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Clinical Trials |
Thoracic Oncology Service [M. N. D., D. J. M., V. A. M., M. G. K., S. C. G.] and Clinical Immunology Service [G. R., N. X. L., C. M., P. O. L.], Department of Medicine, Memorial Sloan-Kettering Cancer Center and Cornell University Medical College, New York, New York 10021, and Department of Medical Microbiology and Immunology, University of Göteborg, Guldhedsgatan 10, S-413 46 Göteborg, Sweden [F-T. B.]
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ABSTRACT |
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1—2Gal
1—3GalNAc1—4(NeuAc2—3)Gal1—4Glc1—1Cer (Fuc-GM1), a ganglioside expressed on the SCLC cell surface, was selected as a target for active immunotherapy. Fuc-GM1 is present on most SCLCs but on few normal tissues. SCLC patients achieving a major response to initial therapy were vaccinated s.c. on weeks 1, 2, 3, 4, 8, and 16 with Fuc-GM1 (30 µg) conjugated to the carrier protein keyhole limpet hemocyanin and mixed with the adjuvant QS-21. Ten patients received at least five vaccinations and are evaluable for response. All patients demonstrated a serological response, with induction of both IgM and IgG antibodies against Fuc-GM1, despite prior treatment with chemotherapy with or without radiation. Posttreatment flow cytometry demonstrated binding of antibodies from patients’ sera to tumor cells expressing Fuc-GM1. In the majority of cases, sera were also capable of complement-mediated cytotoxicity. Mild transient erythema and induration at injection sites were the only consistent toxicities. The Fuc-GM1-KLH + QS-21 vaccine is safe and immunogenic in patients with SCLC. Continued study of this and other ganglioside vaccines is ongoing. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Potential targets for immunotherapy have been identified on the cell surface of SCLC. These include the gangliosides GM2, GD2, GD3, 9-O-acetyl GD3, and Fuc-GM1, as well as the polysialic acid epitope characteristic of the embryonic neural-cell adhesion molecule, the carbohydrate Globo H, and the glycoprotein KSA (3, 4, 5, 6, 7, 8, 9, 10) . Of these antigens, the ganglioside Fuc-GM1 is the most restricted in its expression on normal tissues (4 , 5) . The importance of gangliosides as targets for immunotherapy has been demonstrated by clinical responses observed in melanoma patients after passive immunotherapy with monoclonal antibodies against GM2, GD2, and GD3 (11, 12, 13, 14) . In addition, the presence of either naturally occurring antibodies or actively induced antibodies directed against gangliosides has been associated with an improved prognosis (15, 16, 17) . Previously, we immunized SCLC patients after initial chemotherapy with BEC2, an anti-idiotypic monoclonal antibody that mimics GD3 (18) . Patients developed anti-GD3 antibodies and had prolonged survival compared with historical controls. With these encouraging results, we investigated Fuc-GM1 as a target for immunotherapy.
Fuc-GM1 was initially identified and isolated from the bovine thyroid gland (19) . With the use of a highly specific mouse monoclonal antibody, F12, the ganglioside Fuc-GM1 was identified in the majority of SCLC tissue samples and the serum of patients with the disease (4 , 5 , 20 , 21) . Fuc-GM1 was not detected in normal lung and bronchus but was sparsely distributed on occasional small round cells in the thymus, spleen, pancreatic islet cells, lamina propria, and intramural ganglionic cells of the small intestine, as well as on a small subset of peripheral sensory neurons and dorsal root ganglia (4 , 5 , 22) .
Serum antibodies against Fuc-GM1 have been described in a few patients with sensory neuropathies but not in other settings, suggesting that this antigen is poorly immunogenic (22) . We have explored a variety of approaches for augmenting the immunogenicity of poorly immunogenic antigens. The most effective of these methods has been chemical conjugation to KLH, a shellfish-derived protein, followed by mixture with the immunological adjuvant QS-21 (23, 24, 25, 26, 27) . In the present Phase I/II study, 10 patients with SCLC achieving a major response to standard therapy received at least five vaccinations with a Fuc-GM1-KLH conjugate vaccine. The primary end points of the trial were the assessment of the ability to induce an antibody response and to monitor patients for toxicity. The reactivity of the induced antibody response has been evaluated, and the patients have been observed for relapse-free survival.
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PATIENTS AND METHODS |
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70%; age 18; total WBC 3.0 x 106 cells/µl; total lymphocyte count 0.5 x 106 cells/µl; serum bilirubin 
1.5 mg/dl; and serum aspartate aminotransferase and alkaline phosphatase 1.5 x upper limit of normal. Patients with a history of seafood allergy, clinically significant peripheral neuropathy, immunodeficiency or autoimmune disease, splenectomy or splenic radiation, current use of corticosteroids, or other active malignancies within the past 5 years were excluded. All patients signed an informed consent that had been approved by the Institutional Review Board at Memorial Sloan-Kettering Cancer Center.
Patient Characteristics.
Thirteen patients received the Fuc-GM1-KLH conjugate plus QS-21 vaccine. Patient characteristics are listed in Table 1
. Nine patients had extensive stage SCLC, and four patients had limited stage disease. The median age was 52 years (range, 43–76 years), with a median KPS of 90%. Eleven patients received cisplatin plus etoposide chemotherapy, one received carboplatin plus etoposide, and one received cyclophosphamide, doxorubicin, and etoposide. Six patients received thoracic radiation, and four patients were treated with prophylactic cranial radiation as part of the initial planned therapy. Vaccination was started >12 weeks after completing initial therapy in one patient with the approval of the Institutional Review Board. Of the 13 patients on study, 3 patients relapsed prior to receiving the fifth vaccination, and therefore only 10 patients are evaluable for serological response. Of these 10 patients, four received only five vaccinations secondary to relapsed SCLC prior to completing the protocol therapy. At the time of disease progression, patients were taken off study, and further therapy was at the discretion of the patient’s physician.
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Patients received a series of s.c. vaccinations administered on weeks 1, 2, 3, 4, 8, and 16. Blood was drawn for serological testing before each vaccination, and 2 weeks after the fourth, fifth, and sixth vaccinations. A history, physical examination, and chest X-ray were performed at week eight and eighteen. Complete blood count, chemistries, and amylase were drawn on weeks 3, 8, and 18. Patients were monitored for toxicity by history and physical examination and with patient-completed diaries. The NCI common toxicity scale was used to grade toxicity except for symptoms of myalgias, fatigue, and chills, which were graded using the Cancer and Leukemia Group B common toxicity scale.
Serological Assays.
ELISA assays were performed to detect IgM and IgG antibody responses (25)
. Nunc microwell plates (Nunc, Inc., Naperville, IL) were coated with purified Fuc-GM1 ganglioside at 0.2 µg/well in 50 µl of ethanol and incubated at room temperature overnight. In the morning, plates were incubated with 3% HSA at 37°C for 2 h. Serial dilutions of patient sera were added to the plates. For IgM assays, the plates were incubated for 1 h at room temperature, washed, and then alkaline-phosphatase-conjugated goat anti-human IgM (Southern Biotechnology Associates, Inc., Birmingham, AL) was added and incubated for an additional hour at room temperature. For IgG assays, goat anti-human IgG unlabeled antibody (Southern Biotechnology Associates) was added and incubated for 1 h. Mouse anti-goat alkaline-phosphatase-conjugated antibody (Southern Biotechnology Associates) was added and incubated for 45 min. Determination of IgG subclass was performed by ELISA using subclass-specific secondary mouse anti-human IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies (Zymed Laboratories, Inc., San Francisco, CA). Alkaline-phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) was used as a third antibody at a dilution of 1:200. All plates were washed and developed with Sigma 104 phosphatase substrate (Sigma Diagnostics, St. Louis, MO) in 10% diethanolamine. Absorbance was measured at 414 nm, and the highest dilution with an absorbance of at least 0.100 was defined as the antibody titer. To confirm the specificity of the assay, ELISA was also performed using the purified antigen GM1. To control for nonspecific binding, patient sera were also tested on plates that were processed identically but to which no ganglioside had been added, and this reading was subtracted from the value obtained in the presence of the ganglioside.
FACS were performed on the human SCLC cell line H146 and the rat hepatoma cell line H4IIE, both of which express Fuc-GM1 (H4IIE expresses Fuc-GM1 more than H146). Single-cell suspensions of tumor cells (3 x 105 cells/tube) were washed with 3% FCS in RPMI 1640 medium. Patient sera were added to the cell pellets at a 1:10 dilution and then mixed and incubated for 30 min on ice. The cells were washed once with 3% FCS-RPMI and were incubated with either 20 µl of 1:25-diluted FITC-labeled goat anti-human IgM (Zymed) or 1:25-diluted FITC-labeled goat anti-human IgG (Southern Biotechnology Associates) on ice for 30 min. The percentage of positive cell population and the mean fluorescence intensity of the stained cells were analyzed by flow cytometry (FACScan, Becton Dickinson, CA). The mouse anti-Fuc-GM1 monoclonal antibody, F12, was used as a positive control, and pretreatment sera were used as negative controls. FACS inhibition studies were performed on select sera using the Fuc-GM1 antigen, and the GD3 ganglioside was used as a negative control.
CDC assays were performed by a 2-h chromium release assay. The Fuc-GM1-positive cell lines H146 and H4IIE served as target cells. Approximately 107 cells were labeled with 100 µCi of Na251CrO4 (New England Nuclear, Boston, MA) in 3% HSA for 2 h at 37°C, shaking every 15 min. The cells were washed four times and brought to a concentration of 106 live cells/ml. Fifty µl of labeled cells were mixed with 50 µl of undiluted pre- or post-vaccination serum or with medium alone in 96-well, round-bottomed plates (Corning, New York, NY) and incubated at 4°C on a shaker for 45 min. Human complement (Sigma Diagnostics, St. Louis, MO) diluted 1:5 with 3% HSA was added, at 100 µl/well, and incubated at 37°C for 2 h. The plates were spun at 100 x g for 5 min, and an aliquot of 100 µl of supernatant from each well was read by a gamma counter to determine the amount of 51Cr released. All samples were performed in triplicate and included control wells for maximum release and for spontaneous release in the absence of complement. Maximum release was the amount released by target cells after a 2-h incubation with 1% Triton X-100 (Sigma Diagnostics) and 100 µl of human complement. Spontaneous release (the amount released by target cells incubated with complement alone) was subtracted from both experimental and maximal release values. Specific release was equal to corrected experimental release divided by corrected maximal release:
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Patients were considered evaluable for immunological response if they had received at least five vaccinations. Sera were considered positive by ELISA if the titer of reactivity was at least 1:40, by FACS if the percent of positive cells doubled in posttreatment sera compared with pretreatment sera, and by CDC assay if there was 10% or more specific release.
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RESULTS |
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and Fig. 1
). IgG antibodies were primarily of the IgG1 subclass (Table 3)
. Of the three evaluable patients with limited stage disease, each received all of the six planned vaccinations, with induction of IgM and IgG antibody titers of 1:320–1:2560 and 1:40–1:2560, respectively. Although four patients with extensive stage disease relapsed prior to receiving the sixth vaccination, the majority of these patients demonstrated both an IgM and IgG response. The interval between diagnosis of SCLC and first immunization did not appear to affect the antibody titers. Specificity of the antibody response to the Fuc-GM1 antigen was confirmed by ELISA performed with GM1, GM2, and Globo H. Posttreatment sera demonstrated minimal reactivity (two or three titers of 1:20 and one titer of 1:40 against each antigen). All other sera were nonreactive.
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. Results using the human SCLC cell line H146, which expresses lower levels of Fuc-GM1 than H4IIE, showed posttreatment IgM and IgG antibodies that bound to tumor cells from 6 of 10 patients and 3 of 10 patients, respectively (Table 4)
. Addition of the Fuc-GM1 antigen to selected patient sera inhibited subsequent binding of antibody to tumor cells, whereas binding to tumor cells was not inhibited by the addition of GD3 (Table 5
and Fig. 2
). Post-vaccination sera from 9 of 10 patients evaluable for response induced CDC of the Fuc-GM1-positive tumor cell lines (Table 6)
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2–5 days and was most pronounced after the second or third vaccination. Mild, transient, flu-like symptoms including low-grade fever, myalgias, headache, and chills occurred after a minority of vaccinations. Diarrhea was observed after 9 of 60 (15%) vaccinations (eight grade 1 and one grade 2). Grade 1 fatigue was observed after 13 of 60 (22%) vaccinations. Worsening of chemotherapy-induced sensory neuropathy by one toxicity grade was observed in six patients (Table 7)
. Three patients had worsening of sensory neuropathy from grade 0 to grade 1, two patients from grade 1 to grade 2, and one patient from grade 2 to grade 3. No motor neuropathy was observed. Of the six patients who completed the full course of six immunizations, three remain relapse free, 18, 24, and 30 months from diagnosis.
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DISCUSSION |
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Median peak ELISA antibody titers against Fuc-GM1 after immunization with the Fuc-GM1-KLH plus QS-21 vaccine were 1:320 for IgM and 1:480 for IgG. These titers are similar to the titers induced against GM2 with the GM2-KLH plus QS-21 vaccine in previous trials in melanoma patients (25 , 26) , but the melanoma patients were free of detectable disease and had not received previous chemotherapy or radiation therapy. The majority of the SCLC patients treated in this trial had recently completed treatment with chemotherapy with or without radiation therapy and continued to have radiological evidence of evaluable disease. Despite the greater extent of disease and prior therapy, the Fuc-GM1-KLH conjugate vaccine consistently induced IgM and IgG antibody responses.
On the basis of these results, Fuc-GM1 appears to be the most immunogenic of the gangliosides we have tested, clearly more immunogenic than GD2 and GD3, and at least as immunogenic as GM2. Reactivity of the sera was demonstrated by both flow cytometry and CDC assay using the rat hepatoma cell line H4IIE, with extensive cell surface expression of Fuc-GM1, and the SCLC cell line H146, with more modest cell surface expression of Fuc-GM1. Eight of 10 patients and 6 of 10 patients showed at least a doubling of the percentage of positive cells bound by IgM and IgG flow cytometry against H4IIE, respectively. Specificity of these reactions for cell surface Fuc-GM1 was demonstrated in most cases by inhibition of reactivity of post-immunization sera after the addition of purified Fuc-GM1, but not GD3, to the reactions (Table 5)
. At least a doubling of CDC against H4IIE was seen in 8 of 10 patients, and two-thirds of the patients had at least 70% cytotoxicity induced by post-vaccination sera. Increases were seen against both H146 and H4IIE but were more significant against H4IIE. Because both IgM and IgG antibodies against Fuc-GM1 were induced, competition between the two antibody classes undoubtedly occurred, as we have described previously with fractionated immune sera against GM2. However, because the IgG antibodies against GM2 were of the IgG1 subclass, both FACs and CDC reactivities against GM2-positive tumor cells were greater with the IgG and IgM fractions combined than they were with either fraction tested separately. The anti-Fuc-GM1 IgG antibodies were also of the IgG1 subclass and therefore able to activate complement. Consequently, we expect that CDC induced by IgM and IgG antibodies will be greater (although less than additive because of the competition) than with either class alone (26)
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The Fuc-GM1-KLH + QS-21 vaccine was generally well tolerated. Mild transient erythema and induration at the injection sites were observed in most patients, associated with occasional flu-like symptoms. Slight increases in the severity of sensory neuropathy (by one National Cancer Institute toxicity grade) were observed in six patients during the course of the study. This was in the setting of an objective but not clinically significant baseline neuropathy in 8 of 13 patients (62%) entering the study, presumably related to prior platinum-based chemotherapy. Binding of antibody to Fuc-GM1 expressed on peripheral sensory neurons is a possible explanation for the observed changes; however, the changes were mild, with the majority of patients reporting no change in functional capacity or progressive worsening of symptoms over time. There was no evidence for diabetes mellitus, gastrointestinal or immunological dysfunction, or other problems to suggest potential autoimmunity based on Fuc-GM1 distribution on normal tissues.
We have demonstrated that despite chemotherapy, these SCLC patients exhibited a good response against the Fuc-GM1-KLH conjugate vaccine, producing antibodies that were reactive by ELISA against purified Fuc-GM1 and by flow cytometry and CDC assays against Fuc-GM1 expressed on tumor cells. The antibodies induced were specific for Fuc-GM1, showing no reactivity with GM1. Immunization with Fuc-GM1 was well tolerated and was not associated with significant toxicity. In addition, Fuc-GM1 is at least as immunogenic in humans as the other gangliosides that we have tested previously (GM2, GD2, GD3, and 9-O-acetyl GD3; Refs. 17 , 23, 24, 25, 26 , and 30, 31, 32 ).
Although Fuc-GM1 is expressed on most SCLC cells in most specimens, it is not expressed in every cell or in every specimen. To effectively target every SCLC cell, a polyvalent vaccine containing multiple consistently immunogenic antigens will be required. Our study demonstrates that Fuc-GM1 should be a key component of any polyvalent vaccine against SCLC, and we continue to evaluate other vaccines directed against antigens expressed by SCLC.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by PO1 CA 33049 and The Milstein Family Foundation. 
2 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8800; Fax: (212) 717-3619.
3 The abbreviations used are: SCLC, small cell lung cancer; Fuc-GM1, Fuc1—2Gal1—3GalNAc1—4(NeuAc2–3)Gal1—4Glc1—1Cer; KLH, keyhole limpet hemocyanin; CDC, complement-dependent cytotoxicity; FACS, fluorescence-activated cell sorter assays; HSA, human serum albumin.
Received 2/22/99; revised 7/12/99; accepted 7/24/99.
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REFERENCES |
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