qs-21 and Melanoma

The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation.. A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat.. After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity.. GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Cancer Vaccines; Disease-Free Survival; Female; G(M2) Ganglioside; Hemocyanins; Humans; Kaplan-Meier Estimate; Lymph Nodes; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Neoplasm Staging; Saponins; Skin Neoplasms; Watchful Waiting

The importance of CD8(+) cytolytic T cells for protection from viral infection and in the generation of immune responses against tumors has been well established. In contrast, the role of CD4(+) T-helper cells in human infection and in cancer immunity has yet to be clearly defined. In this pilot study, we show that immunization of three resected, high-risk metastatic melanoma patients with a T-helper epitope derived from the melanoma differentiation antigen, melanoma antigen recognized by T cells-1, results in CD4(+) T-cell immune responses. Immune reactivity to that epitope was detected by DR4-peptide tetramer staining, and enzyme-linked immunospot assay of fresh and restimulated CD4(+) T cells from patients over the course of the 12-month vaccine regimen. The postvaccine CD4(+) T cells exhibited a mixed T-helper 1/T-helper 2 phenotype, proliferated in response to the antigen and promiscuously recognized the peptide epitope bound to different human leukocyte antigen-DRbeta alleles. For 1 DRbeta1*0401(+) patient, antigen-specific CD4(+) T cells recognized human leukocyte antigen-matched antigen-expressing tumor cells, secreted granzyme B, and also exhibited cytolysis that was MHC class II-restricted. These data establish the immunogenicity of a class II epitope derived from a melanoma-associated antigen and support the inclusion of class II peptides in future melanoma vaccine therapies.

Topics: Age Factors; Alleles; Antigens, Neoplasm; Cancer Vaccines; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Flow Cytometry; Granzymes; HLA Antigens; HLA-DR Antigens; Humans; Immunotherapy; Leukocytes, Mononuclear; Lipid A; Lymphocytes; Male; MART-1 Antigen; Melanoma; Microscopy, Fluorescence; Neoplasm Proteins; Peptides; Pilot Projects; Saponins; Serine Endopeptidases; T-Lymphocytes; Th1 Cells; Th2 Cells; Time Factors; Treatment Outcome

We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.

Topics: Adult; Aged; Aged, 80 and over; CD8-Positive T-Lymphocytes; Female; Freund's Adjuvant; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-A2 Antigen; Humans; Lymphocyte Count; Male; Melanoma; Middle Aged; Monophenol Monooxygenase; Peptide Fragments; Saponins; T-Lymphocytes; Treatment Outcome; Vaccination

Vaccine alternatives to high-dose interferon alfa-2b therapy (HDI), the current standard adjuvant therapy for high-risk melanoma, are of interest because of toxicity associated with HDI. The GM2 ganglioside is a well-defined melanoma antigen, and anti-GM2 antibodies have been associated with improved prognosis. We conducted a prospective, randomized, intergroup trial to evaluate the efficacy of HDI for 1 year versus vaccination with GM2 conjugated to keyhole limpet hemocyanin and administered with QS-21 (GMK) for 96 weeks (weekly x 4 then every 12 weeks x 8).. Eligible patients had resected stage IIB/III melanoma. Patients were stratified by sex and number of positive nodes. Primary end points were relapse-free survival (RFS) and overall survival (OS).. Eight hundred eighty patients were randomized (440 per treatment group); 774 patients were eligible for efficacy analysis. The trial was closed after interim analysis indicated inferiority of GMK compared with HDI. For eligible patients, HDI provided a statistically significant RFS benefit (hazard ratio [HR] = 1.47, P = .0015) and OS benefit (HR = 1.52, P = .009) for GMK versus HDI. Similar benefit was observed in the intent-to-treat analysis (RFS HR = 1.49; OS HR = 1.38). HDI was associated with a treatment benefit in all subsets of patients with zero to > or = four positive nodes, but the greatest benefit was observed in the node-negative subset (RFS HR = 2.07; OS HR = 2.71 [eligible population]). Antibody responses to GM2 (ie, titers > or = 1:80) at days 29, 85, 365, and 720 were associated with a trend toward improved RFS and OS (P2 = .068 at day 29).. This trial demonstrated a significant treatment benefit of HDI versus GMK in terms of RFS and OS in melanoma patients at high risk of recurrence.

Topics: Adult; Aged; Cancer Vaccines; Disease-Free Survival; Female; G(M2) Ganglioside; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Interferon alpha-2; Interferon-alpha; Male; Melanoma; Middle Aged; Neoplasm Staging; Prospective Studies; Recombinant Proteins; Saponins; Vaccination; Vaccines, Conjugate

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Cancer Vaccines; CD4-Positive T-Lymphocytes; Cell Division; Cell Line; Cytokines; Epitopes, T-Lymphocyte; Female; Follow-Up Studies; gp100 Melanoma Antigen; Headache; HLA-A2 Antigen; Humans; Hypersensitivity, Delayed; Interferon-gamma; Lymph Nodes; Male; Melanoma; Membrane Glycoproteins; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Neoplasm Staging; Pain; Peptide Fragments; Peptides; Saponins; Skin; Skin Diseases; Skin Tests; Survival Analysis; T-Lymphocytes, Cytotoxic; Tetanus Toxoid; Th1 Cells; Time Factors; Treatment Outcome

The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the immunological adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 microg of ganglioside plus 100 microg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40-1/1,280 in all patients and IgG titers of 1/160-1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28.

Topics: Adjuvants, Immunologic; Antibody Formation; Autoantibodies; Cancer Vaccines; Carbohydrate Sequence; Enzyme-Linked Immunosorbent Assay; Gangliosides; Humans; Immunoglobulin M; Melanoma; Molecular Sequence Data; Neoplasm Staging; Saponins; Time Factors; Vaccines, Conjugate

Increasing doses of saponin fraction QS-21 were administered as immunological adjuvant in a Phase 1 trial with a constant dose of the melanoma ganglioside GM2 covalently attached to keyhole limpet haemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IV melanoma who were free from disease after surgery were treated with six vaccinations administered subcutaneously over a 5-month period. Local and systemic reactions were QS-21 dose-related. Doses of < or = 100 micrograms induced mild local tenderness and inflammation at vaccination sites lasting 2-4 days and occasional brief low-grade fever and malaise, but no significant incapacitation. The 200 micrograms dose induced low-grade fever and malaise after 30% of vaccinations and local reactions as large as 20 cm in diameter were seen in all patients, resulting in discomfort with usage of the injected extremity for 5-10 days. The titres of IgM and IgG antibodies against GM2, and IgG antibodies against KLH, were highest at the 100 and 200 micrograms QS-21 doses. No antibodies against QS-21 were detected. This trial identifies the 100 micrograms dose of QS-21 as the optimal well tolerated dose for induction of antibodies against both the melanoma ganglioside/GM2 and the protein KLH in melanoma patients.

Topics: Adjuvants, Immunologic; Antibodies, Neoplasm; G(M2) Ganglioside; Hemocyanins; Humans; Hypersensitivity, Delayed; Melanoma; Saponins

The saponin fraction QS-21 from Quillaja saponaria has been demonstrated to be a potent immunological adjuvant when mixed with keyhole limpet hemocyanin conjugate vaccines, as well as with other classes of subunit antigen vaccines. QS-21 adjuvant is composed of two isomers that include the apiose and xylose forms in a ratio of 65:35, respectively. The chemical syntheses of these two isomers in pure form have recently been disclosed. Herein we describe detailed in vivo immunological evaluations of these synthetic QS-21 isomeric constituents, employing the GD3-KLH melanoma antigen. With this vaccine construct, high antibody titers against GD3 ganglioside and KLH were elicited when GD3-KLH was co-administered with adjuvant, either as the individual separate synthetic QS-21 isomers (SQS-21-Api or SQS-21-Xyl), or as its reconstituted 65:35 isomeric mixture (SQS-21). These antibody titer levels were comparable to that elicited by vaccinations employing naturally derived QS-21 (PQS-21). Moreover, toxicities of the synthetic saponin adjuvants were also found to be comparable to that of naturally derived PQS-21. These findings demonstrate unequivocally that the adjuvant activity of QS-21 resides in these two principal isomeric forms, and not in trace contaminants within the natural extracts. This lays the foundation for future exploration of structure-function correlations to enable the discovery of novel saponins with increased potency, enhanced stability, and attenuated toxicity.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Cancer Vaccines; Drug Evaluation, Preclinical; Female; Gangliosides; Hemocyanins; Melanoma; Mice; Mice, Inbred C57BL; Molecular Structure; Saponins

Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH.. Eighteen patients with melanoma were vaccinated s.c. in the adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 micro g) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the immunological adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response.. The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30- micro g dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30- micro g GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100.. GD2L-KLH conjugate vaccine plus adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.

Topics: Adjuvants, Immunologic; Antibody Formation; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Cancer Vaccines; Enzyme-Linked Immunosorbent Assay; Gangliosides; Hemocyanins; Humans; Immunoglobulin G; Immunoglobulin M; Lactones; Melanoma; Saponins; Skin Neoplasms; Vaccination; Vaccines, Conjugate

Cell surface gangliosides show altered patterns of expression as a consequence of malignant transformation and have therefore been of interest as potential targets for immunotherapy, including vaccine construction. One obstacle has been that some of the gangliosides that are overexpressed in human cancers are poorly immunogenic in humans. A case in point is GD3, a prominent ganglioside of human malignant melanoma. Using an approach that has been effective in the construction of bacterial carbohydrate vaccines, we have succeeded in increasing the immunogenicity of GD3 in the mouse by conjugating the ganglioside with immunogenic carriers. Several conjugation methods were used. The optimal procedure involved ozone cleavage of the double bond of GD3 in the ceramide backbone, introducing an aldehyde group, and coupling to aminolysyl groups of proteins by reductive amination. Conjugates were constructed with a synthetic multiple antigenic peptide expressing repeats of a malarial T-cell epitope, outer membrane proteins of Neisseria meningitidis, cationized bovine serum albumin, keyhole limpet hemocyanin, and polylysine. Mice immunized with these conjugates showed a stronger antibody response to GD3 than mice immunized with unconjugated GD3. The strongest response was observed in mice immunized with the keyhole limpet hemocyanin conjugate of the GD3 aldehyde derivative and the adjuvant QS-21. These mice showed not only a long-lasting high-titer IgM response but also a consistent high-titer IgG response (predominantly IgG1), indicating recruitment of T-cell help, although the titers of IgM and IgG antibodies following booster immunizations were not as high as they are in the response to classical T-cell-dependent antigens. This method is applicable to other gangliosides, and it may be useful in the construction of immunogenic ganglioside vaccines for the immunotherapy of human cancers expressing gangliosides on their cell surface.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neoplasm; Female; Gangliosides; Hemocyanins; Immunotoxins; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oligosaccharides; Saponins; Vaccines