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Clinical Cancer Research Vol. 10, 6094-6100, September 15, 2004
© 2004 American Association for Cancer Research

Clinical Trials

Vaccination of Patients with Small-Cell Lung Cancer with Synthetic Fucosyl GM-1 Conjugated to Keyhole Limpet Hemocyanin

Lee M. Krug1, Govind Ragupathi2, Chandra Hood2, Mark G. Kris1, Vincent A. Miller1, Jennifer R. Allen3, Stacy J. Keding3, Samuel J. Danishefsky3, Jorge Gomez1, Leslie Tyson1, Barbara Pizzo1, Valerie Baez1 and Philip O. Livingston2

1 Thoracic Oncology Service and 2 Laboratory of Tumor Vaccinology, Department of Medicine, and 3 Laboratory of Bioorganic Chemistry, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Immunotherapy directed toward cell surface antigens may provide a novel approach to the eradication of chemoresistant micrometastatic disease in patients with small-cell lung cancer (SCLC). Studies in SCLC cell lines and human tissues suggest that the ganglioside fucosyl GM1 is an abundant yet specific target. A prior clinical study demonstrated the potent immunogenicity of fucosyl GM-1 derived from bovine thyroid gland, conjugated to keyhole limpet hemocyanin (KLH) and administered with QS-21 adjuvant.

Experimental Design: We tested the immunogenicity of three different doses of a synthetic version of fucosyl-GM1 in patients with SCLC after a major response to initial therapy. The primary end point was to establish the lowest effective dose capable of inducing antibody production.

Results: Five of six patients at the 30-µg dose and three of five patients at the 10-µg dose mounted IgM responses of 1:80 or greater. These antibodies were confirmed by flow cytometry in seven of eight cases. None of the patients at the 3-µg dose had titers above 1:80. One patient at the 30-µg dose had an IgG response with a titer of 1:80. The sera from six of the eight responders induced potent complement-mediated cytotoxicity of tumor cells.

Conclusions: Vaccination with the synthetic fucosyl GM1-KLH conjugate induces an IgM antibody response against fucosyl GM1 and tumor cells expressing fucosyl GM1, comparable with the response induced by the bovine derivative. We plan to combine synthetic fucosyl GM1 vaccine at a dose of 30 µg with vaccines against three other antigens—GM2, Globo H, and polysialic acid—to test in patients with SCLC after initial chemotherapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The currently accepted treatment for small-cell lung cancer (SCLC) involves four to six cycles of etoposide plus cisplatin or carboplatin, with concurrent thoracic radiation added for patients with limited-stage disease. In the majority of cases, patients derive substantial tumor reduction with this treatment, and for about 20 to 25% of patients with limited-stage disease, this can represent curative therapy. However, the remaining patients with limited-stage disease, and essentially all patients with extensive-stage disease, still die from chemoresistant disease that often returns just months after initial therapy is completed. Because our currently available treatments do not eradicate residual disease, different strategies need to be explored. One approach involves the induction of a host immune response to attack chemoresistant tumor cells.

Because of its neuroectodermal origin, SCLC has a number of specific antigens that could serve as immune targets. To identify the most appropriate carbohydrate antigens to use, we analyzed a series of tumor samples and normal tissues by immunohistochemistry (1) . With the use of a specific mouse monoclonal antibody, F12, (2) fucosyl-GM1 (Fuc{alpha}1–2Galß1–3GalNAcß1–4[NeuAc{alpha}2–3]-Galß1–4Glcß1–1Cer) was identified in nearly all cases of SCLC (3 , 4) . In contrast, fucosyl GM1 was detected in only 2 of 10 squamous cell tumors, 1 of 5 large-cell tumors, 0 of 8 adenocarcinomas, and 0 of 3 carcinoids. Uptake was also noted in small round cells of the thymus, spleen, small intestine, and pancreatic islet cells. No fucosyl GM1 was detected in normal lung or other normal tissues. Fucosyl GM1 is expressed more frequently and more abundantly on SCLC tumors than are two other gangliosides, GD3 and GM2 (5) . The presence of fucosyl GM1 has been demonstrated by high-performance thin-layer chromatography immunostaining in culture media from SCLC cell lines, and in tumor extracts and serum of nude mouse xenografts (6) . Furthermore, it was detected in the serum of 4 of 20 SCLC patients, all of whom had extensive-stage disease. Fucosyl GM1 was not detected in the serum of 12 patients with non–SCLC or in 20 healthy volunteers (6) .

A prior clinical study demonstrated the potent immunogenicity of bovine fucosyl GM1 (7) . Thirteen SCLC patients (4 limited stage, 9 extensive stage) who had achieved a major response to initial chemotherapy were immunized with fucosyl-GM1 (30 µg) extracted from bovine thyroid gland, conjugated to keyhole limpet hemocyanin (KLH), and administered with the adjuvant QS-21. Six vaccinations were planned over 16 weeks. All 10 patients who completed five vaccinations mounted both IgM and IgG titers of ≥1:40 against Fuc-GM1 as measured by ELISA. Toxicities were mild and transient and included local skin reactions, flu-like symptoms, diarrhea, and fatigue. Six patients had sensory peripheral neuropathy worsen by one grade. Three patients with limited-stage SCLC were relapse-free at 18, 24, and 30 months at the time this trial was reported.

Fucosyl-GM1 was subsequently synthesized with a glycal assembly method similar to that used previously for the synthesis of other carbohydrate antigens (8, 9, 10) . The construct was armed with a terminal pentenyl group in place of ceramide or allyl glycoside. In the presently reported trial, we tested the immunogenicity of the synthetic version of fucosyl-GM1 in patients with SCLC after a major response to initial therapy. As in prior trials, we conjugated fucosyl GM1 to the carrier protein KLH, and coadministered it with the immunologic adjuvant QS-21 to overcome immunologic tolerance (11, 12, 13, 14) .


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Selection.
We enrolled adult patients with SCLC, limited or extensive stage, who had completed initial therapy with chemotherapy (and radiation if needed) at least 4, but not more than 12 weeks previously. Patients needed to have a Karnofsky performance status of 70% or greater. Required hematologic and biochemical measurements included a total white blood count ≥3.0 x 106 cells/µL, a total lymphocyte count ≥0.5 x 106 cells/µL, aspartate aminotransferase level ≤1.5 x upper limit of normal, serum bilirubin ≤1.5 mg/dl, and serum alkaline phosphatase level ≤1.5 x upper limit of normal. Patients with immune deficiency or autoimmune disease, prior splenectomy or splenic radiation, or patients on oral corticosteroids were excluded. Patients could not have peripheral neuropathy greater than grade 1 at baseline. Because of the potential cross-reaction with islet cells, patients with type II diabetes mellitus were excluded. Pregnant or lactating women, patients with New York Heart Association class III or IV heart failure, or patients with another active malignant disease in the last 5 years were also excluded.

Within 3 weeks of starting treatment, all of the patients underwent a history and physical examination including neurologic examination, chest X-ray, complete blood count, and biochemical profile, including amylase. Premenopausal women were required to have a negative pregnancy test. A computed tomography scan was done after completion of chemotherapy to document disease status. For patients lost to follow-up, survival status was determined by using the Social Security Death Index.4

Immunization.
To establish the lowest effective dose capable of inducing antibody production, three dose levels of fucosyl GM1-KLH conjugate (30, 10, and 3 µg) were studied. The initial dose level of 30 µg was chosen based on the prior study of the fucosyl-GM1 vaccine extracted from bovine thyroid gland. The dose of QS-21 was 100 µg at all dose levels. Vaccinations were administered intradermally on weeks 1, 2, 3, 4, 8, and 16.

Vaccine Preparation.
Fucosyl-GM1 was synthesized as a pentenyl glycoside with the glycal assembly method (8 , 10) . The pentenyl group of the fully synthetic fucosyl-GM1 construct was converted to an aldehyde group by ozonolysis and linked to the -NH2 group of the heterobifunctional cross-linker 4-(4-N-maleimidomethyl) cyclohexane-1-carboxyl hydrazine (MMCCH) as previously described (15) . The maleimide group of MMCCH was then reacted with thiolated KLH. The schema of synthesis and the structure of the synthetic fucosyl GM1 compared with the previously tested extracted fucosyl GM1 are demonstrated in Fig. 1Citation . In the previous study, the pentenyl in fucosyl-GM1 isolated from bovine extracts was converted to an aldehyde group by ozonolysis and treatment with methyl sulfide, which was followed by direct linkage to NH2 groups on KLH with sodium cyanoborohydride. KLH was obtained from Progenics (Tarrytown, NY). The fucosyl-GM1-KLH conjugate was washed and filtered to confirm sterility, and conjugate containing 30 µg of fucosyl-GM1 was aliquoted into individual vials containing saline with 100 µg of the immunologic adjuvant QS-21 [obtained from Antigenics Pharmaceuticals (New York, NY)].



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  		Fig. 1. Schema for synthesis of synthetic Fucosyl GM1 and comparison of different forms of Fucosyl GM1

 
Safety Testing.
Samples from the vial materials were tested for sterility and safety, and immunogenicity was confirmed in mice.

Serologic Assays.
Techniques for ELISA assays, flow cytometry, and complement-dependent cytotoxicity assays, detailed previously (7) , are briefly described below. All of the data points were repeated once on a separate day, and three or more times in some cases.

ELISA.
Serial dilutions of patient sera were placed on Nunc microwell plates coated with purified natural Fuc-GM1 ganglioside. Alkaline-phosphatase-conjugated goat antihuman IgM or IgG was added. After incubation and washing, plates were developed with Sigma 104 phosphatase substrate (Sigma Diagnostics, St. Louis, MO). Absorbance was measured at 414 nm, and the highest dilution with an absorbance of at least 0.100 was defined as the antibody titer.

Fluorescence-Activated Cell Sorting.
Fluorescence- activated cell sorting (FACS) was done on the human SCLC cell line DMS 79. Patient sera were added to cell pellets and were incubated with either FITC-labeled goat antihuman IgM or FITC-labeled goat antihuman IgG. The percentage of positive cell population and the mean fluorescence intensity of the stained cells were analyzed by flow cytometry (FACScan, Becton Dickinson, CA). Pre- and postvaccination sera were analyzed together. Prevaccination sera were used to set the FACScan result at 10% as background for comparison with percentage positive cells with postvaccination sera. The mouse anti-Fuc-GM1 monoclonal antibody, F12, was used as a positive control.

Complement-Dependent Cytotoxicity Assays.
Complement-dependent Cytotoxicity Assay (CDC) was done by a 2-hour chromium release assay with serum diluted at 1:4, 1:100, and 1:1000 with DMS 79 cells and human complement.

Immune Thin-Layer Chromatography.
Immune staining by thin layer chromatography (ITLC) was done with patient sera before and after immunization. Purified gangliosides and tumor extracts were spotted on silica gel glass plates as described previously (16) . After plexigum coating, blocking, and washing, horseradish peroxidase-conjugated antihuman IgM and IgG antibodies were added for 2 hours, and the plates were developed with 4-chloro-1-naphthol with H2O2.

Statistical Considerations.
The primary end point of this study was to determine the immunogenicity of synthetic fucosyl GM1-KLH conjugate plus QS-21 in patients with SCLC who have had a major response after initial chemotherapy or chemoradiotherapy. We planned to enroll up to six patients at each dose level. The lowest dose level at which four of the six patients developed a significant immune response was to be selected as the phase II dose. Because of low drug supply, only five patients were enrolled at the two lower dose levels.

A significant immune response was defined as (a) an antibody titer of ≥1:80 by ELISA against Fucosyl-GM1 confirmed by ITLC and a titer of 1:20 against tumor cells expressing Fucosyl-GM-1 for patients with no detectable baseline titer; or (b) an antibody titer ≥8-fold increase over baseline for patients with a detectable baseline titer.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Six patients were enrolled at the 30-µg dose level, and five patients were enrolled at the 10- and 3-µg dose levels. Baseline patient characteristics are specified in Table 1Citation . Three patients at the 30-µg dose level and one patient at the 3-µg dose level received five of six planned injections, and two patients at the 3-µg dose level received four injections. All of these patients were taken off study for disease progression. The remaining 10 patients received all six vaccinations.


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  		Table 1 Patient characteristics

 
No grade 3 or 4 toxicity occurred. The most common toxicity, injection site reaction, was observed in 87% of patients (56% grade 1, 31% grade 2). Five patients (31%) experienced grade 1 myalgias. Grade 1 flu-like symptoms, arthralgias, fever, or chills were reported by one patient each. Three patients (19%) experienced peripheral sensory neuropathy, one of which was grade 2 (from a grade 1 background). Other symptoms reported by more than one patient included cough (31%) and fatigue (25%). One patient had a grade 2 elevation of amylase and one patient had grade 1 hyperglycemia with no clinical sequelae.

The peak responses for each patient are summarized in Table 2Citation . Five of six patients at the 30-µg dose and three of five patients at the 10-µg dose mounted IgM responses of 1:80 or greater. The IgM ELISA titers for all six of the patients vaccinated at the 30-µg dose are shown in Fig. 2Citation . These antibodies were confirmed against synthetic- and tumor-extracted Fucosyl GM1 by ITLC (data not shown) and against fucosyl GM1-positive tumor cells by flow cytometry in seven of eight cases. Flow cytometry results against the DMS-79 SCLC cell line for all six patients treated at the 30-µg dose level are shown in Fig. 3Citation . Only patient 6, who had low-level ELISA and ITLC reactivity, was negative. However, none of the patients at the 3-µg dose had titers above 1:80. This suggests a dose response with the 10- and 30-µg doses superior to the 3-µg dose. One patient at the 30-µg dose had an IgG response with a titer of 1:80. Whereas these IgG ELISA titers are lower than those seen with the previous bovine fucosyl GM1-KLH conjugate vaccine, the IgM titers were similar, and sera from six of the eight responders induced potent complement-mediated cytotoxicity of tumor cells, a result that was comparable with the CDC observed in the previous trial.


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  		Table 2 Summary of immunology results for patients vaccinated with fucosyl GM1-KLH + QS21

 


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  		Fig. 2. IgM antibody ELISA responses in six SCLC patients vaccinated with fucosyl GM1-KLH plus QS-21 at the first dose level (30 µg). Arrows, the time of vaccinations.

 


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  		Fig. 3. IgM FACS results before and after immunization with synthetic fucosyl GM1-KLH plus QS-21 for the six patients who received the 30-µg fucosyl GM1 dose. Target, DMS-79 SCLC cell line. Percentage positive cells (mean fluorescence intensity)

 
The clinical outcomes, including time to progression and survival from the start of vaccination, are shown in Table 3Citation . No radiologic responses were observed.


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  		Table 3 Time to progression (TTP) and survival from start of vaccination

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There are advantages and disadvantages to the use of synthetic fucosyl GM1 for vaccine production. Whereas the synthesis itself is technically demanding, it simplifies vaccine production rather than obtaining it from bovine tissues. Nuclear magnetic resonance and mass spectrometry analyses provided assurance that the carbohydrate portion of the synthetic fucosyl GM1 is identical to that which is found expressed on tumor cells. However, it is possible that the different functionalities present at the reducing end of the oligosaccharide (pentenyl versus ceramide) may alter the architecture or three-dimensional shape of the entire fucosyl GM1 construct. We demonstrate here that vaccination with the synthetic fucosyl GM1-KLH conjugate induces an IgM antibody response against fucosyl GM1 and tumor cells expressing fucosyl GM1, which is comparable with the response induced by fucosyl GM1-KLH conjugate originating from bovine thyroid tissues (7) . This was confirmed by complement-dependent cytotoxicity against SCLC cells, which was at least as potent as that seen after immunization with the bovine-derived fucosyl GM1.

Unexpectedly, the IgG response to vaccination with the synthetic conjugate was lower than seen with the bovine-derived conjugate. Only 1 of 6 patients at the highest dose had an IgG antibody titer of 1:80 or greater, compared with 7 of 10 patients immunized with the naturally derived conjugate. Neither vaccine induced IgG antibodies with frequent reactivity to SCLC cells by FACS (0 of 6 were positive with the synthetic fucosyl GM1; 3 of 10 were marginally positive with the bovine-derived fucosyl GM1), and we have been unable to demonstrate antibody-dependent cellular cytotoxicity against SCLC with sera from either trial (data not shown); therefore, the apparent superiority of the bovine-derived conjugate may be of little consequence.

Our observation with carbohydrate epitopes in a variety of glycolipids and glycoproteins has been that, even when high-titer, vaccine-induced IgG antibodies are demonstrated by ELISA, cell-surface reactivity demonstrable by FACS is rarely seen. This is probably a consequence of the relatively low affinity of these IgG antibodies, which is substantially augmented in the pentameric configuration of IgM antibodies. It is likely that, as a consequence of the carbohydrate and autoantigen status of their targets, affinity maturation of these antibodies does not occur. The difference between the IgG ELISA reactivity in the two trials is probably a consequence of the structural difference between the two antigens. Although the hexasaccharide primary fucosyl GM1 epitope is the same in both, synthetic fucosyl GM1 lacks the ceramide tail that is present on natural fucosyl GM1 (Fig. 1)Citation used both for vaccine construction in the initial trial and as target in ELISA assays for both trials. It may be that a portion of the epitope recognized by these IgG antibodies includes the proximal portion of the sphingolipid tail. The use of an ineffective dose could also explain the findings.

On the basis of the hypothesis that a broader immune response would more likely result in immunologic activity against this heterogeneous tumor, we plan to combine the synthetic fucosyl GM1 vaccine with vaccines against three other antigens that are prevalent in SCLC—GM2, and Globo H, and polysialic acid (polySA; ref. 1 ). PolySA is a negatively charged side chain on the neural cell adhesion molecule with highly selective expression in SCLC (1 , 17 , 18) . PolySA was shown to be a poor immunogen (19) . However, vaccination with polySA, modified by N-propionylation (NP-polySA) and conjugated to KLH, induced IgM antibodies to NP-polySA in six of six patients, and these cross-reacted with unmodified polySA in all but one case (20) . IgG antibodies to NP-polySA were observed in five patients, but these did not cross-react with polySA. The other antigens, GM2 and Globo H, have demonstrated immunogenicity in melanoma, breast, and prostate cancer trials (9 , 21 , 22) . Preclinical data further support the use of this "tetravalent" vaccine (23) . Ten SCLC cell lines were tested with monoclonal antibodies against seven target antigens individually or pooled in different combinations. No single monoclonal antibody bound to more than 6 or 4 of the 10 cell lines by FACS or CDC. However, combining monoclonal antibodies against GM2, fucosyl GM1, Globo H, and polySA resulted in strong reactivity against 8 of 10 cell lines tested by flow cytometry and 9 of 10 cell lines tested by CDC. The addition of mAbs against GD2, GD3, and sLea increased reactivity only slightly. A randomized phase II trial testing this polyvalent vaccine in patients who have completed usual initial therapy for SCLC will begin next year.


   FOOTNOTES
 
Grant support: Supported by NIH grants PO1CA33049 (P. Livingston), GM19578 (J. Allen), and AI16943 and CA28824 (S. Danishefsky); an American Society of Clinical Oncology Career Development Award (L. Krug); and the Lawrence and Selma Ruben Foundations.

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.

Requests for reprints: Lee M. Krug, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 327, New York, NY 10021. Phone: 212-639-8420; Fax: 212-794-4357; E-mail: krugl{at}mskcc.org

4 www.ssdi.genealogy.rootsweb.com. Back

Received 3/ 9/04; revised 5/21/04; accepted 6/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  16. Ragupathi G, Livingston PO, Hood C, et al Consistent antibody response against ganglioside GD2 induced in patients with melanoma by a GD2 lactone-keyhole limpet hemocyanin conjugate vaccine plus immunological adjuvant QS-21. Clin Cancer Res 2003;9:5214-20.[Abstract/Free Full Text]
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