Identification of a novel HLA-C allele, C*02:10:03, in a Venezuelan patient.

HLA-C*02:10:03 identified in a Venezuelan patient and characterized using next generation sequencing.

Molecular requirements for attachment of the glycosylphosphatidylinositol anchor to the human alpha folate receptor.

The alpha isoform of the folate receptor (FR) is a 38-KDa glycosylphosphatidylinositol (GPI) protein which mediates the internalization of folates. The FR amino acid sequence has features typical of GPI-linked proteins, including the presence of a hydrophobic carboxyl-terminus, a hinge region, and a stretch of small and uncharged amino acids. Substitution of predicted cleavage/attachment Ser234 with arginine or threonine, or replacement of Gly235 with proline by site-directed mutagenesis had no effect on GPI processing. In fact, CHO cells transfected with each of the three cDNA variants or with FR wild-type showed comparable amounts of phosphatidylinositol-specific phospholipase C-resistant FR in double-determinant radioimmunoassay. Western blot analysis of total cell lysates from all transfectants consistently revealed the 38-KDa FR band. Deletion of residues 233-237 in the amino-terminal portion of the FR cDNA constructs derived by a polymerase chain reaction strategy abrogated GPI processing, with only a small proportion of the FR remaining in the cytoplasm in four of the five clones tested. This finding suggests that FR residues 233-237 are essential in properly juxtaposing the FR hydrophobic domain. Together, these data support the hypothesis that the postulated Ser234 is not the only potential cleavage/attachment site of the alpha isoform of FR.

A novel approach to human anti-HLA mABs production: use of phage display libraries.

The production of human monoclonal antibodies was previously limited to very laborious and time-consuming processes involving EBV-transformation and/or hybridoma generation. Due to the development of molecular cloning techniques, it is now possible to produce human monoclonal antibody fragments quickly by panning phage display libraries against predefined antigenic specificities. Therefore, we tested this technology for producing human single chain Fv fragments (scFvs) against HLA-DR1 purified molecules immobilized on solid phase. Enrichment of DR1-specific phages was measured through five selection rounds of a synthetic library and revealed a 100-fold amplification. Soluble antibody fragments were then expressed and 7 out of 48 clones were found to secrete scFvs which specifically bind to DR1 molecules in ELISA. Further analysis revealed binding of the scFvs also to DR3 but not to DR5 or DR7 molecules correlating with the presence of particular polymorphic aminoacid residues in the DR beta chain. Western blot analysis indicated that the 7 scFvs react with the DR1 alpha/beta-dimer but not with free alpha- or beta- chains. This study shows that the innovative approach of phage display libraries can efficiently provide scFv fragments as useful reagents for the identification and dissection of HLA polymorphic epitopes.

Role of membrane folate-binding protein in the cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cell lines in vitro.

Lometrexol (5,10-dideazatetrahydrofolic acid; DDATHF), is a specific inhibitor of glycinamideribonucleosyl (GAR) transformylase with anti-tumour activity in murine and human carcinomas. The cytotoxicity activity of DDATHF was evaluated in vitro in NIH/3T3 cells transfected with human alpha-folate-binding protein (FBP) complementary DNA to examine the role of the receptor. In FBP-transfected NIH/3T3 (FBP-tNIH/3T3) cells, which internalised about three times more 5-methyltetrahydrofolic acid than the mock-transfected cells, the cytotoxtic potential of DDATHF showed a clear increase. Subsequently, we analysed four ovarian carcinoma cell lines (OVCAR3, IGROV1, SKOV3, and SW626) expressing different amounts of FBP. Cells were conditioned to grow in medium depleted of folic acid then tested by MOv18 and folic acid binding. Only SKOV3 and SW626 cells grown in folic acid-depleted medium showed increased FBP expression, about 3- and 8-fold respectively. The cytotoxic potential of DDATHF was evaluated by a standard clonogenic assay. In a medium containing 2.27 microM folic acid the DDATHF IC50 values were 50 nm on OVCAR3, 500 nM on SW626 and 1000 nM on IGROV1. In folic acid-free medium IC50 values were 2 nM on OVCAR3 and Sw626 and 40 nM on IGROV1. Only on SKOV3 cells was DDATHF cytotoxicity the same regardless of the amount of folic acid in the medium (IC50 8 nM). Thus, DDATHF did not inhibit the growth of IGROV1 cells depleted of folic acid after stripping FBP with phosphatidylinositol-phospholipase C, even at a dose toxic for cells constitutively expressing FBP. Although FBP expression is certainly one of the parameters affecting drug toxicity, taken alone it is not a sufficiently reliable predictor of cancer cell sensitivity to DDATHF.

Growth of ovarian-carcinoma cell lines at physiological folate concentration: effect on folate-binding protein expression in vitro and in vivo.

Ovarian-carcinoma cell lines (OVCAR3, IGROVI, OVCA432, SW626 and SKOV3), grown in standard medium containing supra-physiological (2.3 microM) folate concentration, display different levels of reactivity with the anti-folate-binding-protein (FBP) monoclonal antibody MOv18, which recognizes the alpha-isoform of the protein. Gel-filtration and absorption experiments indicated that on IGROVI cells this molecule accounts for all folic-acid binding at nanomolar concentrations. The aim of the study was to investigate the effect of extracellular folate levels on cells adapted to grow in medium containing physiological folate concentration (20 nM). By the ternary complex assay, all cell lines showed a marked depletion of intracellular reduced folates, compared with those in standard folate medium. The monitoring of FBP by MOv18 showed on IGROVI cells a transient up-regulation of the protein, whereas on the other cell lines, except SKOV3, no changes were detected. These data suggest that in these cells further over-expression of the molecule cannot generally be induced by lowering the extracellular folate concentration. On SKOV3, Scatchard analysis of 125I-MOv18 binding, as well as the evaluation of total folate binding capacity, showed a 2- to 3-fold stable increase of FBP expression after long-term growth in low-folate medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated in these cells a 1.5-fold increase in alpha-FBP mRNA. SKOV3 cells, maintained in vitro in medium containing supraphysiological and physiological (i.e., low-folate) concentrations were injected into nude mice. Weight differences, though not statistically significant, were observed in favour of low-folate-derived tumors. Immunohistochemical and immunochemical analysis of the tumor samples showed that in SKOV3 cells the receptor modulation can also be induced by restoring the physiological folate concentration in vivo.

Folate binding protein distribution in normal tissues and biological fluids from ovarian carcinoma patients as detected by the monoclonal antibodies MOv18 and MOv19.

Folate-binding proteins (FBP), which are molecules relevant in folate metabolism, are overexpressed in ovarian carcinomas, as detected by the monoclonal antibodies (MAb) MOv18 and MOv19, which recognise two different epitopes of the gp38/FBP. In this paper, features of the FBP such as the distribution on normal tissues and the release in biological fluids of normal and tumour origin have been investigated. Immunohistochemical analyses on frozen sections of normal tissues showed the presence of the gp38/FBP on some epithelia. The reactivity of both the MAb on Fallopian tubes was intense and comparable to that observed on ovary carcinoma sections. The kidney, bronchial glands, alveolar epithelium of the lung, oesophagus, stomach, pancreas, breast and thyroid showed different levels of staining. By MOv18/MOv19 double-determinant immunoradiometric assay (DDIRMA), the gp38/FBP was found in soluble form in ascitic fluid, serum and urine of nude mice in which the human ovary carcinoma cell line IGROV1 grew as ascitic carcinomatosis. In human biological fluids, the gp38/FBP was detected in ascites of 60% of ovarian carcinoma patients, and in 29% of those with other carcinomas, but not in patients with non-epithelial tumours or with other non-tumoral pathologies. The mean serum arbitrary units (a.u.)/ml values of ovary carcinoma patients were significantly different to those of healthy donors or patients with endometriosis (P < 0.005 and P < 0.01, respectively), but not when compared to the sera of lung carcinoma patients. In addition, the sensitivity of DDIRMA was poor, since only 24% of the ovary carcinoma patients were positive with this assay. When a restricted number of cases selected for the presence of tumour cells in the ascites was examined, the percentage of DDIRMA-positive sera and ascites rose to 41 and 94%, respectively. In the urine, a strong reactivity was observed in the samples of both normal and tumour origin.

Gene transfection and expression of the ovarian carcinoma marker folate binding protein on NIH/3T3 cells increases cell growth in vitro and in vivo.

The glycoprotein gp38 is overexpressed in 90% of ovarian carcinomas and recognized by monoclonal antibodies MOv18 and MOv19. This molecule is a high affinity folate binding protein (FBP) and a potential marker for ovarian carcinoma. We have developed a model to investigate the biochemical and biological properties of this folate receptor by transfecting NIH/3T3 cells, which do not endogenously express FBP, with a vector containing the complementary DNA for the gp38 cloned from the ovarian carcinoma cell line IGROV1. The FBP expressed shows features identical to those of the protein produced by IGROV1 cell. The FBP is expressed on the cell membrane in a glycosylphosphatidylinositol-linked form, since it is released by treatment with phosphatidylinositol-specific phospholipase C, and is shed into the culture medium of the NIH/3T3 transfectants. Immunoblot analysis with MAbs MOv18 and MOv19 showed that both the glycosylphosphatidylinositol-linked and the soluble FBP migrate at the same apparent molecular weight as the respective IGROV1 proteins. The FBP-transfected NIH/3T3 cells bound folic acid and internalized about 30-fold more folic acid than mock-transfected cells. Growth analysis revealed that FBP-transfected NIH/3T3 cells like IGROV1 maintained their growth rate after 10 days of culture in medium containing physiological or low folate concentration, and tumors arising after transplanting FBP-tNIH/3T3 cells in nude mice were 3-fold heavier than those arising after transplantation of non-FBP-expressing NIH/3T3 cells. These results suggest a correlation between human ovarian carcinoma growth and FBP overexpression.

Conventional antibodies: requirements and methods for their optimization.

Eighteen years after the discovery of monoclonal antibodies (MAbs), the debate as to whether these reagents have fulfilled their promise must now be addressed. When MAbs were studied as tools for diagnosis and therapy, problems arose regarding the reagents themselves and the recognized target antigens. Because most MAbs are of murine origin, they usually evoke an immune response in human patients, which reduces their effectiveness. Moreover, their bulky size and dispersal via the blood prevent most of the injected antibodies from substantially penetrating solid, poorly vascularized tumors. However, MAbs that are adequately selected and manipulated to circumvent these problems have been found to successfully image tumors and to be therapeutically active.