[Show abstract][Hide abstract] ABSTRACT: Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1-seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1-seronegative patients (P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1-specific CD4(+) and CD8(+) T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1-seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1-seropositive patients with associated CD8(+) T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8(+) T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage (P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer.
Full-text · Article · Sep 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8(+) T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED).
Human leukocyte antigen (HLA)-A*0201(+) disease-free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 microg or 2,000 microg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201-restricted gp100 epitopes [gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA)].
Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer(+)CD8(+) T cells, all carrying the CCR7(lo)CD45RA(lo) effector-memory phenotype. Five of 27 patients generated IFN-gamma(+)CD8(+) T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-gamma(+)CD8(+) T-cell generation (P = 0.07).
A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection.
Full-text · Article · Aug 2010 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Melanoma patients treated with anti-CTLA-4 have shown a range of anti-tumor responses. In this report, we describe the response of a single patient to anti-CTLA-4, with individual lesions disappearing, others stabilizing, and others progressing. These responses can be viewed as a clear manifestation of cancer immunoediting and its three phases of elimination, equilibrium and escape, with each tumor in this patient being at a discrete stage in the process. The patient's course and associated immunological monitoring and other laboratory data are presented in an immunogram, a way to visualize temporal associations between the multiple clinical and laboratory parameters.
Full-text · Article · Jan 2010 · Cancer immunity: a journal of the Academy of Cancer Immunology
[Show abstract][Hide abstract] ABSTRACT: Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines.
We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a.
Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method.
Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.
[Show abstract][Hide abstract] ABSTRACT: How the immune system recognizes and responds to mutations expressed by cancer cells is a critical issue for cancer immunology. Mutated self-polypeptides are particularly strong tumor-specific rejection antigens for natural tumor immunity, but we know remarkably little about T-cell responses to mutated self during tumor growth in vivo, including levels of response, kinetics, and correlates that predict tumor rejection. To address these questions, a mutated self-antigen, designated tyrosinase-related protein 1 (Tyrp1)-WM, derived from Tyrp1 was expressed in the poorly immunogenic, spontaneously arising B16 melanoma and the immunogenic, chemically induced LiHa fibrosarcoma. Syngeneic mice challenged with LiHa fibrosarcoma cells expressing Tyrp1-WM, but not native Tyrp1, induced specific CD8(+) and CD4(+) T-cell responses against defined mutated epitopes in tumor-draining lymph nodes and in tumors. Subsequently, specific CD8(+) T-cell responses contracted as a minority of tumors progressed. B16 melanomas expressing Tyrp1-WM induced minimal T-cell responses, and no tumor immunity was detected. Treatment with an agonist monoclonal antibody against glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) increased the level of CD8(+) T cells recognizing a peptide derived from the Tyrp1-WM sequence and the proportion of mice rejecting tumors. These results show that B16 tumors expressing mutations that generate strongly immunogenic epitopes naturally induce T-cell responses, which are insufficient to reject tumors. Immune modulation, such as inducing GITR signaling, is required to enhance CD8(+) T-cell responses to specific mutations and to lead to tumor rejection.
[Show abstract][Hide abstract] ABSTRACT: A differentiation antigen commonly expressed on melanoma cells, gp100 is the target of infiltrating T cells. We conducted a phase I randomized cross-over trial of melanoma patients with either xenogeneic (mouse) or human gp100 plasmid DNA injected intramuscularly at three dosages (100, 500 or 1,500 microg) every three weeks for three doses. After the first three injections, patients were then immunized three times with gp100 from the other species. Peripheral blood samples were analyzed at various time points following 10-day culture with gp100 peptides using multi-parametric flow cytometry. A total of 19 patients were enrolled, with 18 assessable for immune function and survival. 14 (74%) were male, with a median age of 56 years (range, 20-82). All patients had no evidence of disease; 10 (53%) had stage III disease, 3 each (16%) had stage IIB and IV disease, 2 (11%) had choroidal and 1 (5%) had anal mucosal involvement. With a median follow-up of 30 months, median progression-free survival (PFS) is 44 months. Median survival is not reached. There was no grade 3/4 toxicity; the most common grade 1/2 toxicity was an injection site reaction in 12 patients (63%, all grade 1). Five patients developed CD8+ cells binding gp100(280-288) HLA-A2-restricted tetramer. One patient had an increase in CD8+ IFN-gamma+ cells. This xenogeneic immunization strategy was safe and associated with minimal toxicity. There was also evidence of immune response.
Preview · Article · Feb 2009 · Cancer immunity: a journal of the Academy of Cancer Immunology
[Show abstract][Hide abstract] ABSTRACT: Blockade of inhibitory signals mediated by cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to enhance T cell responses and induce durable clinical responses in patients with metastatic melanoma. The functional impact of anti-CTLA-4 therapy on human immune responses is still unclear. To explore this, we analyzed immune-related adverse events and immune responses in metastatic melanoma patients treated with ipilimumab, a fully human anti-CTLA-4 monoclonal antibody. Fifteen patients were selected on the basis of availability of suitable specimens for immunologic monitoring, and eight of these showed evidence of clinical benefit. Five of the eight patients with evidence of clinical benefit had NY-ESO-1 antibody, whereas none of seven clinical non-responders was seropositive for NY-ESO-1. All five NY-ESO-1 seropositive patients had clearly detectable CD4(+) and CD8(+) T cells against NY-ESO-1 following treatment with ipilimumab. One NY-ESO-1 seronegative clinical responder also had a NY-ESO-1 CD4(+) and CD8(+) T cell response, possibly related to prior vaccination with NY-ESO-1. Among five clinical non-responders analyzed, only one had a NY-ESO-1 CD4(+) T cell response and this patient did not have detectable anti-NY-ESO-1 antibody. Overall, NY-ESO-1-specific T cell responses increased in frequency and functionality during anti-CTLA-4 treatment, revealing a polyfunctional response pattern of IFN-gamma, MIP-1beta and TNF-alpha. We therefore suggest that CTLA-4 blockade enhanced NY-ESO-1 antigen-specific B cell and T cell immune responses in patients with durable objective clinical responses and stable disease. These data provide an immunologic rationale for the efficacy of anti-CTLA-4 therapy and call for immunotherapeutic designs that combine NY-ESO-1 vaccination with CTLA-4 blockade.
Full-text · Article · Jan 2009 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Passive immunization with monoclonal antibody TA99 targeting melanoma differentiation antigen tyrosinase-related protein-1 (Tyrp1; gp75) and active immunization with plasmid DNA encoding altered Tyrp1 both mediate tumor immunity in the B16 murine melanoma model. We report here that TA99 enhances Tyrp1 DNA vaccination in the treatment of B16 lung metastases, an effect mediated by immunologic mechanisms as Tyrp1 has no known role in regulating tumor growth. TA99 is shown to increase induction of anti-Tyrp1 CD8+T-cell responses to DNA vaccination against Tyrp1 as assessed by IFN-gamma ELISPOT assays. Immunohistochemistry studies reveal that TA99 localizes rapidly and specifically to B16 lung nodules. Augmentation of T-cell responses is dependent on the presence of tumor as well as on activating Fc receptors. Furthermore, TA99 enhances DNA vaccination against a distinct melanoma antigen, gp100(pmel17/silver locus), improving antitumor efficacy, augmenting systemic CD8+ T-cell responses to gp100, and increasing CD8+ T-cell infiltration at the tumor site. Epitope spreading was observed, with CD8+ T-cell responses generated to Tyrp1 peptide in mice receiving gp100 DNA vaccination in the presence of TA99. Finally, we show that TA99 improves therapeutic efficacy of DNA vaccination combined with adoptive T-cell transfer in treatment of established subcutaneous B16 melanoma. In conclusion, TA99 enhances DNA vaccination against both the target antigen Tyrp1 and a distinct melanoma antigen gp100 in an Fc receptor-dependent mechanism, consistent with enhanced cross-presentation of tumor-derived antigen. Monoclonal antibodies should be tested as vaccine adjuvants in the treatment of cancer.
[Show abstract][Hide abstract] ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances immune responses by inducing proliferation, maturation, and migration of dendritic cells (DCs) as well as expansion and differentiation of B and T lymphocytes. The potency of DNA vaccines can be enhanced by the addition of DNA encoding cytokines, acting as molecular adjuvants. We conducted a phase I/II trial of human GM-CSF DNA in conjunction with a multipeptide vaccine (gp100 and tyrosinase) in stage III/IV melanoma patients. Nineteen human leukocyte antigen (HLA)-A*0201+ patients were treated. Three dose levels were studied: 100, 400, and 800 microg DNA/injection, administered subcutaneously every month with 500 microg of each peptide. In the dose-ranging study, three patients were treated at each dose level. The remaining patients were then treated at the highest dose. Most toxicities were grade 1 injection-site reactions. Eight patients (42%) developed CD8+ T-cell responses, defined by a > or =3 SD increase in baseline reactivity to tyrosinase or gp100 peptide in tetramer or intracellular cytokine staining (ICS) assays. There was no relationship between dose and T-cell response. Responding T cells had an effector memory cell phenotype. Polyfunctional T cells were also demonstrated. At a median of 31 months follow-up, median survival has not been reached. Human GM-CSF DNA was found to be a safe adjuvant.
Full-text · Article · Oct 2008 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: The literature contains several publications describing the use of visual arts to develop observational skills in medical students. Portraits of individuals of the Italian Renaissance can be used to enhance these skills and stimulate the development of differential diagnoses in medical students. The Duke of Urbino, Federico di Montefeltro (1422-1482), lost his right eye and nasal bridge during a jousting accident in 1450. Consequently, almost every profile of him in existence today depicts his face in a left lateral view. Although some authors have described the Duke's missing nasal bridge, none have described his prominent thoracic hyperkyphosis, which is clearly discernible in two paintings by Piero della Francesca. The purpose of this report is to describe the Duke's hyperkyphosis, develop relevant differential diagnoses, and suggest a possible etiology of the convexity.
We have examined two paintings of the Duke by Piero della Francesca - the diptych, The Duke and Duchess of Urbino (1465), and the Madonna of the Egg (1472). A MEDLINE search revealed 2 articles that were relevant to this study. This search was complemented by a search of the collection at the library of Seton Hall University, and the first author's experience studying at the University of Urbino. The historical data obtained from these searches were incorporated with the visual analysis to formulate a plausible etiology of the Duke's thoracic hyperkyphosis.
Differential diagnoses of the Duke's thoracic hyperkyphosis include Scheuermann disease, osteoporosis, and trauma-related spinal changes. Based on the available evidence, the Duke's thoracic hyperkyphosis could have been caused by repetitive trauma to the spine due to numerous hours on horseback with heavy armor. The role that osteoporosis played in the development of the hyperkyphosis is unclear, as is whether the Duke had the convexity during childhood. The hyperkyphosis as a stylistic variant by Piero della Francesca is unlikely. This report is an example of a teaching strategy that can be used to enhance the observational skills of medical students in evidence-based medical education.
Full-text · Article · Feb 2008 · Journal of Medical Case Reports
[Show abstract][Hide abstract] ABSTRACT: Immunity to self antigens on cancer is constrained by tolerance/ignorance. DNA vaccines encoding xenogeneic differentiation antigens, such as tyrosinase (TYR), mediate tumor protection and regression in implantable mouse models, and dogs with spontaneous melanoma. We conducted a trial of mouse and human TYR DNA vaccines in stage III/IV melanoma patients. Eighteen human leukocyte antigen (HLA)-A*0201(+) melanoma patients were randomized as follows: one group received three mouse TYR DNA injections followed by three human TYR DNA injections; the other group received the same vaccines in opposite sequence. The study was conducted at three dose levels: 100, 500, and 1,500 microg DNA/injection, administered intramuscularly (IM) every 3 weeks. Most toxicities were grade 1 injection site reactions. Seven patients developed CD8(+) T-cell responses, defined by a >3 SD increase in baseline reactivity to TYR peptide in tetramer or intracellular cytokine staining (ICS) assays. There was found to be no relationship between dose, assigned schedule, and T-cell response. At a median of 42 months follow-up, median survival has not been reached. Mouse and human TYR DNA vaccines were found safe and induced CD8(+) T-cell responses in 7 of 18 patients. T cells recognizing a native TYR peptide had a phenotype consistent with that of effector memory cells.
Full-text · Article · Nov 2007 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: This nation--and in fact the world--is currently facing a crisis in the form of a growing antibacterial drug resistance. In the 60 or so years since the discovery of penicillin, physicians and pharmaceutical companies have been constantly challenged to stay a step ahead of the bacteria that constantly adapt to the drugs used to control them. These magic bullets were at one time expected to eliminate the concern over infectious disease and have been relied upon heavily. But their effectiveness has been steadily waning in recent years as, more and more, strains of bacteria emerge that are resistant to multiple drugs and, in some cases, have become nearly "panresistant." Nosocomial infections with these resistant strains were once confined to hospitals but new community-acquired infections are an ominous portent. Meanwhile, perhaps equally as distressing, many pharmaceutical companies are discontinuing efforts to develop new antimicrobial drugs for a variety of reasons and few new agents are currently in the pipeline. The selective pressure triggering these bacterial mutations are complex, but they can be shared by healthcare workers, hospitals, long-term care facilities, the agriculture industry, and even healthcare consumers themselves. In this paper, these topics are discussed, in turn, and the paper concludes with an apologia for change that can and should be equally shared by these stakeholders.
Full-text · Article · Jul 2007 · Medical science monitor: international medical journal of experimental and clinical research