[Cancer Research 60, 2136-2139, April 15, 2000]
© 2000 American Association for Cancer Research
cdr2, a Target Antigen of Naturally Occurring Human Tumor Immunity, Is Widely Expressed in Gynecological Tumors1
Jennifer C. Darnell,
Matthew L. Albert and
Robert B. Darnell2
Laboratory of Molecular Neuro-Oncology, Rockefeller University, New York, New York 10021
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ABSTRACT
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The paraneoplastic neurological disorders provide perhaps the best known
example of naturally occurring tumor immunity in humans. For example,
patients with paraneoplastic cerebellar degeneration (PCD) appear to
suppress the growth of occult breast or ovarian tumors that express a
neuronal antigen termed cdr2. PCD patients harbor cdr2-specific CTLs in
their peripheral blood, and these cells are likely mediators of the
tumor suppression. Whereas cdr2 therefore appears to be the target of
an effective immune response in patients with PCD, the general
relevance to cancer patients has been unclear, due in part to reports
indicating that cdr2 is not expressed in tumors obtained from
neurologically normal patients. We have reexamined this question, and
we find that cdr2 is widely expressed in such tumors, indicating that
cdr2 is in fact an important tumor antigen in the general population of
breast and ovarian cancer patients.
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Introduction
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Given recent efforts to boost the immune system for purposes of
immunotherapy, identification and understanding of naturally occurring
tumor immune responses in humans are increasingly important. One
difficulty in this endeavor is that patients with successful immune
responses to cancer are not likely to come to medical attention. An
important exception is patients with
PND.3
In these individuals, it is believed that tumor cells expressing
neuron-specific proteins successfully trigger an effective antitumor
immune response. However, PND patients go on to develop an autoimmune
neuronal degenerative disorder, which brings them to clinical
attention. Studies of PND patients have led to insights regarding the
body’s ability to mount an immune response to tumor-restricted
antigens (1
, 2)
. For example, in patients with PCD, breast
or ovarian tumors express a Purkinje neuronal protein termed cdr2
(3, 4, 5)
. Recently, cells capable of mediating tumor
immunity (expanded populations of cdr2-specific CTLs) were detected in
five of five HLA-A2.1+ PCD patients (6
, 7)
. In contrast, tumor-specific CTLs have not been found to be
expanded in patients with actively growing tumors, even in cases where
tumor-restricted antigens are known to be expressed (e.g.,
the melanoma MAGE/MART antigens; Ref. 8
). Studies
on PCD patients also demonstrated that dendritic cells were capable of
cross-presenting tumor antigen from apoptotic cells, resulting in the
activation of class I-restricted tumor-specific CTLs (7)
and suggesting that apoptotic tumor cells may be an important source of
antigen that triggers tumor immunity. In this study, we examine
whether the cdr2 antigen is expressed in tumors obtained from
neurologically normal breast and ovarian cancer patients.
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Materials and Methods
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Reagents.
Reagents were obtained from Sigma unless otherwise noted.
Tissue Lysate Preparation.
Tumor samples and pathology reports were obtained from the Memorial
Sloan-Kettering Cancer Center Tissue Procurement Service. Tissue
lysates were prepared by pulverizing tissues in a liquid
nitrogen-cooled mortar and pestle, followed by homogenization in lysis
buffer [10 mM Tris-HCl (pH 7.4), 15 mM EDTA,
50 mM KF, 50 mM
NaH2PO4, 10 mM
Na PPi, and 1% NP40 with Complete protease
inhibitors (Boehringer Mannheim)] using a Wheaton homogenizer. Protein
concentrations were determined by Bio-Rad Protein Assay using BSA as a
standard. Purkinje cell extract was prepared as described previously
(5)
. Normal ovary extract was purchased from Clontech.
Preclearing Tumor Lysates.
Tumor lysates were microfuged at 10,000 rpm for 10 min, adjusted to 375
µg of protein in 0.55 ml of lysis buffer, and cleared of IgG three
times by adding 75 µl of a 50:50 protein A-Sepharose:protein
G-Sepharose bead slurry (prepared as a 50% slurry in PBS), followed by
shaking at 4°C for 1 h. The final supernatant was analyzed by
SDS-PAGE or IEF/SDS-PAGE.
SDS-PAGE and IEF/SDS-PAGE.
For one-dimensional analysis, 10% SDS-PAGE gels were prepared and run
by standard methods. Twenty µg of protein were loaded per lane, and
gels were transferred to nitrocellulose (S&S BA85) for Western
blotting.
For two-dimensional gel electrophoresis, tumor lysate or mouse
cerebellar lysates were diluted 1:1 with two-dimensional lysis buffer
[9.5 M urea (Pharmacia Biotech PlusOne), 2% NP40
(Calbiochem), 5% 2-mercaptoethanol (Pharmacia PlusOne), and 2%
Biolyte ampholytes (Bio-Rad) consisting of 75% 3/5 range and 25%
3/10 range Biolytes). IEF gels were performed by the method of
O’Farrell (9)
. IEF slab gels [0.75 mm; 9.2 M
urea, 4% acrylamide (Ready Sol IEF; acrylamide:bis ratio of 19:1;
Pharmacia), 2% NP40, and 5% Biolyte ampholytes] were loaded with 40
µg of total protein per lane, and the sample was overlaid with
buffer (7% urea, 2.5% ampholytes, and 5% 2-mercaptoethanol). The
gels were run using 0.01 M
H3PO4 and 0.02
M NaOH buffers at 4 W for 1800 V/h. Lanes were equilibrated
in SDS sample buffer for 5 min and loaded horizontally (with the acidic
end on the right) on 1 mm 10% SDS-PAGE gels prepared with a 1-cm flat
3% acrylamide stacking gel.
Biotinylation of PCD Sera.
Antibodies were purified from PCD patient and normal human sera,
adjusted to pH 8.0 with 1.0 M Tris, on protein A-Sepharose
(Sigma) columns, and eluted with 100 mM glycine (pH 3.0)
into 0.10 volume 1 M Tris (pH 8.0) to neutralize
them. Immunoglobulin-containing fractions were identified by Bio-Rad
protein assay and pooled. Pooled antibody was dialyzed against 0.1
M sodium borate buffer (pH 8.8).
N-Hydroxysuccinimide biotin at 10 mg/ml in DMSO was added at
a ratio of 150 µg ester/mg antibody and incubated at RT for 4 h.
Twenty µl of 1 M ammonium chloride were added
per 250 µg of ester for 10 min at RT. Antibody was dialyzed against
PBS extensively to remove uncoupled biotin and stored at a final
concentration of 3 mg/ml IgG at 4°C.
Western Blotting.
Filters were blocked in 5% nonfat dry milk/PBS (milk block) for 1 h at RT and probed with human sera at a 1:300 dilution overnight at
4°C or for 1–2 h at 25°C. Filters were washed according to
Amersham enhanced chemiluminescence protocols and incubated with
HRP-conjugated antihuman or mouse secondary antibodies (Jackson
ImmunoResearch Laboratories, Inc.; 1:5000) or blotting grade avidin-HRP
(Bio-Rad; 1:3000) for 1 h at RT. After a second wash, reactive
proteins were detected according to the enhanced chemiluminescence kit
protocol.
Anti-ß-tubulin antibody (Boehringer Mannheim) was used according to
the manufacturer’s specifications. Biotinylated antibodies were used
at 30 µg IgG/ml milk block.
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Results and Discussion
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Whereas PCD presents an important example of tumor immunity, its
general relevance to cancer patients has been questioned following
reports that the cdr2 antigen is only expressed in tumors obtained from
PCD patients (4)
. We have reexamined the relevance of cdr2
as a tumor antigen by carefully evaluating cdr2 expression in
gynecological tumors obtained from neurologically normal cancer
patients. Whereas cdr2 mRNA is widely expressed, the protein is
absolutely restricted at a posttranscriptional level to expression in
the brain and testis (5)
. Therefore, we have examined cdr2
antigen expression by Western blot analysis. Initial attempts to
separate cellular proteins using SDS-PAGE gels failed because of high
levels of IgG present in the tumors, which reacted with the antihuman
secondary antibody and obscured cdr2 reactivity at
Mr 52,000. To overcome this
problem, lysates of tumor specimens were depleted of IgG by incubation
with protein A and protein G coupled beads, run on SDS-PAGE gels,
probed with biotinylated sera, and visualized with HRP-avidin as a
secondary reagent. Using this method, we were able to clearly identify
a cdr2-reactive band (Mr 52,000) that
was not detected when probing with normal human serum or
secondary reagents (Fig. 1B)
. A Mr 69,000 band evident
in some samples was reactive with HRP-avidin alone and was therefore
unrelated to cdr2 (Fig. 1B
, right panel).
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Fig. 1. Expression of cdr2 in ovarian tumors. A,
protein extracts from nine human ovarian tumors were run on Western
blots and probed with biotinylated PCD antisera. Strong cdr2 reactivity
was evident in tumors 1, 2, 6, 7, and 9 as well as in extracts of human
Purkinje. An anti-tubulin antibody was used as a loading control. An
upper band present in Purkinje extracts and ovarian tumors
(NS) was further characterized (B).
Identical cdr2 reactivity was seen in two independent experiments in a
total of 13 of 21 ovarian tumor specimens (Table 1
; data not shown).
Each tumor that did not express cdr2 was re-evaluated by Western blot
analysis of an independent fragment of the tumor, which confirmed the
lack of cdr2 reactivity (data not shown). B, extracts of
normal ovary and cdr2-positive and cdr2-negative ovarian tumors were
either run directly on SDS-PAGE or first precleared of IgG with protein
A and protein G beads, as indicated. Western blots were probed with
biotinylated PCD antisera (anti-cdr2) or normal human
sera (NHS), followed by avidin-HRP or, as a control,
avidin-HRP alone. A Mr 52,000 antigen was
specifically recognized by anti-cdr2 antisera, and an unrelated
Mr 69,000 band reacted with avidin-HRP
alone. Preclearing with protein A/G depleted a faintly detectable
Mr 50,000–55,000 species in the
cdr2-negative uncleared lane of the anti-cdr2 blot (compare
Lanes 3 and 4 of the left
panel).
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Primary ovarian tumors from 21 neurologically normal individuals were
examined (Table 1A)
. Fig. 1A
shows representative results from nine tumors.
Human Purkinje cell extract served as a positive control, and normal
ovary tissue served as a negative control. Five of nine tumors clearly
express a Mr 52,000 antigen, which
comigrates with the cdr2 from Purkinje cells (Fig. 1A)
. To
confirm that the reactive antigen is cdr2, mouse cerebellum and ovarian
tumor extracts were resolved side by side on two-dimensional gels (Fig. 2A)
. The reactive species at
Mr 52,000 exactly comigrated by
isoelectric point as well, indicating that the ovarian tumors
express cdr2.
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Table 1 cdr2 expression in gynecologic tumors
The indicated tumor samples were analyzed by SDS-PAGE and probed with
PCD antisera. cdr2 reactivity was confirmed by two-dimensional
IEF/SDS-PAGE and by probing with multiple PCD and negative control
antisera.
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Fig. 2. IEF-SDS/PAGE analysis of cdr2 in cerebellum and ovarian
tumor. A, extracts of mouse cerebellum and human ovarian
tumor were run side by side on two-dimensional gels and probed with PCD
antisera and HRP-antihuman IgG secondary antibody. Tumor or
cerebellar lysate was loaded in the right-hand lane as a
control (1-D). An immunoreactive band of identical
molecular weight and isoelectric point is present in both
extracts. The mouse cdr2 cDNA encodes a protein that is 87% identical
with human cdr2, and this protein migrates identically with cdr2
detected in human Purkinje extracts (data not shown). B,
mouse cerebellar extract was resolved on four identical two-dimensional
gels and probed with three PCD antisera or antiserum from a POMA
patient, which recognizes a characteristic set of
Mr 52,000–69,000 bands. A sample of extract
was loaded in the right-hand lane as a control
(1-D).
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Because PCD antisera from any one patient is a hyperimmune polyclonal
serum that could contain multiple antibodies, we probed cerebellar
extract and tumor samples with several different PCD sera. As a
negative control, antiserum from a patient with a distinct PND, POMA,
was used. In cerebellum, three different PCD antisera were strongly
reactive with the Mr 52,000 species on
two-dimensional gels (Fig. 2B)
. In contrast, POMA antiserum
as reactive with the characterized Nova antigens (10)
in
the cerebellar extract (Mr
46,000–60,000 on Lane 1-D) but were not reactive
with the cdr2 spot. When ovarian tumor extract was run on five
duplicate two-dimensional gels, two different PCD antisera detected
cdr2, whereas unrelated antisera did not detect cdr2 (Fig. 3)
.
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Fig. 3. The cdr2 antigen in ovarian and breast tumors is
recognized by multiple PCD antisera. Lysates from an ovarian tumor and
a breast tumor were run on duplicate two-dimensional gels, transferred
to nitrocellulose, and probed with two different PCD antisera. As
controls, blots were probed with normal human serum or POMA or
an uncharacterized PND antiserum that recognizes distinct antigens (see
Lane 1-D) but fail to react with cdr2. Breast tumor gels
were not loaded with one-dimensional samples. Identical results were
seen with two independent breast and ovarian tumors, four different PCD
antisera, and four different hyperimmune control antisera (data not
shown).
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We also examined nine breast tumor samples from neurologically normal
individuals. Two of nine tumors were clearly cdr2 positive (Table 1)
.
Again, expression was determined using two-dimensional gels and
multiple PCD antisera and negative controls, confirming that the
antigen was cdr2 (Fig. 3
; data not shown).
Our results demonstrate that a significant subset of breast and ovarian
tumors up-regulates cdr2 expression. Given the recent observation that
cdr2 interacts with c-myc (11)
, the prevalence of cdr2
expression in gynecological tumors may be relevant to tumorigenesis.
Additionally, this finding has great immunological significance because
it establishes cdr2 as a potential target tumor antigen for the
treatment of neurologically normal patients with breast and ovarian
cancer. The finding that PCD patients harbor cdr2-specific CTLs that
are capable of targeting tumor cells expressing cdr2 epitopes
underscores the importance of cdr2 as a potent tumor antigen. An
additional question raised by the data presented here is whether some
patients with cdr2-positive gynecological tumors might harbor
cdr2-specific CTLs and thereby have an improved clinical outcome. This
suggestion stems in part from the finding that 15% of neurologically
normal small cell lung cancer patients have low titer antibodies to the
Hu onconeural antigen, and this evidence of an immune response
correlates with an improved clinical course (12
, 13)
.
Notably, all small cell lung cancers express the Hu onconeural antigen
(14)
. This observation, together with our finding of cdr2
expression in a significant percentage of gynecological tumors,
suggests that tumor cell expression of onconeural antigens is necessary
but not sufficient for the induction of antigen-specific tumor immune
responses.
Several factors may contribute to the development of PND
antigen-specific immunity, including tumor cell expression of MHC class
I (demonstrated in a high percentage of PND-associated tumors; Ref.
15
); the proximity of dendritic cells to apoptotic tumors,
which may be important in tumor antigen presentation (16)
;
and the lack of FasL or other suppressive molecules on the surface of
the tumor cell (17)
. Analysis of cdr2-expressing
gynecological tumors as well as an investigation of immunity to cdr2 in
PCD patients may determine which, if any, of these factors may be
important to the development of naturally occurring tumor immunity.
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FOOTNOTES
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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.
1 Supported by Department of Defense Breast Cancer
Research Award DAMD017-94-J-4277, NIH Medical Scientist Training
Program Grant GM-07739, American Cancer Society Research Award
RPG-98–033-CIM, and Irma T. Hirschl Career Scientist Award. 
2 To whom requests for reprints should be
addressed, at Laboratory of Molecular Neuro-Oncology, Box 226, 1230
York Avenue, New York, NY 10021-6399.
3 The abbreviations used are: PND, paraneoplastic
neurological disorder; PCD, paraneoplastic cerebellar degeneration;
POMA, paraneoplastic opsoclonus-myoclonus; IEF, isoelectric focusing;
RT, room temperature; HRP, horseradish peroxidase.
Received 12/21/99.
Accepted 2/29/00.
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ABSTRACT
Introduction
