Clin Cancer Res J Lister, W B Rybka, A D Donnenberg, et al. immediate posttransplant period.

theadoptive immunotherapy with activated natural killer cells in Autologous peripheral blood stem cell transplantation and

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