Interleukin (IL)-18, a biomarker of human ovarian carcinoma, is predominantly released as biologically inactive precursor.

Interleukin (IL)-18 is a proinflammatory and immune-enhancing cytokine, which exerts antitumor effects in vivo, mediated by the induction of interferon (IFN)γ. We previously reported that IL-18 processing is defective in epithelial ovarian carcinoma (EOC) cells, which secrete an inactive precursor (pro-IL-18) in vitro. In addition, IL-18 was reported as a potential biomarker of EOC. Here, we further investigated its role as a serological marker in human EOC and addressed its possible biological activity in vivo. Our data indicate that immunoreactive IL-18 is increased in EOC patients' sera at diagnosis as compared with age-matched healthy women. IL-18 levels were higher in the ascitic fluids than in sera, suggesting a local production in the peritoneal cavity. Indeed, immunohistochemical analysis of tumors showed IL-18 expression in cytokeratine-positive neoplastic cells, although also scattered histiocytes and some lymphoid cells stained for IL-18. The detection of human IL-18 in sera and ascitic fluids of immunodeficient mice, orthotopically implanted with human EOC cells, further suggested that circulating IL-18 is tumor-derived. However, IL-18 is not an EOC specific biomarker, as increased serum levels were found also in some endometrial cancer patients. By means of a new monoclonal antibody, we characterized IL-18 present in the ascitic fluid as pro-IL-18, which is biologically inactive. Accordingly, IFNγ was not increased in EOC patients' sera and ascitic fluids and showed no correlation with IL-18 levels. Altogether these data indicate that IL-18 in EOC fluids is predominantly tumor-derived and that its lack of biological activity may represent a mechanism of tumor-escape.

Shed HER2 extracellular domain in HER2-mediated tumor growth and in trastuzumab susceptibility.

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.

Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression.

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Role of IL-21 in immune-regulation and tumor immunotherapy.

IL-21, the most recently discovered member of the IL-2 cytokine family, is an attractive subject for research due to its involvement in experimental models of autoimmunity, its ability to down-regulate IgE production, and its anti-tumor properties. Its interest for cancer immunotherapy stems from its physiological immune-enhancing functions. These include regulation of T, B and NK cell proliferation, survival, differentiation, and effector functions. IL-21's functional activities partially overlap those of IL-2. Both cytokines display similar structural features and use the common gamma-chain receptor and its downstream signaling pathways. Besides its activities on normal lymphoid cells, IL-21 is an in vitro growth factor for myeloma and acute-T cell leukemia cells, whereas it induces the apoptosis of B-CLL (chronic lymphocytic leukemia) cells. These findings indicate that the IL-21/IL-21R system exerts opposite functions in different lymphoid neoplasias, and suggest its employment in B-CLL therapy. Since IL-2, but not IL-21, is specifically required for the development of regulatory T (Treg) cell immune-suppressive functions, IL-21 may be a new tool for cancer immunotherapy. It is, in fact, a powerful anti-tumor agent in a variety of murine experimental tumor models through its activation of specific or innate immune responses against neoplastic cells. The preliminary data from phase-I clinical studies suggest that the use of IL-21 is feasible and may result in immune-enhancing effects.

The ALCAM shedding by the metalloprotease ADAM17/TACE is involved in motility of ovarian carcinoma cells.

Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA-reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential.

Targeting tumor-related immunosuppression for cancer immunotherapy.

Tumors produce several factors, such as Prostaglandins (PGs), Interleukin (IL)-10, Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor (TGF)-beta, which may directly or indirectly inhibit the immune response and may hamper immunotherapy. Furthermore, cells of innate or adaptive immunity, recruited by tumor-derived factors, may contribute in immunosuppression. Regulatory T (Treg) cells such as the "naturally occurring" CD4(+)/CD25(+) Treg and the IL-10-induced Tr1 cells are major players in this arena. Paradoxically Treg cells are stimulated by IL-2, which is used in tumor immunotherapy. Treg cells suppress T cell responses through soluble factors or by contact-dependent mechanisms, such as the Cytotoxic T Lymphocyte Antigen (CTLA)-4-mediated induction of Indoleamine 2,3-Dioxygenase (IDO) in dendritic cells (DC). IDO inhibits T cell responses by depleting Tryptophan and producing Kynurenine, which is toxic to lymphocytes. Macrophages, granulocytes or myeloid suppressor cells (MSC) suppress immunity by other enzymatic mechanisms, involving Arginase and Nitric Oxide Synthase (NOS). Subversion of tumor immunosuppression is required for successful immunotherapy. Attempts to block or eliminate Treg cells have been made by the use of chemotherapy, anti-CD25 or anti-CTLA-4 antibodies, IL-2-toxin chimeric proteins or Glucocorticoid-induced TNF-like Receptor (GITR) and CD134/OX-40 ligands. Tumor cells genetically modified to secrete IL-21 (an immune-stimulatory "IL-2-like" cytokine, which is not involved in immune regulation) cured experimental metastases in combination with anti-CD25 monoclonal antibodies (mAbs). Also strategies aimed at blocking enzyme-based immune-suppressive mechanisms are suitable, as suggested by experimental evidences in mouse tumor models.

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

Internalization and recycling of ALCAM/CD166 detected by a fully human single-chain recombinant antibody.

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, promotes heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. Here we describe a fully human single-chain antibody fragment (scFv) directed to ALCAM/CD166. We selected the I/F8 scFv from a phage display library of human V-gene segments by cell panning and phage internalization into IGROV-I human ovary carcinoma cells. The I/F8 specificity was identified as ALCAM/CD166 by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) peptide mass fingerprinting of the I/F8-immunoprecipitated protein. The I/F8 scFv reacts with the human, monkey and murine ALCAM/CD166 molecule, indicating that the recognized epitope is highly conserved. The I/F8 scFv completely abolished binding of both ALCAM/Fc and CD6/Fc soluble ligands, whereas it did not compete with the anti-ALCAM/CD166 murine monoclonal antibodies J4-81 and 3A6 and therefore recognizes a different epitope. Engagement through I/F8 scFv, 3A6 monoclonal antibody or CD6/Fc ligand induced ALCAM/CD166 internalization, with a kinetics slower than that of transferrin in the same cells. Newly internalized I/F8-ALCAM complexes colocalized with clathrin but not with caveolin and we demonstrated, using surface biotinylation and recycling assays, that endocytosed ALCAM/CD166 recycles back to the cell surface. Such an endocytic pathway allows the efficient delivery of an I/F8 scFv-saporin immunotoxin into tumor cells, as the conjugates are able to selectively kill cell lines expressing ALCAM/CD166. Altogether these data provide evidence of the suitability of the I/F8 scFv for further functional analysis of ALCAM/CD166 and intracellular delivery of effector moieties.

A novel isoform of pro-interleukin-18 expressed in ovarian tumors is resistant to caspase-1 and -4 processing.

Interleukin-18 (IL-18) is a proinflammatory cytokine synthesized as a 24 kDa inactive precursor (pro-IL-18) by several cell types, and is processed to a bioactive molecule of 18 kDa by the proteinases caspase-1 or caspase-4. All ovarian carcinoma cell lines express pro-IL-18, only in some instances coexpress caspase-1, and always express caspase-4; in any case, they display a defective processing of IL-18. We analysed whether pro-IL-18, present in two ovarian carcinoma cell lysates, could be processed 'in vitro' by recombinant active caspase-1. While most of pro-IL-18 could be cleaved by caspase-1, a residual of pro-IL-18 appeared to be resistant. Cloning and sequence analysis of the whole pro-IL-18 open reading frame demonstrated the existence of an alternatively spliced mRNA variant, which lacked exon-3 (Delta3pro-IL-18). The 12 bp exon-3 encodes for the AEDD amino-acid sequence, which is N-terminal with respect to the cleavage site of caspase-1. Both pro-IL-18 and Delta3pro-IL-18 mRNA isoforms were detected in all ovarian cancer cell lines analysed, while Delta3pro-IL-18 mRNA was undetectable in normal ovarian epithelial cells. The Delta3pro-IL-18 cDNA induced synthesis of an alternative Delta3pro-IL-18 protein upon transfection into a murine cell line. The Delta3pro-IL-18 protein was resistant to proteolytic activation by caspase-1 and -4, although it was capable to bind caspase-1. Aternative splicing of pro-IL-18 exon-3 may represent a novel mechanism of regulation of bioactive IL-18 production in human ovarian tumors.

Tumor cells engineered with IL-12 and IL-15 genes induce protective antibody responses in nude mice.

IL-12 and IL-15 stimulate T, B, and NK cell functions through independent mechanisms, and cooperative effects of these cytokines have been reported. The human MHC class I-negative small cell lung cancer cell line, N592, genetically engineered to secrete IL-15, N592/IL-15, showed a reduced tumor growth rate, while N592 cells engineered with IL-12, N592/IL-12, grew similarly to the wild-type N592, N592 parental cells (N592pc), in nude mice. However, N592 cells coexpressing both cytokines, N592/IL-12/IL-15 cells, were completely rejected by 100% of nude mice. Here we show that 60% of nude mice rejecting N592/IL-12/IL-15 cells were resistant to N592pc rechallenge. SCID mice rejected N592/IL-12/IL-15 cells, but did not develop resistance to N592pc rechallenge, suggesting a role of Ab responses. Among nude mice rejecting N592/IL-12/IL-15 cells, those developing resistance to N592pc rechallenge had significantly higher titers of anti-N592 IgG2b Abs than nonresistant nude mice. Induction of an Ig class switch in nude mice was related to the expression of IFN-gamma and CD40 ligand in the draining lymph nodes. An IgG2b, anti-N592 mAb, derived from N592/IL-12/IL-15-immunized nude mice splenocytes induced significant protection against N592pc, while an IgM mAb was ineffective. The protective IgG2b mAb, but not the IgM mAb, triggered Ab-dependent cell-mediated cytotoxicity by nude mouse splenocytes against N592pc. These data indicate that IL-12 and IL-15 synergistically trigger innate, immunity-mediated, anti-tumor effects, resulting in cytotoxic IgG Ab responses in T cell-deficient mice. Protective Ab responses may relate to both direct actions of IL-12 and IL-15 on B cells and to the activation of an innate immunity-B cell cross-talk.