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© 2002 American Society for Clinical Oncology Vaccination of Metastatic Melanoma Patients With Autologous Tumor-Derived Heat Shock Protein gp96-Peptide Complexes: Clinical and Immunologic FindingsByFrom the Units of General Surgery 2, Immunotherapy of Human Tumors, Immunohematology and Blood Bank, Pathology, Diagnostic Radiology, and Pharmacy, Istituto Nazionale Tumori; Divisions of Surgery and Pathology, Istituto Europeo di Oncologia, Milan; Units of Cancer Biotherapy, Anesthesiology, and Surgery, Centro di Riferimento Oncologico, Aviano; Divisions of Medical Oncology and Surgery, Istituto Scientifico Tumori, Genoa; and Sigma Tau, i.f.r. S.p.A., Rome, Italy; Antigenics, Inc, Woburn, MA; and Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT. Address reprint requests to Giorgio Parmiani, MD, Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Via Venezian, 1-20133 Milan, Italy; email: parmiani{at}istitutotumori.mi.it
PURPOSE: To determine the immunogenicity and antitumor activity of a vaccine consisting of autologous, tumor-derived heat shock protein gp96-peptide complexes (HSPPC-96, Oncophage; Antigenics, Inc, Woburn, MA) in metastatic (American Joint Committee on Cancer stage IV) melanoma patients. PATIENTS AND METHODS: Sixty-four patients had surgical resection of metastatic tissue required for vaccine production, 42 patients were able to receive the vaccine, and 39 were assessable after one cycle of vaccination (four weekly injections). In 21 patients, a second cycle (four biweekly injections) was given because no progression occurred. Antigen-specific antimelanoma T-cell response was assessed by enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) obtained before and after vaccination. Immunohistochemical analyses of tumor tissues were also performed. RESULTS: No treatment-related toxicity was observed. Of 28 patients with measurable disease, two had a complete response (CR) and three had stable disease (SD) at the end of follow-up. Duration of CR was 559+ and 703+ days, whereas SD lasted for 153, 191, and 272 days, respectively. ELISPOT assay with PBMCs of 23 subjects showed a significantly increased number of postvaccination melanoma-specific T-cell spots in 11 patients, with clinical responders displaying a high frequency of increased T-cell activity. Immunohistochemical staining of melanoma tissues from which vaccine was produced revealed high expression of both HLA class I and melanoma antigens in seven of eight clinical responders (two with CR, three with SD, and the three with long-term disease-free survival) and in four of 12 nonresponders. CONCLUSION: Vaccination of metastatic melanoma patients with autologous HSPPC-96 is feasible and devoid of significant toxicity. This vaccine induced clinical and tumor-specific T-cell responses in a significant minority of patients.
NO EFFECTIVE THERAPY for metastatic melanoma is currently available. Several phase II studies of biochemotherapy have reported 40% to 50% clinical response rates.1 Recently, one2 but not another3 phase III trial confirmed a significantly higher response rate of biochemotherapy as compared with chemotherapy alone.2 However, these treatments were associated with high toxicity and did not result in concurrent improvement in overall survival.2,3 Several vaccine approaches have been tested. Most recently, patients have been vaccinated with peptides (with or without adjuvants, or pulsed on autologous dendritic cells) derived from proteins expressed by melanoma cells. The clinical response rate in these studies has ranged between 5% and 30%.4-6 In one trial where patients received concurrent administration of high-dose interleukin-2,7 a response rate of 42% was achieved. In virtually all of these studies, complete responses are rare and, with an exception,5 of short duration, frequently less than 6 months. As another approach, the use of gene-modified autologous or allogeneic melanoma cell vaccines has resulted in response rates of 10% to 20%.8 In many trials, patients have been monitored for tumor-specific T-cell responses to vaccination by a variety of assays. Such immunologic responses have been usually detected in a low percentage of patients, and when they have been detected, no correlation between immunologic responses and clinical outcomes has been observed.5,7,9 In this study, we have immunized melanoma patients with vaccines consisting of heat shock protein-peptide complexes. Heat shock proteins (HSPs) are the most common and abundant proteins in all forms of life. They come in different families characterized by members of similar molecular mass (such as hsp70 and hsp90). Previous work has demonstrated that purified, apparently homogeneous preparations of gp96, hsp70, hsp90, and certain other HSPs are actually noncovalent complexes of HSPs and peptides (see Srivastava10). The peptides are derived from the proteins expressed in the cells from which the HSPs are purified and include normal self-peptides and antigenic peptides. This phenomenon has been demonstrated in mouse11,12 and human tumors.13 In the latter, hsp70-peptide complexes extracted from melanoma cells have been found to contain well-known peptides on the basis of their ability to stimulate antigen-specific CD8+ T cells from melanoma patients’ peripheral blood mononuclear cells (PBMCs).13 Vaccination with autologous tumor-derived HSP-peptide complexes has been shown to result in both prophylactic and therapeutic antitumor activity in multiple animal tumor models in multiple genera.14,15 Furthermore, a pilot clinical study of vaccination with HSP peptide complex-96 (HSPPC-96) isolated from autologous nonmelanoma tumors suggests that a specific antitumor immunity can be generated, even in advanced cancer patients.16 The immunogenicity of tumor-derived HSP-peptide complexes, like the immunogenicity of experimentally induced tumors of mice and rats, has been shown to be individually tumor specific and not tumor type specific. These observations have led to the conclusion that the relevant tumor-antigenic, immunoprotective peptides are derived from unique rather than shared tumor antigens.17 This is the basis of the vaccination protocol in this study, where each patient is vaccinated with HSP-peptide complexes isolated from his or her own tumor. In the present study, we have evaluated whether vaccination with autologous, tumor-derived HSPPC-96 (Oncophage; Antigenics, Inc, Woburn, MA) is feasible and safe, and whether such vaccination elicits immunologic and/or clinical response in the autologous host.
Patients and Treatment Between February 1999 and January 2000, 64 stage IV (American Joint Committee on Cancer) metastatic melanoma patients were enrolled on the study and had surgical resection of tumor tissue for vaccine production. Patients were enrolled at four Italian centers: the Istituto Nazionale Tumori (n = 32) and the European Institute of Oncology of Milan (n = 21), the Regional Center of Oncology of Aviano (n = 7), and the Scientific Institute of Tumors of Genoa (n = 6). Of these patients, 39 received at least one cycle of vaccination, and were assessable. Eligibility criteria for vaccination included the following: (1) histologically verified melanoma with resectability of at least one lesion that was able to provide the necessary amount of nonnecrotic neoplastic tissue (3 g for vaccine preparation and 1 g for immunologic assays); (2) a performance status (Zubrod) of 2 or less; (3) life expectancy of at least 20 weeks; (4) normal WBC and platelet counts, and hemoglobin level greater than 10 g/L; (5) bilirubin less than 1.5 times normal, ALT less than four times normal, and adequate renal function with serum creatinine of less than two times normal; (6) fully recovered from prior anticancer therapy with at least a 4-week interval from the last administration of prior anticancer treatment; and (7) a positive response to recall antigens (Multitest Mérieux; Imtix, Milan, Italy) as a sign of sufficient immune function. All patients underwent clinical and radiographic staging before treatment. Patients were excluded from the study if they had active brain metastases, had concomitant autoimmune or malignant diseases or were receiving concurrent anticancer therapy, required steroid drugs, or had a history of serious intercurrent medical illnesses. Women of childbearing potential required a negative serum pregnancy test before entry onto the study and agreed to use an effective method of contraception while on treatment. All patients gave written informed consent to participate in the study. The clinical protocol was approved by the Internal Scientific Review Board and by the Independent Ethics Committee of the Istituto Nazionale Tumori of Milan and of the other participating centers. Patients were vaccinated with autologous HSPPC-96 starting 5 to 8 weeks after resection of tumor metastases with 5 or 50 µg of vaccine given at weekly intervals either intradermally or subcutaneously. Patients were randomly assigned to either dose or route of administration of the vaccine. After four vaccinations (first cycle), and if no progression occurred, patients could receive four additional injections once every 2 weeks (second cycle) if vaccine was available. This occurred in 21 patients; in addition, four patients (02-007, 02-021, 03-003, and 03-005) underwent two to three additional monthly immunizations of HSPPC-96 as a third cycle of vaccination. Patients were monitored for toxicity, including complete clinical evaluation, standard blood tests including differential blood counts, serum chemistry, urinalysis, cardiac, liver and renal functions, and autoimmune reactions (antimicrosomal and antithyroglobulin antibodies, T3, free T4, rheumatic factor, and antinucleoprotein and antimitochondrial antibodies). Ophthalmologic examinations were carried out at 4, 8, and 12 weeks to assess possible autoimmune reactions caused by melanoma/retina cross-reacting differentiation antigens. Tumor evaluation was performed before resection of metastatic melanoma, at the pretreatment visit (baseline evaluation or visit 1 [V1]), at weeks 4 and 8 of the study, and thereafter every 3 months and as indicated. This was by the same sequential diagnostic imaging method. The size of tumor lesions was evaluated as the product of the largest perpendicular diameters of the lesion and recorded. Clinical responses were defined as less than progressive disease, which spans the spectrum from stable disease (SD) to complete response (CR). Disease-free survival (DFS) was measured from the day of onset of vaccination until relapse in patients that were rendered disease-free by surgery.
Preparation of Vaccine
Evaluation of Immune Response Skin response (delayed-type hypersensitivity). Skin reaction at the site of vaccination was assessed 1 hour after every injection by the attending physician and after 24 and 48 hours by the patient on instruction. Delayed-type hypersensitivity was carried out by subcutaneous injection of 3 x 104 or 10 x 105 autologous tumor cells (when available) derived from melanoma lesions by mechanical processing. Tumor cells were then irradiated (150 Gy) and tested for sterility by routine microbiologic assays. Cell suspensions were aliquoted, stored in liquid nitrogen, and thawed before injection. Autologous irradiated (50 Gy) and autologous PBMCs, obtained from the blood by Ficoll (Pharmacia Biosystems, Uppsala, Sweden) gradient, were also injected as negative control immediately before the first, fourth, and fifth vaccinations. Eighty to 100 mL of heparinized blood for immunologic assays was obtained by preparation of a buffy coat from each patient before vaccination (V1) and at V5 (fourth vaccination) and V6 (4 weeks afterward). In patients who received additional vaccinations, blood was obtained also at V7, V9, V10, or V11. The PBMCs were isolated by Ficoll gradient centrifugation and frozen in aliquots in liquid nitrogen. The following tests were performed at the Istituto Nazionale Tumori for all patients to assess their melanoma-specific immune response. PBMCs of only 23 subjects could be analyzed in part for lack of availability of PBMCs, and in part for the lack of appropriate melanoma cell target because of the rare HLA allele of patients.
Enzyme-Linked Immunospot Assay
To control the ELISPOT assay, the anti-Melan-A/MART-127-35 cytotoxic T-lymphocyte clone A4222 in the presence or absence of relevant targets (T2 cells pulsed with Melan-A/MART-127-35 peptide and the Melan-A/MART-127-35+ melanoma line 501mel) was always included at the concentration of 400 cells/well. The HLA-A2+ lymphoblastoid cell line T2 carries a TAP mutation, which prevents endogenous peptide presentation but allows external peptides to be presented by its class I HLA empty molecules. After an additional incubation for 20 hours at 37°C and 5% CO2, plates were washed with filtered phosphate-buffered saline (PBS). Wells were then incubated for 2 to 4 hours at room temperature with 50 µL/well of biotinylated mouse antihuman interferon gamma (IFN-
Indicated spot numbers per seeded PBMCs represent mean values of three or six replicates. To calculate the number of PBMCs responding to the antigen (tumor cells, peptide or HSPPC-96/pulsed monocytes or T2 cells) by IFN-
Melanoma and Other Cells or Cell Lines Tumor cell suspensions were obtained by mechanical processing of melanoma lesions. Enzymatic digestion with a pool of DNAse I, collagenase type IV (1 µg/mL) (Biowhittaker Europe, Belgium), hyaluronidase type IV (Sigma) (1 µg/mL), and trypsin-EDTA (Biowhittaker) was performed in some cases to achieve higher cell recovery. Tumor cells were then aliquoted, frozen, and stored in liquid nitrogen. Melanoma cells expressing different HLA alleles and used as targets in the ELISPOT assay were derived from patients accrued onto the study. In addition, the melanoma lines 501mel and 624.38mel were used as HLA-A2+ target cells,22 whereas Me15932 was used as an HLA-A3+ tumor cell line.23 The colon carcinoma cell line Colo206, expressing both HLA-A2 and HLA-A1 alleles, was a gift from C. Scheibenbogen, MD (B. Franklyn University, Berlin). Tumor cell lines were maintained in RPMI 1640 with 10% FCS unless otherwise indicated.
Immunopathology of the Resected Melanoma Metastases
Patients and Vaccination Of 64 patients (Table 1 lists demographics and clinical data) who underwent surgery for removal of melanoma lesions, 39 received one (n = 18) or two (n = 21) complete cycles of vaccination. Others (n = 25) did not receive a complete cycle of vaccination for reasons listed in Table 2. Of the 39 patients who completed one or two cycles of vaccination, 26 were heavily pretreated with a variety of systemic treatments (Table 1). Others (n = 13) underwent surgery alone. Classified by another parameter, 28 patients had residual measurable disease after surgery and 11 were rendered disease-free (Table 1). As mentioned in Patients and Methods, patients were randomized onto 5 or 50 µg HSPPC-96 per vaccination and onto intradermal and subcutaneous vaccination groups.
Toxicity No toxicity attributable to Oncophage was observed in any of the 39 patients, with the exception of small (less than 5 mm) erythemas and/or induration at vaccination sites, all of which disappeared in less than 24 hours and were attributed to the syringe trauma.
Overall Clinical Response Of the 11 patients rendered disease-free by surgery, DFS ranged from 29 to 642 days (median, 117 days). Three patients (01-017, 01-019, and 03-003) remained disease-free for 252, 366, and 642 days, respectively. A clear correlation between dose/route and clinical responses could not be discerned.
CRs
Patient 02-003. This patient (a 73-year-old female) had resection of a primary melanoma in 1996 and of locoregional recurrence in January 1999. She was then accrued onto a randomized trial and treated for 1 year with intermediate-dose IFN-  . In May 1999, she developed diffuse subcutaneous, cutaneous, and mucosal metastases. During staging, metastases were detected also in the lung, liver, spleen, and mediastinal and retroperitoneal lymph nodes. None of the metastatic lesions exceeded 2 cm in maximum diameter. In July 1999, surgery was performed to remove some of the larger and more accessible subcutaneous lesions, and vaccine was produced. In September 1999, she began the first cycle of vaccination with 50 µg of HSPPC-96 delivered subcutaneously. At the end of the first cycle (four injections), the patient presented with a clear CT scan of the lung, and all cutaneous and mucosal lesions appeared flattened. The patient completed treatment up to the second cycle (March 2000), as no more vaccine was available; 2 months later, while still in CR, she began a daily treatment with IFN- at low dose, which was continued up to January 2001, when it was interrupted because of the patient’s low compliance. At the end of vaccination, two lesions were biopsied and no residual neoplastic cells were present: only S-100+ and CD68+ pigment-rich macrophages were found. CT scans demonstrated the early reduction in size and then the complete disappearance of all the remaining visceral lesions. The patient remained without evidence of disease until May 2001, when she developed a brain metastasis, although no other visceral or cutaneous lesions were evident. She then died in August 2001.
Stable Disease
Re-presentation of gp96-Chaperoned Melanoma Antigens by Monocytes to Patients’ Lymphocytes
Immunologic Response in HSPPC-96 Vaccinated Patients: Increased Recognition of Autologous or HLA-A–Matched Allogeneic Melanoma Cells by Patients’ PBMCs
As further evidence of antigen-specific T-cell involvement in increased tumor recognition, fresh PBMCs of the 11 immunologically responding patients were tested by ELISPOT with irradiated autologous or HLA-matched melanoma cells in the presence of anti–class I (W6.32) or anti–class II (L243) HLA antibodies. In all cases, T-cell cultures recognized autologous and/or HLA-matched melanoma cells, and such recognition was significantly inhibited with W6.32 but not L243 antibody, indicating an anti–class I HLA-restricted, CD8+-mediated, T-cell recognition of melanoma antigens. For the 11 subjects, the inhibition of spot formation ranged from 40% (patient 01-025) to 93% (patient 02-003), with nine of 11 cases in which inhibition was higher than 60%. It is of note that the frequency of clinically responding subjects, broadly considered to include also those with SD and long-term DFS, displaying a T-cell response was higher than that of the group of patients whose disease progressed (Table 3). No delayed-type hypersensitivity reactions were detected, as also reported by Janetzki et al16 during vaccination of 12 nonmelanoma patients given autologous HSPPC-96.
Immunopathology of the Resected Melanoma Metastases Among the 21 samples of assessable patients examined, there was a good correlation between the hematoxylin and eosin evaluation and CD3 staining for TILs. In the cases labeled as absent, immunohistochemistry showed either absence of or only rare lymphocytes within the tumor, whereas the brisk cases displayed a diffuse distribution of CD3+ cells (Table 4). In the nonbrisk cases, the TIL population was somewhere between the two ends of the spectrum and were labeled as scattered. In almost all cases (19 of 21), the intratumoral lymphocytes showed a cytotoxic (CD8+) and memory (CD45RO) phenotype. In two cases (01-001 and 01-022), the intratumoral lymphocytes marked only for CD45RO. The remaining two cases (01-027 and 01-006) showed complete absence or few intratumoral lymphocytes. Therefore, a percentage could not be estimated. In the cases where CD4+ cells were seen, they were either at low percentage (20% to 30% of TILs) or the sample was a lymph node, in which case the CD4+ population was interpreted as being part of the "host" lymph node rather than true intratumoral lymphocytes (data not shown). As for melanoma cells, HLA-A, HLA-B, and HLA-C were well expressed (  50% positive cells) in 12 patients and downregulated ( 20% positive cells) in the remaining eight cases. Melan-A/MART-1 was highly expressed ( 50% positive cells) in 12 cases, with two tumors showing 40% stained cells and the remaining six cases displaying low expression (< 20% positive cells). Tumors of seven of eight clinical responders (including two with CR, three with SD, and three long-term disease-free individuals) showed high expression of HLA class I (70% to 100% positive cells) and either Melan-A/MART-1 or of gp-100 (HMB-45), whereas among the nonresponders, class I HLA was downregulated ( 20% positive cells) or absent in seven of 12 cases (patient 01-006 was not assessable) (Table 4) and Melan-A/MART-1 and/or gp100 in three of 12 cases examined.
Our results show that vaccination of metastatic melanoma patients with autologous tumor-derived HSPPC-96 is feasible and well tolerated. In addition, vaccination resulted in some clinical responses apparently associated with T-cell response against melanoma-associated antigens. In the large majority of cases (n = 39), 3 to 4 g of fresh tumor tissue could provide enough HSPPC-96 to vaccinate patients for at least one cycle. Twenty-one of the 39 assessable patients were also given a second cycle of vaccination with four biweekly injections of Oncophage. No treatment-related toxicity (grade I to IV) was found, with the exception of a few instances of local transient erythema caused by injection trauma. Adverse events occurred in six patients, but all were judged to be unrelated to treatment with HSPPC-96.
There were two CRs (patients 01-007 and 02-003), one still in remission 38 months after vaccination and the other recurring after 24 months (as of April 2002). It should be noted, however, that patient 02-003, after 2 months of CR, received low-dose IFN- We obtained immunologic evidence (in two patients) that the HSPPC-96 used for vaccination contained melanoma-derived antigenic peptides. This was shown through a re-presentation assay13 and is the first demonstration of re-presentation of tumor antigens by gp96 in a human system. Because of the lack of autologous melanoma lines, we could not assess whether recognition of autologous HSPPC-96 in these patients involved unique or shared antigens. Such studies are now ongoing. Evidence for association of HSPs with antigenic peptides has been obtained for a large number of antigens10 including, recently, for HBV-specific epitopes.29
In 11 of 23 cases examined, a class I HLA-restricted immunization likely directed to melanoma antigens was shown to occur after vaccination with HSPPC-96, as evaluated by a significant increase or de novo appearance of IFN-
Immunohistochemical analysis of the melanomas revealed downregulation of expression of HLA-A, HLA-B, or HLA-C in 46% (nine of 21) of the assessable patients examined, a figure consistent with data reported in the literature for metastatic melanoma.33 A sample that showed The expression of melanoma antigens was less frequently reduced, with two groups of seven tumors each, out of 21 samples examined, showing less than 20% of cells positive for Melan-A/MART-1 and gp100, respectively. It was of interest that high HLA-A and Melan-A/MART-1 expression was associated with clinical response, suggesting that patients whose tumors bear the appropriate target molecules (ie, HLA-antigen complexes) may become immunologically primed during early tumor growth and able to develop a recall response on vaccination with HSPPC-96. This study was initiated as part of a clinical program to translate into human trials some of the extensive data on tumor-protective immunogenicity of tumor-derived HSP-peptide complexes. It has shown that in addition to being feasible, safe, and well-tolerated, immunization of cancer patients with bulky advanced disease with HSPPC-96 is associated with clinical and measurable immunologic responses in a significant minority of patients. These data, although limited in number and scope, are consistent with the murine experience that therapy of mice with progressively growing cancers with gp96-peptide complexes leads to regression of tumors in a small number of mice and stabilization of disease in additional mice.14
We thank Giuseppe Viale, MD, and Giovanni Mazzarol, MD, of the European Institute of Oncology (Milan) for providing tissue sections of a patient, Paola Squarcina and Ilaria Bersani for technical help, Mary Gios and Rosella Dragonetti for secretarial help, and Grazia Barp for editorial assistance.
Supported by a grant from Sigma Tau i.f.r. S.p.A. (Rome). P.S. is supported by National Institutes of Health grant no. 84479 and a sponsored research agreement with Antigenics, Inc., in which he has a significant financial interest. R.C. and M.C. are employees of the Sigma Tau i.f.r. S.p.A., and J.J.L. is an employee of Antigenics, Inc. Presented in part at the Thirty-Seventh Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001. © 2002 by American Society of Clinical Oncology. 0732-183X/02/2020-4169/$20.00
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2. Eton O, Legha SS, Bedikian AY, et al: Sequential biochemotherapy versus chemotherapy for metastatic melanoma: Results from a phase III randomized trial. J Clin Oncol 20: 2045-2052, 2002
3. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al: Prospective randomized trial of the treatment of patients with metastatic melanoma using chemotherapy with cisplatin, dacarbazine, and tamoxifen alone or in combination with interleukin-2 and interferon alfa-2b. J Clin Oncol 17: 968-975, 1999 4. Nestle FO, Alijagic S, Gilliet M, et al: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4: 328-332, 1998[CrossRef][Medline] 5. Marchand M, Van Baren N, Weynants P, et al: Tumor regression observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 80: 219-230, 1999[CrossRef][Medline]
6. Thurner B, Haendle I, Roder C, et al: Vaccination with Mage-3A1 peptide-pulsed mature monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190: 1669-1678, 1999 7. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al: Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 4: 321-327, 1998[CrossRef][Medline] 8. Parmiani G, Rodolfo M, Melani C: Immunological gene therapy with ex vivo gene-modified tumor cells: A critique and a reappraisal. Hum Gene Ther 11: 1269-1275, 2000[CrossRef][Medline] 9. Cormier JN, Salgaller ML, Prevette T, et al: Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART1/Melan-A. Cancer J Sci Am 3: 37-44, 1996[Medline] 10. Srivastava PK: Interaction of heat shock proteins with peptides and with antigen presenting cells: Chaperoning of the innate and adaptive immune responses. Annu Rev Immunol 20: 395-425, 2002[CrossRef][Medline] 11. Suto R, Srivastava PK: A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 269: 1585-1588, 1996
12. Ishii T, Udono H, Yamano T, et al: Isolation of MHC class I-restricted tumor antigen peptide and its precursors associated with heat shock proteins hsp70, hsp90, and gp96. J Immunol 162: 1303-1309, 1999
13. Castelli C, Ciupitu A-MT, Rini F, et al: Human HSP70 peptide complexes specifically activate anti-melanoma T-cells. Cancer Res 61: 222-227, 2001
14. Tamura Y, Peng P, Liu K, et al: Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 278: 117-120, 1997 15. Robert J, Menoret A, Basu S, Cohen N, Srivastava PK: Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70. Eur J Immunol 31: 186-95, 2001[CrossRef][Medline] 16. Janetzki S, Palla D, Rosenhauer V, et al: Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: A pilot study. Int J Cancer 88: 232-238, 2000[CrossRef][Medline] 17. Srivastava PK: Do human cancers express shared protective antigens? or the necessity of remembrance of things past. Semin Immunol 8: 295-302, 1996[CrossRef][Medline] 18. Srivastava PK, Jaikaria NS: Methods of purification of heat shock protein-peptide complexes for use as vaccines against cancers and infectious diseases. Methods Mol Biol 156: 175-186, 2001[Medline] 19. Pass HA, Schwarz SL, Wunderlick JR, Rosenberg SA: Immunization of patients with melanoma peptide vaccines: Immunologic assessment using the ELISPOT assay. Cancer J Sci Am 4: 316-323, 1998[Medline] 20. Lewis JJ, Janetzki S, Schaed S, et al: Evaluation of CD8+ T-cell frequencies by the ELISPOT assay in healthy individuals and in patients with metastatic melanoma immunized with tyrosinase peptide. Int J Cancer 87: 391-398, 2000[CrossRef][Medline]
21. Scheibenbogen C, Romero P, Rivoltini L, et al: Quantification of antigen-reactive T cells in peripheral blood by IFN
22. Kawakami Y, Eliyahu S, Delgado CH, et al: Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci U S A 91: 3515-3519, 1994 23. Mazzocchi A, Storkus WJ, Traversari C, et al: Multiple melanoma-associated epitopes recognized by HLA-A3-restricted CTLs and shared by melanomas but not melanocytes. J Immunol 157: 3030-3038, 1996[Abstract]
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
) mAb (Mabtech, Nacka, Sweden) at the concentration of 1 µg/mL in filtered PBS with 0.5% FCS. Wells were then washed with PBS and 100 µL/well of streptavidin alkaline phosphatase (Mabtech) diluted 1:1,000 in filtered PBS–0.5% FCS was then added. After 1 hour incubation at room temperature, plates were washed extensively and 100 µL/well of substrate (BioRad, Hercules, CA) was added. Color development was stopped by washing in tap water when dark spots emerged (up to 30 minutes). Plates were then left to dry overnight at room temperature and spots were counted by a computer-assisted ELISPOT reader (Bioline, AID, Torino, Italy). For HLA-blocking experiments, PBMCs were thawed 18 hours before their use and depleted from monocytes by overnight adherence in 10% FCS RPMI 1640 at 37°C; nonadherent cells were used as effectors at 2 x 105 cells/well. Autologous or allogeneic HLA-matched melanoma cells or HSPPC-96/pulsed monocytes were preincubated with either W6.32 or L243 antibodies for 30 minutes on ice and used as targets (2 x 104 cells/well). 
