Active Immunotherapy with Ultraviolet B-irradiated ... · Vol. 4, 6/9-627, Marc/i 1998 Clinical...

Vol. 4, 6/9-627, Marc/i 1998 Clinical Cancer Research 619

Active Immunotherapy with Ultraviolet B-irradiated Autologous

Whole Melanoma Cells plus DETOX in Patients with

Metastatic Melanoma1

Omar Eton,2 Dmitri D. Kharkevitch,

Mary Ann Gianan, Merrick L Ross, Kyogo Itoh,

Michael W. Pride, Cherrie Donawho,

Antonio C. Buzaid, Paul F. Mansfield,

Jeffrey E. Lee, Sewa S. Legha, Carl Plager,

Nicholas E. Papadopoulos, Agop Y. Bedikian,

Robert S. Benjamin, and Charles M. Balch

Departments of Melanoma/Sarcoma Medical Oncology [0. E..M.A.G.,A.C.B.,S.S.L.,C.P..N.E.P..A.Y.B.,R.S.B.].Surgical Oncology [D. D. K., M. I. R.. P. F. M., J. E. L.], and

Immunology [M. W. P.. C. D.]. The University of Texas M. D.

Anderson Cancer Center, Houston, Texas 77030: Department of

Immunology. Kurume University School of Medicine. Kurume,

Fukuoka 830, Japan [K. I.]: and City of Hope National MedicalCenter and Beckman Research Institute, Duane, California 91010

[C.M.B.]

ABSTRACT

Our objective was to determine the clinical activity,toxicity, and immunological effects of active immunotherapy

using UVB-irradiated (UVR) autologous tumor (AT) cells

plus adjuvant DETOX in metastatic melanoma patients.

Eligibility included nonanergic patients fully recovered after

resection of 5 or more grams of metastatic melanoma. Treat-

ment consisted of intradermal injections of iO� UVR-AT

plus 0.25 ml of DETOX every 2 weeks x 6, then monthly.

Peripheral blood mononuclear cells (PBMCs) were har-vested for cytotoxicity assays, and skin testing was per-

formed for delayed-type hypersensitivity (DTH) determina-tions before the first, fourth, seventh, and subsequent

treatments. Forty-two patients were treated, 18 in the adju-

vant setting and 24 with measurable disease. Among thelatter group, there were two durable responses in soft-tissue

sites and in a bone metastasis. Treatment was well tolerated.

Thirty-five patients were assessable for immunological pa-

rameters; 10 of these patients, including the 2 responders,demonstrated early induction of PBMC cytotoxicity against

AT cells that persisted up to 10 months on treatment beforefalling to background levels. In five of seven patients, the

Received 6/12/97: revised 12/5/97: accepted 12/15/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported in part by NIH Grant RR-551 1.

2 To whom requests for reprints should be addressed, at The Universityof Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,

Box #77, Houston, TX 77030. Phone: (713) 794-4633: Fax: (713) 794-1934.

fall-off heralded progressive disease. Late induction of aweak DTH reaction to AT cells was observed in eight pa.

tients. Active immunotherapy with UVR-AT + DETOX hadmodest but definite clinical activity in advanced melanoma.The induction of both PBMC cytotoxicity and DTH reactiv-ity to AT cells supported a specific systemic immune effect of

treatment, although the former more closely followed dis-ease course in this study.

INTRODUCTION

Malignant cells must elude any host immune surveillance

to grow and overcome the host. Hewitt et a!. ( I ) have shown that

spontaneous tumors in aging mice do not induce a detectable

host immune response. Nevertheless, the hypothesis that those

same cells that escape immune surveillance in vivo might be

manipulated in vitro to become more immunogenic is a basis for

tumor immunotherapy. Methybcholanthrene and UVB-induced

tumors, when excised and reimplanted in mice, have resulted in

varying degrees of specific immune protection (2-5). In pa-

tients, tumor burden, immunosebection (6), tolerance induction

(7-I 0), and biophysical barriers ( 1 1 ) all present challenges to

developing effective antitumor immunotherapy programs.

Efforts to circumvent these obstacles in the 1990s are

focused on identifying specific factors that either drive or inhibit

antitumor immunity (12). However, while the relative impor-

tance of these factors continues to evolve almost on a daily

basis, cruder vaccine preparations from the patients’ tumors

continue to have a role in providing a plethora of characterized

and as yet unidentified targets. These vaccines have resulted in

objective response rates under 20%, including responses in

visceral organ metastases consisting of billions of tumor cells

(13-15). Such clinical activity supports the continued develop-

ment of active immunotherapy in the multidisciplinary antican-

cer armamentarium. Viable autobogous whole tumor cells tested

in melanoma vaccine preparations have some advantages: (a)

the relative safety of intradermab injection of autobogous rather

than ablogeneic material; (b) the potential for inducing as yet

uncharacterized immune responses to altered self-antigens in

common with the patient’s tumor; and (c) the diminution of

some artifacts of vaccine preparation. Disadvantages include:

(a) the difficulty in obtaining and preparing individualized vac-

cines; (b) the limited source material; and (c) the inability to

control for intervaccine variation important in clinical trials.

At M. D. Anderson Cancer Center, Hostetler et a!. (16-18)

demonstrated that UVR3 (280-320 nm) of the weakly immu-

3 The abbreviations used are: UVR, UVB irradiation; AV, antigenicvariant; PBMC, peripheral blood mononuclear cell; DTH, delayed-type

hypersensitivity; AT, autologous tumor.

Research. on March 21, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

620 Active Immunotherapy: Autologous Melanoma Cells, DETOX

nogenic melanoma cell line K1735P (C3H/HeN; MTV) re-

sulted in the generation of a high frequency of highly immuno-

genic AVs. Immunizing syngeneic mice with AV clones

induced protective immunity in in vivo challenge assays against

the immunizing AV clone and the untreated parental tumor line

but not against unrelated tumors (16-18). The parental tumor

did not induce such protective immunity. These AV clones grew

in immunosuppressed mice but not in immunocompetent mice,

and they induced cytotoxic/helper I-lymphocyte activity that

cross-reacted with parental tumor cells (18).

Based on these data, it was hypothesized that the poly-

cbonab population of UVR-parental cells might provide a mul-

titude of immunogenic AVs for active immunotherapy without

requiring individual AV clones to be isolated. Umezu et al. (19)

developed conditions for UVR of human melanoma and renal

cell carcinoma cells to optimize inducing cytotoxic activity of

PBMCs against irradiated- and nonirradiated parental cells. Us-

ing in vivo immunization/challenge and DTH experiments, Pride

et a!. (20, 2 1) demonstrated that UVR of the K! 735-M2 cell line

greatly increased its immunogenicity. Pride et a!. (21) also

found that UVR not only up-regulated expression of MHC

classes I and II molecules but also induced expression of murine

B7.l on Kl735-M2 melanoma.

Mycobacteriab preparations are useful adjuvants in active

immunotherapy (22). In melanoma, Bacillus Calmette-Gu#{233}rin

induces responses in up to 90% of locally injected and 15% of

adjacent lesions but has had no effect on survival in Phase III

trials and can cause disseminated infection. DETOX (Ribi Im-

munoChem Research, Inc. , Hamilton, MI) is a novel adjuvant

available in a lyophilized oil droplet emulsion consisting of both

cell wall cytoskeleton from Mycobacterium phlei and mono-

phosphoryl lipid A from Salmonella minnesota R595. DETOX

can augment I cell and humoral immunity. In the murine

experiments by Pride et a!. (20), DETOX was effective in

significantly augmenting immunoprotection and DTH. In pa-

tients, DETOX has been used only as an adjuvant, and dosing

has been empiric because its effects have been more host than

dose rebated (23, 24).

We undertook the present study to determine the clinical

activity and toxicity of active immunotherapy with UVR-AT

cells + DETOX in patients with metastatic melanoma. We used

serial monitoring of PBMC cytotoxicity and DTH reactions

against AT cells with controls to detect and track any evidence

of a specific systemic immune response to local intradermal

therapy during the course of vaccine treatment.

PATIENTS AND METHODS

Patients. Patients > 15 years with metastatic melanoma

pathologically confirmed at M. D. Anderson Cancer Center and

from whom more than S g of nonnecrotic tumor had been

resected as a part of routine management were candidates for

treatment. Patients were enrolled only after full postoperative

recovery and with: a Karnofsky performance status >70%; life

expectancy >3 months; DIH reaction positive to foreign anti-

gens (see below); results of complete blood counts, serum

chemistry studies, immunogbobulins (IgG, IgA, and 1gM), com-

plement (C3, C4, and CHSO), and I-cell subsets in the clinically

normal range; and signed informed consent forms. Patients were

required to avoid immunomodubatory agents (e.g., corticoste-

roids, nonsteroid anti-inflammatory agents, antihistamines, es-

trogens, high doses of vitamins, and concurrent specific antitu-

mon therapy). All sites of clinically detectable disease were

objectively evaluated at baseline using computed tomography,

magnetic resonance imaging, sonography, photographs, and, for

s.c. disease, multiple investigator assessment.

Vaccine Preparation. Fresh tumor was collected at the

time of surgery from the frozen section laboratory and frag-

mented by slicing. Aliquots of 106 cells each were frozen for

immunoassays. To maximize yield of viable tumor cells for

vaccine preparation, the bulk of tumor was dissociated using

colbagenase type I (2 mg/mb) and type IV DNase (0.4 mg/ml;

Sigma Chemical Co., St. Louis, MO; Ref. 25). These enzymes

can alter the immunogenicity of the resulting cell preparation

(26-29). In control flow cytometry experiments, Itoh et a!. (26)

did not find enough significant alteration to obviate the use of

the enzymes; furthermore, incubation of the tumor cells for 24 h

before vaccine administration, as described below, resulted in

reexpression of specific suppressed antigens (26, 29).

The dissociated cells were washed in sterile HBSS with

gentamicin and resuspended in equal volumes of HBSS and

chilled 10% DMSO + 4% human serum albumin. Aliquots

containing 1.5-2 x i0� viable tumor cells were stored under

liquid nitrogen. For some specimens, an aliquot of fresh tumor

was also put into culture to establish a tumor cell line.

Twenty-four h before patient arrival, an abiquot of tumor

cells was thawed in a 37#{176}Cwater bath, washed twice, resus-

pended in 5 ml of PBS in a 10-cm Petni dish, and exposed to

UVR (2.2 J/m2/s) for 30 s using an FS4O sunlamp (Westing-

house Electric Corp., Bboomfield, NJ; Refs. 18 and 19). Incu-

bation in macrophage-/serum-free medium (Life Technologies,

Inc.) for 24 h resulted in >80% cell viability. Cell suspensions

were checked for bacterial contamination and for Mycoplasma

contamination using the Gene-Probe rapid detection system

(Gene Probe, Inc., San Diego, CA). After incubation, cells were

detached by scraper, washed, resuspended in 0.75 ml of PBS,

and X-irradiated (20,000 cGy) to prevent propagation in vivo

and insure sterility (30, 31). Concurrently, DETOX (each vial

stored at 2-7#{176}Cconsisted of 500 p.g of cell wall cytoskeleton, 50

p�g of monophosphoryl lipid A, 2% squalene, and 0.4% Tween

80 in 2X normal saline) was reconstituted by repeated aspiration

with 0.5 ml of sterile water. In a total volume of 1 ml, approx-

imateby 10� viable replication-deficient UVR autologous mela-

noma cells in PBS were mixed with 0.25 ml of DETOX just

before each treatment (24).

Treatment Plan. Active immunotherapy was adminis-

tered at M. D. Anderson Cancer Center by intradermal injection

into the anteromedial deltoid and femoral regions near intact

lymph node basins, every 2 weeks X 6 and then every 4-6

weeks. Treatment ended with progression of disease (25%

growth) or exhausted supply of vaccine material. Response was

assessed initially at 6 weeks and then at 8-12-week intervals

during long-term follow-up. Toxicity was graded according to

National Cancer Institute guidelines.

Cytotoxicity Assays. For uniformity, all cytotoxicity as-

says were performed by a single investigator (D. K.) throughout

the study. At baseline and before the fourth, seventh, and sub-

sequent vaccinations, PBMCs were isolated by Ficoll-Paque



Clinical Cancer Research 621

Tab le I Pretreatme nt patient and tumor characteristics

n % of total Indicator lesions Adjuvant setting

All patients 42Age (yr): median, 52: range, 27-81

Male 24 57Prior treatment

None 15 36

Biotherapy 14 33

Chemotherapy ± biotherapy 21 50

Regional perfusion 2 5

Karnofsky performance status

90% 36 86

80% 6 14Normal serum level of LDH” 38 90Normal serum level of albumin 40 95

Tumor stage and sites

All patients 42 24 18

AJCC stage III N2 (regional) 6 1 5

AJCC stage IV (disseminated) 36 23 (55%) 13 (3 1%)

Soft-tissue only 16 8 8

Visceral metastases 20 15 5

One visceral organ 12 8 4Lung alone 7 5 2Brain 6 5 1Liver 4 3 1

Spleen 4 2 2Adrenal 2 1 1

Gastrointestinal 2 0 2

C, LDH, lactate dehydrogenase: AJCC, American Joint Committee on Cancer.

gradient (Pharmacia AB, Uppsala, Sweden) from 30 ml of fresh

heparinized peripheral blood at room temperature (22). These

PBMCs were assayed fresh and were also cryopreserved until

sufficient specimens could be obtained over 2 or more months

of treatment to perform a single experiment, thereby minimizing

interassay variability. Four-h � ‘Cr-release assays were used to

detect cytotoxic activity, as described previously (32). Cryopre-

served, uncultured autobogous and albogeneic melanoma cells,

renal cell carcinoma cells, cultured tumor cell lines, and natural

killer-sensitive tumor line K562 were used as the targets in these

assays. Frozen aliquots of uncultured tumor cells were thawed

on the day of the assay; adherent cell lines were trypsinized.

Target cells were labeled with 50 p.Ci/million cells of

Na25tCrO4 (Amersham Corp., Arlington Heights, IL) at 37#{176}C

for 1 h. After washing twice, labeled cell suspensions were

passed through a cotton column to remove cell aggregates and

debris. Labeled target cells at a concentration of 5000 cells/well

in triplicate were added to effector lymphocytes at each of three

E:T ratios, 40: 1, 20: 1, and 10: 1, in 96-well, round-bottomed

plates. The plates were incubated for 4 h at 37#{176}C,after which

100 p.1 of supematant/well was harvested, and radioactivity was

measured in a gamma counter. In all experiments performed, the

spontaneous release did not exceed 20% of the maximum

release. Cytotoxicity assays were also performed with ad-

herent melanoma cell targets in 96-well, flat-bottomed plates.

The percentage of specific lysis was calculated as: (Xa x��)/

(Xmax x��) X 100, where Xa �5 the mean � ‘ Cr release at a

specified E:I ratio; x� is the mean spontaneous 51Cr release (no

effector cells); Xmax �5 the maximum 5tCr release by target cells

treated with 0. 1 N HC1. Positive cytotoxicity as a result of

treatment was defined as > 10% specific lysis and at beast 2-fold

higher specific bysis than at baseline.

Antibody-blocking Experiments. To confirm and char-

acterize any positive cytotoxicity results detected, blocking ex-

periments were performed using antibodies to CD3, CD4, CD8,

DR. DP, DQ (Becton Dickinson, Sunnyvale, CA), and class I

MHC (W6/32; DAKO, Copenhagen, Denmark). Monoclonal

antibodies were mixed with � ‘Cr-labeled AT targets in a volume

of 50 p.1 each for 1 h at 4#{176}Cto try to abrogate lysis. Effector cells

were added at a 40: 1 E:T ratio, and cytotoxicity was measured

as described above. Controls were murine IgG and medium

alone.

DTH Assays. Intradermal skin testing on the vobar

surface of the forearm was performed at baseline against:

foreign recall antigens (Merieux Skintest Multitest Device:

tetanus, diphtheria, strep, tuberculosis, Candida, trichophy-

ton, and proteus); l0� X-irradiated AT, and i05 autobogous

PBMCs (negative control). AT and PBMC skin tests were

repeated before the fourth, seventh, and subsequent vaccina-

tions. A positive DTH reaction consisted of induration over 2

mm in diameter at 48 h. Hypoergy to foreign antigens using

the Merieux skin-test device was defined as two or fewer

positive DTH reactions or sum of induration diameters under

1 cm. In this study, patients were not asked to undergo punch

biopsy of skin test sites to detect subclinical evidence of a

DTH reaction.

Statistical Considerations. Complete or partial re-

sponse required disappearance or >50% decrease, respec-

tively, in the sum of the products of largest diameters of all

indicator lesions lasting at beast 1 month. Stable disease



�- -.�


A

B

Fig. 1 Clinical responses to active immunotherapy using autologous UVR-melanoma cells + DETOX. A, partial response in bone 6 months from

the start of treatment; this patient also had a complete response in s.c. nodules. B, single view of some of several dozen in-transit lower-extremity

metastases which, after an initial flare at 10 weeks, disappeared over a period of 17 months from the start of treatment; surgery to harvest more cells

for vaccine preparation resulted in this patient’s formal response being characterized as mixed to vaccine and complete to vaccine plus surgery.

required less than 25% change in the sum of the products of

diameters lasting at least 8 weeks. Survival from start of

treatment was plotted by the method of Kaplan-Meier and

evaluated using the bog-rank test. PBMC cytotoxicity and

DTH reaction to AT were assessable only after six or more

treatments. A modification of Simon’s design for Phase II

clinical trials was used; assuming a 10% significance level

and 90% power, I 2 patients were enrolled in stage I and 23

additional patients in stage II (32). Induction of PBMC

cytotoxicity in 6 of 35 patients would support further inves-

tigation, assuming that response rates of 30 and 10%, respec-

tively, would and would not be of interest. Enrollment would

have terminated early if none of 12 patients achieved a

clinical response or if 6 of 9 patients treated in an adjuvant

setting had progressive disease within 3 months.

RESULTS

Between September 1992 and May 1995, 42 patients re-

ceived a median of eight treatments (range, 3-21) with UVR-

autobogous melanoma cells plus DETOX. Pretreatment charac-

teristics are summarized in Table I . All patients were assessable

for clinical response and toxicity and 35 for biological response.

Seven patients had disease progression before completing the

first six treatments.

Clinical Responses. Among 24 patients with indicator

lesions, two had major responses and six showed stabilization in

soft-tissue metastases. One 39-year-old woman treated previ-

ously with chemo/biotherapy underwent resection of painful s.c.

nodules for vaccine preparation. Her indicator lesions consisted

of nine s.c. nodules, each 1 cm or smaller, and a large sympto-



40 A

I!11 12

.�‘ 30

#{149}0

� 20

0

a� 10

Before 0Vaccine #: I

Months: �

40

�‘ 30U

� 20

0

a� 10

Before 0 -

Vaccine #: IMonths: 0

4 7 8 9 10

3 6 9

B

In�jI

4


IlLI12

- 40:1 I- 20:1 I

10:1]

Li � Icil � I7 9 10 11 12 14 15 1617 18 LAK

3 6 9 12 15

Fig. 2 Mean PBMC cytotoxicity over time against autologous tumor atthree E:T ratios for each of the two responders (A and B) shown in Fig.

I. L4K, bymphokine-activated killer activity (positive control).

matic lytic bone metastasis that had progressed radiographically

1 year after radiation therapy. Four days after her first treatment,

she noted soreness at all tumor sites. Three skin nodules initially

enlarged by less than 20% with minimal inflammatory changes,

three remained stable, and three regressed; however, by the

seventh treatment (at 4 months), all s.c. lesions had completely

regressed as had her bone pain, with subsequent confirmation of

major radiological response (Fig. 1A). Nine months after her last

treatment, she developed new s.c. nodules and recurrence in

bone (time to progression, 21 months). From a negative base-

line, her unstimubated PBMCs demonstrated persistently posi-

tive cytotoxicity against AT cells (Fig. 2A). She had an unusu-

ally large DTh reaction (4 cm with 10 cm of surrounding

inflammation) at the first vaccine intradermal injection site,

smaller with subsequent treatments. No DTH reaction to the i0�

AT control was detected during 1 year of treatment.

The second responder was a 63-year-old man with exten-

sive, progressing, lower-extremity, in-transit nodules, despite

regional and systemic chemo/biotherapy and radiation therapy.

Resected symptomatic lesions were used for vaccine prepara-

tion. Innumerable in-transit nodules, 0.5-2 cm in caliber, were

used as indicator lesions. By 10 weeks, most lesions had flat-

tened or remained stable; however, a minority appeared more

prominent, although by <20%. Because of early resection of

these lesions to make more vaccine, this was strictly a “mixed”

response. However, after 19 treatments for >20 months, to-

gether with surgery, he ultimately achieved a complete response

lasting 5 months (Fig. 1B). From a negative baseline and ex-

tending through the 16th treatment, his unstimulated PBMCs

demonstrated cytotoxicity against AT cells (Fig. 2B). Fall-off in

cytotoxicity coincided with progressive soft-tissue disease. He

93 consistently had a large DTH reaction (3 cm) to vaccine but

never to the AT control.

Survival. The median survival of the stage IV patients

was 16 months from start of treatment, 20 months from initial

diagnosis of stage IV disease. Review of pretreatment prognos-

tic factors revealed a high frequency of favorable factors (nor-

mal serum levels of lactate dehydogenase; Refs. 33 and 34),

metastases in soft tissue sites alone, or metastases in one visceral

LAK organ such as lung (33), in this study.

Toxicity. Grade I toxicities were limited to local irrita-

tion at the vaccine injection site and in 10% of patients. with

malaise lasting 24-48 h. There was no incidence of skin ulcer-

ation or other grades I1-IV toxicities. Palpable residual granu-

bomas at the injection sites measuring <2 cm became less

apparent over weeks to months. In one-half of the patients,

routine evaluation by an ophthalmologist yielded no abnormal-

ities attributable to treatment. Five of 15 patients (33%) purified

protein derivative negative at baseline converted to purified

protein derivative positive (asymptomatic) using the Merieux

skin-test device, possibly a result of repeated exposure to DE-

lOX (or to the anergy panel).

Detection of PBMC Cytotoxicity against Autologous

Tumor. During the course of treatment, 10 of 35 assessable

patients had an early rise in mean PBMC cytotoxicity against

AT cells within 6 weeks, all from a negative baseline of under

10% specific lysis (Table 2). Lytic activity remained detectable

up to 10 months, later falling back to background bevels in 7 of

the 10 patients. In five patients, the fall-off coincided with

progression of disease within 3 months. For the three patients

without fall-off, autologous material was insufficient to continue

bong-term treatment and monitoring.

Table 3 illustrates the individual spectrum of specificity for

PBMC cytotoxicity. The selective rather than general positivity

in the cytotoxicity assay results against a panel of allogeneic

tumor cells, and K562 does not support a non-specific effect of

vaccine preparation, such as the use of collagenase. In four

patients, the level of lysis of K562 cells was minimal, indicating

that the cytotoxicity was not mediated by natural killer cells.

Table 4 shows that lysis could be abrogated by anti-CD3,

anti-class I, and anti-CD8 but not by anti-class II and anti-CD4

antibodies, indicating that the observed cytolytic activity was

restricted by class I and was mediated by CD8 + I cells.

Univariate analysis indicated that patients with stage IV

disease and positive cytotoxicity had both a prolonged median

time to progression and overall survival duration compared with

those with negative cytotoxicity (Table 5 and Fig. 3). Although

time bias was minimized because PBMC cytotoxicity was al-

ways detected early, the positive cytotoxicity group also had a

lower disease burden, confounding the results of the analysis.

DTH. Among 42 nonanergic patients, 21 (50%) were

hypoergic to foreign recall antigens at baseline, a finding that

did not correlate with any pre- or posttreatment factors. Baseline

hypoergy was evident in 4 of the 10 patients with positive

PBMC cytotoxicity results (Table 5).

Among the 35 assessable patients, 8 (23%) developed a bate

DIH reaction to iO� AT cells (and no reaction to PBMC

control); indurations under I cm first appeared after a median of

8 courses (4-15 courses). Neither responder and only three

patients with positive PBMC cytotoxicity also had a DTH



Patient

Table 2 Ten patients with specific lysis > 10% and rising over 2 X baseline during the course of treatment

E:T 15” 16”16” 17b

2 02 32 3

4 0

2 03 0

9 0

8 0

0 3

Table 3 SpecifIcity of positive PBMC cytotoxicity in four patients

Cytotoxicity against targets


I” 4” 7”

0” 1b 4/’

40:1 8 14 21

20:1 4 14 21

10:1 12 3 19

2 40:1 4 15 1420:1 5 13 17

10:1 4 10 3

3 40:1 6 22 23

20:1 0 15 23

10:1 0 10 15

4 40:1 0 10 0

20:1 1 9 1

10:1 0 9 0

5 40:1 2 6 10

20:1 5 0 5

10:1 1 0 0

6 40:1 4 24 18

20:1 0 17 3

10:1 0 8 0

7 40:1 8 9 12

20:1 5 10 8

10:1 0 0 3

8 40:1 0 1 2

20:1 I 0 1

10:1 0 0 29 40:1 2 19 24

20:1 0 7 18

10:1 0 1 6

10 40:1 1 0 0

20:1 1 3 1

10:1 0 3 0

‘I Vaccine number.

I, Months.

9” ioa6”

7b

49

8” 11”

_�t48 31

28 3429 22

15 20 10

5 17 6

0 5 8

28 30 31 4717 23 23 15

8 2 5 15

6 21 24

5 23 12

5 15 2

15 15 10 13

5 10 9 4

I 7 3 0

38 46 15 1

21 18 8 0

11 13 8 0

11 16 12 8

0 6 14 1

0 0 5 3

16 20 16 23

6 14 11 12

2 7 1 2

25 13 26 3619 4 17 19

3 4 3 4

27 30 28 823 33 17 0

9 10 5 0

12” 13” 14”

11” 12” 14”

27 55 5122 47 8

15 22 6

9 2 5

6 I 3

5 0 1

16 10 5

19 7 4

I 1 4

30 23 15

17 11 10

2 0 8

7 3 0

1 3 2

0 2 1

12 13 5

2 1 3

0 0 6

18 28

12 16

8 7

2717

50

0

0

0 3

0 0

0 0

Patient Autologous Allogeneic Renal cell NK”-sensitive

no. melanoma melanoma carcinoma K562

I + + - -

4 + - - -

10 + ± ± ±

11 + + - -

“ NK, natural killer.

reaction to AT cells. Skin testing using UVR-AT was not

performed. Contaminants in preparation could have resulted in

a high frequency of false-positive DTH reactions to both AT and

PBMC control skin tests; this was not observed. No tests, such

as punch biopsy, were done to evaluate for false-negative

results.

The size of the DTH induration to the vaccine itself (UVR-

AT + DETOX), measuring primarily response to adjuvant, was

above the average and median values in 7 of the 10 patients with

positive cytotoxicity, including the two responders and the three

patients with a DTH reaction to AT. No consistent trend in the

size of the DTH reaction to vaccine was observed in individuals

or in population groups over time. Biopsy and staining of a

1-month-old indurated injection site in one patient revealed

heavy infiltration by histiocytes and I-cells but not by B cells

(data not shown).

DISCUSSION

Among 24 patients with indicator lesions, intradermal ac-

tive immunotherapy with UVR-AT + DETOX resulted in two

major responses in soft-tissue sites and in a bone metastasis,

supporting clinically meaningful systemic activity, albeit in

<20% of patients with 95% confidence. This is no different

from the results reported in other vaccine trials and does not

support using UVR alone to enhance the clinical activity of

autologous whole-cell vaccines plus adjuvant. As in other such

trials, the overall survival data were encouraging but likely

reflect selection bias. The present American Joint Committee on

Cancer criteria for stage IV disease only distinguish between

soft-tissue and visceral metastases in patients having a median

overall of survival of 8 months. Newer, more effective stratifi-

cation parameters for clinical trials using pretreatment serum

levels of lactate dehydogenase and albumin (33-34) and the

presence of visceral disease in zero-to-one organ versus more-

than-one organ (33) have identified prognostic groups with

median survivals of over 12 months versus <6 months. Until a

regimen with a high response rate is found, stratification might

help determine which active immunotherapy trials warrant con-

firmation of “encouraging” survival data in a Phase III trial

setting.



Table 5 Characteristics of patients with positive and negative

detection of PBMC cytotoxicity against autologous tumor

0.8

0.6zC

C

Q.0.4

0.2


4 M. Pride, unpublished data.

Table 4 Antibody inhibitio n of PBMC cyto toxicity against aut ologous melanoma

Patient Media

no. alone IgG anti-CD3 anti-CD8 anti-class I anti-CD4 anti-DR anti-DP

1 50” 56 14 13 12 54 55 52

4 47 53 0 1 1 45 46 48

11 46 43 7 4 6 42 41 45

a Percentage of cytotoxicity.

PBMC c

Positive

ytotoxicity

Negative

Patients (n = 35) 10 25

Male 8 14

Median age (yr) 53 52

Prior treatmentNone 50% 36%

Biotherapy alone 10% 8%

Chemotherapy ± biotherapy 40% 56%

Tumor stage and site(s):AJCC� stage III I 5

AJCC stage IV 9 (90%) 20 (80%)

Adjuvant 6 6Soft-tissue only or one visceral organ 8 13

Two or more visceral organs 1 1

Major objective response 2 0Median time to progression (mo) 18 3

Median survival (mo)

All patients 28+ 16Stage IV patients 28+ 14

DTH

Hypoergy to foreign antigens 40% 68%

Positive reaction to 10� AT 30% 20%

Larger than average reaction to vaccine 70%

a AJCC, American Joint Committee on Cancer.

38%

With regard to laboratory tests that might also guide such

determination, none to-date has proven reliable, in part because

of the dearth of correbatable clinical responses and in part

because of the inability to demonstrate reproducible dose-

response relationships in a heterogeneous patient population.

Posttreatment laboratory results cannot be stratified prospec-

tively in Phase I or II studies because of time bias and other

factors that complicate any analysis of such data (35).

The serial detection of PBMC cytotoxicity against AT in

10 of 35 assessable patients (Tables 2 and 5) supported induc-

tion of a specific systemic immune response to intradermal

treatment, a primary end point of this study. There are no data

to permit a comparison of the frequency of induction of positive

cytotoxicity with that using a non-UVR-AT + DETOX vaccine

because no patient received such treatment in this pilot study.

Among the stage IV patients, almost uniformly with favorable

clinical pretreatment prognostic features, detection of PBMC

cytotoxicity against AT cells correlated significantly with sur-

vival (P = 0.009; Fig. 3). Different patterns of specificity were

observed, suggesting that the positive results were not likely to

be an artifact of the assays themselves (Table 3). Also, MHC

class I-restricted CD8+ I cells and not natural killer cells were

the primary mediators of this activity in the patients tested

I : � � H�1 �,

4---+ -�

PBMC Cytotoxicity Total Dead

Positive 9 3

Negative 20 12 P a 0.009

0 12 24 36 48

MONThS

Fig. 3 Kaplan-Meier survival curves for 29 assessable patients with

stage IV melanoma and similar pretreatment prognostic features dis-

tinguished by the presence or lack of detectable PBMC cytotoxicity

against autologous tumor.

(Table 4). The fall-off of detection of PBMC cytotoxicity con-

current with tumor progression in some patients suggested in-

duction of tolerance. It must be noted, however, that the periph-

eral blood is only one pharmacological compartment (36), and

this study did not account for efficient trafficking of cytotoxic I

cells to metastatic deposits and any resulting immunoselection

(37).

The induction of a DTH reaction to AT cells was not

observed in clinical responders. Subclinical reactivity could

have been evaluated further by punch biopsy of skin test sites;

this would require an informed consent from the patients. The

prolonged time to first clinically apparent DTH reaction to AT

cells (median of 8 courses) introduced a substantial “time bias”

precluding developing this test for prognostic purposes (35).

Among all DTH assays performed, only above average DTH

reaction to vaccine, largely a response to the adjuvant DETOX,

correlated favorably with the clinical course of disease. The

study was too small to evaluate the effect of concurrent detec-

tion of both PBMC cytotoxicity and DTH reactivity to AT cells,

observed in only three patients.

Demonstration of UVR-induced expression of B7. 1 on

human melanoma, as reported recently on murine melanoma

(21) as well as on CT-26 (colon carcinoma) and HCA (hepato-

cellular carcinoma) cell lines,4 could support using UVR to

study the antigen-presenting capability of tumor cells without

resorting to gene transfection. Besides the ongoing character-



626 Active Immunotherapy: Autologous Melanoma Cells. DETOX

ization of the positive cellular immune responses as presented

above and in cytokine elaboration experiments, future efforts

will be geared to testing cytokines that drive TH’ immune

responses (25) or that recruit dendritic cells or other professional

antigen-presenting cells to the vaccine injection site (15).

Suto and Srivastava (38) has been able to extract from

autobogous tumor cells heat shock protein-96, a chaperon that

complexes with the cells’ unique repertoire of soluble immuno-

genic peptides (HSPPC-96). He has shown that HSPPC-96 can

elicit antigen-specific CILs. Treatment with HSPPC-96 could

provide an array of autobogous tumor-specific peptide targets for

CIL activation without the need to characterize each peptide or

to exclude patients based on human leukocyte antigen pheno-

type. This may prove to be a far more exploitable and versatile

strategy for immunotherapy than trying to convert an autologous

tumor cell that has already eluded host immune surveillance into

a better antigen-presenting cell. Autologous tumor HSPPC-96

could be used in the future to prime dendritic cells activated or

expanded in vitro with cytokines such as granubocyte/macro-

phage colony-stimulating factor, interleukin 4, or flt3 bigand.

No active immunotherapy program will be optimized, how-

ever, until methods are developed in individual patients to

circumvent immunoselection (6), local secretion of tolerizing

molecules by tumors (7-10), and biophysicab barriers that make

metastases sanctuary sites from the immune system ( I I).

ACKNOWLEDGMENTS

We thank Dr. Margaret Kripke for helpful suggestions and thought-

ful review of the manuscript.

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1998;4:619-627. Clin Cancer Res O Eton, D D Kharkevitch, M A Gianan, et al. melanoma.whole melanoma cells plus DETOX in patients with metastatic Active immunotherapy with ultraviolet B-irradiated autologous

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