Showing papers by "Francesco M. Marincola published in 1997"

Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A.

Abstract: PURPOSE—In this study, we tested the effectiveness of a melanoma-associated antigen–derived peptide, MART-127–35, in eliciting cellular immune responses in vivo in the context of a phase I active immunization protocol. This peptide (AAGIGILTV) corresponds to residues 27–35 from the nonmutated melanoma-associated antigen MART-1/Melan A and is recognized by most melanomaspecific, HLA-A* 0201–restricted, tumor-infiltrating lymphocytes. To test the in vivo induction of cytotoxic T lymphocyte (CTL) sensitization, we compared CTL reactivity in vitro from peripheral blood mono-nuclear cell (PBMC) pools obtained before and after vaccination. PATIENTS AND METHODS—MART-127–35 was administered to HLA-A*0201 melanoma patients subcutaneously in an emulsification with incomplete Freund’s adjuvant. A vaccination course included four inoculations of peptide at 3-week intervals. PBMC collected by leukapheresis and separated by Ficoll-Hypaque gradient before and after vaccination were analyzed in 18 patients by in vitro sensitization with MART-127–35– To induce MART-127–35–specific CTL, PBMC were incubated with 1 μM peptide (on day 0) and interleukin-2 (IL-2) (300 IU/mL, on days 1 and 4 after each stimulation). At weekly intervals, cells were harvested and an aliquot was cryopreserved for later analysis. The remaining cells were replated and restimulated using irradiated autologous PBMC pulsed with 1 μM of relevant peptide. After three restimulations, all samples from one patient were tested simultaneously for HLA-A*0201-restricted anti-MART-127–35 reactivity by microcytotoxicity and cytokine (IFN-γ) release assays. RESULTS—Toxicities were minimal and consisted of local irritation at the site of vaccine administration. None of the patients sustained a clinical response. The first eight patients were monitored by inducing CTL reactivity from PBMC obtained preimmunization and after two and four vaccinations. Only two prevaccination cultures were reactive to MART-1, compared with five and seven cultures from PBMC obtained after two and four vaccinations, respectively. Thus, an enhancement in cytotoxic activity could be detected in postvaccination CTL cultures, and serial vaccine administrations appeared to boost the detectability of cytotoxicity in vitro. For completeness, the analysis compared prevaccination with postvaccination PBMC cultures. Specific anti– MART-127–35 cytotoxicity (≥ 10 lytic units) could be detected in two prevaccination and 12

Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine

Abstract: Recombinant poxviruses encoding tumor-associated antigens (TAA) are attractive as candidate cancer vaccines. Their effectiveness, however, will depend upon expression of the TAA in appropriate antigen-presenting cells. We have used a murine model in which the TAA is β-galactosidase (β-gal) and a panel of recombinant vaccinia viruses (rVV) in which β-gal was expressed under early or late promoters at levels that varied over 500-fold during productive infections in tissue culture cells. Remarkably, only those rVV employing early promoters were capable of prolonging the survival of mice bearing established tumors expressing the model TAA. Late promoters were ineffective regardless of their determined promoter strength. The best results were obtained when β-gal was regulated by a strong early promoter coupled to a strong late promoter. When a variety of cell types were infected with the panel of viruses in vitro, dendritic cells were found to express β-gal only under the control of the early promoters even though late promoters were intrinsically more active in other cell types. Furthermore, in a functional assay, dendritic cells infected in vitro with rVV encoding β-gal regulated by an early promoter activated β-gal-specific cytotoxic T lymphocytes, whereas similar rVV with a late promoter-regulated gene did not. These data indicate that promoter strength per se is not the most critical quality of a recombinant poxvirus-based tumor vaccine and that the use of promoters capable of driving the production of TAA in “professional” antigen presenting cells may be crucial.

Dendritic Cells Infected with Poxviruses Encoding MART-1/Melan A Sensitize T Lymphocytes In Vitro

Abstract: Most melanoma-associated antigens (MAA), including MART-1/Melan A, are nonmutated proteins processed intracellularly to yield peptide fragments presented by major histocompatibility complex (MHC) class I molecules and thus recognized by cytotoxic T lymphocytes (CTL) (1–4). Repetitive in vitro sensitization of peripheral blood mononuclear cells (PBMC) with synthetic peptides representing sequences derived from these epitopic determinants can induce potent CTL that recognize naturally processed antigen on the surface of melanoma cells (5,6). Furthermore, we have shown that multiple in vivo administrations of the same synthetic peptides emulsified in incomplete Freund’s adjuvant (IFA) can specifically enhance CTL precursor reactivity in the peripheral circulation (7,8). This enhanced immunologic reactivity, however, is not sufficient to induce clinical responses in most patients. Preclinical models have shown that the administration of peptide alone is not very efficient (9), and the choice of an appropriate adjuvant, including dendritic cells (DC), may be critical in enhancing antigen presentation in vivo (10). DC are antigen-presenting cells that are useful in the initiation of cellular immune responses (11). First described by Steinman and Cohn in 1973 (12,13), DC possess a special capacity to circulate and migrate to secondary lymphoid organs, where they exert their effects by stimulating resting T lymphocytes in a MHC class I (14,15) and class II (16–20) restricted manner. Clinically, the presence of DC in tumors has been correlated with improved prognosis in a variety of human cancers (21). Previously, DC have been isolated from progenitor cells of human bone marrow (22) or cord blood (23). It was not until recently that significant quantities of DC could be generated from human peripheral blood (24). Sallusto et al. (24) have shown that monocytes derived from the peripheral circulation of humans can be activated into DC by in vitro culture with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). DC can function as an efficient adjuvant for MHC class I restricted antitumor sensitization in vivo (15,18,25–30). Bone marrow–derived murine DC pulsed with MHC class I restricted soluble β-galactosidase (28), ovalbumin-derived peptides (15,27), or unfractionated acid-eluted tumor peptides (26) could induce long-lasting immunity against challenges with tumors. These studies have shown that peptide-pulsed DC are more efficient in inducing antitumor protection than immunization with peptide alone (27,28) or emulsified in IFA (29). However, exogenous loading of peptide on HLA molecules has two major disadvantages: first, the stability of the peptide binding to the HLA molecule will affect its immunogenicity (31), and second, the amino acid sequence appropriate for each HLA allele must be known. These limitations may be circumvented by a system that allows endogenous presentation of antigens, such as the infection of DC with recombinant viral vectors. Bhardwaj et al. (32) have shown that DC are infectable by influenza virus. Infected DC express viral proteins and are capable of generating anti-viral specific CD8+ CTL in humans (32). In this study, we tested whether the infection of human DC with viral vectors encoding MAA genes could induce significant expression of the relevant antigen to allow endogenous presentation in association with HLA class I molecules and induce anti-MAA CTL reactivity in vitro.

Effusion cytology of malignant melanoma. A morphologic and immunocytochemical analysis including application of the MART-1 antibody.

Abstract: BACKGROUND Malignant effusions are complications of metastatic malignant melanoma (MM). Differential diagnosis often involves distinguishing MM from adenocarcinoma and reactive mesothelial cells. Descriptions in the literature of the morphologic and immunocytochemical (IM) staining characteristics of MM in effusions are sparse. A combination of morphology and immunocytochemistry should yield the most accurate diagnostic results. The MART-1 antigen, a transmembrane protein, is specifically expressed in melanocytes and MM. A recently developed monoclonal antibody to the MART-1 antigen may represent a useful marker for the identification of MM in effusions. METHODS The authors conducted a retrospective review of 32 effusion samples diagnosed as MM. The review consisted of morphologic and IM analyses of the effusion samples with antibodies to MART 1, HMB45, S-100, and cytokeratins (AE1/AE3).IM stains were performed on cell block or cytospin material, depending on availability. In the morphologic review, emphasis was placed on Diff Quik-stained material, due to its enhanced cytoplasmic volume and detail. RESULTS Predominant cytologic features noted were lack of cellular cohesion (in 100% of cases), large eccentric nuclei with prominent nucleoli (in 100%), multinucleation (in 84%), variable cytoplasmic vacuolization (in 75%), pigment (in 72%), and cell-in-cell engulfment (in 47%). All immunoreactive cases with sufficient material stained with at least one of the markers used. Tumor cells were positive with IM stains to MART-1 in 78% of cases, HMB45 in 81%, and S-100 in 81%. Coexpression of MART-1, HMB45, and S-100 was noted in 63% of cases. Of cases that showed expression for only 1 of the 3 antigens, the MART-1 was positive in 1 case, and HMB45 and S-100 were positive in 2 cases each. Three cases showed immunoreactivity for cytokeratins in the melanoma cells. CONCLUSIONS The diagnosis of MM in effusions can be made reliably through a combination of morphologic and IM features. Differential diagnosis often involves distinguishing MM from adenocarcinoma or reactive mesothelial cells. Cytoplasmic vacuolization, multinucleation, prominent nucleoli, and cell-in-cell engulfment are cytologic features common to all three. The lack of IM staining for cytokeratins alone cannot reliably distinguish MM; 11% of cases showed positive staining with this antibody in the melanoma cells. The use of a panel of antibodies increases the accuracy of diagnosing MM. In this study, MART-1 proved a useful adjunct to the HMB45/S-100/cytokeratin panel for the diagnosis of MM in effusions, staining 78% of the immunoreactive cases, with positivity in 1 case that was negative for HMB45 and S-100. Cancer 1997; 81:57-63. © 1997 American Cancer Society.

Cytotoxic T‐cell clone against rectal carcinoma induced by stimulation of a patient's peripheral blood mononuclear cells with autologous cultured tumor cells

Abstract: In an effort to establish cytolytic T lymphocytes (CTLs) against colorectal carcinoma (CRC) by stimulating patients' lymphocytes with autologous tumor cells, we used peripheral blood mononuclear cells (PBMC) from a patient with minimal residual rectal carcinoma following removal of the primary lesion and involved regional lymph nodes as a source to generate CTLs in culture. A CTL line and clone were established from the patient's PBMC following stimulation of PBMC with autologous, cultured tumor cells and interleukin-2. The CTL line and the clone consisted predominantly of CD4+ lymphocytes. The CTL clone expressed two T-cell receptor variable alpha chains (V alpha11 and V alpha22) and one beta chain (Vbeta14). The cytokine secretion pattern of the CTL line was of the Th1-type. Both the CTL line and the clone lysed the autologous rectal carcinoma cells, but not the allogeneic, partially human lymphocyte antigen (HLA)-matched or nonmatched CRC cells, autologous Epstein-Barr virus-transformed B cells, K562 (natural killer target) cells or Daudi (lymphokine-activated killer target) cells. Lysis of autologous tumor cells most likely was HLA class I-restricted. Our unique success in generating CTLs against this tumor type may rest in the inclusion of a patient with minimal residual, rather than advanced, disease.

A prospective randomized evaluation of the prophylactic use of low-dose dopamine in cancer patients receiving interleukin-2.

Abstract: The administration of high-dose interleukin-2 (IL-2) causes tumor regression in 17-25% of patients with metastatic melanoma or renal cell carcinoma. Renal dysfunction is a common dose-limiting toxicity of IL-2 administration, limiting 26% of treatment cycles. We have conducted a prospective randomized trial to evaluate whether the prophylactic administration of low-dose dopamine (2 mg/kg/min) can minimize renal toxicity and thus affect the amount of IL-2 administered. Forty-two patients were randomly assigned to receive systemic high-dose IL-2 with standard supportive measures (group A = 21 patients) or with the addition of prophylactic dopamine (group B = 21 patients) at 2 mg/kg/min. For patients in group B, dopamine was instituted 1 h before the initiation of IL-2 administration and was discontinued 6-12 h after the maximum number of doses of IL-2 were given. There was no difference in the amount of IL-2 administered for each course of therapy for groups A and B. Despite differences in urine flow (milliliters per kilogram per day), fluid balance (liters per day), and overall weight gain, prophylactic low-dose dopamine did not significantly alter maximum plasma urea or creatinine levels in group B when compared with the control group (group A). The overall toxicity profile considering all grade 3 and 4 toxicities for patients in groups A and B was comparable. Thus, there is no evidence to support the routine use of prophylactic low-dose dopamine in patients receiving high-dose IL-2.

Melan-A/MART-1 antigen expression in cutaneous and ocular melanomas.

Abstract: SummaryThe human immune repertoire appears to be capable of recognizing normal antigens expressed by tumor cells. Among these antigens, those of differentiation, characterized by a restricted tissue expression, could be of clinical interest since they may represent a target for immunotherapeutic pro