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Vaccine therapy for small cell lung cancer
Vaccine Therapy for Small Cell Lung Cancer Lee M. Krug One novel approach to the treatment of lethal residual disease in patients with small cell lung cancer (SCLC) relies on 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 be capitalized on as immune targets. This article reviews two vaccine strategies currently in clinical study. The anti-idiotype vaccine to the GD3 ganglioside, BEC-2, has recently been tested in a phase III trial. In this trial, patients with SCLC who had completed initial chemotherapy were randomized to observation or vaccination with BEC-2 plus bacillus Calmette-Guerin adjuvant. A series of other trials have established the immunogenicity of several keyhole limpet hemocyanin conjugate vaccines relevant to SCLC, including GM2, Globo H, fucosyl GM1, and polysialic acid. To optimize an immune response against a broad range of tumor phenotypes, these components will be combined into a polyvalent vaccine. A randomized phase II trial of this polyvalent vaccine is planned to start in 2004. Semin Oncol 31 (suppl 1):112-116. © 2004 Elsevier Inc. All rights reserved.
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FTER two decades of randomized clinical trials, a simplified algorithm for the treatment of small cell lung cancer (SCLC) has emerged. The currently accepted treatment course involves four to six cycles of etoposide plus cisplatin or carboplatin, with concurrent thoracic radiation added for patients with limited-stage disease. For patients with a complete response, prophylactic cranial irradiation is also recommended. In the majority of cases, patients can 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
From the Division of Thoracic Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, NY. Dr Krug has received research grant support from ImClone Systems Inc. Address reprint requests to Lee M. Krug, MD, Memorial SloanKettering Cancer Center, 1275 York Ave, New York, NY 10021. © 2004 Elsevier Inc. All rights reserved. 0093-7754/04/3101-0115$30.00/0 doi:10.1053/j.seminoncol.2003.12.022 112
completed. The question of how to eradicate the residual tumor cells after standard therapy remains. The success that chemotherapy initially affords provides incentive to study various permutations of chemotherapy regimens to improve outcomes. Investigators have attempted to continue chemotherapy for a longer period of time (maintenance therapy), to use alternating non– cross-resistant regimens, to add extra drugs, to administer consolidation chemotherapy, and even to use such highdose chemotherapy that autologous stem cell transplant is necessary for recovery. In randomized trials, all of these approaches have consistently failed to improve survival.1-5 As a result, the current recommendation upon completion of the usual initial therapy is expectant observation. This is clearly not acceptable. An effective method of eradicating residual disease is desperately needed. Only with unique biology-based therapies are we likely to improve treatment of SCLC. Investigators at the Memorial Sloan-Kettering Cancer Center (MSKCC) (New York, NY) are pursuing immune therapies as a way of attacking residual disease. Passive and actively induced immunity has been shown to eradicate circulating tumor cells and micrometastases in mouse tumor models, which seems an appropriate approach in SCLC. Furthermore, because of its neuroectodermal origin, SCLC has a number of specific antigens that could be capitalized on as immune targets (Fig 1). A vaccine approach has a low toxicity potential, which is ideal for patients who have just completed a course of chemotherapy. Research at the MSKCC has led to the development of an anti-idiotype antibody to the ganglioside GD3 called BEC-2, and has also included a program to develop a polyvalent vaccine of keyhole limpet hemocyanin (KLH) conjugates against SCLC antigens. The status of these investigations is detailed below. BEC-2 AND THE SILVA TRIAL
In the 1980s, gangliosides were identified as a potential target for immunotherapy to treat melanoma.6 Gangliosides are glycolipids with a ceramide group anchored to the cell membrane and an extracellular sugar moiety that is the unique Seminars in Oncology, Vol 31, No 1, Suppl 1 (February), 2004: pp 112-116
VACCINE THERAPY FOR SMALL CELL LUNG CANCER
Fig 1. Cell surface antigens of small cell lung cancer. (Printed with permission from P. O. Livingston.)
identifier. To overcome the natural immunologic tolerance to gangliosides, an anti-idiotype platform was tested and found effective in inducing an immune response initially in animal models7 and subsequently in humans.8 This involved a multistep process. An anti-GD3 antibody was raised (R24) that, when injected into syngeneic mice, induced anti–anti-GD3 antibodies, termed BEC-2.7 When BEC-2 was injected intradermally into the subject along with an immunologic adjuvant, such as bacillus Calmette-Guerin (BCG),9 the immune response resulted in antibodies to BEC-2 that then cross-reacted with GD3. The fact that most SCLC cell lines and tumors express GD310 prompted the testing of BEC-2 in SCLC patients. A pilot trial was conducted in which BEC-2 was administered with the immunologic adjuvant BCG9 to 15 patients with SCLC who had completed initial chemotherapy.11 Patients received five intradermal injections over 10 weeks. All patients developed anti–BEC-2 antibodies, and five patients developed anti-GD3 an-
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tibodies. The results of this pilot trial were considered promising because the survival of these vaccinated patients was better than expected, especially for those patients with limited-stage disease. The overall median survival was 21 months, which is much longer than that of historic controls. Many of the patients who achieved the longest survival were those who had generated antiGD3 antibodies. The only definitive way to confirm these observations was in a randomized trial. The European Organization for the Research and Treatment of Cancer elected to sponsor the necessary trial, which was titled the “Survival in an International Phase III Prospective Randomized LD Small Cell Cancer Vaccination Study with Adjuvant BEC-2 and BCG.” This trial enrolled only patients with limited-stage SCLC who had completed appropriate treatment with chemotherapy and thoracic radiation. Prophylactic cranial irradiation was recommended but not mandatory. Four to 10 weeks after completion of treatment, patients were stratified by complete or partial response, performance status, and study site, and then randomized to observation or vaccination with BEC-2 plus BCG in the same manner as in the pilot trial. Accrual was completed in October 2002 with 515 patients randomized. The trial was powered to detect a 40% increase in survival. The results can be analyzed after 376 events (patient deaths), which will likely occur in 2004. BEC-2 plus BCG is currently licensed by ImClone Systems Incorporated in North America, and by Merck Oncology in Europe, Australia, and New Zealand. POLYVALENT VACCINE
A series of trials have tested other vaccines against antigens relevant to SCLC. Based on prior immunohistochemical analyses of tumor samples, four antigens emerged as lead targets for immunotherapy in SCLC: GM2, Globo H, fucosyl GM1, and polysialic acid.12 The immunogenicity of GM2 and Globo H has been established in studies of other malignancies.13-15 However, fucosyl GM1 and polysialic acid have highly restricted expression in SCLC, thus these vaccines have been tested only in SCLC patients (Table 1).13-18 For the SCLC vaccine trials, eligibility criteria have been relatively consistent. Patients with limited- or extensive-stage SCLC are enrolled 4 to 12
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Table 1. Proposed Components for a Polyvalent Vaccine in Small Cell Lung Cancer
Antigen GM2 Globo H Fucosyl GM1 Bovine Thyroid Synthetic Polysialic acid Unmodified N-Propionylated
Tumor Type Tested
Study
Melanoma Breast Prostate
Helling et al, 199513 Gilewski et al, 200114 Slovin et al, 199915
SCLC
Dickler et al, 199916 Krug et al, 200217
SCLC
Ng et al, 200118 Krug, IRB 02-097
NOTE. All of the antigens have been tested as conjugates with keyhole limpet hemocyanin with QS-21 adjuvant. Abbreviation: SCLC, small cell lung cancer.
weeks after completion of their usual initial chemotherapy and radiation. Patients must not be taking systemic corticosteroids or have an underlying immune deficiency. Because of the potential cross-reaction of the vaccines with neural tissue, patients must not have peripheral sensory neuropathy greater than grade 1. To overcome immunologic tolerance, all of these trials use antigen conjugates with the carrier KLH.19 QS-21, a saponin tree bark extract,20 is added as an immunologic adjuvant. This compound has previously been shown to induce high antibody titers when compared with other adjuvants.13 The vaccinations are administered subcutaneously on weeks 1, 2, 3, 4, 8, and 16. Blood is drawn for immunology studies before each vaccination and on weeks 10 and 18. The primary endpoint of these trials is immune response defined as an antibody titer of ⱖ1:80 by enzymelinked immunosorbent assay (ELISA) and confirmed by flow cytometry using tumor cells expressing the target. If five of nine patients develop a significant immune response, the vaccine warrants further study.21 Fucosyl GM1 The first small cell-specific antigen tested in our vaccine clinical trials was fucosyl GM1. Fucosyl GM1 is a ganglioside that was initially isolated from the bovine thyroid gland.22 Several lines of evidence support targeting fucosyl GM1 in pa-
tients with SCLC. Using the monoclonal antibody F12, fucosyl GM1 was identified by immunofluorescence in 19 of 21 human SCLC tumors.23 It was also detected in culture media from SCLC cell lines and in tumor extracts and serum of nude mice xenografts.24 Furthermore, fucosyl GM1 was detected in the serum of six of 20 patients with extensive-stage SCLC but not in 12 non–small cell lung cancer patients or 20 healthy volunteers. In the first clinical trial of this vaccine, fucosyl GM1 was directly obtained from cow thyroid gland.16 Ten patients received at least five vaccinations with fucosyl GM1-KLH 30 g plus QS-21 100 g and were considered evaluable for immune response. All patients had serologic immune response by ELISA with IgM titers ranging from 1:320 to 1:2,560 and IgG titers ranging from 1:40 to 1:2,560. Most patients mounted significant antibody titers by week 4, and these titers were sustained. Flow cytometry demonstrated antibody binding to fucosyl GM1 expressing rat hepatoma and SCLC cell lines in eight of 10 and six of 10 cases, respectively. Posttreatment sera induced complement-mediated cytotoxicity in nine of 10 cases. To allow easier manufacturing and to decrease the risk of prion infection, a synthetic version of fucosyl GM1 was formulated. A trial with this synthetic fucosyl GM1 was then conducted.17 Three dose levels of fucosyl GM1 in the KLH conjugate (30, 10, and 3 g) were studied with 100 g QS-21. After vaccination, five of the six patients treated at 30 g and three of the five patients who received 10 g mounted an IgM response with titers ⱖ1:80 (range, 1:80 to 1:2,560). In seven of these patients, specificity of the response was confirmed by immune thin-layer chromatography with purified gangliosides. Cell surface reactivity was detected by fluorescent-activated cell sorting in seven patients. Polysialic Acid The next target that was studied was polysialic acid. Polysialic acid is a long polymer of negatively charged sialic acid residues that binds restrictively to a neural cell adhesion molecule.25 Expression is normally restricted to the surface of group B meningococcus and, in humans, to embryonic neural tissues.26 Its large size physically inhibits the affinity of neural cell adhesion molecule to the receptors of adjacent cells and thereby promotes cell
VACCINE THERAPY FOR SMALL CELL LUNG CANCER
motility. Several investigators have found immunostaining for polysialic acid in nearly every SCLC tumor tested.12,27,28 Because it inhibits cell– cell interactions, polysialic acid may contribute to the high metastatic potential of SCLC.29 Because of its presence in the embryo and to a limited extent in adult brain tissues, humans have an immunologic tolerance to polysialic acid.30 This may, in fact, explain the lack of clearance of group B meningococcus and the subsequent development of meningitis caused by this organism. In an attempt to overcome this tolerance, MSKCC investigators have modified polysialic acid by Npropionylation. This technique, which involves replacement of acetyl groups with propionyl groups, has been shown to boost the IgG response to meningococcal group B polysaccharide in mice.31 The MSKCC group conducted a study in which seven patients received unmodified polysialic acid, and six patients received N-propionylated polysialic acid conjugated with KLH and administered with QS-21.18 Serologic analysis of serum IgM and IgG titers showed no reactivity after treatment with the unmodified polysialic acid vaccine. With the N-propionylated version, five of six patients showed augmentation of IgM antibodies against polysialic acid as well as the H345 SCLC cell line. A follow-up dose de-escalation study with N-propionylated polysialic acid has been initiated to better assess the immunogenicity, toxicities, and optimal dose of this vaccine. The ultimate plan of the MSKCC vaccine program is to combine these components into a polyvalent vaccine. The hypothesis is that a broader immune response would more likely result in immunologic activity against this heterogenous tumor. Preclinical data support this approach. Nine SCLC cell lines were tested with monoclonal antibodies against seven target antigens individually or pooled in different combinations.32 No single monoclonal antibody bound to more than five of the nine cell lines. However, combining monoclonal antibodies against GM2, fucosyl GM1, Globo H, and polysialic acid resulted in strong reactivity against seven of nine cell lines tested by flow cytometry and all six cell lines tested by complement-dependent cytotoxicity. The addition of monoclonal antibodies against GD2, GD3, and sLea increased reactivity only slightly.
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CONCLUSIONS
In summary, an immune approach to the treatment of residual disease in SCLC appears logical and feasible. Small cell tumors express a number of antigens that can be targeted for immunotherapy. The anti-idiotype antibody to the ganglioside GD3, termed BEC-2, has now completed phase III testing in patients with limited-stage disease, and the results of that trial are pending. Smaller trials of KLH conjugate vaccines have established the immunogenicity of GM2, Globo H, fucosyl GM1, and polysialic acid. A randomized phase II trial testing a tetravalent vaccine of these components in patients with SCLC who have completed initial therapy is planned to start in 2004. Other biologicbased therapies relevant to SCLC, such as monoclonal antibodies, tyrosine kinase and cell cycle inhibitors, and antisense drugs, are also being developed that, hopefully, may be used to prevent disease relapse. REFERENCES 1. Sculier JP, Berghmans T, Castaigne C, et al: Maintenance chemotherapy for small cell lung cancer: A critical review of the literature. Lung Cancer 19:141-151, 1998 2. Niell H, Herndon JE, Miller A, et al: Randomized phase III Intergroup trial (CALGB 9732) of etoposide (VP-16) and cisplatin (DDP) with or without paclitaxel (TAX) and G-CSF in patients with extensive stage small cell lung cancer (EDSCLC). Proc Am Soc Clin Oncol 21:293a, 2002 (abstr 1169) 3. Roth BJ, Johnson DH, Einhorn LH, et al: Randomized study of cyclophosphamide, doxorubicin, and vincristine versus etoposide and cisplatin versus alternation of these two regimens in extensive small-cell lung cancer: A phase III trial of the Southeastern Cancer Study Group. J Clin Oncol 10:282-291, 1992 4. Schiller JH, Adak S, Cella D, et al: Topotecan versus observation after cisplatin plus etoposide in extensive-stage small-cell lung cancer: E7593–A phase III trial of the Eastern Cooperative Oncology Group. J Clin Oncol 19:2114-2122, 2001 5. Humblet Y, Symann M, Bosly A, et al: Late intensification chemotherapy with autologous bone marrow transplantation in selected small-cell carcinoma of the lung: A randomized study. J Clin Oncol 5:1864-1873, 1987 6. Livingston PO, Calves MJ, Natoli EJ Jr: Approaches to augmenting the immunogenicity of the ganglioside GM2 in mice: Purified GM2 is superior to whole cells. J Immunol 138:1524-1529, 1987 7. Chapman PB, Houghton AN: Induction of IgG antibodies against GD3 ganglioside in rabbits by an anti-idiotypic monoclonal antibody. J Clin Invest 88:186-192, 1991 8. Yao TJ, Meyers M, Livingston PO, et al: Immunization of melanoma patients with BEC2-keyhole limpet hemocyanin plus BCG intradermally followed by intravenous booster im-
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munizations with BEC2 to induce anti-GD3 ganglioside antibodies. Clin Cancer Res 5:77-81, 1999 9. McCaffery M, Yao TJ, Williams L, et al: Immunization of melanoma patients with BEC2 anti-idiotypic monoclonal antibody that mimics GD3 ganglioside: Enhanced immunogenicity when combined with adjuvant. Clin Cancer Res 2:679-686, 1996 10. Fuentes R, Allman R, Mason MD: Ganglioside expression in lung cancer cell lines. Lung Cancer 18:21-33, 1997 11. Grant SC, Kris MG, Houghton AN, et al: Long survival of patients with small cell lung cancer after adjuvant treatment with the anti-idiotypic antibody BEC2 plus Bacillus CalmetteGuerin. Clin Cancer Res 5:1319-1323, 1999 12. Zhang S, Cordon-Cardo C, Zhang HS, et al: Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int J Cancer 73:42-49, 1997 13. Helling F, Zhang S, Shang A, et al: GM2-KLH conjugate vaccine: Increased immunogenicity in melanoma patients after administration with immunological adjuvant QS-21. Cancer Res 55:2783-2788, 1995 14. Gilewski T, Ragupathi G, Bhuta S, et al: Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial. Proc Natl Acad Sci U S A 98:3270-3275, 2001 15. Slovin SF, Ragupathi G, Adluri S, et al: Carbohydrate vaccines in cancer: Immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man. Proc Natl Acad Sci U S A 96:5710-5715, 1999 16. Dickler MN, Ragupathi G, Liu NX, et al: Immunogenicity of a fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer. Clin Cancer Res 5:2773-2779, 1999 17. Krug L, Ragupathi G, Livingston P, et al: Pilot trial of a synthetic fucosyl-GM1 ganglioside vaccine in patients with small cell lung cancer (SCLC). Proc Am Soc Clin Oncol 21:4b, 2002 (abstr 1824) 18. Ng K, Kris M, Miller V, et al: Vaccination with polysialic acid (polySA)-KLH or N-propionylated (NP) polysialic acid-keyhole limpet hemocyanin (KLH) conjugate plus QS-21 in patients with small cell lung cancer (SCLC) after a major response to therapy. Proc Am Soc Clin Oncol 20:223b, 2001 (abstr 2644) 19. Musselli C, Livingston PO, Ragupathi G: Keyhole limpet hemocyanin conjugate vaccines against cancer: The Memorial Sloan Kettering experience. J Cancer Res Clin Oncol 127:R20-R26, 2001 (suppl 2) 20. Kensil CR, Patel U, Lennick M, et al: Separation and
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characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J Immunol 146:431-437, 1991 21. Yao TJ, Begg CB, Livingston PO: Optimal sample size for a series of pilot trials of new agents. Biometrics 52:992-1001, 1996 22. Macher BA, Pacuszka T, Mullin BR, et al: Isolation and identification of a fucose- containing ganglioside from bovine thyroid gland. Biochim Biophys Acta 588:35-43, 1979 23. Brezicka FT, Olling S, Nilsson O, et al: Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49:1300-1305, 1989 24. Vangsted AJ, Clausen H, Kjeldsen TB, et al: Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum. Cancer Res 51:28792884, 1991 25. Finne J, Finne U, Deagostini-Bazin H, et al: Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule. Biochem Biophys Res Commun 112:482-487, 1983 26. Rutishauser U: Polysialic acid at the cell surface: Biophysics in service of cell interactions and tissue plasticity. J Cell Biochem 70:304-312, 1998 27. Komminoth P, Roth J, Lackie PM, et al: Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids. Am J Pathol 139:297-304, 1991 28. Lantuejoul S, Moro D, Michalides RJ, et al: Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors. Am J Surg Pathol 22:1267-1276, 1998 29. Daniel L, Durbec P, Gautherot E, et al: A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process. Oncogene 20:997-1004, 2001 30. Finne J, Leinonen M, Makela PH: Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2:355-357, 1983 31. Jennings HJ, Roy R, Gamian A: Induction of meningococcal group B polysaccharide- specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 137:1708-1713, 1986 32. Livingston P, Hood C, Krug L, et al: Antigen expression on small cell lung cancer (SCLC) cell lines confirms selection of a tetravalent vaccine against SCLC containing GM2, fucosyl GM1, Globo H, and polysialic acid. Proc Am Assoc Cancer Res 44:1090, 2003 (abstr 4754)
A
FTER two decades of randomized clinical trials, a simplified algorithm for the treatment of small cell lung cancer (SCLC) has emerged. The currently accepted treatment course involves four to six cycles of etoposide plus cisplatin or carboplatin, with concurrent thoracic radiation added for patients with limited-stage disease. For patients with a complete response, prophylactic cranial irradiation is also recommended. In the majority of cases, patients can 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
From the Division of Thoracic Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, NY. Dr Krug has received research grant support from ImClone Systems Inc. Address reprint requests to Lee M. Krug, MD, Memorial SloanKettering Cancer Center, 1275 York Ave, New York, NY 10021. © 2004 Elsevier Inc. All rights reserved. 0093-7754/04/3101-0115$30.00/0 doi:10.1053/j.seminoncol.2003.12.022 112
completed. The question of how to eradicate the residual tumor cells after standard therapy remains. The success that chemotherapy initially affords provides incentive to study various permutations of chemotherapy regimens to improve outcomes. Investigators have attempted to continue chemotherapy for a longer period of time (maintenance therapy), to use alternating non– cross-resistant regimens, to add extra drugs, to administer consolidation chemotherapy, and even to use such highdose chemotherapy that autologous stem cell transplant is necessary for recovery. In randomized trials, all of these approaches have consistently failed to improve survival.1-5 As a result, the current recommendation upon completion of the usual initial therapy is expectant observation. This is clearly not acceptable. An effective method of eradicating residual disease is desperately needed. Only with unique biology-based therapies are we likely to improve treatment of SCLC. Investigators at the Memorial Sloan-Kettering Cancer Center (MSKCC) (New York, NY) are pursuing immune therapies as a way of attacking residual disease. Passive and actively induced immunity has been shown to eradicate circulating tumor cells and micrometastases in mouse tumor models, which seems an appropriate approach in SCLC. Furthermore, because of its neuroectodermal origin, SCLC has a number of specific antigens that could be capitalized on as immune targets (Fig 1). A vaccine approach has a low toxicity potential, which is ideal for patients who have just completed a course of chemotherapy. Research at the MSKCC has led to the development of an anti-idiotype antibody to the ganglioside GD3 called BEC-2, and has also included a program to develop a polyvalent vaccine of keyhole limpet hemocyanin (KLH) conjugates against SCLC antigens. The status of these investigations is detailed below. BEC-2 AND THE SILVA TRIAL
In the 1980s, gangliosides were identified as a potential target for immunotherapy to treat melanoma.6 Gangliosides are glycolipids with a ceramide group anchored to the cell membrane and an extracellular sugar moiety that is the unique Seminars in Oncology, Vol 31, No 1, Suppl 1 (February), 2004: pp 112-116
VACCINE THERAPY FOR SMALL CELL LUNG CANCER
Fig 1. Cell surface antigens of small cell lung cancer. (Printed with permission from P. O. Livingston.)
identifier. To overcome the natural immunologic tolerance to gangliosides, an anti-idiotype platform was tested and found effective in inducing an immune response initially in animal models7 and subsequently in humans.8 This involved a multistep process. An anti-GD3 antibody was raised (R24) that, when injected into syngeneic mice, induced anti–anti-GD3 antibodies, termed BEC-2.7 When BEC-2 was injected intradermally into the subject along with an immunologic adjuvant, such as bacillus Calmette-Guerin (BCG),9 the immune response resulted in antibodies to BEC-2 that then cross-reacted with GD3. The fact that most SCLC cell lines and tumors express GD310 prompted the testing of BEC-2 in SCLC patients. A pilot trial was conducted in which BEC-2 was administered with the immunologic adjuvant BCG9 to 15 patients with SCLC who had completed initial chemotherapy.11 Patients received five intradermal injections over 10 weeks. All patients developed anti–BEC-2 antibodies, and five patients developed anti-GD3 an-
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tibodies. The results of this pilot trial were considered promising because the survival of these vaccinated patients was better than expected, especially for those patients with limited-stage disease. The overall median survival was 21 months, which is much longer than that of historic controls. Many of the patients who achieved the longest survival were those who had generated antiGD3 antibodies. The only definitive way to confirm these observations was in a randomized trial. The European Organization for the Research and Treatment of Cancer elected to sponsor the necessary trial, which was titled the “Survival in an International Phase III Prospective Randomized LD Small Cell Cancer Vaccination Study with Adjuvant BEC-2 and BCG.” This trial enrolled only patients with limited-stage SCLC who had completed appropriate treatment with chemotherapy and thoracic radiation. Prophylactic cranial irradiation was recommended but not mandatory. Four to 10 weeks after completion of treatment, patients were stratified by complete or partial response, performance status, and study site, and then randomized to observation or vaccination with BEC-2 plus BCG in the same manner as in the pilot trial. Accrual was completed in October 2002 with 515 patients randomized. The trial was powered to detect a 40% increase in survival. The results can be analyzed after 376 events (patient deaths), which will likely occur in 2004. BEC-2 plus BCG is currently licensed by ImClone Systems Incorporated in North America, and by Merck Oncology in Europe, Australia, and New Zealand. POLYVALENT VACCINE
A series of trials have tested other vaccines against antigens relevant to SCLC. Based on prior immunohistochemical analyses of tumor samples, four antigens emerged as lead targets for immunotherapy in SCLC: GM2, Globo H, fucosyl GM1, and polysialic acid.12 The immunogenicity of GM2 and Globo H has been established in studies of other malignancies.13-15 However, fucosyl GM1 and polysialic acid have highly restricted expression in SCLC, thus these vaccines have been tested only in SCLC patients (Table 1).13-18 For the SCLC vaccine trials, eligibility criteria have been relatively consistent. Patients with limited- or extensive-stage SCLC are enrolled 4 to 12
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LEE M. KRUG
Table 1. Proposed Components for a Polyvalent Vaccine in Small Cell Lung Cancer
Antigen GM2 Globo H Fucosyl GM1 Bovine Thyroid Synthetic Polysialic acid Unmodified N-Propionylated
Tumor Type Tested
Study
Melanoma Breast Prostate
Helling et al, 199513 Gilewski et al, 200114 Slovin et al, 199915
SCLC
Dickler et al, 199916 Krug et al, 200217
SCLC
Ng et al, 200118 Krug, IRB 02-097
NOTE. All of the antigens have been tested as conjugates with keyhole limpet hemocyanin with QS-21 adjuvant. Abbreviation: SCLC, small cell lung cancer.
weeks after completion of their usual initial chemotherapy and radiation. Patients must not be taking systemic corticosteroids or have an underlying immune deficiency. Because of the potential cross-reaction of the vaccines with neural tissue, patients must not have peripheral sensory neuropathy greater than grade 1. To overcome immunologic tolerance, all of these trials use antigen conjugates with the carrier KLH.19 QS-21, a saponin tree bark extract,20 is added as an immunologic adjuvant. This compound has previously been shown to induce high antibody titers when compared with other adjuvants.13 The vaccinations are administered subcutaneously on weeks 1, 2, 3, 4, 8, and 16. Blood is drawn for immunology studies before each vaccination and on weeks 10 and 18. The primary endpoint of these trials is immune response defined as an antibody titer of ⱖ1:80 by enzymelinked immunosorbent assay (ELISA) and confirmed by flow cytometry using tumor cells expressing the target. If five of nine patients develop a significant immune response, the vaccine warrants further study.21 Fucosyl GM1 The first small cell-specific antigen tested in our vaccine clinical trials was fucosyl GM1. Fucosyl GM1 is a ganglioside that was initially isolated from the bovine thyroid gland.22 Several lines of evidence support targeting fucosyl GM1 in pa-
tients with SCLC. Using the monoclonal antibody F12, fucosyl GM1 was identified by immunofluorescence in 19 of 21 human SCLC tumors.23 It was also detected in culture media from SCLC cell lines and in tumor extracts and serum of nude mice xenografts.24 Furthermore, fucosyl GM1 was detected in the serum of six of 20 patients with extensive-stage SCLC but not in 12 non–small cell lung cancer patients or 20 healthy volunteers. In the first clinical trial of this vaccine, fucosyl GM1 was directly obtained from cow thyroid gland.16 Ten patients received at least five vaccinations with fucosyl GM1-KLH 30 g plus QS-21 100 g and were considered evaluable for immune response. All patients had serologic immune response by ELISA with IgM titers ranging from 1:320 to 1:2,560 and IgG titers ranging from 1:40 to 1:2,560. Most patients mounted significant antibody titers by week 4, and these titers were sustained. Flow cytometry demonstrated antibody binding to fucosyl GM1 expressing rat hepatoma and SCLC cell lines in eight of 10 and six of 10 cases, respectively. Posttreatment sera induced complement-mediated cytotoxicity in nine of 10 cases. To allow easier manufacturing and to decrease the risk of prion infection, a synthetic version of fucosyl GM1 was formulated. A trial with this synthetic fucosyl GM1 was then conducted.17 Three dose levels of fucosyl GM1 in the KLH conjugate (30, 10, and 3 g) were studied with 100 g QS-21. After vaccination, five of the six patients treated at 30 g and three of the five patients who received 10 g mounted an IgM response with titers ⱖ1:80 (range, 1:80 to 1:2,560). In seven of these patients, specificity of the response was confirmed by immune thin-layer chromatography with purified gangliosides. Cell surface reactivity was detected by fluorescent-activated cell sorting in seven patients. Polysialic Acid The next target that was studied was polysialic acid. Polysialic acid is a long polymer of negatively charged sialic acid residues that binds restrictively to a neural cell adhesion molecule.25 Expression is normally restricted to the surface of group B meningococcus and, in humans, to embryonic neural tissues.26 Its large size physically inhibits the affinity of neural cell adhesion molecule to the receptors of adjacent cells and thereby promotes cell
VACCINE THERAPY FOR SMALL CELL LUNG CANCER
motility. Several investigators have found immunostaining for polysialic acid in nearly every SCLC tumor tested.12,27,28 Because it inhibits cell– cell interactions, polysialic acid may contribute to the high metastatic potential of SCLC.29 Because of its presence in the embryo and to a limited extent in adult brain tissues, humans have an immunologic tolerance to polysialic acid.30 This may, in fact, explain the lack of clearance of group B meningococcus and the subsequent development of meningitis caused by this organism. In an attempt to overcome this tolerance, MSKCC investigators have modified polysialic acid by Npropionylation. This technique, which involves replacement of acetyl groups with propionyl groups, has been shown to boost the IgG response to meningococcal group B polysaccharide in mice.31 The MSKCC group conducted a study in which seven patients received unmodified polysialic acid, and six patients received N-propionylated polysialic acid conjugated with KLH and administered with QS-21.18 Serologic analysis of serum IgM and IgG titers showed no reactivity after treatment with the unmodified polysialic acid vaccine. With the N-propionylated version, five of six patients showed augmentation of IgM antibodies against polysialic acid as well as the H345 SCLC cell line. A follow-up dose de-escalation study with N-propionylated polysialic acid has been initiated to better assess the immunogenicity, toxicities, and optimal dose of this vaccine. The ultimate plan of the MSKCC vaccine program is to combine these components into a polyvalent vaccine. The hypothesis is that a broader immune response would more likely result in immunologic activity against this heterogenous tumor. Preclinical data support this approach. Nine SCLC cell lines were tested with monoclonal antibodies against seven target antigens individually or pooled in different combinations.32 No single monoclonal antibody bound to more than five of the nine cell lines. However, combining monoclonal antibodies against GM2, fucosyl GM1, Globo H, and polysialic acid resulted in strong reactivity against seven of nine cell lines tested by flow cytometry and all six cell lines tested by complement-dependent cytotoxicity. The addition of monoclonal antibodies against GD2, GD3, and sLea increased reactivity only slightly.
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CONCLUSIONS
In summary, an immune approach to the treatment of residual disease in SCLC appears logical and feasible. Small cell tumors express a number of antigens that can be targeted for immunotherapy. The anti-idiotype antibody to the ganglioside GD3, termed BEC-2, has now completed phase III testing in patients with limited-stage disease, and the results of that trial are pending. Smaller trials of KLH conjugate vaccines have established the immunogenicity of GM2, Globo H, fucosyl GM1, and polysialic acid. A randomized phase II trial testing a tetravalent vaccine of these components in patients with SCLC who have completed initial therapy is planned to start in 2004. Other biologicbased therapies relevant to SCLC, such as monoclonal antibodies, tyrosine kinase and cell cycle inhibitors, and antisense drugs, are also being developed that, hopefully, may be used to prevent disease relapse. REFERENCES 1. Sculier JP, Berghmans T, Castaigne C, et al: Maintenance chemotherapy for small cell lung cancer: A critical review of the literature. Lung Cancer 19:141-151, 1998 2. Niell H, Herndon JE, Miller A, et al: Randomized phase III Intergroup trial (CALGB 9732) of etoposide (VP-16) and cisplatin (DDP) with or without paclitaxel (TAX) and G-CSF in patients with extensive stage small cell lung cancer (EDSCLC). Proc Am Soc Clin Oncol 21:293a, 2002 (abstr 1169) 3. Roth BJ, Johnson DH, Einhorn LH, et al: Randomized study of cyclophosphamide, doxorubicin, and vincristine versus etoposide and cisplatin versus alternation of these two regimens in extensive small-cell lung cancer: A phase III trial of the Southeastern Cancer Study Group. J Clin Oncol 10:282-291, 1992 4. Schiller JH, Adak S, Cella D, et al: Topotecan versus observation after cisplatin plus etoposide in extensive-stage small-cell lung cancer: E7593–A phase III trial of the Eastern Cooperative Oncology Group. J Clin Oncol 19:2114-2122, 2001 5. Humblet Y, Symann M, Bosly A, et al: Late intensification chemotherapy with autologous bone marrow transplantation in selected small-cell carcinoma of the lung: A randomized study. J Clin Oncol 5:1864-1873, 1987 6. Livingston PO, Calves MJ, Natoli EJ Jr: Approaches to augmenting the immunogenicity of the ganglioside GM2 in mice: Purified GM2 is superior to whole cells. J Immunol 138:1524-1529, 1987 7. Chapman PB, Houghton AN: Induction of IgG antibodies against GD3 ganglioside in rabbits by an anti-idiotypic monoclonal antibody. J Clin Invest 88:186-192, 1991 8. Yao TJ, Meyers M, Livingston PO, et al: Immunization of melanoma patients with BEC2-keyhole limpet hemocyanin plus BCG intradermally followed by intravenous booster im-
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characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J Immunol 146:431-437, 1991 21. Yao TJ, Begg CB, Livingston PO: Optimal sample size for a series of pilot trials of new agents. Biometrics 52:992-1001, 1996 22. Macher BA, Pacuszka T, Mullin BR, et al: Isolation and identification of a fucose- containing ganglioside from bovine thyroid gland. Biochim Biophys Acta 588:35-43, 1979 23. Brezicka FT, Olling S, Nilsson O, et al: Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies. Cancer Res 49:1300-1305, 1989 24. Vangsted AJ, Clausen H, Kjeldsen TB, et al: Immunochemical detection of a small cell lung cancer-associated ganglioside (FucGM1) antigen in serum. Cancer Res 51:28792884, 1991 25. Finne J, Finne U, Deagostini-Bazin H, et al: Occurrence of alpha 2-8 linked polysialosyl units in a neural cell adhesion molecule. Biochem Biophys Res Commun 112:482-487, 1983 26. Rutishauser U: Polysialic acid at the cell surface: Biophysics in service of cell interactions and tissue plasticity. J Cell Biochem 70:304-312, 1998 27. Komminoth P, Roth J, Lackie PM, et al: Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids. Am J Pathol 139:297-304, 1991 28. Lantuejoul S, Moro D, Michalides RJ, et al: Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors. Am J Surg Pathol 22:1267-1276, 1998 29. Daniel L, Durbec P, Gautherot E, et al: A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process. Oncogene 20:997-1004, 2001 30. Finne J, Leinonen M, Makela PH: Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2:355-357, 1983 31. Jennings HJ, Roy R, Gamian A: Induction of meningococcal group B polysaccharide- specific IgG antibodies in mice by using an N-propionylated B polysaccharide-tetanus toxoid conjugate vaccine. J Immunol 137:1708-1713, 1986 32. Livingston P, Hood C, Krug L, et al: Antigen expression on small cell lung cancer (SCLC) cell lines confirms selection of a tetravalent vaccine against SCLC containing GM2, fucosyl GM1, Globo H, and polysialic acid. Proc Am Assoc Cancer Res 44:1090, 2003 (abstr 4754)