Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes
(CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs
with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous
antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA)
was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to
these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of
tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1+ antibody had NY-ESO-1+ tumors, and no
patients with NY-ESO-1
tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1+ tumors have antibody,
this would suggest that a high percentage of patients with NY-ESO-1+ tumors develop an antibody response to NY-ESO-1.
 |
Introduction |
Analysis of the human immune response to cancer has
had a long and complex history (1). Although serological methods dominated initial efforts to find evidence for
immune recognition of cancer, advances in analyzing cell-mediated immunity have now permitted exploration of T
cell recognition of human cancer (2). Interpreting the specificity of an observed humoral or cellular immune response to cancer cells has always been the critical issue in human
tumor immunology. Test systems restricting the analysis to
autologous systems, i.e., antibody or T cells from the same
patient, eliminated the contribution of alloantigens and
provided provocative evidence for humoral (3) and cellular
(4) immunity to human cancer cells. However, the molecular cloning of tumor antigens recognized by CTLs (2) and
antibodies (5) has opened a new era in tumor immunology,
and the list of defined immunogenic human tumor antigens is growing rapidly. These antigens fall into one of the following categories: (a) cancer-testis (CT) antigens, e.g.,
MAGE (2), SSX2 (HOM-MEL-40; reference 5), and NY-ESO-1 (6), which are expressed in a variable proportion of
a wide range of human tumors, but show a highly restricted
expression pattern in normal tissues, namely testis; (b) antigens coded for by mutated genes, e.g., p53 (7) and CDK4
(8); (c) differentiation antigens, e.g., tyrosinase (9, 10) and
Melan-A (11); (d) amplified gene products, e.g., HER2/
neu (12) and carbonic anhydrase (5); and (e) viral antigens,
e.g., retrovirus (13), HPV (14), and EBV (15). Although
some of these antigens, e.g., MAGE and tyrosinase, were
initially detected by CTLs and cloned via their CTL-recognized epitopes, they also elicit humoral immunity and
can be identified and cloned by using antibodies from cancer patients (5, 16). This study initiates a survey of the human humoral immune response to a panel of those recently
defined autoimmunogenic human tumor antigens.
 |
Materials and Methods |
Tissues and Sera.
Tumor tissues were obtained during routine surgical procedures, frozen in liquid nitrogen, and stored at
80°C until use. Human sera were obtained from patients with
various tumor types and from normal blood donors and were
stored at
80°C (IRB No. 87-20; Memorial Sloan-Kettering
Cancer Center, New York; and IRB No. 0596-336, Cornell
University Medical College, New York). The patients with melanoma, breast, and ovarian cancer had metastatic disease, whereas
the majority of patients with lung and colon cancer had primary
operable disease.
ELISA.
10 µl/well of a 1 µg/ml recombinant protein in
coating buffer (15 mM Na2CO3, 30 mM NaHCO3, pH 9.6, with
0.02% NaN3) was adsorbed to TC microwell plates 60 × 10 (Nunc, Roskilde, Denmark) overnight at 4°C. Plates were washed
with PBS and blocked overnight at 4°C with 10 µl/well of 2%
BSA/PBS. After washing, 10 µl/well of serum dilutions in 2%
BSA was added and incubated for 2 h at room temperature. Plates
were washed and 10 µl/well diluted secondary antibody/2% BSA
was added (Goat anti-human IgG-AP; Southern Biotechnology, Birmingham, AL) and incubated for 1 h at room temperature.
Plates were washed, incubated with 10 µl/well of substrate solution (Attophose substrate; JBL Scientific, San Louis Obispo, CA)
for 25 min at room temperature, and immediately read (CytoFluor 2350; Millipore, Bedford, MA). For the serological survey
of human sera, sera were tested over a range of serial fourfold dilutions from 1:100 to 1:100,000. A positive reaction is defined as
an OD value of a 1:400 diluted serum that exceeds the mean OD
value of sera from normal donors (n = 70) by three standard deviations. Fig. 2 shows characteristic titration curves of negative and
positive sera.

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Fig. 2.
Representative results of ELISA reactivity with sera from
melanoma patients NW29, NW38, and NW33, against a panel of seven
recombinant tumor antigens.
|
|
Reverse Transcription PCR.
Messenger RNA (mRNA) expression of NY-ESO-1 in normal and malignant tissues was evaluated by RT-PCR assays as previously described (6, 17).
Expression of Recombinant Tumor Antigens in Escherichia coli.
The tumor antigens listed in Table 1 were expressed in E. coli using histidine-tag-containing vector pQE9 (Qiagen, Chatsworth, CA). Various cDNA amplification primers were designed to encompass entire or partial coding sequences of these genes, corresponding to amino acid positions shown in Table 1. The induction of recombinant protein synthesis and subsequent purification
by Ni+2 column were performed as described (17).
Immunoblotting Analysis.
Serum antibody responses against
the purified recombinant protein were tested by standard Western blot analysis (22) using 1 µg of the purified protein and human serum at 1:1,000, 1:10,000, and 1:100,000 dilutions, or with
1:50 diluted mouse mAb supernatants (see below). Goat anti-human
IgG (Fc specific; Sigma Chemical Co., St. Louis, MO) diluted
1:10,000 and goat anti-mouse IgG (Bio-Rad, Hercules, CA) diluted 1:3,000 were used as secondary reagents.
Monoclonal Antibodies.
A series of mAbs were generated
against NY-ESO-1, MAGE-3, and SSX2 (HOM-MEL-40) using
methods previously described (18). Three representative mAbs
were used in this study, E978 for NY-ESO-1, M3-6 for MAGE-3,
and HM498 for SSX2.
Expression of NY-ESO-1 in Baculovirus.
The NY-ESO-1 cDNA
insert from the pQE9 recombinant clone was released and subcloned in pBlue BacHis2A vector (Invitrogen, Carlsbad, CA) and
positive clones were isolated. Transfection of Sf9 cells with
pBlueBacHis2A/NY-ESO-1 and isolation of recombinant viruses
were accomplished following the protocol from Invitrogen. Infection of insect cells was performed in IPL-41 medium with 10%
fetal calf serum at a multiplicity of infection (MOI) of 20. Expression of recombinant NY-ESO-1/His-tag protein was confirmed by Western blot analysis with NY-ESO-1 E978 mAb, and purification was by Ni2+ affinity chromatography.
 |
Results |
Establishment of ELISA Systems for Tumor Antigen Typing.
Table 1 lists the seven protein antigens included in
the typing panel. NY-ESO-1 (full length), Melan-A, and
SSX2 represent full-length products, whereas NY-ESO-1
(long and short), MAGE-1, MAGE-3, tyrosinase, and carbonic anhydrase represent truncated products involving the
internal region. These expressed proteins were used to generate mAbs, and prototype mAbs for each antigen were selected by specific reactivity in ELISA and Western blots. mAbs against MAGE-1, tyrosinase, and Melan-A have
been previously reported (17), and detailed characterization of NY-ESO-1, MAGE-3, SSX2, and carbonic anhydrase mAbs will be published elsewhere. Each of the
mAbs showed specificity for the immunizing antigen and
did not cross-react with the other antigens in the panel. Fig. 1 shows the reactivity pattern of the NY-ESO-1,
MAGE-3, and SSX2 mAbs used as typing reagents for standardizing and optimizing conditions for ELISA, e.g., protein concentration, conditions for antigen adsorption, test
antigen stability, blocking, and washing conditions.

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Fig. 1.
Western blot analysis of mouse mAbs against recombinant
tumor antigens. NY-ESO-1 (full length), SSX2, MAGE-3, and carbonic
anhydrase (lanes a, b, c, and d) were purified and reacted with mAbs
against NY-ESO-1 (mAb E978), SSX2 (mAb HM498), and MAGE-3
(mAb M3-6), respectively. Arrowheads, the main reactive protein species
in each lane, migrating at the expected mol wt of these proteins (see Table 1).
|
|
Survey of Human Sera for Antibodies to the Panel of Human
Tumor Antigens.
Table 2 summarizes the results with 234 sera from patients with cancer and 70 from normal individuals and Fig. 2 illustrates titrations of sera from selected individuals. Positive sera were tested at least three times on
the seven antigens, and most negative sera were tested
twice. Absorbing reactive sera with lysates of E. coli and
bacteriophages did not reduce serum titers, nor did it affect
the background reactivity of unreactive sera. A small fraction of sera in this series (one colon cancer, one ovarian cancer, four melanomas, and two normal blood donors)
showed a nonspecific reactivity pattern with the entire antigen panel and were easily distinguished and eliminated.
These non-specifically reactive sera also bound strongly to
the assay plates in the absence of adsorbed protein. Our
survey showed that 9.4% (12/127) of melanoma patients,
12.5% (4/32) of ovarian cancer patients, 4.2% (1/24) of patients with lung cancer, and 7.7% (2/26) of patients with breast cancer have antibody against NY-ESO-1. No specific antibody reactivity to NY-ESO-1 was detected in sera
of 25 patients with colon cancer and in 70 normal human
sera. MAGE-1 antibodies were found in three patients in
this study, one with melanoma, one with ovarian cancer,
and one with lung cancer, MAGE-3 antibody was found in
two patients with melanoma, and SSX2 antibody was found in one patient with melanoma. No antibody against
Melan-A, tyrosinase, or carbonic anhydrase was found.
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Table 2
Survey of Sera from 70 Normal Blood Donors and 234 Cancer Patients: ELISA Reactivity with Recombinant Tumor Antigens
|
|
Reactivity of Human NY-ESO-1 Antibodies with Recombinant NY-ESO-1 Protein Produced in Baculovirus.
NY-ESO-1
produced by baculovirus was as reactive as NY-ESO-1 of
bacterial origin in tests with human sera. Reactivity with mouse mAb against NY-ESO-1 also showed that the bacterial and baculovirus NY-ESO-1 products were equally
recognized.
Correlation of NY-ESO-1 Expression and Presence of NY-ESO-1 Antibodies.
Fresh-frozen tumor specimens and serum samples were available from 62 patients with melanoma. All patients had a history of metastatic melanoma for
>2 yr before serum and tumor samples were collected, and
had undergone different chemo- and immunotherapeutic regimens. Tumors were typed for NY-ESO-1 expression
by RT-PCR and sera were assayed for NY-ESO-1 antibody by ELISA and by Western blotting (Table 3 and Fig.
3). In this series of 62 patients, 15 had NY-ESO-1 positive
tumors and 8 of these patients had NY-ESO-1 antibodies.
The two NY-ESO-1 detection systems, ELISA and Western blotting, gave identical results. No NY-ESO-1 antibody was detected in patients with NY-ESO-1 negative
tumors. Seven patients had NY-ESO-1 positive tumors
and no detectable NY-ESO-1 antibody. Although the
RT-PCR analysis was not designed to be a quantitative assay, the PCR signals in this group of tumors appeared to be lower than the signal in NY-ESO-1 positive tumors from
NY-ESO-1 antibody positive patients.

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Fig. 3.
RT-PCR analysis
of NY-ESO-1 expression in tumor specimens and Western blot
analysis for anti-NY-ESO-1 antibodies in patient sera. Of the
five cases illustrated, three (lanes
a, b, and c, NW178, NW33, and
NW38, respectively) were NY-ESO-1 mRNA positive, showing the expected 355-bp RT-PCR product (top; m, molecular
standard). Of these three cases,
two were anti-NY-ESO-1 antibody positive, showing 22-kD
NY-ESO-1 recombinant protein
on Western blot (bottom, lanes a
and c, 1:1000 serum dilution), whereas one (lane b) was negative. Positive
control on Western blot was provided by using anti-NY-ESO-1 mouse
mAb (lane Co). Two cases (lanes d and e, NW309 and NW145) were
negative for both NY-ESO-1 mRNA and anti-NY-ESO-1 antibody.
ELISA and Western blotting gave identical results.
|
|
 |
Discussion |
Based on the recognition by humoral or cellular immune
responses in the autologous human host, a number of human antigens have been identified (2, 5, 6, 16, 23). These
antigens provide attractive new targets for vaccine-based
therapies, and a range of different strategies including peptide, protein, RNA, DNA, and viral vector vaccines are
being pursued. The availability of these cloned tumor antigens also permits the development of serological or cell-based assays for screening human populations for specific
antibody or T cell responses. Because a number of these
antigens appear to be recognized by humoral and/or cellular immune reactions, we have chosen serological assays to
initiate this survey because of their greater simplicity and
speed. The ELISA screening system established in this study
has been standardized using mouse mAbs with specificity
for each of the antigens. In tests of human sera, the use of
different antigens prepared in the same bacterial expression
system provides a critical internal specificity control to
eliminate reactions directed against contaminating bacteria
and phage proteins. To control for the influence of protein folding and glycosylation on antibody detection, the reactivity of tumor antigens purified from mammalian sources
will be compared with the corresponding antigens produced in bacterial systems.
Of the seven antigens tested in this study, antibodies to
NY-ESO-1 were observed most frequently. Reactivity to
NY-ESO-1was found in ~10% of patients with melanoma
and ovarian cancer and in lung and breast cancer with a
lower frequency. No reactivity was found in the sera of patients with colon cancer. MAGE-1 reactivity was found in
one patient each with melanoma, ovarian, or lung cancer,
MAGE-3 reactivity was found in two patients with melanoma, and SSX2 was found in one patient with melanoma.
In the study of Sahin et al., SSX2 antibodies were found in 2 of 11 patients with melanoma using a plaque assay for antibody detection (5), and we are currently comparing the sensitivity of ELISA and plaque assays. The sera from 70 healthy
blood donors were negative with all seven antigens. Hoon et
al. (24) reported that MAGE-1 antibodies were frequently
found in normal individuals as well as in melanoma patients,
and that the titer of MAGE-1 increases after vaccination with MAGE-1+ tumor cells. This discrepancy between our
results and Hoon et al. (24) may reflect differences in the assay system or the MAGE-1 antigen constructs.
In our initial study, we found NY-ESO-1 mRNA in
20-40% of melanomas (6). As 10% of unselected melanoma
patients have NY-ESO-1 antibody (Table 2), this would
suggest that ~50% of patients with NY-ESO-1 positive
melanomas develop NY-ESO-1 antibodies at some time
during the course of their disease. To make a direct test of
this relationship, we studied a series of 62 patients with
stage IV melanomas, where tumor and serum specimens
from the same patient were available and compared the
NY-ESO-1 antibody status with NY-ESO-1 mRNA expression in the autologous tumor. The results showed that
NY-ESO-1 antibodies were found in patients with NY-ESO-1 positive tumors, and that no patients with NY-ESO-1 negative tumors had NY-ESO-1 antibody. However, there were seven patients with NY-ESO-1 positive
tumors with no detectable antibody, suggesting that these
patients did not form antibody, that they made antibody
not detectable by ELISA or immunoblotting, or that they
developed cellular but not humoral immunity against NY-ESO-1. Analyses of NY-ESO-1 antibody status and patient
characteristics (sex, stage, state, and extent of disease and
previous therapies) has not revealed any correlation with
serological status, but this will need more extensive study,
particularly with sera from patients with less advanced disease.
To explore the relationship between humoral and cellular immune recognition of NY-ESO-1, CD4 and CD8 T
cell responses to NY-ESO-1 also need to be evaluated. We
have recently described a patient with high NY-ESO-1 antibody titers and strong CTL reactivity against autologous
melanoma cells (25). Transfection experiments showed that
NY-ESO-1 coded for the CTL-recognized antigen, and
using motif analysis, NY-ESO-1-related peptides were
synthesized that were efficiently recognized by the CTLs.
This patient showed that strong humoral and cellular immunity to a tumor antigen can coexist in the same patient.
One of the major challenges confronting the clinical
testing of vaccines is the availability of reliable assays that
can monitor specific immune responses to the vaccine as a
way to guide the development of maximally immunogenic
vaccines. With regard to vaccines aimed at eliciting cytotoxic antibodies to cell surface antigens, such as GM2, sensitive and specific assays have been developed (26). However, monitoring the immune response to vaccines aimed
at eliciting cellular immunity, particularly CTL generation, has been problematic and difficult to interpret. No CTL
responses were seen in vaccine trials with MAGE peptides
(27), and even though CTL responses can be elicited in patients immunized with peptides derived from melanoma-associated differentiation antigens such as Melan-A or tyrosinase, nonimmunized normal individuals can also generate
CTL responses to these antigens (28, 29). The presence of
high titered NY-ESO-1 antibodies in patients with melanoma, ovarian, lung, and breast cancer indicates CD4 recognition of NY-ESO-1, and serological tests therefore provide a way to monitor the CD4 repertoire to tumor antigens.
Should other patients with NY-ESO-1 antibody also have
a specific CTL response, as we have already shown in one
patient (25), serological tests may be useful as a way to
identify patients with a CTL response to NY-ESO-1.
Address correspondence to Elisabeth Stockert, Ludwig Institute for Cancer Research, New York Branch at
Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021. Phone: 212-639-7542;
Fax: 212-717-3100; E-mail: stockere{at}mskcc.org
We thank Drs. Nasser K. Altorki, Jonathan S. Cebon, John M. Kirkwood, Antonio I. Picon, and Özlem
Türeci for providing us with serum and tissue samples, and Allison Sweeney for excellent technical assistance.
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