Biomedical subjects

Suzanne L Topalian

Publications and source records attributed to Suzanne L Topalian.

18 recordsLinked to original sources

PubMed2004

CD4(+) T-cell recognition of mutated B-RAF in melanoma patients harboring the V599E mutation.

The potential of antigen-directed cancer immunotherapy has not been fully realized, perhaps because many commonly targeted tumor associated proteins are not essential to maintaining the malignant cell phenotype. A constitutively activating mutation in the signaling molecule BRAF is expressed frequently in melanomas and may play an important role in the biology of this disease. A 29-mer B-Raf peptide incorporating the V599E mutation was used for in vitro stimulation of lymphocytes derived from melanoma patients, generating MHC class II-restricted CD4(+) T cells specific for this peptide as well as for melanoma cells expressing B-Raf V599E. Mutated B-Raf exemplifies targets that may be ideal for immunotherapy.

Alleles↗

PubMed2004

Lentivirus vector-mediated expression of tumor-associated epitopes by human antigen presenting cells.

Directing the human immune system to recognize and eliminate tumor cells is the ultimate goal of cancer immunotherapy. Vaccinating patients with autologous antigen presenting cells (APC) expressing tumor-associated antigens (TAA) represents a promising approach for activating tumor-reactive T cells in vivo. In addition, APC expressing TAA provide a means of generating tumor-specific T cells in vitro, for therapeutic and diagnostic applications. Lentiviral vectors are attractive vehicles for introducing TAA-encoding genes into APC. In this study, lentiviral vectors expressing the reporter gene GFP or the melanoma-associated antigen tyrosinase were used to transduce three different kinds of human APC: monocyte-derived dendritic cells (DC), CD40L-activated B lymphocytes, and Epstein Barr virus (EBV)-transformed B lymphocytes. Using optimized transduction conditions for each cell type, tyrosinase was expressed at levels sufficient to stimulate antigen-specific major histocompatibility complex (MHC) class I-restricted T cells from melanoma patients. While transduced EBV-B cells demonstrated the highest level of transgene expression, optimal T-cell recognition was achieved with transduced DC. Substituting the CAG promoter for PGK in lentiviral constructs enhanced transgene expression in DC and EBV-B cells, amplifying T cell recognition. Lentiviruses inducing sustained transgene expression with relatively low cellular toxicity and background viral gene expression may be ideal vectors for immunotherapeutic applications.

Antigen-Presenting Cells↗

PubMed2004

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR.

Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Animals↗

PubMed2003

Randomized study of high-dose and low-dose interleukin-2 in patients with metastatic renal cancer.

PURPOSE: This three-arm randomized study compares response rates and overall survival of patients with metastatic renal cell cancer (RCC) receiving high-dose or one of two low-dose interleukin-2 (IL-2) regimens. PATIENTS AND METHODS: Patients with measurable metastatic RCC and a good performance status were randomized to receive either 720,000 U/kg (high-dose [HD]) or 72,000 U/kg (low-dose [LD]), both given by intravenous (IV) bolus every 8 hours. After randomly assigning 117 patients, a third arm of low-dose daily subcutaneous IL-2 was added, and an additional 283 patients were randomly assigned. RESULTS: A total of 156 patients were randomly assigned to HD IV IL-2, and 150 patients to LD IV IL-2. Toxicities were less frequent with LD IV IL-2 (especially hypotension), but there were no IL-2-related deaths in any arm. There was a higher response proportion with HD IV IL-2 (21%) versus LD IV IL-2 (13%; P =.048) but no overall survival difference. The response rate of subcutaneous IL-2 (10%, partial response and complete response) was similar to that of LD IV IL-2, differing from HD IV (P =.033). Response durability and survival in completely responding patients was superior with HD IV compared with LD IV therapy (P =.04). CONCLUSION: Major tumor regressions, as well as complete responses, were seen with all regimens tested. IL-2 was more clinically active at maximal doses, although this did not produce an overall survival benefit. The immunological factors which constrain the curative potential of IL-2 to only a small percentage of patients need to be further elucidated.

Carcinoma, Renal Cell↗

PubMed2003

Recombinant fowlpox viruses encoding the anchor-modified gp100 melanoma antigen can generate antitumor immune responses in patients with metastatic melanoma.

PURPOSE: The purpose of this study was to evaluate the immunological responses and therapeutic effectiveness of immunization with fowlpox vaccines encoding the gp100 melanoma antigen in patients with metastatic melanoma. EXPERIMENTAL DESIGN: In three consecutive clinical trials, patients were immunized with recombinant fowlpox viruses encoding three different forms of the melanoma/melanocyte-associated antigen gp100: (a) the native, full-length gp100 molecule; (b) the gp100 molecule with two amino acids modified to increase binding to HLA-A*0201 molecules; and (c) a "minigene" construct encoding a single, modified epitope gp100:209-217(210M) targeted to the endoplasmic reticulum. The immunogenicity of these constructs was studied using peripheral blood mononuclear cells to measure epitope-specific release of IFN-gamma. RESULTS: Reactivity against gp100 was not seen in any patient before receiving fowlpox immunization. Whereas just one of seven patients developed reactivity after receiving fowlpox encoding native gp100, 10 of 14 patients who received fowlpox encoding the anchor modified full-length gp100 exhibited reactivity against the native gp100 molecule, and 12 of 16 patients were successfully immunized after inoculation with the modified minigene construct (p2 = 0.02). There was no difference in the latter group between those randomized to vaccination by i.v. or i.m. routes. There was one partial cancer regression in the group of 46 patients receiving virus in the absence of interleukin (IL)-2. Once patients showed evidence of progressive disease, they were eligible for "cross-over" treatment to IL-2 alone or with the fowlpox virus. None of the 13 patients receiving the full-length or modified full-length forms of gp100 responded when receiving IL-2, whereas 6 of 12 patients who received the fowlpox containing the minigene construct and then received IL-2 showed objective cancer regressions, including three patients with complete regression. CONCLUSIONS: These data underscore the importance of modifying anchor residues of nonmutated self-antigen peptides to generate cellular immune responses after immunization and support the further investigation of recombinant fowlpox viruses encoding modified epitopes administered in combination with IL-2.

Adult↗

PubMed2003

A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer.

BACKGROUND: Mutations in the tumor-suppressor gene VHL cause oversecretion of vascular endothelial growth factor by clear-cell renal carcinomas. We conducted a clinical trial to evaluate bevacizumab, a neutralizing antibody against vascular endothelial growth factor, in patients with metastatic renal-cell carcinoma. METHODS: A randomized, double-blind, phase 2 trial was conducted comparing placebo with bevacizumab at doses of 3 and 10 mg per kilogram of body weight, given every two weeks; the time to progression of disease and the response rate were primary end points. Crossover from placebo to antibody treatment was allowed, and survival was a secondary end point. RESULTS: Minimal toxic effects were seen, with hypertension and asymptomatic proteinuria predominating. The trial was stopped after the interim analysis met the criteria for early stopping. With 116 patients randomly assigned to treatment groups (40 to placebo, 37 to low-dose antibody, and 39 to high-dose antibody), there was a significant prolongation of the time to progression of disease in the high-dose--antibody group as compared with the placebo group (hazard ratio, 2.55; P<0.001). There was a small difference, of borderline significance, between the time to progression of disease in the low-dose--antibody group and that in the placebo group (hazard ratio, 1.26; P=0.053). The probability of being progression-free for patients given high-dose antibody, low-dose--antibody, and placebo was 64 percent, 39 percent, and 20 percent, respectively, at four months and 30 percent, 14 percent, and 5 percent at eight months. At the last analysis, there were no significant differences in overall survival between groups (P>0.20 for all comparisons). CONCLUSIONS: Bevacizumab can significantly prolong the time to progression of disease in patients with metastatic renal-cell cancer.

Adenocarcinoma, Clear Cell↗

PubMed2003

Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule (expressed on activated T cells and a subset of regulatory T cells) capable of down-regulating T cell activation. Blockade of CTLA-4 has been shown in animal models to improve the effectiveness of cancer immunotherapy. We thus treated 14 patients with metastatic melanoma by using serial i.v. administration of a fully human anti-CTLA-4 antibody (MDX-010) in conjunction with s.c. vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209-217(210M) and gp100:280-288(288V). This blockade of CTLA-4 induced grade III/IV autoimmune manifestations in six patients (43%), including dermatitis, enterocolitis, hepatitis, and hypophysitis, and mediated objective cancer regression in three patients (21%; two complete and one partial responses). This study establishes CTLA-4 as an important molecule regulating tolerance to "self" antigens in humans and suggests a role for CTLA-4 blockade in breaking tolerance to human cancer antigens for cancer immunotherapy.

Adult↗

PubMed2003

Inability to immunize patients with metastatic melanoma using plasmid DNA encoding the gp100 melanoma-melanocyte antigen.

Immunization with plasmid DNA represents a theoretically attractive method for increasing T cell responses against cancer antigens. We administered plasmid DNA encoding the gp100 melanoma-melanocyte differentiation antigen to 22 patients with metastatic melanoma and evaluated immunologic and clinical responses. Patients were randomized to receive plasmid DNA either intradermally (n = 10) or intramuscularly (n = 12). One patient (4.5%) exhibited a partial response of several subcentimeter cutaneous nodules. All other patients had progressive disease. Of 13 patients with cells available before and after immunization, no patient exhibited evidence of the development of anti-gp100 cell responses using in vitro boost assays. The same assays were capable of demonstrating immunologic precursors after immunization with fowl poxvirus encoding gp100 or with gp100 peptides. We were thus unable to demonstrate significant clinical or immunologic responses to plasmid DNA encoding the "self" nonmutated gp100 tumor antigen.

Adult↗

PubMed2003

Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.

The use of lentiviral vectors for basic research and potential future clinical applications requires methodologies that can accurately determine lentiviral titers and monitor viral transgene expression within target cells, beyond the context of reporter genes typically used for this purpose. Here we describe a quantitative RT-PCR (qRT-PCR) method that achieves both goals using primer sequences that are specific for the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an enhancer contained in many retroviral vectors and that is incorporated in the 3' UTR of nascent transgene transcripts. Quantitation of titers of three recombinant lentiviruses, genetically identical except for the transgene, demonstrated consistent differences in titer that were likely due to transgene-associated toxicity in producer cells and target cells. Viruses encoding the tumor-associated antigens tyrosinase and neo-poly(A) polymerase yielded reproducibly lower titers than a virus encoding enhanced green fluorescent protein (GFP) at the viral RNA, integrated DNA, and transgene mRNA levels, as measured by WPRE qPCR. Furthermore, the magnitude of differences in expression of the three transgenes in transduced target cells could not have been predicted by measuring vector DNA integration events. Since transgene expression in target cells is the most common goal of lentiviral transduction, and since methods to quantify transgene expression on the protein level are not always readily available, qRT-PCR based on a nucleotide sequence included in the transcript provides a useful tool for titering novel recombinant lentiviruses.

3' Untranslated Regions↗

PubMed2002

Revelation of a cryptic major histocompatibility complex class II-restricted tumor epitope in a novel RNA-processing enzyme.

CD4+ T-cell responses against human tumor antigens are a potentially critical component of the antitumor immune response. Molecular methods have been devised for rapidly identifying MHC class II-restricted tumor antigens and elucidating the recognized epitopes. We describe here the identification of neo-poly(A) polymerase (neo-PAP), a novel RNA processing enzyme overexpressed in a variety of human cancers, by screening a melanoma-derived invariant chain fusion cDNA library with tumor-reactive CD4+ T lymphocytes. A cryptic nonmutated HLA-DRbeta1*0701-restricted neo-PAP epitope was processed through the endogenous MHC class II pathway. A unique point mutation effected a nonconservative substitution of a leucine for a proline residue at a structurally important site in neo-PAP that was remote from the recognized peptide, revealing a normally silent epitope for immune recognition. Genetic aberrations such as the described point mutation can have unexpected immunological consequences, in this case leading to immune recognition of a distant normal self epitope.

Adult↗

PubMed2002

Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes.

We report here the adoptive transfer, to patients with metastatic melanoma, of highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen. This approach resulted in the persistent clonal repopulation of T cells in those cancer patients, with the transferred cells proliferating in vivo, displaying functional activity, and trafficking to tumor sites. This led to regression of the patients' metastatic melanoma as well as to the onset of autoimmune melanocyte destruction. This approach presents new possibilities for the treatment of patients with cancer as well as patients with human immunodeficiency virus-related acquired immunodeficiency syndrome and other infectious diseases.

Adolescent↗

PubMed2002

Quantitative real-time RT-PCR as a method for monitoring T lymphocyte reactivity to full-length tyrosinase protein in vaccinated melanoma patients.

The major goal of therapeutic cancer vaccine trials is to mediate tumor regression. However, it is critically important to devise in vitro immunological assays that correlate with clinical outcome, for use as surrogate markers of vaccine efficacy. To date, clinical emphasis has been placed on peptide vaccines, but trends towards the use of more complex immunogens such as whole proteins require the development of efficient and sensitive methods for monitoring their immunological effects. In the context of a vaccination trial using full-length tyrosinase (Ty) to immunize patients with metastatic melanoma, a monitoring technique was developed in which autologous dendritic cells (DC) infected with a recombinant adenovirus encoding the Ty protein were used to assess the Ty-specific reactivity of fresh peripheral blood lymphocytes (PBL) collected from patients at different intervals during therapy. Quantitative real-time RT-PCR (qRT-PCR) was used to measure the production of cytokine mRNA by T cells following a 2.5-h incubation with Ty-expressing DC. Two out of ten patients studied demonstrated Ty protein-specific reactivity that increased during and after the period of vaccination. While one of these patients also reacted to an HLA-A1-compatible Ty peptide, the second did not recognize any of the known Ty epitopes, highlighting the importance of this technique for monitoring the effects of complex vaccines.

Adenoviridae↗

PubMed2002

Tumor-specific CD8+ T lymphocytes derived from the peripheral blood of prostate cancer patients by in vitro stimulation with autologous tumor cell lines.

To identify tumor-associated antigens as putative targets for developing immunotherapies against prostate cancer, we investigated the ability of T cells derived from the peripheral blood lymphocytes of prostate cancer patients to recognize autologous tumor cells. The technical challenge of growing in vitro carcinoma cell lines from small prostate cancer samples was previously addressed by immortalization of early epithelial cell cultures with the HPV16 transforming proteins E6 and E7 and by genetic characterization of the carcinoma and normal prostate cell lines. In our study, peripheral blood lymphocytes were stimulated in vitro using autologous IFNgamma-treated prostate carcinoma cells transduced with the B7.1 molecule as a source of T-cell costimulation. Tumor-specific CD8+ T lymphocytes were obtained from 3 of 6 prostate cancer patients tested and included T cells restricted by classical and nonclassical HLA molecules. In 1 case, we demonstrated that the prostate cancer-reactive T cells were TCRalpha/beta+ and recognized autologous tumor cells but not autologous normal cells in the context of HLA-B or -C molecules. These results validate the approach of in vitro stimulation of peripheral blood lymphocytes from prostate cancer patients with autologous tumor cell lines to isolate prostate cancer-specific T cells and demonstrate the existence of a functionally diverse immune response against prostate cancer.

Antigens, Neoplasm↗

PubMed2002

Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma.

PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS: The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.

Adult↗

PubMedDate not supplied

Immunization of patients with metastatic melanoma using both class I- and class II-restricted peptides from melanoma-associated antigens.

Cancer vaccines targeting CD8+ T cells have been successful in eliciting immunologic responses but disappointing in inducing clinical responses. Strong evidence supports the importance of CD4+ T cells in "helping" cytotoxic CD8+ cells in antitumor immunity. We report here on two consecutive clinical trials evaluating the impact of immunization with both human leukocyte antigen class I- and class II-restricted peptides from the gp100 melanoma antigen. In Protocol 1, 22 patients with metastatic melanoma were immunized with two modified class I A*0201-restricted peptides, gp100:209-217(210M) and MART-1:26-35(27L). In Protocol 2, 19 patients received the same class I-restricted peptides in combination with a class II DRB1*0401-restricted peptide, gp100:44-59. As assessed by in vitro sensitization assays using peripheral blood mononuclear cells (PBMC) against the native gp100:209-217 peptide, 95% of patients in Protocol 1 were successfully immunized after two vaccinations in contrast to 50% of patients in Protocol 2 (P(2) < 0.005). Furthermore, the degree of sensitization was significantly lower in patients in Protocol 2 (P = 0.01). Clinically, one patient in Protocol 2 had an objective response, and none did in Protocol 1. Thus, the addition of the class II-restricted peptide gp100:44-59 did not improve clinical response but might have diminished the immunologic response of circulating PBMC to the class I-restricted peptide gp100:209-217. The reasons for this decreased immune reactivity are unclear but may involve increased CD4+CD25+ regulatory T-cell activity, increased apoptosis of activated CD8+ T cells, or the trafficking of sensitized CD8+ reactive cells out of the peripheral blood. Moreover, the sequential, nonrandomized nature of patient enrollment for the two trials may account for the differences in immunologic response.

Adult↗

PubMedDate not supplied

Cell transfer therapy for cancer: lessons from sequential treatments of a patient with metastatic melanoma.

The development of effective autologous cell transfer therapies for the treatment of patients with cancer has been difficult, in part because the cells used to treat each patient are different, as are the patient's tumor and immune status. Much can thus be learned by sequential treatments of the same patient with the same cells, making single modifications in the treatments to determine which factors are critical. The authors have treated a single patient with five sequential administrations of the same cells with minor modifications in the mode of administration and the immune status of the patient. The treatment of this patient strongly suggested that 1) the highly avid recognition of tumor antigens in vitro by a transferred lymphocyte population does not necessarily predict in vivo antitumor activity; 2) the administration of highly avid antitumor autologous lymphocyte populations can be far more active in mediating tumor regression in vivo when administered after nonmyeloablative chemotherapy than when administered without this prior chemotherapy; 3) intra-arterial administration of highly avid antitumor autologous lymphocytes into the blood supply of the tumor can be more effective in mediating tumor regression than the intravenous administration of these same tumor infiltrating lymphocytes; 4) one mechanism of tumor escape from immunotherapy is loss of class I MHC antigen expression by the tumor due to mutation of the beta-2 microglobulin gene.

Adult↗

PubMedDate not supplied

Immunization of HLA-A0201 and/or HLA-DPbeta104 patients with metastatic melanoma using epitopes from the NY-ESO-1 antigen.

HLA class I-restricted peptides are often used in peptide vaccine regimens. There is strong evidence that many of these peptides can generate specific CD8 T-cell responses in vivo; however, only occasional objective clinical responses have been reported. To test whether provision of "help" would enhance antitumor immunity, the authors initiated a clinical trial in which patients with metastatic melanoma were immunized against the NY-ESO-1 tumor antigen, using an HLA-A2-restricted peptide (ESO-1:165V), an HLA-DP4-restricted peptide (NY-ESO-1:161-180), or both peptides given concomitantly. The first cohorts received only ESO-1:165V, using three vaccination schedules. Immunologically, most patients developed immune responses to the HLA-A2-restricted native ESO-1 epitope after vaccination. Peptide vaccine given daily for 4 days appeared to induce immunologic responses more rapidly than if given once a week or once every 3 weeks. In contrast, vaccination using the NY-ESO-1:161-180 peptide induced immune responses in only a few patients. Clinically, one patient who received NY-ESO-1:161-180 peptide alone had a partial response lasing 12 months. Concomitant vaccination with the HLA class II-restricted peptide did not alter the immune response to the HLA class I-restricted peptide form NY-ESO-1. However, vaccination with the HLA-A2-restricted epitope generated primarily T cells that did not recognize tumor after in vitro sensitization. This result raises questions about the use of synthetic peptides derived from NY-ESO-1 as a sole form of immunization.

Antigens, Neoplasm↗

PubMedDate not supplied

A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma.

This report describes a phase I clinical trial using nonmyeloablative, lympho-depleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma. The chemotherapy-conditioning schedule that induced transient lymphopenia consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m(2) per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded, tumor-reactive, autologous T-cell clones selected for high avidity recognition of melanoma antigens. Cohorts of three to six patients each received either no interleukin (IL)-2, low-dose IL-2 (72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2 (720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. High dose intravenous IL-2 was better tolerated by patients after chemotherapy than during previous immunotherapy cycles without chemotherapy. No patient exhibited an objective clinical response to treatment, although five patients demonstrated mixed responses or transient shrinkage of metastatic deposits. This study established a nonmyeloablative-conditioning regimen that could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma.

Adolescent↗