Showing papers by "Francesco M. Marincola published in 2001"

Cytokines and Immune Response in the Tumor Microenvironment.

Abstract: SUMMARY: Over the last few decades a wealth of evidence has been gathered on the potential role that the immune system (IS) can play in the fight against cancer. Together with cell surface adhesion molecules, cytokines (CKs) mediate the activities of IS cells. Therefore, CK kinetics may represent a mirror of the immunologic phenomena occurring in the tumor microenvironment, where immune and malignant cells interact. Yet, CKs are currently used in a clinical setting to polarize the immune response against cancer. Despite the large amount of information available on IS physiology, little is known about the role of CKs in modulating the effectiveness of immunotherapy clinical trials aimed at the treatment of patients with cancer. This underscores our relative ignorance about the complex cascade of events that lead to tumor rejection. Here, we review the properties of some CKs believed to be particularly relevant to tumor immunology (i.e., interleukin [IL]-10, transforming growth factor-beta, interferon-gamma, IL-2, IL-4, and IL-12). We summarized the experience gained with these CKs in vitro, in animal models, and in human beings to illustrate the achievements, the controversies, and the challenges that characterize this fascinating field of oncology. In addition, we added a short section in which a broad view of CKs released in the tumor microenvironment is proposed to underline the variety of factors that contribute to the complexity of tumor-IS interactions.

Antigenicity of Fusion Proteins from Sarcoma-associated Chromosomal Translocations

Abstract: Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Kinetics of cytokine expression in melanoma metastases classifies immune responsiveness.

Abstract: Production of cytokines (CKs) in the tumor micro-environment may modulate tumor–host interactions. However, pre-clinical models often provide conflicting data and there is no established role for CKs as modulators of the natural or treatment-related behavior of tumors. Serial sampling by fine-needle aspirates (FNAs) of identical metastases from patients affected with metastatic melanoma and undergoing IL-2–based vaccination allowed prospective measurement of IL-10, TGF-β1, TGF-β2 and IFN-γ transcriptional levels assessed by quantitative real-time PCR. Thus, it was possible to prospectively document the expression of markers relevant to a given treatment and follow at the same time the clinical outcome of the lesions left in place. Eight of 27 metastatic lesions completely regressed in response to the treatment and 1 demonstrated >50% shrinkage. These regressions occurred after the follow-up FNA had been obtained. IL-10 transcript was differentially expressed in pre-treatment FNA of responding lesions (t-test p2 = 0.002). During treatment, INF-γ transcript levels significantly increased in regressing compared to non-regressing lesions (t-test p2 = 0.03). These data suggest that the pre-treatment CK profile of the tumor micro-environment may determine clinical responsiveness to immune therapy. Furthermore, temporal changes in CK expression during treatment might describe the biological characteristics of an effective immune response. © 2001 Wiley-Liss, Inc.

Kinetics of TCR Use in Response to Repeated Epitope-Specific Immunization

Abstract: Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209-217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient's kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-gamma ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies.

Unmasking cryptic epitopes after loss of immunodominant tumor antigen expression through epitope spreading.

Abstract: The basis of intra-tumoral and systemic T cell reactivity toward cancer remains unclear. In particular the role that peripheral stimuli, whether endogenous or exogenous, play in shaping acquired immune response toward cancer remains poorly understood. In this study we document the surfacing of systemic immune reactivity toward a cryptic epitope from the MAGE-12 gene (MAGE-12:170-178), after temporary regression of a single melanoma metastasis, in response to gp100/PMel17-specific vaccination. This emergence was unlikely related to unusually high expression of MAGE-12 by the tumor, by the influence of analog epitopes to MAGE-12:170-178. Because MAGE-12 was unlikely to be expressed at sites other than the tumor, the demonstration of MAGE-12:170-178 reactivity in post- but not pre-vaccination circulating lymphocytes suggests that the systemically observed immune response was influenced by events induced by the vaccine at tumor site or draining lymph nodal areas. Possibly, as suggested by pre-clinical models, immunologic ignorance is the default response toward cancer in humans unless unusual stimulatory conditions occur in peripheral tissues. Surfacing of MAGE-12 specificity occurred in association with loss of gp100/PMel 17 targeted by the vaccine. This finding suggests that vaccinations might have effects beyond their intrinsic specificity and may trigger broader immune responses through epitope spreading by inducing changes within the tumor microenvironment. This may have important practical implication for the development of immunization strategies. Published 2001 Wiley-Liss, Inc.

Discordant cellular and humoral immune responses to cytomegalovirus infection in healthy blood donors: existence of a Th1-type dominant response

Abstract: Previous studies have documented discordant cellular and humoral immune responses to subjects exposed to HIV-1, and that the nature of such responses may determine susceptibility and resistance to disease. We determined whether there is a spectrum of cellular versus humoral immunodominant responses to cytomegalovirus (CMV) infection. Blood samples from 50 healthy blood donors were tested for anti-CMV IgG antibodies and for proliferative responses of peripheral blood mononuclear cells (PBMC) to CMV antigens. Four patterns of immune responses to CMV were found: no detectable response (30%, Ab(-)/Tc(-)), anti-CMV IgG only (28%, Ab(+)/Tc(-)), both anti-CMV IgG and T lymphocyte proliferation to CMV antigens (18%, Ab(+)/Tc(+)), and, interestingly, T lymphocyte proliferation to CMV only (24%, Ab(-)/Tc(+)). To determine whether these immunodominant phenotypes correlate with the ability of PBMC to secrete IL-2 and IFN-gamma in response to CMV antigens, we found that a greater percentage of individuals with a T cell proliferative response to CMV antigens (Ab(-)/Tc(+) and Ab(+)/Tc(+)) responded with increased IL-2 (P = 0.001) and IFN-gamma levels (P = 0.002), compared to those without a proliferative response (Ab(-)/Tc(-) and Ab(+)/Tc(-)). Our data therefore demonstrate that different individuals exhibit different immunodominant patterns of response to CMV. In particular, some individuals who are exposed to CMV fail to develop an antibody response but do develop cellular immunity. Whether these different patterns predict susceptibility or resistance to CMV-induced disease remains to be determined.

T-cell-directed cancer vaccines: the melanoma model.

Abstract: Significant advances in the understanding of the molecular basis for tumour/host interactions in humans have occurred in the last decade through studying patients with metastatic melanoma. This disease is characterised by its tendency to be modulated by immunologic factors. Furthermore, immunologic manipulation of the host with various systemic agents, in particular IL-2, frequently affects this natural phenomenon and can lead to complete rejection of cancer. By studying the cellular immunology occurring in patients undergoing immunotherapy, several tumour antigens (TA) and their epitopes recognised by human leukocyte antigen (HLA) class I-restricted cytotoxic T-lymphocytes (CTL) have been identified. Most of these TA are non-mutated molecules expressed by the majority of melanoma in vivo and most melanoma cell lines. In addition, unique minimal epitopic sequences play an immunodominant role in the context of specific HLA class I alleles. Since melanoma lesions from different patients often share expressi...

T-cell-directed cancer vaccines: mechanisms of immune escape and immune tolerance

Abstract: Recent clinical trials using vaccines directed toward tumour-associated antigens (TA) have shown the increasing capacity of vaccines to cause immunologic responses. In fact, strongly reactive TA-specific cytolytic T-lymphocytes and tumour-infiltrating lymphocytes (TIL) can be identified and expanded ex vivo from patients with metastatic melanoma vaccinated with melanoma-associated antigens. Paradoxically, this strong immunological response does not correlate with clinical tumour regression. Proposed mechanisms responsible for this glaring inconsistency are numerous and varied; systemic immunosuppressive as well as local mechanistic factors are implicated. In this review we will critically evaluate the possible mechanisms that allow tumours to escape immune destruction and be tolerated by the immune system. In addition, strategies that may allow further insight into the biology of tumour rejection are discussed, in the hope of deepening the understanding of this phenomenon and enhancing its therapeutic potential.

High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer.

Abstract: Aims and background The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). Methods The SBT method described utilizes an ABI Prism 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. Results Data from one 96-well capillary run on the ABI Prism 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. Discussion The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

Gene profiling arrays

Abstract: Ordered arrays of mixtures of nucleic acid molecules are provided, which mixtures reflect the expression profile of one or more specimens, such as different cells or tissues. In particular embodiments, complete mRNA mixtures from specimens are separately arrayed on a substrate. Specimens from which such mixtures of nucleic acid molecules are produced can be taken from any source, including animal, plant and/or microbial cells, and can be assembled in any collection desired. The collections can, for instance, include different cell types, different phenotypes, cells grown under different conditions, cells of different ages or developmental stages, and so forth. The nucleic acid arrays are provided in both macro- and microarray formats, and are suitable for gene profiling in which relative quantitative expression from a single source or multiple sources may be determined. Techniques are also disclosed for producing high-fidelity, amplified mixtures of nucleic acid molecules using a combination of anti-sense RNA amplification and template-switching synthesis. Amplified mixtures produced using this method can, for instance, be applied to the disclosed arrays. The disclosed arrays allow high throughput analysis of differential gene expression in a specimen (such as a tumor) or a variety of specimens (such as a variety of tumors), and are suitable for automated preparation and analysis.

A prospective, randomized, double-blind, placebo-controlled trial evaluating the effect of nystatin on the development of oral irritation in patients receiving high-dose intravenous interleukin-2.

Abstract: Interleukin-2 (IL-2) has been used to treat patients with metastatic melanoma and renal cell cancer for nearly two decades, and much progress has been made in ameliorating its adverse effects. One bothersome adverse effect, oral pain or oral irritation, is usually treated with an oral antifungal antibiotic, nystatin. The authors performed a prospective, randomized, double-blind, placebo-controlled trial involving 64 patients to evaluate the effect of prophylactic administration of nystatin or placebo on the development of oral irritation in patients receiving high-dose intravenous IL-2. No difference was found between patients randomized to receive nystatin or placebo in their rates of development of oral irritation, the severity of IL-2 adverse effects, the duration of their treatment, the rate of development of positive studies for oral yeast, or their pattern of experiencing other adverse effects. Thus, patients who receive high-dose intravenous IL-2 should not be treated prophylactically with nystatin to prevent oral irritation, and clinicians should seek evidence of the presence of oral thrush before using antifungal agents to treat oral pain in these patients.

T-cell epitope of mage-12 and related nucleic acids, vectors, cells, compositions and methods of inducing an immune response to cancer

Abstract: The present invention provides an isolated and purified polypeptide consisting essentially of the amino acid sequence VRIGHLYIL, an isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding the amino acid sequence VRIGHLYIL, such as SEQ ID NO: 2, a vector comprising and expressing such an isolated and purified nucleic acid molecule and a cell comprising such a vector. The present invention further provides a composition comprising the isolated and purified polypeptide or the vector in an amount effective to induce an immune response to the polypeptide in a mammal as well as a method of inducing an immune response to a cancer in a mammal having cancer, such as melanoma. The method comprises administering the composition to the mammal, which preferably has HLA-Cw*0702, in an amount effective to induce an immune response to the cancer in the mammal. The method can further comprise administering a cytokine, such as interleukin-2.