Randomized trial of an allogeneic melanoma lysate vaccine with low-dose interferon Alfa-2b compared with high-dose interferon Alfa-2b for Resected stage III cutaneous melanoma.

PURPOSE: To compare the overall survival (OS) of patients with resected stage III melanoma administered active specific immunotherapy and low-dose interferon alfa-2b (IFN-alpha-2b) with the OS achieved using high-dose IFN-alpha-2b. PATIENTS AND METHODS: An Ad Hoc Melanoma Working Group of 25 investigators treated 604 patients from April 1997 to January 2003. Patients were stratified by sex and number of nodes and were randomly assigned to receive either 2 years of treatment with active specific immunotherapy with allogeneic melanoma lysates and low-dose IFN-alpha-2b (arm 1) or high-dose IFN-alpha-2b alone for 1 year (arm 2). Active specific immunotherapy was injected subcutaneously (SC) weekly for 4 weeks, at week 8, and bimonthly thereafter. IFN-alpha-2b SC was begun on week 4 and continued thrice weekly at 5 MU/m2 for 2 years. IFN-alpha-2b in arm 2 was administered according to the Eastern Cooperative Oncology Group 1684 study regimen. RESULTS: Median follow-up time was 32 months for all patients and 42 months for surviving patients. Median OS time exceeds 84 months in arm 1 and is 83 months in arm 2 (P = .56). Five-year OS rate is 61% in arm 1 and 57% in arm 2. Estimated 5-year relapse-free survival (RFS) rate is 50% in arm 1 and 48% in arm 2, with median RFS times of 58 and 50 months, respectively. The incidence of serious adverse events as a result of treatment was the same in both arms, but more severe neuropsychiatric toxicity was seen in arm 2. CONCLUSION: OS and RFS achieved by active specific immunotherapy and low-dose IFN-alpha-2b were indistinguishable from those achieved by high-dose IFN-alpha-2b. Long RFS and OS times were observed in both treatment arms.

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2.

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (10(9)) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2(+) MUC1(+) MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were "consensus" agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-alpha, IFN-gamma, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.

Phase I trial of large multivalent immunogen derived from melanoma lysates in patients with disseminated melanoma.

PURPOSE: The purpose of this research was to determine the toxicity and immunological activity of large multivalent immunogen (LMI), a preparation of tumor cell membranes affixed to amorphous silica microbeads, in patients with melanoma. EXPERIMENTAL DESIGN: Nineteen patients with metastatic (stage IV) melanoma were entered into the study, of whom 15 received the full 3 months of treatment with LMI. LMI was administered without adjuvant, one-half intradermally (i.d.) and the other half s.c. Because we expected little toxicity, we first treated 2 patients at each dose level, 10-, 30-, or 100-million tumor cell equivalents on weeks 0, 4, and 8, and subsequently randomized the remaining 13 patients to receive treatment with one of those dosage schedules, for a total of 19 patients. Two patients who were registered were found to be ineligible because of brain metastases, and 2 others did not complete the course of treatment for reasons other than toxicity. Thus, 15 patients were fully evaluable. Patients with evidence of a clinical response (at least stable disease at the 12-week checkpoint) had the option of continuing treatment at 4-week intervals. Frequencies of cytolytic T cell precursors against HLA-A2 matched melanoma cells, and delayed-type hypersensitivity to a melanoma cell membrane preparation from a component melanoma cell line were performed to measure immunological efficacy, and serum chemistries and complete blood counts were performed every 2 weeks throughout the study to measure possible toxicity. Computed tomography scans were performed pretreatment and at week 12 to measure possible beneficial effects on known lesions. RESULTS: Eight of the 15 evaluable patients had an increase in cytolytic T-cell precursors during the course of therapy, usually by day 42. No patient had demonstrable delayed-type hypersensitivity to a melanoma membrane preparation before or after treatment. No toxicity of any kind was observed. A degree of clinical effectiveness of LMI was suggested by the elicitation of stable disease in 5 patients at 12 weeks. One patient had >50% regression of a lung nodule but progression of disease to the brain, whereas a second patient had a bona fide partial remission of a 3-cm diameter solitary lung nodule. CONCLUSIONS: LMI was nontoxic, improved immunological reactivity to melanoma cells, and showed evidence of clinical effectiveness (shrinkage of tumor) in 1 patient. Additional studies with LMI with added adjuvant materials, in melanoma and other cancers, appear warranted.

Combinations of anticancer drugs and immunotherapy.

Immunotherapy (biological therapy) comprises such things as active specific immunotherapy ("cancer vaccines"), nonspecific immunostimulation with cytokines, and the inhibition of suppressor influences exerted or elicited by the tumor. Just as cancer chemotherapy began with the use of single agents and evolved into combination therapy, so immunotherapeutic agents have been combined with each other and with chemotherapy. The alkylating agent cyclophosphamide (Cytoxan; CYC) has been used for many years to inhibit tumor-derived suppressor influences in rodents, and has been exploited for the same use in humans. Combinations of CYC and cancer vaccines such as autologous tumor cells, Melacine, large multivalent immunogen (LMI), and Theratope have been tested with some success in humans for more than a decade. In this use, the CYC is a biological response modifier rather than an antitumor agent, intended to inhibit suppressor influences. CYC and low- to moderate-dose IL-2 has also been a useful regimen in treating human melanomas. IL-2 is itself a useful component of combination immunotherapy, such as with melanoma peptide vaccines, or with interferon alpha-2b, (IFN-alpha), as a dual combination or part of a biochemotherapy regimen. Several different combinations of drugs and biological agents have been used as biochemotherapy for melanoma, but although there are higher response (regression) rates the long-range survival benefits have been marginal, not justifying the severe toxicity. Combinations of 5-fluorouracil (5-FU) and IFN-alpha or levamisole have had efficacy in colon and head and neck cancers, but here the biological agents have been biochemical modulators, not immunotherapy. Although experience with combinations of monoclonal antibodies and chemotherapy has been limited, it appears that trastuzumab (Herceptin) potentiates antitumor therapy in breast cancer but also increases the cardiotoxicity of those regimens.

Immunotherapy as part of combinations for the treatment of cancer.

Immunotherapy (biological therapy) comprises such things as active specific immunotherapy ("cancer vaccines"), nonspecific immunostimulation with cytokines, and the inhibition of suppressor influences exerted or elicited by the tumor. Just as cancer chemotherapy began with the use of single agents and evolved into combination therapy, so immunotherapeutic agents have been combined with each other and with chemotherapy. The alkylating agent cyclophosphamide (Cytoxan; CY) has been used for many years to inhibit tumor-derived suppressor influences in rodents, and has been exploited for the same use in humans. Combinations of CY and cancer vaccines such as autologous tumor cells, Melacine, large multivalent immunogen (LMI), and Theratope have been tested with some success in humans for more than a decade. In this use, the CY is a biological response modifier rather than an antitumor agent. Delayed treatment with CY in treating mouse plasmacytomas has proved more effective than immediate treatment, probably because it allows immunity to develop in the host. CY and moderate-dose interleukin-2 (IL-2) have also been a useful regimen in treating human melanomas. IL-2 is itself a useful component of combination immunotherapy, such as with melanoma peptide vaccines, or with interferon-alfa-2b, (IFN-a), as a dual combination or part of a biochemotherapy regimen. IL-2 and histamine, to block reactive oxygen species, may be a more useful combination for treatment of liver metastases of melanoma than IL-2 alone. In this combination, the histamine may permit continued, unimpeded activity of cytolytic T lymphocytes. Several different combinations of drugs and biological agents have been used as biochemotherapy for melanoma, but although there are higher immediate response rates, the long-range survival benefits have been marginal, not justifying the severe toxicity. Combinations of 5-fluorouracil (5-FU) and IFN-a or levamisole have had efficacy in colon and head and neck cancers, but here the biological agents acted as biochemical modulators. Trials of antibodies and chemotherapy have been limited. It appears that trastuzumab (Herceptin) potentiates antitumor therapy in breast cancer and also increases the cardiotoxicity of those regimens.

Radiation improves intratumoral gene therapy for induction of cancer vaccine in murine prostate carcinoma.

Our goal was to convert murine RM-9 prostate carcinoma cells in vivo into antigen-presenting cells capable of presenting endogenous tumor antigens and triggering a potent T-helper cell-mediated immune response essential for the generation of a specific antitumor response. We showed that generating the major histocompatibility complex (MHC) class I+/class II+/Ii- phenotype, within an established subcutaneous RM-9 tumor nodule, led to an effective immune response limiting tumor growth. This phenotype was created by intratumoral injection of plasmid cDNAs coding for interferon gamma, MHC class II transactivator, and an antisense reverse gene construct (RGC) for a segment of the gene for Ii protein (-92,97). While this protocol led to significant suppression of tumor growth, there were no disease-free survivors. Nevertheless, irradiation of the tumor nodule on the day preceding initiation of gene therapy yielded 7 of 16 mice that were disease-free in a long-term follow up of 57 days compared to 1 of 7 mice receiving radiotherapy alone. Mice receiving radiotherapy and gene therapy rejected challenge with parental RM-9 cells and demonstrated specific cytotoxic T-cell activity in their splenocytes but not the mouse cured by radiation alone. These data were reproduced in additional experiments and confirmed that tumor irradiation prior to gene therapy resulted in complete tumor regression and specific tumor immunity in more than 50% of the mice. Increasing the number of plasmid injections after tumor irradiation induced tumor regression in 70% of the mice. Administering radiation before this novel gene therapy approach, that creates an in situ tumor vaccine, holds promise for the treatment of human prostate carcinoma.

Active immunotherapy of metastatic melanoma with allogeneic melanoma lysates and interferon alpha.

PURPOSE: A therapeutic lyophilized melanoma vaccine consisting of two mechanically disrupted allogeneic melanoma cell lines and the immunological adjuvant Detox-PC (Melacine) has demonstrated encouraging activity in metastatic malignant melanoma, often in regimens containing pretreatment with low-dose cyclophosphamide. In addition, IFN-alpha2b (INTRON A; Schering-Plough Corporation, Kenilworth, NJ) has shown efficacy in melanoma refractory to Melacine. In this Phase II trial, the combination of cyclophosphamide, Melacine, and IFNalpha was tested in metastatic malignant melanoma. EXPERIMENTAL DESIGN: Eligibility criteria included measurable disease, no prior systemic therapy for a minimum of 4 weeks, and adequate marrow, renal, and hepatic function. Cyclophosphamide was administered once at a dose of 300 mg/m(2) i.v. on day -3 before the first dose of Melacine. Melacine was administered at a dose of 2 x 10(7) tumor cell equivalents per dose admixed with 0.25 ml of Detox-PC s.c. once a week on weeks 1-4 and week 6. Melacine maintenance was then given monthly from the 8th week, until progression or intolerable toxicity. IFN was started in the evening after the fourth dose of Melacine at a dose of 5,000,000 units/m(2) 3 times a week, and continued until progression. RESULTS: Forty-seven patients were enrolled, of whom 39 completed the full course and were considered evaluable. The toxicity of the regimen was minimal and consisted mainly of pain at injection sites and granulomas caused by Detox-PC, and constitutional symptoms attributable to IFN. In 39 evaluable patients, the overall objective response rate was 10.2%, but 64% of patients had stabilization of their disease for at least 16 weeks. The median time to disease progression in evaluable patients was 8 months [95% confidence interval (CI), 6-13 months]. Median survival time for all of the 47 patients enrolled was 12.5 months (95% CI, 8-15 months) with a median time to disease progression of 4 months (95% CI, 3-7 months). CONCLUSION: Despite a low objective response rate, this combination holds great promise because of its tolerability and the high proportion of prolonged durations of remission or disease stabilization that it elicited.

Cancer vaccines, a critical review--Part I.

Cancer vaccines are more properly referred to as 'active specific immunotherapy', and are used to treat cancers rather than to prevent them, at least at present. Vaccines augment already established tumor immunity, are far more specific against the tumor than cytokines, have little or no toxicity, and thus may easily be combined with other types of immunotherapy. They also elicit immunological memory, which may check recurrence of the tumor. Melanoma vaccines have received the most attention thus far. Among the several vaccines in clinical trials are whole cell lysates, such as Melacine, hapten-treated autologous melanoma cells (M-Vax) and irradiated allogeneic cells (CancerVax). Regressions of metastatic nodules have been noted with each preparation. Controlled trials of Melacine indicate prolongation of survival in patients with resected stage IIB disease, particularly those with one or more of the following HLA class I alleles: HLA-A2 or -A28 (-A6802), HLA-B12, -44 or -45, and HLA-C3. A combination of interferon-alpha2b and Melacine appears to enhance the anti-tumor response in advanced (stage IV) disease, and is being tested in a large randomized controlled trial in resected stage III disease. An irradiated autologous colon carcinoma vaccine has improved relapse-free survival in resected stage II disease (Dukes B) in a controlled trial. Second-generation whole cell vaccines include those incorporating genes such as GM-CSF or CD80 (B7-1) to improve immunogenicity, and the use of immunogenic cell membranes such as large multivalent immunogen (LMI). Upregulation of HLA class II molecules and concomitant inhibition of the Ii molecule are also being explored as a strategyfor improved presentation of tumor-associated antigens in vaccines. Complex whole cell-derived vaccines have given clinically superior responses compared to vaccines containing well-defined antigens, such as peptides or gangliosides; however, well-defined vaccines are theoretically more desirable because of their reproducibility.

Cancer vaccines, a critical review--Part II.

Cancer vaccines have been explored clinically against melanomas, adenocarcinomas and lymphomas. Breast cancer vaccines include Theratope, MUC1 mucin peptides and HER-2/neu peptide vaccines. Phase II trials suggest prolongation of survival of advanced breast cancer patients who generate high titers of antibody to Theratope. In contrast, melanoma ganglioside vaccines, which also elicit only antibodies, have not been effective in improving survival in controlled trials. Anti-idiotype vaccines for solid tumors, which depend upon mimicry of the tumor-associated antigens, have also had limited success. In lymphomas, where the idiotypes are the tumor-associated antigens, greater success has been achieved. A number of tumor-associated antigens have been identified in melanoma, such as the lineage related cancer-testis group (MAGE) and tyrosinase-related antigens. Non-lineage related antigens shared among a variety of very different tumors have recently been demonstrated too, which may permit immunization against more than one tumor group. Telomerase and MG50, one of several interleukin-1 receptor antagonist molecules, are both immunogenic and widespread in their representation. Carcinoembryonic antigen is the basis for vaccines against many adenocarcinomas. Both viral and non-viral vectors are being used to improve the reactivity to peptides in adenocarcinomas. Dendritic cell-carried vaccines, which package the antigens ex vivo rather than depending upon in vivo uptake, are being extensively explored in clinical models to improve the effectiveness of defined vaccines, such as peptides and RNA. 'Naked' DNA vaccines injected intramuscularly also have their advocates. Among the most recent attempts to improve the immunogenicity of vaccines is the use of antigens newly identified by genomic techniques and 'superagonist' peptide mimics, selected from combinatorial peptide libraries. These modern biochemical and molecular biological methods may greatly expand our ability to immunize against tumor antigens, which are essentially 'self' molecules. Finally, a greater understanding of ways in which tumors escape immunological detection or thwart immunological responses should lead to improved strategies against the tumor to augment the effect of vaccination.

Phase I trial of adoptive immunotherapy with cytolytic T lymphocytes immunized against a tyrosinase epitope.

PURPOSE: To study distribution and toxicity of cytolytic T lymphocytes (CTLs) against a single melanoma epitope. PATIENTS AND METHODS: CD8(+) T cells obtained by leukapheresis from 10 patients with disseminated HLA-A2.1(+), tyrosinase-positive melanomas were immunized in vitro against tyrosinase(369-377) (YMNGTMSQV). Drosophila cells transduced with HLA-A2.1, CD80, and CD54 (intracellular adhesion molecule-1) were used for priming, followed by two rounds of immunization with mononuclear cells as antigen-presenting cells. 1 x 10(8) CTL were infused intravenously (IV) on day 1. CTL frequency was measured by limiting dilutions in five patients. (111)In labeling and scintigraphy measured distribution of CTL in next five. Five days later, 1 x 10(8) CTLs were infused on 4 successive days to both groups. Immunohistology of response was judged by biopsies. RESULTS: Infusions were nontoxic. CTLs were undetectable in the blood, going to lungs within 5 minutes. At 4, 24, and 72 hours, they were found in liver and spleen. Lesions were visualized by scintiscans in one responding patient where two subcutaneous nodules were noted at 4 and 24 hours. A second patient had a partial response and remains alive with disease 2 years later. CD8(+) T cells were found in lesions of responders, associated with the presence of HLA-A2 molecules and tyrosinase. Two nonresponders without tyrosinase and HLA-A2 molecules had a paucity of CD8(+) T cells in their lesions. Whether the CD8(+) T cells in lesions of responders were those we had reinfused is uncertain. CONCLUSION: CTLs immunized against a single melanoma epitope were nontoxic but did not specifically localize to tumor sites. Nevertheless, two patients had disease regression. Additional therapeutic studies with specifically immunized CTL seem justified.