A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.

Abstract

The objectives of this study were to assess the toxicity and immunological response induced by the intradermal (i.d) administration of tumor lysate-pulsed dendritic cells (DCs).
Patients with stage IV solid malignancies were treated in cohorts that received 10(6), 10(7), and 10(8) DCs i.d. every 2 weeks for three vaccines. Each vaccine was composed of a mixture of half DCs pulsed with autologous tumor lysate and the other half with keyhole limpet hemocyanin (KLH). Peripheral blood mononuclear cells (PBMCs) harvested 1 month after the last immunization was compared with pretreatment PBMCs for immunological response. Delayed-type hypersensitivity reactivity to tumor antigen and KLH was also assessed.
Fourteen patients received all three vaccines and were evaluable for toxicity and/or immunological monitoring. There were no grade 3 or 4 toxicities associated with the vaccines or major evidence of autoimmunity. Local accumulation of CD4(+) and CD8(+) T cells were found at the vaccination sites. There was a significant proliferative response of PBMCs to KLH induced by the vaccine. In 5 of 6 patients, the vaccine resulted in increased IFN-gamma production by PBMCs to KLH in an ELISPOT assay. Using the same assay, 3 of 7 patients' PBMCs displayed increased IFN-gamma production in response to autologous tumor lysate. One patient with melanoma also was observed to have an increased frequency of MART-1- and gp100-reactive CD8(+) T cells after vaccination. By delayed-type hypersensitivity testing, 8 of 9 and 4 of 10 patients demonstrated reactivity to KLH and autologous tumor, respectively. Two patients with melanoma experienced a partial and a minor response, respectively.
The administration of tumor lysate-pulsed DCs is nontoxic and capable of inducing immunological response to tumor antigen. Additional studies are necessary to improve tumor rejection responses.

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A Phase I Trial of Tumor Lysate-pulsed Dendritic Cells in the
Treatment of Advanced Cancer1
Alfred E. Chang,2Bruce G. Redman,
Joel R. Whitfield, Brian J. Nickoloff,
Thomas M. Braun, Peter P. Lee, James D. Geiger,
and James J. Mule ´
Departments of Surgery [A. E. C., J. R. W., J. D. G., J. J. M.], Internal
Medicine [B. G. R., J. J. M.], and Biostatistics [T. M. B.], University
of Michigan, Ann Arbor, Michigan 48109-0932; Department of
Medicine, Stanford University, Stanford, California 94305 [P. P. L.];
and Department of Pathology, Loyola University, Maywood, Illinois
60153 [B. J. N.]
ABSTRACT
Purpose: The objectives of this study were to assess the
toxicity and immunological response induced by the intra-
dermal (i.d) administration of tumor lysate-pulsed dendritic
cells (DCs).
Experimental Design: Patients with stage IV solid ma-
lignancies were treated in cohorts that received 106, 107, and
108DCs i.d. every 2 weeks for three vaccines. Each vaccine
was composed of a mixture of half DCs pulsed with autolo-
gous tumor lysate and the other half with keyhole limpet
hemocyanin (KLH). Peripheral blood mononuclear cells
(PBMCs) harvested 1 month after the last immunization was
compared with pretreatment PBMCs for immunological re-
sponse. Delayed-type hypersensitivity reactivity to tumor
antigen and KLH was also assessed.
Results: Fourteen patients received all three vaccines
and were evaluable for toxicity and/or immunological mon-
itoring. There were no grade 3 or 4 toxicities associated with
the vaccines or major evidence of autoimmunity. Local ac-
cumulation of CD4?and CD8?T cells were found at the
vaccination sites. There was a significant proliferative re-
sponse of PBMCs to KLH induced by the vaccine. In 5 of 6
patients, the vaccine resulted in increased IFN-? production
by PBMCs to KLH in an ELISPOT assay. Using the same
assay, 3 of 7 patients’ PBMCs displayed increased IFN-?
production in response to autologous tumor lysate. One
patient with melanoma also was observed to have an in-
creased frequency of MART-1- and gp100-reactive CD8?T
cells after vaccination. By delayed-type hypersensitivity test-
ing, 8 of 9 and 4 of 10 patients demonstrated reactivity to
KLH and autologous tumor, respectively. Two patients with
melanoma experienced a partial and a minor response,
respectively.
Conclusion: The administration of tumor lysate-pulsed
DCs is nontoxic and capable of inducing immunological
response to tumor antigen. Additional studies are necessary
to improve tumor rejection responses.
INTRODUCTION
DCs3are highly potent antigen-presenting cells of bone
marrow origin that are integral in the stimulation of primary and
secondary T- and B-cell responses. Preclinical studies have
demonstrated that DCs are preferentially responsible for the
sensitization of naive T cells in their first exposure to antigen (1,
2). The ability to generate DCs ex vivo along with pulsing with
antigen has enabled investigators to generate reagents that can
be used for immunization purposes. An important advantage of
using DCs as a mode of tumor vaccination is its ability to induce
immunity to poorly immunogenic tumors (i.e., human malig-
nancies), thus potentially bypassing inherent deficiencies in
antigen presentation within the host (3).
Our preclinical models demonstrated that tumor lysate-
pulsed DCs were capable of mediating the regression of micro-
metastatic disease upon s.c. administration (4). The antitumor
response by tumor lysate-pulsed DCs comprised both CD4?and
CD8?T cells. One advantage of using tumor lysates to pulse
DCs is the multiple antigens that are included in such a prepa-
ration. Use of a tumor lysate may allow for the sensitization of
T cells to a variety of antigens that may be heterogeneously
expressed on growing tumors and is particularly relevant to
those human cancers which to date do not have molecularly
defined tumor-associated antigens.
On the basis of our preclinical animal studies, we have now
completed a Phase I clinical trial of the intradermal administra-
tion of autologous tumor lysate-pulsed DCs in patients with
advanced solid malignancies. Cohorts of patients received 106,
107, and 108DCs pulsed with tumor lysate and KLH every 2
weeks for a total of three injections. Besides toxicity, secondary
end points included immune monitoring of PBMCs for reactiv-
ity to autologous tumor and KLH.
Received 7/25/01; revised 12/27/01; accepted 12/27/01.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1This work was supported in part by NIH Grants PO1 CA59327, R29
CA77471, and M01 RR00042, as well as grants from the Gillson
Longenbaugh Foundation and the Walther Foundation.
2To whom requests for reprints should be addressed, at 3302 Cancer
Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0932.
Phone: (734) 936-4392; Fax: (734) 647-9647; E-mail: aechang@
umich.edu.
3The abbreviations used are: DC, dendritic cell; KLH, keyhole limpet
hemocyanin; PBMC, peripheral blood mononuclear cell; DTH, delayed-
type hypersensitivity; i.d., intradermally; GM-CSF, granulocyte/macro-
phage-colony stimulating factor; IL, interleukin; PE, phycoerythrin;
FACS, fluorescence-activated cell sorter; mAb, monoclonal antibody;
ELISPOT, enzyme-linked immunospot; TSH, thyroid-stimulating hor-
mone; ANA, anti-nuclear antibody; SI, stimulation index.
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PATIENTS AND METHODS
Patients and Study Design
Patient Selection.
cies were eligible if they had a performance status of Eastern
Cooperative Oncology Group 0 or 1 and had measurable or
evaluable disease. Additional eligibility requirements consisted
of absence of brain metastases by computed tomography or
magnetic resonance imaging and absence of anergy to common
recall antigens. Anergy was initially assessed with the Multitest
CMI kit (Merieux Institute, Inc., Miami, FL), which measures
DTH reactivity to seven common recall antigens. However,
midway during the trial, these kits were no longer manufactured,
and an alternate method of DTH testing to 4 antigens (Candida
albicans, mumps, PPD tuberculin, and tetanus toxoid) was
adopted. Reactivity to any one of these recall antigens (indura-
tion ?5 mm) was considered evidence for lack of anergy.
Study Design.
The primary end points for the Phase I
study was to assess feasibility and toxicity of the DC vaccina-
tion regimen. Secondary end points were to assess immune
responses induced by the vaccination procedure to the marker
antigen, KLH, and to autologous tumor. There were three co-
horts of patients who received 106, 107, and 108DCs pulsed
with KLH and autologous tumor lysate. Each cohort of patients
received the DCs i.d. near an inguinal or axillary nodal region
deemed clinically free of disease. A total of three vaccinations
were administered at intervals of 2 weeks in the outpatient clinic
of the General Clinical Research Center. One month after the
third immunization, patients underwent leukapheresis for im-
mune monitoring as well as radiological assessment of tumor
status. If the tumor status was deemed to be stable or responding
to therapy, an additional three vaccines were given as before.
Conventional criteria for tumor response was used. A partial
response was ?50% decrease in the perpendicular diameter of
all measurable lesions without progression of evaluable disease.
Progressive disease was ?25% increase in the perpendicular
diameter of any measurable disease, appearance of a new lesion,
or worsening of evaluable disease. This protocol was approved
by the University of Michigan Institutional Review Board (IRB
97-064; UMCC 9702) and by the Food and Drug Administration
under IND BB6958. Before protocol entry, each subject pro-
vided written informed consent.
Patients with stage IV solid malignan-
Vaccine Toxicity Grading System
For systemic toxicities, the National Cancer Institute Com-
mon Toxicity scale was used. For local vaccine toxicities, the
following scale was used: grade I, erythema and induration ?20
mm; grade II, erythema and induration ?20 mm without ulcer-
ation; grade III, ulceration or painful adenopathy; and grade IV,
permanent dysfunction related to local toxicity.
Tumor Cell Harvest and Cryopreservation
Tumors were harvested surgically for palliative or curative
intent and cryopreserved. Tumors were kept sterile on ice and
transported from the operating room to the laboratory. A single
cell suspension was made by a combination of mechanical and
enzyme dispersion techniques as described previously (5). The
tumor cells were cryopreserved in 90% human AB serum plus
10% DMSO at ?178°C in a liquid nitrogen freezer. Each
freezing vial contained 2–4 ? 107tumor cells.
Leukapheresis and Cryopreservation of PBMCs
In accordance with the University of Michigan Blood Bank
guidelines, protocol patients underwent a 4-h leukapheresis on a
COBE spectrum apheresis system to ensure adequate numbers
of PBMCs for DC culture and for immune monitoring. PBMCs
were obtained by taking the apheresis product, diluting it 4-fold
in HBSS (Life Technologies, Inc., Grand Island, NY), and
overlaying it on Ficoll-Hypaque gradients. The cells were then
centrifuged at 900 ? g for 30 min at room temperature. The
interface representing the PBMCs were then collected and
washed in HBSS twice to reduce platelets. Aliquots of PBMCs
were then cryopreserved in 70% human AB serum 20% X-
VIVO 15 and 10% DMSO for future use in cryopreservation
bags (Baxter Corp., Deerfield, IL) or cryovials.
Vaccination Preparation
DC cultures and antigen pulsing were performed in the
Human Applications Laboratory of the General Clinical Re-
search Center, which is a facility that operates under good
manufacturing procedures. The vaccine for each patient was
prepared from the fresh leukapheresis sample. Subsequent vac-
cines were prepared from cryopreserved PBMCs obtained from
the pretreatment leukapheresis. PBMCs were resuspended in
serum-free X-VIVO 15 medium (BioWhittaker, Walkerville,
MD) at 1 ? 107cells/ml for a total volume of 30 ml in 225-cm2
flasks. The cells were allowed to adhere for 2 h at 37°C in 5%
CO2, and the nonadherent cells were removed after gentle
rocking of the flasks and aspiration of the medium. Immediate
replacement of 30 ml of X-VIVO 15 medium containing GM-
CSF (100 ?g/ml, Schering-Plough, Kenilworth, NJ) and IL-4
(50 ?g/ml; Schering-Plough) was completed, and the cells were
incubated for 6 days at 37°C, 5% CO2before pulsing with tumor
lysate or KLH.
The adherent DCs were harvested from the flasks using 10
ml of EDTA (3 mM) for each flask and allowed to incubate for
10 min. The detached DCs were harvested, washed, and resus-
pended at 1 ? 106cells/ml in fresh X-VIVO 15 medium
containing GM-CSF and IL-4. Ten ml of the cell suspension
were placed in 75-cm2flasks (107DCs/flask) for pulsating with
tumor lysate or KLH. Single cell suspensions of tumor freshly
removed or cultured short-term (?2 weeks to ensure removal of
contaminating enzymes used for disaggregation) were snap
freeze-thawed three times in rapid succession, irradiated at
10,000 cGy, and stored in liquid nitrogen for later use. Tumor
lysate suspension was added to DCs at 1:1 cell equivalent ratio.
Specifically, a volume of tumor lysate equal to 107tumor cells
was added to each flask and incubated for 18 h at 37°C, 5%
CO2. KLH pulsation was performed in separate flasks of DCs.
A volume of 300 ?l of KLH stock solution diluted in PBS (50
?g/ml; Calbiochem, San Diego, CA) was added to each flask
and incubated for 18 h.
After incubation, the tumor lysate-pulsed and KLH-pulsed
DCs were harvested and counted. For each vaccine dose, 50%
was composed of tumor lysate-pulsed DCs admixed with 50%
of KLH-pulsed DCs. The DC suspension was adjusted to a total
1022 DC Vaccination of Advanced Cancer

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volume of 0.5 ml of PBS. For the 108DC dose, the cell
suspension was divided into five separate syringes each con-
taining 0.5 ml for intradermal administration.
Flow Cytometric Analysis of DCs
An aliquot of the vaccine preparation was retained to
examine the expression of extracellular markers. The following
monoclonal antibodies were purchased from Caltag Laborato-
ries (Burlingame, CA): FITC-conjugated CD45, CD83, CD86,
mouse IgG1, and mouse IgG2b; PE-conjugated CD14, mouse
IgG1, and mouse IgG2a. PE-conjugated CD40 and HLA-DR
were purchased from PharMingen (San Diego, CA). Vaccine
cells were washed with FACS buffer (PBS ? 2% fetal bovine
serum, 0.1% sodium azide) and counted. One million cells in
100 ?l were added to culture tubes containing 1 ?g of each
labeled antibody. Cells were incubated on ice for 40 min,
washed two times with FACS buffer, and then suspended in
PBS ? 1% paraformaldehyde and stored at 4°C before FACS
analysis.
Immune Monitoring of PBMCs
PBMCs were harvested pretreatment at the time of leuka-
pheresis for DC generation and 1 month after the third vacci-
nation when tumor response assessment was determined. All of
the assays were done in batch on cryopreserved PBMCs. Cells
were used soon after thawing. Cell viabilities ranged from 67 to
98% between patients, but for a given patient, viabilities be-
tween pre- and posttreatment samples were within 15%. The
following assays were performed by either the Immune Moni-
toring Core of the University of Michigan Comprehensive Can-
cer Center or at Stanford University.
Proliferation Assay.
Cryopreserved
thawed, washed, and suspended in X-VIVO 15 medium supple-
mented with 1% human AB serum (BioWhittaker), 2 mM glu-
tamine, 100 units of penicillin, 10 mg/ml streptomycin, and 50
?M 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO).
Viability was assessed by trypan blue exclusion, and cell con-
centrations were adjusted to 6 ? 106/ml. Cells were added to
96-well, round-bottomed plates (Falcon-BD, Franklin Lakes,
NJ) in 100-?l volumes and incubated in a final volume of 200
?l with either medium alone, KLH (40 ?g/ml), or tumor lysate
(prepared to deliver lysate at tumor cell equivalence) for a total
of 6 days at 37°C, 5% CO2. Phytohemagglutinin (Sigma Chem-
ical Co.) was added to some of the wells as a positive control on
day 3. The cultures were pulsed with 1 ?Ci/well of [3H]thymi-
dine (ICN, Costa Mesa, CA) on day 5 and incubated overnight
before harvest onto glass-fiber filter plates (Millipore, Bedford,
MA). Data were collected on a TopCount NXT scintillation
counter (Meriden, CT). A SI was calculated:
PBMCswere
SI ?Avg. cpm of antigen-stimulated culture
Avg. cpm of unstimulated culture
ELISPOT Assay.
plates (Whatman, Clifton, NJ) were prepared by adding 75
?l/well of anti-IFN-? monoclonal capture antibody (Pierce-
Endogen, Rockford, IL) adjusted at 4 ?g/ml in sterile 0.1 M
carbonate buffer (pH 9.5) and incubated at 4°C overnight.
The day of assay, the plates were washed twice with sterile
One day prior to assay, unfiltered
PBS (Mediatech, Herndon, VA) and then blocked for ?1 h
with X-VIVO 15 medium with 10% AB serum. PBMCs were
prepared as above and adjusted to 1 ? 107/ml. One hundred
?l of PBMCs were added to each well and incubated with
antigen as above. Negative controls for the assay were un-
stimulated PBMCs. Background counts for these samples
were quite low and were subtracted from the counts generated
from stimulated cultures. Positive controls were stimulated with
phytohemagglutinin. Cultures were incubated undisturbed at
37°C, 5% CO2for 24 h.
After 24 h, cells were removed, and the plates were washed
three times with PBS and then three times with Tris-buffered
saline (0.05 M, pH 7.5) TBS-Tween 20 (TBS-T). Biotinylated
secondary antibody (Pierce-Endogen; 2 ?g/ml) in TBS-T plus
10% fetal bovine serum (Life Technologies, Inc.) was added,
and the plates were incubated at room temperature for 2 h,
followed by six TBS-T washes. Streptavidin-alkaline phospha-
tase (Sigma Chemical Co.) was added and incubated for 1 h at
room temperature, followed by eight TBS-T washes. Plates
were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-
indolylphosphate substrate (Sigma Chemical Co.) for 15–45
min and then rinsed extensively with deionized water and al-
lowed to dry. Quantitative analysis of positive spots was per-
formed on an ImmunoSpot Series 1 Analyzer (Cellular Tech-
nology Ltd., Cleveland, OH).
MHC/Peptide Tetramer Analysis.
peptide tetramers was described in detail elsewhere (6). Plas-
mids encoding the A2-BSP fusion protein and human ?2-
microglobulin were kind gifts of Dr. John Altman (Emory
University, Atlanta, GA). These proteins were expressed in
Escherichia coli and refolded in vitro with the specific peptide
ligand. The properly folded MHC-peptide complexes were ex-
tensively purified using fast protein liquid chromatography and
anion exchange and biotinylated on a single lysine within the
BSP using the BirA enzyme (Avidity, Denver, CO). Tetramers
were produced by mixing the biotinylated MHC-peptide com-
plexes with either PE-conjugated avidin or APC-conjugated
avidin (Molecular Probes) at a molar ratio of 4:1. Tetramers
were validated by staining against a CTL line or clone specific
for HLA-A2 in association with the peptide of interest. Speci-
ficity was demonstrated by the lack of staining of irrelevant
CTLs. By titrating positive CTLs into PBMCs from normal
controls, we established our limit of detection to be as low as
0.01% of CD8?cells. Each tetramer reagent was titered indi-
vidually and used at the optimum concentration, generally
10–50 ?g/ml.
Three-color FACS analyses were performed using a Bec-
ton Dickinson FACScan (Mountain View, CA). For phenotypic
analysis of antigen-specific T-cell populations, 10-color FACS
analysis was performed on a 12-parameter FACS instrument
developed by the Stanford Shared FACS Facility. Data were
compensated, analyzed, and presented using FlowJo (Tree Star,
Inc., San Carlos, CA). Except as listed, all fluorescein- or
PE-conjugated as well as unconjugated monoclonal antibodies
were obtained from PharMingen (San Diego, CA). Anti-CD8-
FITC, anti-CD4-Cy5PE, anti-CD13-Cy5PE, and anti-CD19-
Cy5PE antibodies were purchased from Caltag (South San Fran-
cisco, CA).
Production of MHC/
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DTH Testing
In addition to in vitro immune monitoring, we assessed
patients for in vivo immune reactivity to KLH and autologous
tumor by DTH testing. For KLH reactivity, patients were given
intradermal inoculations of 2, 20, and 200 ?g of KLH in 0.2-ml
volumes of PBS. Induration was measured 48 h later in two
perpendicular diameters. For autologous tumor reactivity, pa-
tients were assessed before treatment and 1 month after treat-
ment with irradiated autologous tumor cells (6,000 cGy) at 104,
105, and 106doses i.d. Induration was measured in a similar
fashion as KLH. Positive DTH reactions were scored if the
average perpendicular measurements exceeded 5 mm.
Light Microscopic and Immunohistochemical Analysis of
Vaccine Sites
In select patients, vaccine sites and adjacent skin were
excised 1 week after the third vaccine for histological analysis.
Besides routine light microscopic assessment of formalin-fixed,
paraffin-embedded sections of either tissue excised from the
vaccine site or adjacent non-injected normal skin, stained with
H&E, immunopathological examination was also performed.
Serial sections of the paraffin blocks were cut onto glass slides
and immunostained using a highly sensitive avidin-biotin im-
munoperoxidase technique with diaminobenzidine to produce a
positive brown reaction product. For detection of some antigens,
tissue sections were microwaved in PBS for antigen retrieval
before addition of primary antibodies. The following antibodies
were used to visualize DCs: LN3 mAb (Dako, Carpinteria, CA)
was used to detect HLA-DR and rabbit antisera was used to
detect factor XIIIa (Calbiochem-Novabiochem, San Diego,
CA). DCs were also distinguished by their morphology. Rabbit
anti-S100 was used to detect melanoma cells (Dako) and anti-
MAC387 mAb used to detect macrophages. Lymphocytes were
detected by using an anti-CD3 pan T cell mAb, as well as
anti-CD4 and anti-CD8 mAbs (Becton Dickinson, Mountain
View, CA).
RESULTS
Patient Characteristics.
all three vaccinations and had adequate follow-up to monitor
toxicity, immune response, or tumor response. An additional 10
patients were entered into the study who progressed and did not
complete all of the protocol end points. The study required
autologous tumor harvest, DC generation, three vaccinations,
and a follow-up leukapheresis 1 month after the last vaccine. In
general, these study requirements involved a period of 3.5–4
months. Among the 10 inevaluable patients, 1 patient with
colorectal cancer did not receive any vaccines because of the
onset of brain metastases after tumor harvest. The remaining
patients (3 with breast tumors and 6 with melanoma) progressed
during the course of therapy and became ineligible. The
characteristics of the 14 evaluable patients are summarized in
Table 1. There were 5, 6, and 3 patients in groups where 106,
107, and 108DCs were administered, respectively. The histo-
logical diagnoses included melanoma (n ? 11), colorectal can-
cer (n ? 2), and 1 patient with neuroblastoma. The median age
of the patients was 53 years (range, 36–73 years).
A total of 14 patients received
Table 1
Patient characteristics, therapy, and response
Patient
no.
1
Age
63
Sex
F
Diagnosis
Melanoma
Sites of disease
Retroperitoneal LNa
Prior systemic
treatments
None
DC dose
106
Response
Minor response after 3 vaccines, partial
response after 3 more vaccines; CNS
relapse after 8th vaccine
Progressed2 65M Melanoma Liver, axillary, and
retroperitoneal LNs
Abdominal LNs
Lung, liver, inguinal
LNs, subcutis
Lung, adrenal, axillary
LNs
Liver
Lung, liver, subcutis
None106
3
4
55
40
M
F
Melanoma
Melanoma
None
IFN-?
106
106
Progressed
Progressed
5 66M Melanoma None 106
Progressed
6
7
50
69
M
F
Colorectal cancer
Melanoma
Chemotherapy
None
107
107
Progressed
Stable after 3 vaccines, progressed after
6th vaccine
Progressed
Stable after 6 vaccines, minor response
after 9th vaccine, progressed after
12th vaccine
Progressed
8
9
73
41
M
M
Melanoma
Melanoma
Lung
Retroperitoneal and
pelvic LNs
None
IFN-?
107
107
1040M MelanomaMesenteric LNsIFN-?
IL-2
Chemotherapy
107
1147M Colorectal cancerLiver, retroperitoneal LNs 107
Stable after 3 vaccines, progressed after
6th vaccine
Progressed
Stable after 3 vaccines, progressed after
6th vaccine
Stable after 3 vaccines, progressed after
6th vaccine
12
13
49
66
M
F
Melanoma
Melanoma
Lung, axillary LNs
Lung, breast
IFN-?
None
108
108
1436FNeuroblastoma Lung, thoracoabdominal
wall mass
Chemotherapy 108b
aLN, lymph node; CNS, central nervous system.
bFirst 3 vaccines were at 107/dose; subsequent vaccines at 108/dose.
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DC Culture Results and Phenotype.
tured in serum-free medium containing IL-4 and GM-CSF for 6
days after plastic adherence and removal of nonadherent cells.
At the end of that period, the yield of cells compared with the
starting PBMC population was a mean (SE) of 4.5 ? 0.6%
(range, 2.2–10.4%; n ? 17 cultures). The cells were readjusted
in cell numbers and subsequently pulsed with KLH or autolo-
gous tumor lysate. The yield of cells after antigen pulsation was
a mean (SE) of 70 ? 7.4% (range, 27–100%; n ? 12 cultures).
FACS analysis of the DC phenotypic profile from a represent-
ative culture is illustrated in Fig. 1. Among 5 patients undergo-
ing 15 DC cultures, the mean (SE) percentage of positively
stained cells for various markers was: CD14, 18 ? 3; CD45,
59 ? 5; CD40, 12 ? 4; CD83, 17 ? 4; CD86, 68 ? 6; and
HLA-DR, 62 ? 5. The expression of CD83 was low, indicating
the relative immature status of the DCs.
Toxicity.
There were no significant toxicities associated
with the vaccine administrations. Two of 5 patients who re-
ceived a dose of 106DCs experienced grade 1 local toxicity at
the vaccine site. Grade 2 local toxicity was observed in 1 patient
from each cohort that received 107and 108DCs, respectively.
No patients experienced local tumor growth at the site of the
vaccine injection.
A panel of autoimmune parameters was used before and 1
month after the completion of three vaccinations. Table 2 sum-
marizes the results of the tests. Overall, there was no evidence
of autoimmunity as assessed by these tests. One patient (no. 5)
PBMCs were cul-
with the elevated TSH value after vaccination had an elevated
TSH value before vaccination. One patient (no. 9) with a pos-
itive ANA after vaccination had a negative assessment before
treatment; however, the posttreatment ANA level had a low titer
(1:80). Interestingly, patient 10 experienced the onset of vitiligo
after the vaccinations and demonstrated normal posttreatment
autoimmune parameters and no evidence of melanoma regres-
sion.
Characterization of Vaccine Sites.
tients, the vaccine sites were excised after the last vaccination,
and the intradermal reaction to the injection of lysate-pulsed
In two different pa-
Fig. 1 Two-color FACS analysis of tumor lysate-pulsed DCs. Upper two left panels, background antibody staining controls. Upper right panel,
scatterplot and gated areas (ovals) of cells evaluated. The DCs were positive for HLA-DR, CD86, CD40, and CD45. Markers for monocytes (CD14) and
mature DC (CD83) were low.
Table 2
Autoimmune parameters after DC vaccination
DC dose
106
107
108
Total
No. of patients with abnormal values/Total no. of
patientsa
RF
0/4
0/2
0/3
0/9
ANA
0/4
1/2c
0/3
1/9
DNA binding
0/4
0/2
0/3
0/9
CH50
0/4
0/2
0/3
0/9
T4
0/4
0/2
0/3
0/9
TSH
1/4b
0/2
0/3
1/9
aAutoimmune parameters consist of: rheumatoid factor (RF);
ANA; DNA binding; total complement (CH50); total T4 or free T4; and
TSH.
bOne patient with elevated TSH level (10.38 uu/ml; nl. range,
0.30–6.50 uu/ml).
cOne patient had a positive ANA with a titer of 1:80.
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Clinical Cancer Research