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Vaccination with a Bivalent G_M2 and G_D2 Ganglioside Conjugate Vaccine: A Trial Comparing Doses of G_D2-Keyhole Limpet Hemocyanin¹

Department of Medicine [P. B. C., P. O. L.], Department of Epidemiology and Biostatistics [K. S. P.], Department of Nursing [L. W.], and Department of Surgery [J. J. L.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Progenics Pharmaceuticals, Inc., Tarrytown, New York 10591 [D. M., R. J. I., W. B. H.]

	ABSTRACT

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Immunization with GMK vaccine (G_M2 ganglioside conjugated to keyhole limpet hemocyanin mixed with QS-21 adjuvant) induces anti-G_M2 antibodies in close to 100% of patients. We found previously that anti-G_D2 antibodies could be induced in some patients using G_D2-keyhole limpet hemocyanin + QS-21 (GDK). In this trial, we wished: (a) to determine whether immunization with both GMK and GDK vaccines could induce antibodies against both G_M2 and G_D2; and (b) to determine the optimal dose of GDK. Thirty-one patients with melanoma or sarcoma who had no evidence of disease after complete surgical resection were immunized with both GMK (30 µg of G_M2) and GDK on weeks 1, 2, 3, 4, 12, 24, and 36. Patients were assigned to one of five GDK dose levels (3, 10, 30, 70, or 130 µg of G_D2). Anti-G_M2 IgM or IgG were induced in 97% of patients. The dose of GDK did not affect the anti-G_M2 response, although at the highest GDK dose level, 3 of 7 patients did not make anti-G_M2 IgG. GDK was less immunogenic; overall 45% of patients developed either IgM or IgG against G_D2. At GDK doses of 30 or 70 µg, 8 of 11 patients (73%) made either IgM or IgG anti-G_D2 antibodies. We conclude that both GMK and GDK vaccines can induce antibodies against G_M2 and G_D2 in a majority of patients and are safe. The optimal dose of GDK appears to be either 30 or 70 µg when administered with GMK vaccine.

	INTRODUCTION

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Gangliosides expressed on melanoma are attractive targets for immunotherapy. They are abundantly expressed, are not down-regulated when bound by antibody, do not require HLA molecule coexpression for the immune system to react to them, and may play an important role in melanoma-cell adhesion. In addition, it is clear that patients with melanoma can generate antibodies against ganglioside antigens (1, 2, 3) . Among the gangliosides expressed by human melanomas, G_M2 appears to be the most immunogenic. Immunization with G_M2 and BCG adjuvant induced anti-G_M2 IgM in more than 85% of patients, and the presence of either vaccine-induced or natural antibodies against G_M2 correlated with an improved relapse-free survival (4) . Subsequent studies showed that immunization with G_M2 conjugated to KLH⁴ and mixed with QS-21 (5 , 6) adjuvant (GMK vaccine) induced anti-G_M2 IgM and IgG in close to 100% of patients (3 , 7) . On the basis of these observations, we determined that GMK vaccine resulted in optimal immunogenicity at a G_M2 dose of 30 µg.

Because not all melanomas express G_M2 (8) , it seems likely that a maximally effective ganglioside vaccine will need to include multiple gangliosides. G_D2 ganglioside is expressed in many melanomas and sarcomas, and monoclonal antibodies against G_D2 can induce antitumor effects in patients with melanoma or neuroblastoma. Although antibodies against G_D2 can be induced by immunizing patients with G_D2-KLH plus QS-21 (9) , the optimal G_D2 dose has not been determined, and it is not known whether a bivalent ganglioside vaccine can induce antibodies against both G_M2 and G_D2.

We carried out a trial using a mixed ganglioside conjugate vaccine consisting of both G_M2-KLH and G_D2-KLH with the adjuvant QS-21. G_M2-KLH was administered at a fixed dose of 30 µg of G_M2/immunization with one of five G_D2-KLH doses. The primary goals were to assess the anti-G_M2 antibody responses induced, determine which G_D2-KLH doses induced anti-G_D2 antibodies, and to determine whether this mixed ganglioside vaccine was safe.

	MATERIALS AND METHODS

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Vaccine Preparation
G_M2-KLH and G_D2-KLH were manufactured by Progenics Pharmaceuticals, Inc. (Tarrytown, NY) as described previously (3 , 10) . QS-21 was supplied by Aquila BioPharmaceuticals (Framingham, MA).

Patient Eligibility
Patients with American Joint Committee on Cancer stage III or IV melanoma 2 weeks to 1 year after complete surgical resection were eligible for the study. After the first 24 patients had been entered, the protocol was amended to allow patients with metastatic sarcoma. Patients had to be free of disease, older than 18 years of age, and have a Karnofsky performance status 80%. Previous radiotherapy, chemotherapy, or immunotherapy were permitted, but patients must have completed treatment >4 weeks before starting the protocol. Patients were required to have WBC 3.0 cells/mm³, platelets 100,000/mm³, total bilirubin 2.0 mg/dl, aspartate aminotransferase 74 U/l, lactate dehydrogenase 400 U/l, and alkaline phosphatase within normal limits. All patients signed written informed consent before participating in the study.

Patients were excluded if they had had another malignancy within the past 5 years (other than basal cell, squamous carcinomas of the skin, or cervical carcinoma in situ), or if they had a medical condition which might make it difficult to complete the full course of treatments or to respond immunologically.

Treatment Plan
Before treatment, all patients had a chest X-ray or chest computed tomography showing no evidence of disease. Additional radiographic tests were performed as necessary to confirm that the patient was free of disease. ß-human chorionic gonadotropin was measured within 2 weeks of starting vaccination in women of child-bearing potential to make sure they were not pregnant. All patients had an electrocardiogram within 10 months of the start of treatment.

Patients were injected s.c. with a mixture of G_M2-KLH, G_D2-KLH, and QS-21 (MGV vaccine). Vaccines were administered weekly for 4 weeks, then on weeks 12, 24, and 36. G_M2-KLH was administered at a G_M2 dose of 30 µg; QS-21 was administered at a dose of 100 µg. Initially, patients were randomized to receive one of four G_D2-KLH dose levels: 3, 10, 30, or 70 µg of G_D2. After the first 24 patients, additional patients were accrued into a fifth G_D2-KLH dose level of 130 µg in a nonrandomized manner.

After seven immunizations with G_M2-KLH and G_D2-KLH, patients were observed until week 60. At that time, if they remained free of disease, they were eligible to receive three additional booster immunizations with G_M2-KLH + QS-21 (no G_D2-KLH) at 4-month intervals.

Clinical Evaluation
Patients underwent a history and physical exam at Memorial Hospital on weeks 4, 12, 24, and 48, which corresponded to vaccines 4, 5, 6, and 7. In addition, patients were examined at the times of booster immunization (weeks 60, month 18, and month 22). Chest X-rays or chest computed tomograms were obtained on weeks 12, 36, 48, and at months 18 and 22. Toxicity was scored using standard criteria (11) .

Serological Evaluations
ELISA.
Serum was collected immediately before each vaccination (including pretreatment), as well as 2 and 6 weeks after vaccines four through seven. A final serum was collected on week 60. In patients who received G_M2-KLH booster vaccinations in year 2, serum was collected at the time of each vaccination and 1 month later. Anti-G_M2 and anti-G_D2 antibodies were measured using an ELISA method in which ganglioside is adsorbed to 96-well polystyrene microtiter plates. Remaining binding sites on the plate were blocked by PBS/Casein/Tween 20 buffer. Serially diluted patient sera or controls were added, and bound antibody was detected using a goat antihuman IgM or IgG antibody (heavy chain-specific) conjugated to alkaline phosphatase. Plates were developed using p-nitrophenyl phosphate substrate and absorbance read at 405 nm with a correction of 620 nm. Antibody titer was defined as the highest dilution of patient serum yielding a corrected absorbance of 0.1. Pooled human serum from previously vaccinated patients with a known anti-G_M2 or anti-G_D2 antibody titer or pooled normal human serum with no reactivity were used as positive and negative controls, respectively.

Dot Blot Immunoassay.
The specificity of the ganglioside antibody response was analyzed by dot blot immune staining. Ganglioside standards G_M2, G_M3, and G_D2 (Sigma Chemical Co., St. Louis, MO) were applied to polyvinylidene difluoride membranes using a dot blot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). Nonspecific binding to the membrane was blocked with PBS/casein/Tween 20 buffer. Immune serum was added and then goat antihuman IgM- or IgG-specific antibody conjugated to horseradish peroxidase. Specific binding was visualized by chemiluminescence staining. Serum was considered positive if the staining was dose-dependent and specific compared with normal control human serum.

Flow Cytometry.
Single cell suspensions of G_M2⁺ melanoma cells (SK-MEL-31) or G_D2⁺ melanoma cells (SK-MEL-24) were incubated with pre- or postvaccination serum in PBS with sodium azide on ice for 30 min. The cells were washed with complete culture medium and incubated for 30 min on ice with FITC-conjugated rabbit antihuman IgM or IgG. Cells were washed once, resuspended in paraformaldehyde buffer and analyzed on a FACSCaliber (Becton Dickinson). Patients were considered positive if there was a shift in the relative staining intensity of the postvaccination serum of 20% compared with prevaccination serum.

ITLC.
G_M1, G_M2, G_M3, G_D1b, and G_D2 ganglioside standards were separated on a 10 x 10-cm silica plate in chloroform:methanol:water (65:35:6) solution and allowed to air dry. Plates were coated in 5% methacrylate/chloroform-hexane (10:90) for 1 min and air-dried in a fume hood for 20 min. Plates were then blocked in PBS/BSA buffer. Pre- and postvaccination sera were added and then peroxidase-conjugated goat antihuman immunoglobulin. Plates were washed and developed with New England Nuclear Enhanced Chemiluminescence Reagent. Patients were considered positive if specific ganglioside staining was seen in the postvaccination serum compared with prevaccination serum.

On the basis of our previous experience (4) , a positive serological response against G_M2 ganglioside was defined as an ELISA titer 1:40. For anti-G_D2 responses, a positive serological response was defined as a titer 1:80 as measured by ELISA in the setting of confirmatory positive staining by dot blot, ITLC, or flow cytometry. This more stringent criterion was required for anti-G_D2 responses to be certain that we were not confounded by background binding.

	RESULTS

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Patients Treated.
Between September 1996 and October 1997, 24 patients were entered into the trial and randomized between one of four G_D2-KLH dose levels (3, 10, 30, or 70 µg). After October 1997, an additional seven patients were entered at a G_D2-KLH dose level of 130 µg. Overall, 31 patients were treated (Table 1) . There were 20 men and 11 women. Twenty-four patients had stage III melanoma; six had stage IV. One patient had synovial cell sarcoma metastatic to lung, which had been completely resected.

Of the 27 patients who completed the first seven immunizations, 22 received either one (5 patients), two (4 patients), or three (13 patients) additional vaccinations with GMK alone at 4-month intervals.

Immunogenicity of G_M2 Ganglioside.
Previous studies demonstrated that immunization with GMK at a G_M2 dose of 30 µg or 70 µg induces anti-G_M2 IgM antibodies in 100% of patients and IgG antibodies in 85% of patients (7) . Fig. 1 shows the peak anti-G_M2 titers observed in the patients immunized in the current trial. Before starting immunization, three patients (10%) had detectable anti-G_M2 antibodies (two patients with anti-G_M2 IgM at 1:40 and 1:80 and one patient with anti-G_M2 IgG at 1:40). Although this is a slightly higher incidence of natural anti-G_M2 antibodies than our previous experience of 5% (4) , the difference was not statistically significant. The peak anti-G_M2 titers ranged from 0–1:2560 for IgM and from 0–1:5120 for IgG.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Peak anti-GM2 IgM and IgG antibody titers. Each dot represents peak titers from an individual patient. The horizontal lines represent the median titer for each GD2 dose level. A titer of 1:40 was considered to be a positive anti-GM2 response. One patient each in the 10-µg and the 70-µg dose levels had pretreatment IgM anti-GM2 titers of 1:80 and 1:40, respectively. One patient in the 3-µg dose level had pretreatment IgG anti-GM2 titers of 1:40. These patients are indicated by the encircled dots.

Immunogenicity of G_D2 Ganglioside.
As expected from our previous experience, G_D2 was less immunogenic than G_M2. Six of 31 patients (19%) developed an IgM anti-G_D2 response; 12 patients (39%) developed an IgG anti-G_D2 response. Overall, 14 patients (45%) developed either an IgM or an IgG anti-G_D2 response (Table 3 ). No patient had detectable anti-G_D2 antibodies before immunization. All patients who developed anti-G_D2 antibodies also developed anti-G_M2 antibodies. In three patients, binding to cell-surface G_D2 was confirmed by flow cytometry; 12 of 14 patients had binding confirmed by ITLC, which also demonstrated lack of antibody induction against gangliosides G_M1 and G_D1b. The IgM anti-G_D2 response seemed best at the 70-µg dose level; three of five patients developed IgM anti-G_D2 antibodies at titers of 1:80 (Fig. 2) . The IgG anti-G_D2 response seemed equivalent at doses 30 µg in terms of the proportion of patients developing IgG anti-G_D2 antibodies (range, 43–67%), although the median peak titers of IgG anti-G_D2 seemed to be highest at doses of 30 µg and 70 µg (1:320 and 1:160, respectively). Considering the 30-µg and 70-µg cohorts together, 73% of patients (8 of 11) developed anti-G_D2 IgM or IgG. The median number of vaccinations required to induce peak IgG anti-G_D2 titers at these two dose levels was six immunizations. From these observations, it appears that when administered with G_M2-KLH at a G_M2 dose of 30 µg, the optimal dose of G_D2-KLH vaccine is 30–70 µg.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Peak anti-GD2 IgM and IgG antibody titers. Each dot represents peak titers from an individual patient. The horizontal lines represent the median titer for each GD2 dose level. A titer 1:80 with confirmatory binding demonstrated by either flow cytometry, ITLC, or dot blot was considered to represent an anti-GD2 response.

	DISCUSSION

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In designing a defined, multivalent ganglioside vaccine, we have built upon our experience immunizing against G_M2 ganglioside. When patients were immunized with G_M2 + BCG, 85% developed IgM anti-G_M2 antibodies but only 10% developed IgG anti-G_M2 antibodies (4) . Still, it was possible to show that, even with this level of immunogenicity, antibodies against G_M2 (whether natural or vaccine-induced) correlated with an improved relapse-free survival among patients with stage III melanoma who were free of disease after complete surgical resection. This was consistent with previous observations suggesting that naturally occurring antibodies against G_M2 were associated with improved prognosis (15) . Subsequent studies showed that conjugating G_M2 to KLH and using the adjuvant QS-21 resulted in a more robust, durable, and consistent immune response against G_M2. Using this formulation, essentially all patients develop both IgM and IgG antibodies against G_M2 (7) . The hypothesis that higher titers of anti-G_M2 antibodies and the induction of long-lived IgG anti-G_M2 antibodies will result in an improved therapeutic effect is currently being addressed in an intercooperative group randomized trial in which patients were randomized to receive G_M2-KLH + QS-21 or IFN-2b after surgery for stage III melanoma.

Because G_M2 is not expressed on all melanomas, it was logical to build a multivalent ganglioside vaccine that could induce antibodies against even G_M2-negative tumors. G_D2 ganglioside is the next most immunogenic melanoma ganglioside after G_M2, and so it was logical to try immunizing patients using both G_M2-KLH and G_D2-KLH. It was not obvious, however, that the immune response against this combination would be simply additive. In principle, it was possible that one of the gangliosides would prove to be an immunodominant epitope which could prevent an antibody response against the other ganglioside. Because of this, we wished to determine whether it was possible to immunize patients against both G_M2 and G_D2, and to determine what was the optimal dose of G_D2.

The results from this study confirmed that a 30-µg dose of G_M2 conjugated to KLH is highly immunogenic when given with QS-21; 97% of patients developed antibodies against G_M2. For the most part, the immunogenicity of G_M2 was not affected by the coadministration of G_D2-KLH, although at the highest dose of G_D2-KLH (130 µg), only four of seven patients developed anti-G_M2 IgG.

As expected, GDK was less immunogenic than GMK. Overall, only 14 of 31 (45%) patients developed anti-G_D2 antibodies. It appeared that the 30- and 70-µg dose levels were most efficient in inducing both IgM and IgG anti-G_D2 antibodies (8 of 11 patients or 73%). At these two dose levels, median peak anti-G_D2 titers (1:30–1:80 for IgM and 1:160–1:320 for IgG) were lower than the anti-G_M2 titers. These anti-G_D2 antibody responses were not associated with the toxicities seen in patients infused with anti-G_D2 monoclonal antibodies, such as pain or motor weakness (12, 13, 14) . This was presumably attributable to the much lower antibody levels achieved in our patients immunized with G_D2-KLH compared with patients infused with anti-G_D2 monoclonal antibodies.

We have demonstrated for the first time that it is possible to induce humoral immune responses to two ganglioside targets by simultaneous vaccination in the form of a bivalent ganglioside conjugate vaccine. Although most melanomas express either G_M2 or G_D2 (or both), an optimal multivalent ganglioside vaccine would include G_D3 as well, because essentially all melanomas express G_D3 and at levels much higher than either G_M2 or G_D2. Attempts to immunize patients against G_D3 have shown that G_D3 is far less immunogenic than either G_M2 or G_D2. Despite this, there is evidence that it is possible to induce anti-G_D3 antibodies in a subset of patients by immunizing either with G_D3 lactone-KLH (16) or with BEC2, an anti-idiotypic monoclonal antibody that mimics G_D3 (17) . It is hoped that these studies will lead to a multivalent ganglioside vaccine that induces antibodies against the three major melanoma gangliosides: G_M2, G_D2, and G_D3.

	ACKNOWLEDGMENTS

 
We are grateful to Deirdre McGagh and Sandi Holland for data management and to the Immunology Nurses at the Memorial Sloan-Kettering Cancer Center. Amy J. Chodera and Cenchen Zhan provided expert technical assistance in carrying out the serological analyses.

	FOOTNOTES

 
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 This study was supported by National Cancer Institute PO1 CA33049 and by Progenics Pharmaceuticals, Inc.

² To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center; 1275 York Avenue, Room K718, New York, NY 10021. Fax: (212) 794-4352.

³ P. O. L. is a paid consultant and stockholder in Progenics Pharmaceuticals, Inc.

⁴ The abbreviations used are: KLH, keyhole limpet hemocyanin; ITLC, immuno-thin layer chromatography.

Received 6/ 3/00; revised 9/26/00; accepted 9/26/00.

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