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Departments of Pediatrics [N-K. V. C., H-f. G., I. Y. C.] and Biostatistics [G. H.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Of interest are
anti-ids (Ab2ß) that recognize the antigen-binding site of the
immunizing MoAb and mimic the original antigen. Because of this
mimicry, they form part of a self-regulating network (3)
.
Vaccination with anti-id has induced protective immunity against viral
(4)
, bacterial (5)
, and parasitic
(e.g., trypanosomiasis) infections (6)
. Anti-id
vaccines that mimic carbohydrate or glycolipid antigens have shown
immunological advantages over the natural antigen (7
, 8)
:
(a) unlike natural carbohydrate antigens, anti-id vaccines
can stimulate cellular in addition to humoral immune response
(9)
; and (b) anti-id vaccines may be more
immunogenic than the non-protein antigens they mimic. Several anti-ids
have been raised against MoAbs recognizing cell surface tumor targets.
Immunization of animals with these anti-ids can generate antibodies
(i.e., Ab3') that recognize the original tumor antigen
(10, 11, 12, 13, 14, 15, 16)
; in some reports, specific T-cell-mediated
immunity was also found (13
, 17, 18, 19, 20)
. The potential role
of an anti-idiotypic network in colon cancer was first described in
patients treated with monoclonal antibody CO-17-1A (21)
.
In subsequent studies, Ab2 and Ab3 (8
, 22, 23, 24, 25, 26)
and T-cell
immunity (13
, 18
, 20
, 27)
were demonstrated in responding
patients. Previously, we reported 34 patients with high-risk metastatic neuroblastoma treated with anti-GD2 monoclonal antibody 3F8 at the time of minimal disease. Thirteen patients remained progression free 4–12 years after the initiation of 3F8 treatment. A self-limiting HAMA response was found to be associated with long-term survival (28) . No late (after 4 years) relapse was seen. We postulate that an idiotype network might be responsible for the long-term tumor control. Natural antitumor antibodies have been described previously (29 , 30) , which correlated strongly with clinical outcome in patients with neuroblastoma. The antigen specificities include HUD (29) and a Mr 260,000 molecular weight cell membrane protein (31) . HUD is a nuclear antigen found in 78% of human neuroblastomas, and anti-HUD antibody was present in a small subset of patients. Among patients with small cell lung cancer, those with anti-HUD antibodies typically present with autoimmune paraneoplastic encephalomyelitis and/or paraneoplastic sensory neuronopathy, and these patients had longer survival. If 3F8 enhances tumor antigen processing or presentation, one might expect an enhanced antibody response to HUD, which in turn will have a positive effect on survival. In this report, we measure serum Ab3 (anti-anti-idiotype), Ab3' (anti-antigen), antimouse IgG3, and anti-HUD antibody responses and study their impacts on patient survival.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thirteen patients (group I)
were either in early relapse, second or subsequent remission, or with
stable microscopic disease; 21 patients (group II) were in first
remission and were treated with N6 chemotherapy, of whom 17 received
only N6, whereas 4 had some additional chemotherapy prior to N6
(28)
. Informed consent for 3F8 treatment was obtained in
accordance with Institutional Review Board guidelines. All 13 patients
in group I had failed at least one chemotherapy regimen, and 11 had
progressive or persistent disease after myeloablative therapies. Their
first relapses occurred at a median of 20 months from diagnosis (range,
7–88 months). Two patients were outliers, with biopsy-proven skeletal
relapse at 82 and 88 months from initial diagnosis. Eleven of these 13
patients achieved remission after reinduction therapy. 3F8 was
administered at a median of 8.6 months (range, 1.4 to 18.6 months) from
the time of relapse for these group I patients. For patients in first
remission (group II), 3F8 treatment was initiated at a median of 9.2
months (range, 6.7–14.6 months) from diagnosis after they finished
induction therapy. Because all 34 patients were treated with 3F8 at a
time of near-complete remission and their long-term survivals were
comparable, they were analyzed as one group in this report.
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Quantitation of Anti-GD2 IgG (Ab3') by ELISA.
Ninety-six-well, flat-bottomed polyvinyl microtiter plates (Dynex
Technologies, Chantilly, VA) were coated with antigen ganglioside
GD2 (Advanced Immunochemical, Long Beach, CA) at
20 ng/well. The purity of GD2 was >95%. Control
wells for serum background subtraction were not coated with antigen.
One hundred µl/well of 0.01% gelatin (Sigma Chemical Co., St. Louis,
MO) in PBS was used as filler protein to saturate unbound sites for
1 h at room temperature. After washing with PBS (plates were
washed three times with PBS between steps), patient sera (50 µl) in
duplicates were serially diluted in 0.03% BSA in PBS and were allowed
to react with GD2-coated plates for 2.5 h at
37°C. A standard curve was constructed using serial dilutions of
human-mouse chimeric 3F8. After being washed with PBS, a
peroxidase-conjugated affinity purified goat-anti-human IgG (heavy and
light chains) antibody (Jackson Immunoresearch, West Grove, PA) diluted
(1:1000 to 0.8 µg/ml) in 0.5% BSA in PBS was added to the wells.
After a 1-h incubation at 4°C and washing, a color reaction was
performed at room temperature using hydrogen peroxide as substrate and
o-phenylenediamine (Sigma) as chromogen. Upon stopping the
reaction with 30 µl of 5 N sulfuric acid, the
microtiter plates were analyzed at 490 nm using MRX microplate reader
(Dynex Technologies). Based on the titration curves using human-mouse
chimeric 3F8, anti-GD2 IgG (Ab3') titer was
calculated in units/ml of binding activity (each unit equivalent to 10
ng of chimeric 3F8). Because there was reactivity with light chains, we
could not rule out a low level of IgM in our measurements. Ab3' after
3F8 was expressed both as a ratio of pre-3F8 levels and as increments
of Ab3' over pretreatment levels. Conclusions drawn from either
calculation were similar. The day-to-day variability of these assays
was ±20%. Experiments were generally repeated once with similar
conclusions.
Quantitation of Human Ab3 by ELISA.
Six rat anti-3F8-ids (A1G4, idio2, C4E4, A2A6, C2H7, and C2D8; Ref.
32
) were digested using immobilized ficin (Pierce,
Rockford, IL) according to the manufacturer’s instructions. The size
of the fragment was confirmed by SDS-PAGE. Retention of binding of the
antibody F(ab')2 fragment to 3F8 was measured by
ELISA. Completeness of the digestion was assayed using
peroxidase-conjugated mouse antirat IgG secondary antibodies, specific
for F(ab')2 and Fc (data not shown).
A mixture of equal amounts of anti-idiotype F(ab')2 at 100 µg/ml was used to coat 96-well, flat-bottomed polyvinyl microtiter plates at 50 µl/well. Control wells without antigen were used for serum background subtraction. After incubation at 37°C for 1 h, the antigen was removed. One hundred µl of 0.01% gelatin (Sigma) in PBS were added as filler protein for 1 h at room temperature and then washed three times with PBS. Patient sera in duplicate serial dilutions were preincubated in diluent containing 50 µg/ml of 2E6 (a rat IgG1 specific for the Fc portion of 3F8) in 0.03% BSA in PBS for 1 h at 37°C to inhibit human antibodies directed against non-3F8-idiotypic determinants. Serum samples were then added at 50 µl/well to the microtiter plates. A standard curve was constructed using a human-mouse chimeric 3F8. After a 2.5-h incubation at 37°C and PBS washing, a peroxidase-conjugated goat antihuman IgG (H+L; Jackson Immunoresearch Laboratories) was added at 100 µl/well and allowed to incubate at 4°C for 1 h. Upon washing, color reaction was carried out. Absorbance of the wells were then read using an MRX microplate reader, and antibody titer, calculated in units/ml Ab3 after 3F8 treatment, was expressed as a ratio relative to pretreatment levels. When expressed as increments of Ab3 over pretreatment levels, similar conclusions were found.
Quantitation of Antimouse IgG3 and Anti-HUD by ELISA.
Ten µg/ml of NS.7, a mouse IgG3 myeloma (American Type Culture
Collection, Rockville, MD) was used to coat microtiter plates at 50
µl/well. Patient sera were diluted in 0.03% BSA in PBS, and the
anti-IgG3 titer was assayed using ELISA, as described previously
(28)
. Recombinant HUD protein, kindly provided by Dr.
Josef Dalmau (Department of Neurology, Memorial Sloan-Kettering Cancer
Center, NY), was used as antigen in coating microtiter plates. A
high-titer human anti-HUD serum (gift of Dr. J. Dalmau) was used for
constructing the standard curves. Anti-HUD titer was calculated in
units/ml based on the standard.
Statistical Methods.
The end points for this analysis were PFS and overall survival. PFS was
defined as the interval from first 3F8 treatment to progression, or
last follow-up. Overall survival was defined as the interval from first
3F8 treatment to death, or last follow-up. The score test from the
proportional hazards model was used to determine an association between
the PFS or overall survival rates and Ab3 or Ab3' titers (both titers
being continuous value assay measurements). Patients were deemed
evaluable if the length of their PFS or overall survival exceeded the
time period when antibody titers were measured (i.e., 6 and
14 months, respectively). The test of significance was based on a
permutation distribution of the score statistics. Permutation procedure
was applied because of the small number of subjects in this study
(33)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At diagnosis, distant metastases to bones were
present in 29 patients, and 31 patients had bone marrow disease. Most
patients had high-risk features: unfavorable histology (16 of 17),
serum lactate dehydrogenase >1500 units/ml (9 of 24), serum ferritin
>142 ng/ml (20 of 23), and MYCN >10 copies (7 of 25). Treatment with
3F8 was initiated 1.6–9.1 (median, 3.4) months after the last course
of chemotherapy. Patients received a total of 5–40 days (50–400
mg/m2) of 3F8. Fourteen patients are alive, and
13 (1.8–7.4 years at diagnosis) are progression-free (53–143 months
from the initiation of 3F8 treatment) without further systemic therapy.
Ab3' (Anti-GD2) Response and Clinical Outcome.
Sera collected around 6 and 14 months after the initial 3F8 treatment
were assayed for anti-GD2 antibody (Ab3')
activity by ELISA. Median Ab3' at 6 months was 40% above pretreatment
levels (i.e., in half of the patient group, Ab3' was 
40%
above pretreatment level; range, 40–1450%). When the median is used
as a cutpoint, patients with Ab3' above the median had a significantly
better long-term PFS than those below the median (71%
versus 21%, P = 0.02; Table 2
; Fig. 1A
); overall survival was also
significant (59% versus 25%, P = 0.04;
Table 2
; Fig. 1B
). Similar PFS and overall survival trends
(P = 0.21 and 0.16, respectively) were found for the 14
months post 3F8 titers (Table 2)
. Increasing the Ab3' threshold at
either 6 or 14 months to 300% above pre-3F8 levels increased the
separation between the high versus low Ab3 groups for both
PFS and survival curves (data not shown).
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; Table 2
) and survival
(59% versus 25%, P = 0.03; Fig. 2D
; Table 2
). At 14 months, the median Ab3 was 1.5, and the
correlation of Ab3 and outcome was also significant for both PFS (90%
versus 36%, P = <0.01) and overall
survival (65% versus 19%, P = 0.04). As
one might expect, stepwise increase in the cutpoint of Ab3 in the
statistical analysis (e.g., increasing the Ab3 threshold
from 1.2 to 1.9 in Fig. 2
) widened the separation in the PFS and
survival curves between the Ab3-high and Ab3-low groups. Using the
permutation test based on score statistics from the Cox proportional
hazard model, the Ab3 level was a strong predictor of survival,
especially if measured at 14 months from first 3F8 treatment (Table 3)
. On the other hand, Ab3' was
statistically less significant.
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. The 6-month sera were then assayed for general antitumor activity
using HUD as the target antigen. Previous studies have shown that
patients with neuroblastoma or small cell lung cancer developed
anti-HUD antibodies that appeared to correlate with survival (29
, 34)
Again, the PFS and survival durations between the high- and
low-titer groups were not significantly different (Fig. 3
; Table 2
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although Ab3 titers were measured
within the first 14 months in this study, such titers appeared to
persist for years in the few patients we studied (data not shown).
Because these patients have remained in remission from 4 to 12 years
since, one might hypothesize that such titers may be responsible for
long-term tumor control. Previously, we observed that only the pattern
II response (i.e., transient low HAMA/Ab2 titer) was
associated with superior clinical outcome (28)
. We
interpret these findings to mean that high-titer Ab2 was not inducive
to idiotype network formation. Furthermore, the initiation of the
network appeared relatively early after antibody treatment. Because the
number of cycles of 3F8 was also associated with good outcome
(28)
, we hypothesize that a potential strategy in
anti-GD2 immunotherapy might be to continue to
measure Ab3' and Ab3 responses after each subsequent cycle of 3F8 until
the ratio of post/pre levels above a threshold level is reached. Our observation of the detrimental effect of high-titer HAMA/Ab2 has potential implications in the mechanism of the idiotype network. In contrast to patients with no HAMA/Ab2 response or those with persistently high titer, patients with self-limited HAMA/Ab2 response had significantly better PFS. HAMA formation may reflect an intact immune surveillance system, indirectly responsible for tumor control. However, general measures of global immune function do not correlate with tumor control among patients with neuroblastoma (35) . More importantly, patients with the highest persistent HAMA response were generally not able to benefit from 3F8 treatment compared with those with the self-limited HAMA titer. In our analysis, antimouse and anti-HUD antibody titers (as a general measure of immune function) did not correlate with outcome. The alternative explanation invokes the idiotypic network of Jerne (36) that takes into account this paradoxical dose relationship between high levels of HAMA and failure to mount a substantial Ab3' or Ab3 response. Because GD2 is a self-antigen, one can hypothesize that significant rises in Ab3 or Ab3' would not be possible unless suppressor pathways are removed and naive T or B cells are allowed to repopulate. After intensive chemotherapy that eliminates a large part of the lymphoid system, exposure to tumor-selective Ab1s that induce unique Ab2s may bias the recovering repertoire toward the specific antigen network. One can speculate that a weak HAMA/Ab2 response may be an indirect measure of a relatively "vacant" immune system. A high HAMA/Ab2 titer, on the other hand, may reflect a healthier but saturated lymphoid system with less chance for antigens to create a repertoire bias. The potential for biasing the immune system toward specific antigens has been well documented in murine models (37) and human disease states (38 , 39) . If true, one would expect such idiotype network to be successful only after intensive immuno-/myelosuppressive therapy. We have reported previously the association of Ab3 with prolonged survival among patients after autologous bone marrow transplantation (40) . In other words, such network will not be easily induced if fully immunocompetent patients are treated with Ab1. An alternative explanation may lie in the quality of the HAMA response. In some murine models, only IgM anti-ids can induce a significant Ab3 response, whereas IgG anti-id was suppressive (41) . It was postulated that in a normal antibody response, dominant B-cell clones (Ab2) would preempt Ab3 response against themselves by early switching from IgM to IgG secretion, before the immunogenic IgM Ab had time to activate anti-idiotypic B cells. After treatment with 3F8, two patterns of HAMA were noted (28) . The pattern II HAMA response (low titer and transient) might represent an IgM anti-idiotype response, whereas the high and persistent pattern III HAMA might represent an IgG anti-idiotype response. This model could also explain why in most clinical studies of monoclonal antibodies, such an anti-idiotype network was not observed. Only in patients with heavy prior chemotherapy would T cells be significantly depleted such that class switch (IgM to IgG) would not occur. One can speculate that a protective idiotype network may be possible in patients receiving MoAbs if they were optimally timed with chemotherapy. Alternatively, immune modulators to prevent Ab2 class switch may improve the idiotype network. The suppressive effect of IgG anti-id was consistent with our findings in animals models, where the optimal dose range for anti-GD2 antibody induction was unexpectedly low,4 suggesting that anti-ids can be tolerogenic when administered at conventional/high doses. Clearly, this could be detrimental if the host antitumor response contributed to tumor control.
It is noteworthy that the clinical benefit from Ab1 treatment correlated with antibody titer of Ab3 or Ab3'. As one might expect, the higher the titer at 6 or 14 months resulted in better outcome. For GD2, this is equivalent to breaking self-tolerance by tipping the idiotype network balance toward Ab3' and possibly higher orders of network antibodies. More importantly, this network of antibodies must be sufficiently self-regulated so that clinical signs or symptoms of autoimmunity would not appear, even when tumor control was successful. It is of interest that the timing of the Ab3'/Ab3 peak coincided with a declining or undetectable HAMA/Ab2 titer. A trivial explanation was the interference by Ab2 in Ab3' or Ab3 assays, such that Ab3' or Ab3 always mirrored the rise and the fall of Ab2. However, there were patients who had undetectable or declining HAMA/Ab2, as well as undetectable Ab3' or Ab3 response. We like to interpret this to mean that the disappearance of Ab2, although conducive, is not sufficient for an Ab3'/Ab3 response. It is possible that strategies to break tolerance at the peak of the Ab2 response may facilitate Ab3'/Ab3 formation and render clinical use of anti-GD2 antibodies more effective.
The association of anti-idiotypic response and improved outcome has been described in other tumor systems, e.g., 17-1A for colon cancer (25 , 26 , 42, 43, 44) and Ca125 for ovarian cancer (45, 46, 47, 48) , although the paradoxical dose relationships of high HAMA/Ab2 and outcome have not been described previously. It is also possible that Ab1 was bound to free antigen and became complexed with HAMA, thereby allowing the antigen to be presented more efficiently to the immune system. Clearly, for these complexes to form, significant amounts of free antigens had to be available in circulation. Our patients were in complete or near-complete remission and generally had no detectable circulating GD2 antigens. Alternatively, the idiotype network could be responsible for the induction of anti-GD2 or antitumor immune response (a subset of the Ab3), which may play a key role in long-term tumor control. In our study, the correlation of Ab3' with outcome was generally not as good as Ab3, especially by 14 months after 3F8 treatment. It is possible that Ab3 represents an indirect measure of an antitumor repertoire broader than anti-GD2 (Ab3') alone, thus providing a better association with clinical outcome. Alternatively, because GD2 is expressed on tumors and some normal tissues, the Ab3' level is dependent on the tumor load ("acting as a sink") and antibody avidity (which affects clearance), whereas Ab3 (most of which is not tumor-binding) is free to circulate and thereby easier to quantify. Furthermore, in our Ab3 assay, a panel of six anti-idiotypic specificities were used. It is possible that only certain idiotopes (as defined by anti-idiotypic antibodies) will be correlated with patient outcome, thereby providing a useful roadmap in our choice of anti-idiotypic vaccines. Further analysis will be needed to identify these protective epitope(s).
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FOOTNOTES |
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1 Supported in part by Grant CA61017 from the
National Cancer Institute and grants from the Robert Steel Foundation,
the Justin Zahn Fund, and the Katie-Find-a-Cure Fund. 
2 To whom requests for reprints should be
addressed, at Memorial Sloan Kettering Cancer Center, 1275 York Avenue,
New York, NY 10021. Phone: (212) 639-8401; Fax: (212) 744-2245;
E-mail: cheungn{at}mskcc.org
3 The abbreviations used are: MoAb, monoclonal
antibody; HAMA, human antimouse antibody; ids, idiotypic antibodies;
PFS, progression-free survival.
Received 10/28/99; revised 2/23/00; accepted 3/29/00.
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, high Ab3' group; 
, low Ab3' group. The high
Ab3' group has a titer above the median (post/pre ratio of 1.2).
