Download Identification of Immunogenic Human Melanoma Antigens in a

(CANCER
RESEARCH
52, 5948-5953,
November
1, I992j
Identification of Immunogenic Human Melanoma Antigens in a Polyvalent
Melanoma Vaccine'
Jean-Claude
Bystryn,2
Milagros
Henn, Joseph
Li, and Susan Shroba
Kaplan Comprehensive Cancer Center and the Department ofDermatology,
New York University School ofMedicine, New York, New York 10016
ABSTRACT
An essential element in the developmentof effectivevaccines against
human
malignant
melanoma
is the identification
of antigens
which are
relevant for vaccine construction as evidenced by their ability to stimu
late antimelanoma immune responses in humans. In this study, we
identified immunogenic melanoma antigens using as probes antibodies
induced in patients immunized with a vaccine which contains a broad
range of potential
immunogens.
By immunoprecipitation/sodium
dode
cyl sulfate-polyacrylamide gel electrophoresis analysis of detergent ly
sates of radioiodinated
melanoma
cells, we found that 17 (65%) of 26
patients sequentially immunized with a polyvalent melanoma antigen
vaccine
developed
antibodies
to one or more melanoma
cell surface
antigens with approximate molecular weights of38,000—43,000,
75,000,
110,000, 150,000, and 210,000. The immunodominant antigens which
most frequently stimulated antibody responses were the Mr 110,000
antigen
However, there is a need to develop more potent vaccines,
because the current generation fails to augment immunity to
melanoma or to prevent the progression of this cancer in many
patients. An essential element in this endeavor is the identifi
cation of melanoma antigens which are relevant for vaccine
construction, as evidenced by their ability to stimulate antimel
anoma immune responses in humans and by their expression on
a broad spectrum of different melanomas. The purpose of this
study was to identify such antigens, using as probes antibodies
elicited by active immunization of patients with a vaccine for
mulation containing a broad range of potential immunogens.
followed by the Mr 210,000 and 38,000—43,000 antigens,
which
induced antibody responses in 62%, 27%, and 19% of patients, respec
MATERIALS
AND METhODS
Melanoma Vaccine. A soluble, partially purified, polyvalent, mela
noma antigen vaccinewas prepared from the material shed into culture
medium by four lines of melanoma cells, as previously described (7).
tively. These three antigens were commonly expressed on different mel
anomas but rarely on nonmelanoma
cells and are unrelated to class I or
II human leukocyte antigens or to the previously described p97 or Mr
240,000 proteoglycan melanoma-associated antigens. Thus, these three
antigens are attractive candidates for the construction of melanoma
vaccines, because they are immunogenic in humans and are preferen
flatly expressed on melanomas.
The cells were adapted to long term growth in serum-free medium to
prevent contamination of the vaccine with fetal bovine serum proteins.
For vaccine production, the shed material was collected, concentrated
by vacuum dialysis, pooled, treated with 0.5% NP-403 detergent to
break up aggregates, and ultracentrifuged at 100,000 X g for 90 mm to
remove transplantation alloantigens. The supernatant was concentrated
by vacuum dialysis, filter sterilized, adjusted to a protein concentration
of200 @tg/ml,
bound to alum as an adjuvant, dispensed into sterile glass
INTRODUCTION
There is increasing interest in the development of vaccines to
treat, and possibly to prevent, malignant melanoma. The pro
gression of melanoma depends in part on host immune re
sponses
to melanoma
(1), so that specific stimulation
of anti
melanoma immune responses with melanoma antigen vaccines
may increase resistance to melanoma. The most convincing
evidence that this concept is correct is that immunization to
melanoma vaccines can prevent this cancer in syngeneic mice
(2—4).The protection conferred is specific (2), i.e., mice immu
nized with control vaccine are not protected against melanoma
and melanoma vaccine-immunized mice are not protected
against unrelated syngeneic tumors, indicating that the protec
tion is mediated by immune mechanisms.
Based on these observations a number of first-generation
melanoma vaccines have been developed. These have proven to
be safe to use and are capable of inducing immune responses to
melanoma
in some individuals
(5—9).The clinical effectiveness
of melanoma vaccines is still not established, although several
studies suggest that they can slow the progression
of this cancer
vials, and stored at —4°C
until used. The biochemical and antigenic
properties ofthe vaccine have been published (7). The vaccine is free of
fetal calf serum proteins and of human immunodeficiency virus and
hepatitis virus. All lots were tested for aerobic and anaerobic bacteria,
fungi, and pyrogens and were injected into mice and guinea pigs for
general safety tests prior to use.
Immunization.
The vaccine was used to treat sequentially
26 patients
with surgically resected stage II (regional) malignant melanoma. Eligi
biity criteria included histologically confirmed regional metastases;
adequate bone marrow, renal, and liver function; and the absence of
residual
metastatic
disease as documented
by physical examination
and
normal computerized tomography scans of brain, chest, abdomen, and
pelvis. The patients were older than 18 years and were not receiving
steroids
or other potentially
immunosuppressive
medications.
All pa
tients had intact cellular immunity, as evidenced by skin test reactivity
to at least one of a standard panel of recall antigens (purified protein
derivative intermediate strength, mumps, candida, and str cytokinase
streptodornase) or by the ability to be sensitized to dinitrochloroben
zene. All patients signed written informed consent forms prior to treat
ment, as approved by the New York University Medical Center
Institutional Review Board.
Patients were immunized with 40 @g
of vaccine bound to alum as an
adjuvant. This dose was divided into four equal aliquots and adminis
tered intradermally, in 0.5 ml of saline, into each extremity every 3
or cause the regression of established metastasis in some pa
tients (7—14).
weeks for four cycles.
Sera and Monoclonal Antibodies. Serum was collected from each
Received 4/8/92; accepted 8/21/92.
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 accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was supported
in part
by USPHS
Research
Grants
patient immediately prior to the first and 1 week after the fourth vaccine
immunization.
Murine
MoAb
96.5 to the p97 MAA was kindly pro
vided by Dr. K. E. Hellstrom (University ofWashington, Seattle, WA),
and MoAb 9.2.27 to a Mr 240,000 proteoglycan MAA was provided by
P3OCA16087,
AR39749, FD-R-000632, and A27663 and by grants from the Rose M. Badgeley
ResiduaryTrust.
2 To
whom
requests
3 The abbreviations
for
reprints
should
be addressed,
at Department
of Derma
tology, New York University School of Medicine, 560 First Aye, New York, NY,
10016.
used are: NP-40,
Nonidet
P-40;
BSA,
bovine serum albu
mm; FBS, fetal bovine serum MAA, melanoma-associated antigen; MoAb,
monoclonal antibody, PBS, phosphate-buffered saline; SDS, sodium dodecyl sal
fate; PAGE, polyacrylamide gel electrophoresis; HLA, human leukocyte antigen.
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IMMUNOGENIC
MELANOMAANTIGENS
Dr. R. Reisfeld (Scripps Clinic, La Jolla, CA). Murine MoAb W6/32 to
incubated in the presence of competing proteins with that in a control
class I HLA antigens and MoAb L243 to class II HLA antigens were
sample incubated with normal saline.
prepared
from the corresponding
hybridomas
obtained
Immunodepletion Experiments. These were performed by incubat
from the Amer
icaii Type Culture Collection (Rockville, MD).
Cells. Antibody studies were conducted using as target SK-Mel-28
ing 0.12 ml (containing 1—2x l0@ cpm and approximately 180 @g
protein) of detergent lysate of radioiodinated melanoma (SK-Mel-28)
cells (kindly provided by Dr. J. Fogh, Sloan-Kettering Institute, Rye,
NY), one of the human melanoma cell lines used to construct the
melanoma vaccine. Specificity analysis was conducted using a panel of
control cells consisting of allogeneic pigmented (SK-Mel-23 and SK
Mel-30) and nonpigmented (SK-Mel-28, M14, and M20) human mel
anoma cells; xenogeneic melanoma cells (hamster HM54 and murine
B16 melanoma); normal allogeneic cells (melanocytes and peripheral
blood lymphocytes (HLI and HL2)J; allogeneic unrelated tumors [neu
roblastoma (IMR 32), glioma (u87MG), colon carcinoma (SK-Co-l
and SW620), lung carcinoma (A549), rhabdomyosarcoma (RD), eryth
roleukemia (K562), and breast carcinoma (T47D)J; and xenogeneic
cells with 0.265 ml of monoclonal
baby hamster kidney cells (BHK). M20 and M14 cells were provided by
Dr. Donald Morton (University ofCalifornia, Los Angeles, Los Ange
les, CA), HM54 by Dr. G. Lipkin (New York University School of
Medicine, New York, NY), and u87MG, SW620, and T47D by Dr.
Roberta
Hayes
(New York
University
School
of Medicine).
Human
melanocytes (Clonetics, San Diego, CA) were cultured in melanocyte
growth medium containing basic fibroblast growth factor (1 ng/ml),
bovine pituitary
extract
(0.2%, v/v), insulin
(bovine,
5
@gIml), hydro
cortisone (0.5 @tg/ml),and phorbol myristate acetate (10 ng/ml). Nor
mal human peripheral blood lymphocytes were obtained and separated
by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) on the same day as
used. All other cells were obtained from the American Type Culture
Collection and were cultured in RPM! 1640 supplemented with 10%
FBS and antibiotics.
Radlolodination. Cell surface macromolecules on melanoma and
control cells were radioiodinated by the lactoperoxidase technique, as
previously described (15). The cells were labeled in suspension while in
logarithmic phase growth, washed thoroughly, and then lysed at 10@
cells/ml
in 0.5% NP-40 detergent
containing
0.025 M NaEDTA,
sodium azide, and 0.174 mg/ml phenylmethylsulfonyl
antibody
for 18 h at
gation at 12,000 X g for 20 mm. This procedure was repeated, and the
cleared supernatant was tested by immunoprecipitation/SDS-PAGE
analysis
for the presence
of residual
melanoma
antigens
defined
by
vaccine-induced antibodies, as described above.
RESULTS
Antibodies to Melanoma prior to Vaccine Treatment. The
presence of antibodies to melanoma in 26 patients with post
surgical stage II malignant melanoma was investigated by im
munoprecipitation and SDS-PAGE analysis. In this assay, the
presence of antimelanoma antibodies is evidenced by their abil
ity to precipitate radiolabeled, detergent-soluble, melanoma
macromolecules, and the antigen(s) to which they are directed
are identified by the location of labeled band(s) on the gel, as
illustrated in Fig. 1. Prior to vaccine treatment, antibodies to
melanoma were detected in 8 (31%) ofthe 26 patients (Table 1).
The results obtained for a representative patient with pretreat
ment antimelanoma antibodies to M@75,000, 150,000, and
210,000 antigens are illustrated in Fig. 1 (patient 124). Other
patients were found to have pre-existing antibodies to one or
more other antigens with approximate molecular weights of
38,000—43,000, 90,000, and 210,000. The antibodies were di
rected most often to the Mr 75,000 and the Mr 90,000 antigens,
0.02%
AB
fluoride. Insol
uble material was removed by centrifugation at 12,000 x g for 20 mm.
Total acid-insoluble radioactivity was measured by precipitation with
trichloroacetic
antimelanoma
4°C.Bound antigens were precipitated by incubation with 0.5 ml of
Protein A-Sepharose, diluted 1:2 in PBS, for 2 h at 4C and centrifu
AB
AB
AB
.
I..
acid.
AB
Immunopreclpltatlon/SDS-PAGE Analysis. The presence of anti
@
bodies to melanoma and the identity ofthe antigens to which they were
directed were measured by immunoprecipitation/SDS-PAGE
analysis.
S
*
For assay, 10 @i1
of undiluted human serum were incubated with 0.1ml
of radioiodinated melanoma lysate (diluted to contain approximately
200,000 cpm oftrichloroacetic acid-insoluble radioactivity) in the pres
ence of 0.1 ml of 2% BSA overnight at 4C. Then 0.05 ml of goat
anti-human IgG, 1gM, and IgA (KPL, Gaithersburg, MD) in 0.1 ml of
2% BSA was added to all tubes and incubated at 4C for 1 h, followed
by 0.05 ml ofProtein A-Sepharose (Pharmacia LKB, Uppsala, Sweden)
diluted 1:2 in PBS; incubation continued for 2 h at 4C with continual
mixing. The immunoprecipitates were collected by centrifugation at
12,000 x g for 3 min, washed extensively, extracted by boiling for 5 mm
with 0.05 ml ofmodified Laemmli's buffer (16) containing 5% mercap
toethanol, and run on 8% homogeneous SDS-polyacrylamide gels (15).
Bound labeled antigens were visualized by autoradiography using
Kodak XAR-5 film and Cronex fast screens, at —70C,and the relative
amount present was expressed on a scale ofO to 3+ based on the density
124
ofthe labeled bands. Molecular weight standards were from Sigma (St.
Louis, MO).
Competition Experiments. These experiments were performed by
incubating 0.01 ml of undiluted post-vaccine treatment serum (serum
134)containing antibodies to immunogenic melanoma antigens with 1,
10, or 100 @ig
of concentrated (100-fold) complete culture medium,
lactoperoxidase, BSA, or FBS in 0.1 ml of PBS and with 0.1 ml of a
1:10 dilution of NP-40 lysate (15 gig) of radioiodinated melanoma
(SK-Mel-28)cells
for 18 h at 4C.
Protein
A-Sepharose
was then added,
and the presence of antigens bound by residual melanoma antibodies
was determined by SDS-PAGE analysis as described above. Inhibition
was determined by comparing the presence ofantigen bands in samples
129
133
134
136
Fig. 1. Identification ofmelanoma antigens defined by vaccine-induced human
melanoma antibodies. Five patients (patients 124, 129, 133, 134, and 136) were
immunized 4 times at 3-week intervals by intradermal injections of 40
@gof
vaccinewith alum as an adjuvant.Sara collectedprior to vaccinetreatment (lanes
A) and 1 week after the final immunization (lanes B) were tested concurrently for
antibodies to radiolabeled melanoma antigens (SK-Mel-28 cells) by immuno
precipitation/SDS-PAGE analysis. Induction of antibodies to individual mela
noma antigens is evidenced by the presence of labeled antigen bands precipitated
by post-vaccine treatment serum (lane B) which are absent or present in lesser
amounts in pretreatment serum (lane A). Patient 124 had pre-existingantibodies
to melanoma which were not further increased by vaccinetreatment. The other
four patients developedstrong antibody responses to one or more antigens with
molecular weights of approximately 38,000—43,000, 75,000, 110,000, 150,000,
and 210,000.
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IMMUNOGENIC MELANOMA ANTIGENS
antibodies
to which were present
110,000 antigen, which induced an antibody response in 16
(62%) patients. Antibodies to this antigen were detectable prior
to vaccine treatment in only 2 (8%) patients. The next most
immunogenic antigens were the Mr 38,00043,000
and Mr
210,000 antigens, antibodies to which were induced or aug
mented by vaccine treatment in 7 (27%) and 5 (19%) patients,
respectively.
Specificity of Immunogenic Melanoma Antigens. The speci
ficity of the immunogenic antigens defined by vaccine-induced
antibodies was evaluated by measuring the expression of each
on a panel of control cells consisting of allogeneic pigmented
and nonpigmented human melanomas, xenogeneic melanomas,
unrelated allogeneic tumors, and normal allogeneic cells. The
cells were radioiodinated by lactoperoxidase, solubilized in NP
40, and assayed for expression of melanoma antigens by
immunoprecipitation/SDS-PAGE
analysis using a panel of
post-vaccine treatment sera with vaccine-induced antibodies to
the immunogenic antigens described above and, as controls,
pre-vaccine treatment sera obtained from the same patients. As
in 7 (27%) and in 6 (23%)
patients, respectively (see Table 1). Antibodies to these two
antigens were usually present concurrently in the same patient.
Antibody Response to Melanoma Vaccine Immunization.
The ability of vaccine treatment to stimulate antimelanoma
antibodies
was measured
by comparing
the pattern
of mela
noma antigens bound by sera collected from the same patient
prior to vaccine treatment and 1 week following four vaccine
immunizations.
A vaccine-induced
antibody
response
was evi
denced by the presence of an antigen bound to a greater extent
by post- versus pre-vaccine treatment sera from the same pa
tient, as illustrated in Fig. 1 (lanes A and B, respectively). The
overall results are summarized in Table 1. Some patients had
pre-existing antibodies to melanoma which were not further
augmented by vaccine treatment, as illustrated in Fig. 1 (patient
124). By contrast, other patients developed strong antibody
responses to melanoma antigens following vaccine treatment,
as illustrated in Fig. 1 (patients 134 and 136). Overall, vaccine
treatment induced a new antibody response, or augmented the
level of pre-existing antimelanoma antibodies, in 17 (65%) of
the 26 patients. The antibody response was directed to various
combinations
of one or more antigens,
as described
an additionalcontrol, the panelincludedpre- and post-vaccine
treatment sera collected from a patient (patient 124) who had
pre-existing antibodies to melanoma which were not further
below.
Identification of Immunogenic Melanoma Antigens. The
antigens defined by vaccine-induced antibodies had approxi
mate molecular weights of 38,000—43,000, 75,000, 110,000,
150,000, and 210,000. The immunodominant antigen which
most
frequently
induced
an antibody
response
increased by vaccine treatment. Representative results are illus
trated in Fig. 2. The Mr 38,00043,000,
75,000, 110,000, and
210,000 antigens defined by vaccine-induced antibodies were
strongly expressed on melanoma cells (see Figs. 1 and 2a), but
not on melanocytes (Fig. 2b) or on colon carcinoma cells (Fig.
was the M@
2c). However,the melanocytesand colon carcinomacells did
Table 1 Incidence ofantibodies to melanoma prior to and following
immunization with a polyvalent melanoma antigen vaccine
strongly express antigens defined by pretreatment serum 124,
which served as a positive control for the labeling procedure and
for the antigen assay. The tissue distribution of the antigens
defined by vaccine-induced antibodies in a representative serum
(patient 134) is summarized in Table 2. The M@38,00043,000,
75,000, 110,000, and 210,000 antigens were commonly ex
melanomaaBefore
(%) of patients with antibodies to
byantigenimmunization
MelanomaNo.
After
immunizationMr
Increased
immunization
(27%)Mr
210,0002
(8%)Mr
150,0002
(62%)Mr
110,0002
(8%)
9 (35%)
7
(8%)
(8%)
4 (16%)
18(69%)
2
16
0Mr 90,0006
(8%)Mr
75,0007
(19%)Any
38,000-43,0001
antigen
(23%)
6
(27%)
9
(4%)
6
(31%)
23
a Measuredby immuno8precipitation/SDS-PAGE
(23%)
(35%)
2
(23%)
5
(88%)
17 (65%)
analysis (n = 26).
pressed by human
melanomas,
being expressed
to various
de
grees by the five lines studied. These four antigens were selec
tively associated with melanoma, because they were expressed
on two or fewer of 11 nonmelanoma control cell lines studied.
C
AB
a
AB
AB
AB
AR
b
AB
AB
AB
AB
AB
AB
AB
AB
AB
AB
@@-2OO
V@
@q!
-200
@-2OO
w
..-97.
.._-97
@.
@-68
..-43
w_*
@
SKCO1
124
129
133
134
136
124
129
133
134
136
124
129
133
134
136
Fig. 2. Expression of melanoma antigens on different cells. SDS-PAGE analysis of lactoperoxidase-radioiodinated,
NP-40-soluble antigens on SK-Mel-23 mela
noma cells (a), normal melanocytes (b), and colon carcinoma (SK-CO-l) cells (c) precipitated by pre- (lanes A) and post-vaccine treatment (lanes B) sera from five
patients is shown. The antigens defined by vaccine-induced antibodies in patients 129, 134, and 136 are expressed on melanoma cells but not on the control cells. The
antigens defined by antibodies present prior to vaccine treatment in patient 124 are expressed by control as well as melanoma cells.
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IMMUNOGENIC MELANOMA ANTIGENS
Table 2 Tissuedistributionofmelanoma antigens definedby antibodiesinducedby
vaccineMelanoma
immunizationwithmelanoma
antigenaCell
lines
38,000@43,000Melanoma,
Mr 210,000
humanSK-MeI-23
++SK-Mel-30
++
++++SK-Mel-28
Mr 110,000
++
+
++
++
+
++
++
+
+++
0
0
+
0
00Normal
0
0
00Normallymphocytes (HL1)
00Colon lymphocytes (HL2)
0
0
0
0
0
0
++
0
0
0
0
0
+
+
0
0
0
0
0
0
++M14
++M20
+++++Control
Mr 75,000Mr
cellsXenogeneic
melanomaMurine
+++Hamster
melanoma (B16)
00AllogeneicMelanocytes
melanoma (HM54)
00Colon
carcinoma (5K-Co-I)
00Erythroleukemia
carcinoma (5W620)
++++++Rhabdomyocarcoma
(K562)
++Lung
(RD)
00Breast
carcinoma (A549)
00Glioma
carcinoma (T47D)
00XenogeneicBaby
(u87MG)
00a
hamster kidney (BHK)
Defined by vaccine-induced
134.Table
antibodies from patient
3 Tissuedistributionofmelanoma antigens definedby antibodiespresentprior
treatmentMelanoma
to vaccine
antigenaCell
lines
38,000@43,000Melanoma,
Mr 210,000
Mr 150,000
Mr 110,000
HumanSK-Mel-23
++++SK-Mel-30
+
+
+
++++SK-MeI-28
0
0
++0M20
++0Control
+++0Ml4
0
Mr 75,000Mr
0
+
0
+
0
+
+
0
0
0
0
+
+
0
0
0
cellsXenogeneic
melanomaMurine
+++0Hamster
melanoma (Bl6)
++0AllogeneicMelanocytes
melanoma (HM54)
++0Normal
0
0
+++0Normal
lymphocytes (HL1)
++
++
0
+++0Colon
lymphocytes(HL2)
+++0Colon
carcinoma (SK-Co-l)
+++0Erythroleukemia
carcinoma (SW620)
++++Rhabdomyocarcoma
(K562)
+++0Lung
(RD)
++++Breast
carcinoma (A549)
++
+++
++
0
+
+++
0
++
0
0
+
++
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
+++0Glioma
carcinoma (T47D)
+++0XenogeneicBaby
(u87MG)
+0a
hamster kidney (BHK)
Defined
bynatural
antibodies
frompatient
124.
nonmelanoma cell lines studied. The Mr 110,000 antigen was
The Mr 110,000 antigen was the antigen most selectively asso
expressed, but only weakly, by one melanoma control cell line.
ciated with melanoma, because it was the most strongly cx
The tissue distribution of the Mr 90,000 antigen defined by
pressed on human melanoma cells and the most weakly cx
pre-existing melanoma antibodies could not be studied since
pressed on control cells. The expression of the Mr 150,000
antibodies to it were not present in the serum used for this
antigen on control cells could not be measured reliably because
analysis.
the antibody response to it was weak.
The tissue distributions of the Mr 38,00043,000,
75,000,
The tissue distribution of antigens defined by melanoma an
and 210,000 antigens defined by pre-existing and by vaccine
tibodies present prior to vaccine treatment, using as a probe
induced antibodies differed. As an example, the Mr 75,000 and
serum 124, is summarized in Table 3. The Mr 75,000, 150,000,
and 210,000 antigens defined by this serum were expressed on 210,000 antigens defined by pre-existing antibodies were
most tissues tested, including normal cells. The expression of strongly expressed on lymphocytes and on colon and lung car
cinoma cells, whereas the antigens of similar molecular weight
the Mr 38,00043,000
antigen was more restricted. It was de
tected on two of the five melanoma lines and on two of the 11 defmed by vaccine-induced antibodies were not detectable on
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IMMUNOGENIC MELANOMA ANTIGENS
these cells. Conversely, K562 cells, which expressed the Mr
210,000 antigen defined by vaccine-induced antibodies, did not
express the Mr 210,000 antigen defined by pre-existing anti
bodies. Similarly, M20 cells, which expressed the Mr 38,000
43,000 antigen defined by vaccine-induced antibodies, did not
express the antigen of similar molecular weight defined by pre
existing antibodies.
Relation of Immunogenic Antigens to Culture Medium Corn
ponents
and HLA Antigens.
Because
the M@ 38,00045,000,
110,000, and 210,000 antigens defined by vaccine-induced an
tibodies
were preferentially
expressed
on human
melanoma
cells, their specificity was studied further. They were not related
to FBS proteins, because the vaccine was prepared from cells
which had been maintained in serum-free medium for several
years prior to use for vaccine production,
nor were they related
to components of the medium used to grow cells for vaccine
production, because the binding of these three antigens by spe
cific antisera was not blocked by a large excess of concentrated
(100-fold) complete culture medium (including growth factors
and dyes) used to grow the cells for vaccine production or by a
100-fold excess of unlabeled FBS proteins or bovine serum
albumin.
Immunodepletion of detergent lysates of radioiodinated mel
anoma cells with monoclonal antibody W6/32 to class I HLA
antigens or with MoAb L243 to class II HLA antigens com
pletely removed the respective antigens from the lysate (see Fig.
3, lanes5 and 9) but did not removethe M@38,00043,000,
75,000, 110,000, or 210,000 antigens defined by vaccine-in
duced antibodies (Fig. 3, lanes 4 and 7). In additional experi
ments, the Mr 38,00043,000,
75,000, 110,000, and 210,000
antigens defined by vaccine-induced
antibodies
were found to be
expressed strongly (2+ to 3+) by cells which expressed poorly
or not at all (0 to 1+) the previously described p97 MAA de
fined by MoAb 96.5 or the Mr 240,000 high-molecular weight
proteoglycan MAA defined by MoAb 9.2.2. Conversely, cells
PBS
anti-HLA 1
which expressed the Mr 97,000 and the Mr 240,000 MoAb
defined antigens did not express the M@ 110,000 and Mr
210,000 antigens defined by vaccine-induced antibodies. These
data indicate that the immunogenic Mr 38,00043,0®,
110,000, and 210,000 melanoma antigens defined by vaccine
induced antibodies are not related to FBS proteins, to compo
nents of the culture medium used to grow the cells for vaccine
production, to class I or II HLA antigens, or to previously
described melanoma-associated antigens with closely related
molecular weights.
DISCUSSION
The identification of human melanoma-associated antigens
which are immunogenic in humans is a critical element in the
design of effective vaccines against this cancer. While mela
noma is known to express a broad spectrum ofdistinct antigens,
most of these have been defined by murine monoclonal anti
bodies. Such antibodies identify human antigens which are im
munogenic in mice but reveal little about the immunogenic
potential of the antigens in humans.
One approach to identify immunogenic antigens which are
capable of stimulating antimelanoma immune responses in hu
mans is to utilize as probes antibodies which are induced by
active immunization ofpatients with melanoma vaccines which
contain a broad range of potential immunogens. Using this
approach, and an assay which permits the identification of in
dividual antigens defined by polyvalent antisera, we have pre
viously been able to identify several antigens in murine B16
melanoma which are immunogenic in syngeneic mice (15).
In the current study, we have applied this strategy to identify
human melanoma antigens defined by antibodies induced in
patients immunized with a polyvalent melanoma antigen vac
cine. The melanoma vaccine was made from surface material
shed into culture medium by a pool of four melanoma cell lines
and contains a broad range of potential immunogens (7). Prior
studies have shown that this vaccine is safe to use (5), immu
anti-HLA 2
nogenic in humans (5, 7, 13, 14), and capable of stimulating a
‘-200 cellular
immune
response
toapatient's
owntumor(17).There
is a statistically
@-
A
I
delayed-type
4
5
6
B
7
(P < 0.02) and clinically meaningful
ability
of the
responses
vaccine
and
to induce
a
delay
strong
in
tumor
months,
respectively)
(14,18).ByCoxmultivariate
analysis,
@--@
__
hypersensitivity
the
patients with a strong delayed-type hypersensitivity response to
vaccine immunization was 4.7 years longer than that of nonre
sponders (>72 months versus 15 months, respectively) and
overall survival was 3.7 years longer (>89 months versus 44
..,-43
—
significant
between
@@-68sected
stage
II melanoma,
themedian
disease-free
survival
of
V
@LL
correlation
progression (14). In a study of 94 patients with surgically re
.@
123
.7
8
9
C
Fig. 3. Relation between antigens defined by vaccine-induced antibodies and
HLA antigens.NP-4Olysatesofradioiodinated melanomacells(SK-Mel-28)were
immunodepleted with PBS (A), with MoAb W6/32 to class I HLA antigens (B),
or with MoAb L243 to class II HLA antigens (C). Remaining antigens were
precipitated with vaccine-induced antibodies defined by serum 134 (lanes 1, 4, and
7), by MoAb W6/32 (lanes 2, 5, and 8), or by MoAb L243 (lanes 3, 6, and 9) and
were analyzed by SDS-PAGE and autoradiography. The immunodepletion pro
cedure selectively removed the class I and II HLA antigens defined by W6/32 or
L243, without altering the level ofantigens defined by vaccine-induced antibodies.
these relations are independent of disease severity or of overall
immunological competence, suggesting that this is a vaccine
effect. These observations suggest that the vaccine may be din
ically effective in slowing the progression of melanoma in some
patients.
In this study, we found that antibodies to melanoma were
present in 31% of 26 sequentially tested patients with surgically
resected stage II (regional disease) malignant melanoma prior
to vaccine treatment. The antibodies were directed to one or
more antigens with approximate molecular weights of 38,000—
43,000, 75,000, 90,000, 110,000, 150,000, and 210,000. These
antigens were expressed as often on nonmelanoma tumor cells
as on melanoma cells and were also expressed on some normal
cells, suggesting that they are normal tissue antigens expressed
5952
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research.
IMMUNOGENIC
MELANOMA
on melanoma cells. Because only a limited number of normal
individuals were studied, it is not possible to determine whether
these antibodies represent “natural―
antibodies to melanoma or
reflect patients' spontaneous immune responses to their mela
noma.
The vaccine contains melanoma antigens which are immu
nogenic in humans, since vaccine treatment induced new anti
body responses or augmented the level of pre-existing antibod
ies to antigens expressed by melanoma cells in 65% of the 26
patients. The immunogenic antigens had approximate molecu
lar weights of 38,000—43,000, 75,000, 110,000, 150,000, and
210,000. Because these were antigens defined by polyclonal
sera, there is a possibility that the antigens of similar molecular
weight immunoprecipitated by different sera may not be iden
tical. All antigens appeared to be expressed on the external
surface of melanoma cells, because they were radioiodinated by
the lactoperoxidase technique. The immunogenic potency of
these antigens, as evaluated by the frequency with which they
induced antibody responses in patients, was variable. The most
potent immunogen was the Mr 110,000 antigen, which induced
antibody responses in 62% of patients. Antibody responses in
individual patients were directed to various combinations of
one or more antigens, indicating that the immunogenicity of the
antigens depends in part on host factors, as well as on the
properties
of the antigens.
An interesting
but unexplained
oh
ANTIGENS
and 210,000 melanoma antigens defined by vaccine-induced
antibodies appear to differ from previously described melanoma
antigens of similar molecular weight which are defined by mu
rine monoclonal
antibodies.
In summary, the antigens which appear to be the most at
tractive candidates for the construction of melanoma vaccines
are the Mr 210,000,
1 10,000, and 38,000—45,000 antigens,
be
cause they possess a combination of properties which are par
ticularly desirable for this purpose (1), i.e., the ability to elicit
immune responses in humans, expression on the surface of
melanoma cells where they can be seen by host effector immune
mechanisms on viable cells, selective expression on human mel
anoma, and common expression on different melanoma cells so
that a generic vaccine can be used to treat different patients.
The Mr 110,000 antigen is a particularly attractive candidate
for vaccine construction, because it is the immunodominant
antigen.
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5953
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1992 American Association for Cancer Research.
Identification of Immunogenic Human Melanoma Antigens in a
Polyvalent Melanoma Vaccine
Jean-Claude Bystryn, Milagros Henn, Joseph Li, et al.
Cancer Res 1992;52:5948-5953.
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