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C Melani

Publications and source records attributed to C Melani.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes.

In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-gamma release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients.

B7-1 Antigen↗
PubMed2000

Immunological gene therapy with ex vivo gene-modified tumor cells: a critique and a reappraisal.

Studies using animal models have demonstrated that transduction of genes encoding different cytokines into tumor cells results in a local recruitment of inflammatory cells that in turn can inhibit tumor growth. This is often accompanied by tumor antigen priming of the host immune system, which becomes resistant to subsequent challenge by the parental, untransduced tumor. Gene-transduced tumor cells have therefore been widely used as vaccines, although in the therapeutic setting their antitumor efficacy was limited to a few animal models. On the basis of this rationale, clinical studies were initiated, results of which are evaluated in this review to identify the reasons for their limited efficacy. We point out problems generated by the use of autologous versus allogeneic gene-transduced vaccines, by the choice of the appropriate cytokine(s), and by patient selection. Results of these studies are also compared with those obtained by peptide-based vaccines in similar groups of patients. Altogether, we conclude that improvements can be made in the construction of gene-modified vaccines by (1) using tumor cells known to express molecularly defined antigens, (2) introducing, in addition to genes encoding cytokines, genes encoding T cell costimulatory molecules, (3) increasing the amount of cytokine released locally by irradiated cells, and (4) coadministering adjuvant cytokines (IL-2 and IL-12) systemically in order to expand the T cell pool activated by vaccines.

Adjuvants, Immunologic↗
PubMed2000

Nerve growth factor cooperates with p185(HER2) in activating growth of human breast carcinoma cells.

Nerve growth factor (NGF) is known to exert a mitogenic effect on human breast cancer cells through proto-TrkA activation. Reverse transcriptase-PCR analysis of proto-TrkA expression in human breast carcinoma specimens and cell lines revealed trkA transcript in 12 of 14 human breast carcinoma specimens and in all of four cell lines tested. While cytofluorimetric and Western blot analysis indicated proto-TrkA expression in three of the four cell lines, NGF stimulated growth in only two of the three positive cell lines. Inhibition of NGF-induced MAPK activation by an antibody directed against the extracellular domain of TrkA but not by an inhibitor of TrkA phosphorylation demonstrated the requirement of NGF binding but not of proto-TrkA kinase activity for MAPK activation, suggesting the recruitment of another kinase for transmission of the mitogenic signaling. Indeed, NGF induced tyrosine phosphorylation and stimulated kinase activity of p185(HER2), a kinase receptor of the HER family. A TrkA phosphorylation inhibitor did not affect this activation. Moreover, the two receptors were coprecipitated by antibodies directed against proto-TrkA and p185(HER2). Down-modulation of p185(HER2) expression in a breast carcinoma line transfected with a construct containing an anti-p185(HER2) antibody sequence and expressing proto-TrkA impaired NGF-induced MAPK activation and proliferation. Together these data show that in cells expressing low levels of TrkA such as breast carcinoma cells, NGF must recruit other overexpressed receptors such as p185(HER2) in order to generate a biological signal that can induce breast cancer cell growth.

Blotting, Western↗
PubMed1999

Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells.

A human melanoma line genetically modified to release interleukin 4 (IL-4) was utilized to immunize advanced melanoma patients in order to elicit or increase a specific anti-melanoma immune response, which may affect distant lesions. Twelve metastatic melanoma patients were injected subcutaneously at least three times with 5 x 10(7) IL-4 gene-transduced and irradiated allogeneic melanoma cells per dose. Both systemic and local toxicities were mild, consisting of transient fever and erythema, swelling, and induration at the vaccination site. Two mixed but not complete or partial clinical responses were recorded. To assess the immune response of vaccinated patients, both serological and cell-mediated activities were evaluated. Antibodies to alloantigens could be detected in 2 of 11 patients tested. Mixed tumor-lymphocyte cultures were performed, utilizing autologous and allogeneic HLA-A2-matched melanoma lines as simulators and targets. A significant increase in IFN-gamma release was detected in 7 of 11 cases when postvaccination lymphocytes were stimulated by the untransduced allomelanoma cells. However, induction of a specific recognition of autologous melanoma cells by PBLs was obtained after vaccination in only one of six cases studied. This response involved the melanoma peptide Melan-A/MART-1(27-35) that was recognized in an HLA-A2-restricted fashion. These results indicate that vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although in a minority of patients.

Adult↗
PubMed1999

Cytotoxic T lymphocyte response against non-immunoselected tumor antigens predicts the outcome of gene therapy with IL-12-transduced tumor cell vaccine.

The colon adenocarcinoma C26, carrying two endogenous tumor-associated antigens (TAA) recognized by CTL, has been transduced with the gene coding for the human folate receptor alpha (FR alpha) as an additional antigen in order to study the efficacy of vaccination against a tumor expressing multiple antigens. A dicistronic vector was used to transduce the IL-12 genes to create C26/IL-12/FR alpha that has been used as a cellular vaccine to treat mice bearing lung metastases of C26/FR alpha. After vaccination mice were partially splenectomized and splenic lymphocytes frozen and used retrospectively to study in vitro CD8 T cell response related to the treatment outcome. Vaccination cured 50% of mice and the effect was CD8 T cell dependent. Mice either cured (responders) or not cured (nonresponders) by vaccination developed tumor-specific CTL. However, analysis of CTL specificity and pCTL frequencies revealed that responders had a predominant CTL activity against endogenous C26-related tumor antigens, whereas nonresponders had CTL that recognized preferentially the FR alpha antigen. CD8 from responder mice were characterized to release high levels of granulocyte-macrophage (GM)-CSF upon antigen stimulation. Tumors obtained from mice that died despite vaccination lost expression of the FR alpha transgene but maintained expression of endogenous C26 antigens. Immunoselection against FR alpha antigen was not observed in tumors from non-vaccinated controls and from CD8-depleted vaccinated mice. Down-regulation of FR alpha antigen expression was due, at least in part, to methylation of retroviral vector long terminal repeat promoter since FR alpha expression was partially restored, ex vivo, by treatment with 5-aza-2'-deoxy-cytidine (aza). These results indicate that CD8 T cell-mediated immunoselection and production of GM-CSF are determining factors for the efficacy of tumor vaccines.

Adenocarcinoma↗
PubMed1998

IgG2a induced by interleukin (IL) 12-producing tumor cell vaccines but not IgG1 induced by IL-4 vaccine is associated with the eradication of experimental metastases.

We evaluated whether antibody response correlates with tumor therapy by cytokine gene-modified tumor cell vaccines. To characterize the antibody (Ab) response against a known antigen, colon carcinoma C26 cells and C26 variants engineered to produce interleukin (IL) 12 or IL-4 were further transduced to express the human tumor-associated antigen gp38 folate receptor (FR) alpha. Irradiated IL-12- and IL-4-producing C26/FR alpha cell vaccines cured 50 and 30% of mice bearing C26/FR alpha lung micrometastases. Treatment induced a rapid, CD4-dependent Ab production dominated by IgG2a and IgG1 in response to the IL-12 or IL-4 vaccine, respectively. In contrast, untreated tumor-bearing mice showed a late serological response dominated by IgM. Anti-FR alpha IgG1 and IgG2a were able to suppress tumor metastases upon passive transfer in vivo. Sera from mice cured by the IL-12 vaccine displayed a higher binding activity, a higher anti-FR alpha IgG2a content, and a higher complement-mediated tumor cell lysis in vitro compared to the sera from nonresponder mice. Such a correlation was not found in the sera of mice treated with the IL-4 vaccine. These data indicate that cytokine-producing tumor cell vaccines strongly influence antibody response, and that in the case of the IL-12-based vaccine, the Ab titer correlates with the therapeutic response, thus suggesting its use for monitoring the outcome of vaccination in cancer patients.

Animals↗
PubMed1998

Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody.

To provide a new tool for the immunotherapy of human ovarian carcinoma, we constructed a fusion protein between interleukin-2 (IL-2) and the single-chain Fv (scFv) of MOV19, a monoclonal antibody directed against alpha-folate receptor (alpha-FR), known to be overexpressed on human nonmucinous ovarian carcinoma. This was accomplished by fusing the coding sequences in a single open reading frame and expressing the IL-2/MOV19 scFv chimera under the control of the murine immunoglobulin K promoter in J558L plasmacytoma cells. The design allowed the construction of a small molecule combining the specificity of MOV19 with the immunostimulatory activity of IL-2. This might improve the tissue penetration and distribution of the fusion protein within the tumor, reduce its immunogenicity, and avoid the toxicity related to the systemic administration of IL-2. The IL-2/MOV19 fusion protein was stable on purification from the cell supernatant and was biologically active. Importantly, this construct was able to target IL-2 onto the surface of alpha-FR-overexpressing tumor cells and stimulated the proliferation of the IL-2-dependent CTLL-2 cell line as well as that of human resting peripheral blood lymphocytes. In a syngeneic mouse model, IL-2/MOV19 scFv specifically targeted a-FR gene-transduced metastatic tumor cells without accumulating in normal tissues, due to its fast clearance from the body. Prolonged release of IL-2/MOV19 scFv by in vivo transplanted J558-EF6.1 producer cells protected 60% of mice from the development of lung metastases caused by an i.v. injection of a-FR gene-transduced tumor cells. Moreover, treatment with IL-2/MOV19 scFv, but not with recombinant IL-2, significantly reduced the volume of s.c. tumors. The pharmacokinetics and biological characteristics of IL-2/NMOV19 scFv might allow us to combine the systemic administration of this molecule with the adoptive transfer of in vitro retargeted T lymphocytes for the treatment of ovarian cancer, thereby providing local delivery of IL-2 without toxicity.

Animals↗
PubMed1998

Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice.

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.

Animals↗
PubMed1998

Anti-tumor immunity induced by murine melanoma cells transduced with the Mycobacterium tuberculosis gene encoding the 38-kDa antigen.

The Mycobacterium tuberculosis Ag38 gene, which encodes a highly immunogenic protein, was cloned into a retroviral vector in-frame with the leader and the transmembrane portion of the nerve growth factor receptor, and transduced into murine melanoma cell line B16-B78. Significant protection was observed in mice immunized with the transduced melanoma cells and subcutaneously challenged with parental melanoma cells since only 20% of mice developed tumors. Necroscopy of mice immunized with the transduced melanoma cells revealed dramatic inhibition of experimental metastases induced by intravenous (i.v.) inoculation of parental melanoma cells. Moreover, vaccination with transduced cells significantly prolonged survival of mice challenged i.v. with parental melanoma cells. These data indicate that the presence of the mycobacterial 38-kDa protein greatly enhances immunological recognition of structures expressed by the parental melanoma cells. Comparison of Th1 and Th2 responses in mice immunized with parental melanoma cells versus mice receiving the transduced cells revealed a clear predominance of Th1 responses when the Ag38 protein was endogenously expressed. This transduction approach may represent a promising immunotherapeutic strategy for the treatment of cancer patients.

Animals↗
PubMed1997

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability.

From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2+ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5 x 10(7) and 15 x 10(7) irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5 x 10(7) cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed: in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients.

Adult↗
PubMed1996

Immunotherapy of experimental metastases by vaccination with interleukin gene-transduced adenocarcinoma cells sharing tumor-associated antigens. Comparison between IL-12 and IL-2 gene-transduced tumor cell vaccines.

We have compared the therapeutic activity and characterized the antitumor response induced by IL-12 and IL-2 gene-transduced tumor cell vaccines. Mice bearing lung metastases of the BALB/c colon carcinoma C51 were treated with syngenic, histologically related, and antigenically cross-reacting irradiated IL-12 (C26/IL12) or IL-2 (C26/IL2) gene-transduced C26 tumor cells given s.c. Vaccination with C26/IL12 cells cured 40% of mice, while vaccination with C26/IL2 cells reduced the number of metastatic nodules without affecting survival. Despite this difference, similar antitumor CTL activation was shown in mice treated with C26/IL12 or C26/IL2 cells. The lytic pattern of CTL was shown to be directed to tumor-associated Ags (TAA) shared between the colon carcinomas C51, C26, and CC36 as well as with other syngenic tumors. Both treatments induced anti-TAA Abs, but only sera from mice treated with C26/IL12 contained Ab that lysed tumor cells in a C-dependent cytotoxicity assay. Early infiltration of activated T cells was found in the lungs of mice vaccinated with C26/IL12. CD4+ lymphocytes purified from the lymph nodes draining the vaccination site or from the spleen showed a higher production of IFN-gamma in response to anti-CD3 mAb in C26/IL12 vaccinated mice, while a higher production of IL-4 was shown in mice vaccinated with C26/IL2 cells. These results indicate that the better therapeutic efficacy of vaccination with C26/IL12 is associated with the production of C-binding Ab, an early infiltration of the metastatic lungs by activated T lymphocytes and a predominant systemic activation of Th1 more than Th2 cells.

Adenocarcinoma↗
PubMed1996

Limited antitumor T cell response in melanoma patients vaccinated with interleukin-2 gene-transduced allogeneic melanoma cells.

We have immunized advanced melanoma patients with a HLA-A2-compatible human melanoma line genetically modified to release interleukin-2 (IL-2), to elicit or increase a T cell-mediated anti-melanoma response that may affect distant lesions. Twelve stage-IV patients were injected subcutaneously at days 1, 13, 26, and 55 with IL-2 gene-transduced and irradiated melanoma cells at doses of 5 or 15 x 10(7) cells. Both local and systemic toxicities were mild, consisting of transient erythema at the vaccination site; fever occurred in a minority of patients. Three mixed responses were recorded. Seven patients were evaluable for immunological studies. Mixed tumor-lymphocyte cultures carried out with different allogeneic HLA-A2-matched melanoma lines as stimulators and targets revealed an increase in the MHC-unrestricted, but no changes in the MHC-restricted, cytotoxicity in peripheral blood lymphocytes (PBL) obtained after vaccination as compared with those obtained before vaccination. Increased recognition of the tyrosinase 368-376 peptide occurred in post-vaccination PBL of one patient, whereas a weak increase in recognition of the gp100 280-288 peptide was detectable in another patient; these 2 patients also recognized the gp100 457-466 peptide. After in vitro, stimulation with the only available autologous melanoma line, CD4+ cells with autologous tumor-specific cytotoxicity and ability to release interferon-gamma (IFN-gamma) were found in post- but not in pre-vaccination PBL. In the same patient, as well as in another patient, limiting dilution analysis showed that vaccination resulted in an increased frequency of melanoma-specific cytotoxic T lymphocyte (CTL) precursors. These results indicate that vaccination with cells releasing IL-2 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although this response occurred in a minority of the melanoma patients studied.

Adult↗
PubMed1996

Amount of interleukin 12 available at the tumor site is critical for tumor regression.

The C26 colon carcinoma is resistant to systemic recombinant interleukin 12 (rIL-12) therapy. Transduction of C26 with genes encoding the two subunits of murine IL-12 to release 30-80 pg/ml resulted in delayed tumor onset after injection of 5 x 10(4) cells into syngeneic BALB/c mice and in 40% tumor regression after injection into CD4-depleted mice. Here, we analyzed the activity of rIL-12 (1 microgram/day) against C26 grown into CD4-depleted mice. Like in mice given injections of interleukin 12 (IL-12) gene-transduced C26 cells, depletion of CD4+ cells led to tumor regression in 6 of 14 mice, and immumocytochemical characterization of tumor-infiltrating leukocytes showed abundant infiltration by CD8+ T cells and asialoGM1+ natural killer cells, which were scanty in tumors from nondepleted mice. On the basis of the percentage of tumor regression and leukocyte infiltration we can conclude that, in the C26 system, systemic rIL-12 (1 pmicrogramg/day) produces the same results as 30-80 pg/ml IL-12 released at the tumor site. A new polycistronic retroviral vector was then used to increase the amount of IL-12 produced by C26-transduced cells. C26 cells releasing 5 ng/ml IL-12, nearly 100 times more than the above-mentioned transduced cells, were tumorigenic in less than 50% of the mice given injections of 5 x 10(4) cells. In mice given injections of 5 x 10(5) cells, an initial tumor take of 100% followed by a complete tumor regression. Tumor regression was associated with infiltration of CD8+ and asialoGM1+ cells, and mice that remained tumor free were immune to a rechallenge of nontransduced C26 cells. The results indicate that the amount of IL-12 made available at the tumor site may determine both the type and number of infiltrating leukocytes and the events leading to tumor regression as well as it may overcame host immunosuppression.

Adenocarcinoma↗
PubMed1996

Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2-transduced tumor cells.

The purpose of these studies was to determine whether systemic administration of recombinant interleukin 12 (rIL-12) is able to potentiate an initial, but insufficient T-cell antitumor response. Mice challenged with carcinoma cells engineered to release interleukin 2 (IL-2) and displaying such a response received single or multiple i.p. injections of rIL-12. This combination of systemic rIL-12 and local IL-2 increased the percentage of mice that rejected two different IL-2 gene-transduced tumors. In another set of experiments more closely resembling a clinical situation, IL-2 gene-transduced tumors were used as vaccines in an attempt to cure mice bearing wild-type parental tumors. The combination of these vaccines with systemic rIL-12 cured mice more effectively than rIL-12 and IL-2 gene-transduced tumor vaccines alone.

Adenocarcinoma↗
PubMed1996

Hypoxic tumor cell death and modulation of endothelial adhesion molecules in the regression of granulocyte colony-stimulating factor-transduced tumors.

C-26 colon adenocarcinoma cells transduced with the granulocyte colony-stimulating factor (G-CSF) gene form large tumors when injected into sublethally irradiated mice. These tumors regress when leukocyte function is reconstituted. Electron microscopy and immunocytochemical analysis of regressing C-26/G-CSF nodules indicates that tumor destruction is due mainly to hypoxia resulting from the functional loss of tumor vasculature and is only marginally due to direct cytolysis. Desegregation of basal lamina, cell swelling, and loss of junctions characterized the vessels within regressing tumors. Tumor cells were necrotic or filled with lipid vacuoles regardless of the distance from nearby vessels. Damage of tumor vasculature was dependent on the infiltrating leukocytes and the cytotoxic cytokines they produced. Locally produced interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) induced vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin on tumor vessels. Treatment with monoclonal antibodies to interferon-gamma (IFN-gamma) or TNF-alpha blocked tumor regression by inhibiting VCAM-1 and E-selectin expression on tumor-associated endothelial cells resulting in a reduced number of infiltrating leukocytes. Thus, C-26/G-CSF tumor regression presents features typical of hemorrhagic necrosis that occurs through the cytokines produced by infiltrating leukocytes in response to G-CSF.

Adenocarcinoma↗
PubMed1996

Cytokine-based gene therapy of human tumors. An overview.

This review first summarizes the different strategies of gene therapy of cancer and then focuses on the immunological approach. Several studies in animal models with cytokine gene-transduced tumor cells indicate that local cytokine release usually results in tumor growth inhibition. Moreover, in a number of cases vaccination with such cells can reduce growth of established tumors or even cure the tumor-bearing animals. Translation of such a principle in human clinical setting is reported. We have transduced human melanoma cells with genes coding for interleukin (IL)-2, IL-4 or B7-1 and characterized such lines. The phenotype did not change after gene insertion but the functional, immunostimulatory activity of IL-2 or B7-1 gene-transduced melanoma cells was significantly increased compared to that of parental lines. These-lines were then used to vaccinate melanoma patients. Preliminary results of trials with IL-2 gene-transduced cells are presented which indicate a weak clinical response and the activation of a melanoma-specific cytotoxic T lymphocyte response in a low percentage of patients.

Animals↗