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Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications.

3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4-triazole (ST1959) has shown therapeutic effects in several animal models of autoimmune diseases. In this study the effects of ST1959 were further investigated in a murine model of colitis. The evidence obtained indicates that the beneficial effects exerted by ST1959 rely upon a decreased local immunological response. The cellular effects of ST1959 were additionally investigated on human peripheral blood mononuclear cells and Jurkat T cells by measuring cytokine production, cell proliferation and activation of a set of transcription factors. ST1959 decreases human T cell proliferation and inhibits cytokine expression at the transcriptional level. Moreover, at doses inhibiting cytokine production, ST1959 blocks phorbol 12-myristate 13-acetate (PMA) and ionomycin-induced nuclear factor protein of activated T cell (NFAT1) activity, without impairing AP-1-and NF-κB-dependent transcription. Immunofluorescence data show that ST1959 inhibits the nuclear residency of NFAT1 in both Jurkat and human peripheral blood mononuclear cells activated with PMA/ionomycin. leptomycin B, an inhibitor of CRM1/exportinlα-dependent nuclear export, reverted the inhibitory effect of ST1959 on NFAT1 nuclear localization. This indicates that ST1959 may increase the nuclear export of NFAT1, downregulating NFAT1 activity via a mechanism different from that of cyclosporin A, since it does not affect NFAT phosporylation/dephosphorylation steps. These findings provide new insights into the molecular mechanisms underlying the immunomodulatory activity of ST1959.

ST2146biot is a biotinylated anti-tenascin monoclonal antibody (mAb) to be used for Pretargeted Antibody Guided Radioimmunotherapy (PAGRIT) of solid tumors. In vivo biodistribution studies of 125I-labeled ST2146biot were done in nude mice transplanted with human HT-29 colon carcinoma and/or human U-118MG glioblastoma cells characterized for low and high tenascin expression, respectively. In vitro results show that ST2146 retains immunoreactivity upon biotinylation, in contrast to other anti-tenascin mAbs. In vivo biodistribution of ST2146 shows specific tumor accumulation up to 10 days after the i.v. injection, with no relevant differences between biotinylated and nonbiotinylated ST2146. A dose of 4 μg/mouse saturates the low tenascin-expressing human colon carcinoma HT-29, whereas the high tenascin-expressing human glioblastoma U-118MG seems to be saturated at a ST2146biot dose between 320 and 640 μg/mouse. The percentage of injected dose per gram of tumor ranges from 10% to 30%, corresponding to an amount of ST2146biot/g of tumor of ∼400 ng/g and >200 μg/g for HT-29 and U-118MG, respectively. Tumor to normal organs uptake ratios are between 15 and 60, confirming high tumor selectivity of ST2146biot despite its cross-reactivity with the tenascin expressed at low level in the normal mouse organs. The ST2146biot localization data are substantially confirmed even when both low and high tenascin-expressing tumors are implanted in the same animal. To our knowledge, the absolute amount of ST2146biot, specifically localized in xenotransplanted human tumors, is the highest thus far described and supports the clinical use of this mAb in PAGRIT®.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

The Pretargeted Antibody-Guided RadioImmunoTherapy (PAGRIT) method is based on intravenous, sequential administration of a biotinylated antibody, avidin/streptavidin and (90)Y-labelled biotin. The hybridoma clone producing the monoclonal antitenascin antibody BC4, previously used for clinical applications, was found not suitable for further development because of the production of an additional, nonfunctional light chain. In order to solve this problem, the new cST2146 hybridoma clone was generated. The monoclonal antibody ST2146, produced by this hybridoma, having the same specificity as BC4 but lacking the nonfunctional light chain, was characterised. ST2146 was found able to bind human tenascin at an epitope strictly related, if not identical, to the antigenic epitope of BC4. It showed, compared to BC4, higher affinity and immunoreactivity and similar selectivity by immunohistochemistry. Biodistribution studies of biotinylated ST2146 and three other monoclonal antitenascin antibodies showed for ST2146 the highest and more specific tumour localisation in HT29-grafted nude mice. On the overall, ST2146 appears to be a good alternative to BC4 for further clinical development of PAGRIT.

The efficacy of vaccines can be greatly improved by adjuvants that enhance and modify the magnitude and the duration of the immune response. Several approaches to design rational adjuvants are based on the suppression of regulatory T-cell (Treg) function. Here, we evaluated whether removal or addition of Treg at the time of vaccination with tetanus toxoid and the mucosal adjuvant cholera toxin (CT), would affect immune responses. We found that depletion/inactivation of CD4 + CD25 + Treg, either by treatment of BALB/c mice with anti-CD25 monoclonal antibodies or by adoptive transfer of CD4 + CD25 T lymphocytes depleted of CD4 + CD25 + Treg into nu/nu mice, impaired antibody production after mucosal immunization in the presence of CT. Conversely, transfer of polyclonal, but not Ag-specific, CD4 + CD25 + Foxp3 + Treg to normal BALB/c mice enhanced CT-induced antibody responses. An increased titer of both immunoglobulin IgG1 and IgG2a antibody subclasses was found, however, the ratio between IgG1/IgG2a with or without polyclonal Treg was comparable, suggesting that polyclonal Treg influence the magnitude, but not the quality of the immune response. Recipients of polyclonal Treg that had been immunized with CT had an increased number of Ag-specific CD4 + T cells with an activated phenotype (CD44 hi ) in the draining lymph nodes. This accumulation of Ag-specific CD4 + T lymphocytes could favour the germinal centre formation and may promote T-dependent B-cell responses. Overall, our study indicates that Foxp3 + Treg can not only function as suppressor cells but also as helper T cells, depending on the type of immune response being evaluated and the microenvironment in which the response is generated. Immunology and Cell Biology (2010) 88, 698–706; doi:10.1038/icb.2010.76; published online 29 June 2010

In this study, we reexamined the structural prerequisites for the attachment of P-fimbriated Escherichia coli to human urinary tract epithelial cells. The epithelial cells were obtained from A1P1 nonsecretor individuals, who express the globoseries of glycolipids without the ABH blood group determinants, and from A1P1 secretor individuals, who in addition express globo-A, a receptor for the prsJ96 adhesin. The wild-type E. coli strains J96, AD110, and IA2 and the recombinant clones HB101 papJ96, HB101 prsJ96, HB101 papIA2, and HB101 papAD110 were tested for binding. They expressed P fimbriae, as defined by P blood group-dependent agglutination of human erythrocytes of the globoseries, but differed in reactivity with galactose alpha 1-4galactose beta (Gal alpha 1-4Gal beta)-latex beads, isolated glycolipids of the globoseries, sheep erythrocytes, and uroepithelial cells. Three different patterns of binding were represented among the recombinant clones. HB101 papIA2 and HB101 papAD110 agglutinated sheep erythrocytes and Gal alpha 1-4Gal beta-latex beads and attached to both secretor and nonsecretor epithelial cells. HB101 prsJ96 agglutinated sheep erythrocytes, reacted poorly with Gal alpha 1-4Gal beta-latex beads, and attached to A1 secretor but not to A1 nonsecretor epithelial cells. HB101 papJ96 agglutinated Gal alpha 1-4Gal beta-latex beads but not sheep erythrocytes and attached poorly to human uroepithelial cells. The receptors relevant for adhesion were analyzed by inhibition with glycolipids in suspension. The sheep erythrocyte agglutination and attachment to secretor and nonsecretor epithelial cells of HB101 papIA2 and HB101 papAD110 were inhibited by globotetraosylceramide, while the Forssman glycolipid had no effect. The sheep erythrocyte reactivity and attachment to secretor epithelial cells of HB101 prsJ96 were inhibited by the Forssman glycolipid. These results permitted three conclusions. First, the expression of functionally active Gal alpha 1-4Gal beta-specific adhesins, as in HB101 papJ96, was not sufficient to make E. coli competent to attach to human uroepithelial cells. Attachment required P fimbriae of the papIA2 or papAD110 type. Second, the sheep erythrocyte reactivity of P-fimbriated strains could not be attributed solely to recognition of the Forssman glycolipid and may not be used to define the prsJ96-encoded phenotype. Third, the P-fimbrial adhesins which mediate secretor state-independent attachment to human uroepithelial cells recognized receptor epitopes provided by globotetraosylceramide.

The casein fraction of human milk was found to inhibit the attachment of Streptococcus pneumoniae and Haemophilus influenzae human respiratory tract epithelial cells. The inhibitory activity for S. pneumoniae remained after heat and trypsin treatment of the casein and was found in oligosaccharides released from casein. kappa-Casein, which is the most highly glycosylated casein component, inhibited pneumococcal attachment at concentrations similar to the whole casein fraction. The results are consistent with the known recognition of GlcNAc beta 1-3Gal by S. pneumoniae, since human milk and bovine colostrum, which contain GlcNAc, inhibited attachment, but mature bovine milk lacking GlcNAc did not. The effect on H. influenzae was similar to that on S. pneumoniae in that the attachment was inhibited by human casein and bovine colostrum, but not by either mature bovine milk or by the bovine casein fraction. The kappa-casein component of human milk was a less efficient inhibitor of H. influenzae attachment than the whole casein fraction and the free oligosaccharides were inactive. This anti-microbial effect of human casein represents a new mechanism for the protection by breast-milk against respiratory tract infection.

A novel biotin-DOTA conjugate (r-BHD: reduced biotinamidohexylamine-DOTA) was investigated in order to provide an efficient pretargeted antibody-guided radioimmunotherapy (PAGRIT) application. Preclinical and clinical results are described.(90)Y and (177)Lu were used to label r-BHD. The effect of pH and a wide range of specific activities were studied. Radiolabelled r-BHD was tested for affinity towards avidin and for stability in saline or in human serum with and without ascorbic acid. Pharmacokinetic data were collected and organ biodistribution evaluated in a tumour-bearing pretargeted animal model. A pilot study was performed in a metastatic melanoma patient and dosimetry was estimated.High radiochemical purity (99%) was routinely achieved with (90)Y or (177)Lu in sodium acetate buffer (1.0 M, pH 5.0) at a specific activity of 2.6 MBq/nmol. Both (90)Y- and (177)Lu-r-BHD were also prepared at higher specific activities. Radiolabelled r-BHD was stable up to 96 h in human serum and saline with the addition of ascorbic acid. The structural modifications proposed for the r-BHD stabilised it against enzymatic degradation while retaining high binding affinity for avidin. Renal clearance appeared to be the main route of excretion in animals, and high tumour uptake was observed in the pretargeted animals. The patient study showed a total body clearance of approximately 85% in 24 h, with a kidney absorbed dose of 1.5 mGy/MBq. Tumour uptake was rapid and the calculated dose to a 10-mm tumour lesion was approximately 12 mGy/MBq.These results indicate that the new biotin-DOTA conjugate may be a suitable candidate for pretargeting trials.

BACKGROUND Tenascin-C (TN-C), a large extracellular matrix (ECM) glycoprotein with a molecular weight of 180–250 kilodaltons, is present in several normal adult tissues. TN-C is up-regulated during embryogenesis, in wound healing, and in tumor tissues. Glioblastoma multiforme (GBM) is the most frequent and malignant astrocytic tumor comprised of poorly differentiated, neoplastic astrocytes. Recently, TN-C-based radioimmunotherapy was administered to patients with GBM. METHODS In the current study, the authors used immunohistochemistry to conduct a systematic investigation of TN-C distribution patterns in normal human brain tissue and in a large variety of brain tumors (n = 485 tumors). Immunoreactivity for TN-C was assessed with regard to its localization within tumor cells, blood vessels, and ECM using three different monoclonal antibodies (clones BC2, BC4, and TN2). RESULTS In control human brains, a significant difference was noted in the expression of TN-C when comparing gray with white matter using either Western blot analysis or immunohistochemistry. TN-C was found in the white matter of the frontal, temporal, parietal, and occipital lobes and in the hippocampus, where the immunoreaction was especially strong in the hippocampal formation. In 181 astrocytomas of different grades (World Health Organization [WHO] Grade 2–4), TN-C immunopositivity was seen to varying degrees in the cellular and stromal components of the tumor and in tumor-associated vessels. Glioblastomas (n = 113 tumors) showed strong immunopositivity in the vessels and moderate immunopositivity of the ECM. A statistically significant reduction of TN-C immunopositivity in tumor-associated vessels or ECM was observed in anaplastic astrocytomas (WHO Grade 3) compared with GBM (WHO Grade 4). A Kaplan–Meier analysis showed that patients who had GBM lesions that lacked TN-C immunopositivity in the ECM had a significantly longer survival (median, 28 months; standard error, 7.8 months) (n = 12 patients) compared with patients who had GBM lesions with TN-C immunopositivity (median, 12 months; standard error, 1.6 months) (n = 87 patients). In meningiomas (n = 24 tumors), the neoplastic cells, the ECM of the tumor, and the vessels were TN-C negative. In schwannomas (n = 31 tumors), the tumor cells were TN-C negative; whereas, in > 50% of tumors, the vessels and the ECM of regressively altered tumor areas were positive. In metastatic carcinomas (n = 53 tumors), the tumor cells were negative; seldom were vessels stained positive for TN-C. Focal areas of the ECM, often accompanied with fibrotic changes, were immunopositive for TN-C. CONCLUSIONS The most constant TN-C immunopositivity was noted in the ECM of the fibrotic stroma in highly malignant brain tumors and along the tumor border, especially in high-grade astrocytomas. The current results suggest that TN-C expression may be correlated with the grade of malignancy in astrocytic tumors and that the presence or absence of TN-C expression in the stroma of astrocytic tumors may play a not yet clearly understood role in shortening or prolonging, respectively, the survival of patients. Cancer 2003. © 2003 American Cancer Society.

Activation of T lymphocytes is dependent on multiple ligand-receptor interactions. The possibility that TCR dimerization contributes to T cell triggering was raised by the crystallographic analysis of MHC class II molecules. The MHC class II molecules associated as double dimers, and in such a way that two TCR (and two CD4 molecules) could bind simultaneously. Several subsequent studies have lent support to this concept, although the role of TCR cross-linking in T cell activation remains unclear. Using DRA cDNAs modified to encode two different C-terminal tags, no evidence of constitutive double dimer formation was obtained following immunoprecipitation and Western blotting from cells transiently transfected with wild-type DRB and tagged DRA constructs, together with invariant chain and HLA-DM. To determine whether MHC class II molecules contribute actively to TCR-dependent dimerization and consequent T cell activation, panels of HLA-DR1β and H2-Ek cDNAs were generated with mutations in the sequences encoding the interface regions of the MHC class II double dimer. Stable DAP.3 transfectants expressing these cDNAs were generated and characterized biochemically and functionally. Substitutions in either interface region I or III did not affect T cell activation, whereas combinations of amino acid substitutions in both regions led to substantial inhibition of proliferation or IL-2 secretion by human and murine T cells. Because the amino acid-substituted molecules were serologically indistinguishable from wild type, bound antigenic peptide with equal efficiency, and induced Ag-dependent CD25 expression indicating TCR recognition, the reduced ability of the mutants to induce full T cell activation is most likely the result of impaired double dimer formation. These data suggest that MHC class II molecules, due to their structural properties, actively contribute to TCR cross-linking.

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is a structural analog of ceramide that inhibits glucosylation of this molecule and thus of glucosphingolipid (GSL) expression by living cells. In this study, we used PDMP to slow the synthesis of the globoseries of GSLs (globo-GSLs) (derived from the precursor Gal alpha 1-4Gal beta 1-4Glc-ceramide) by cultured human kidney and large intestinal epithelial cells. The aim was to deplete the cells of receptors for P-fimbriated Escherichia coli and to examine the effects on the bacterially induced cytokine response. The mammalian cells (A498, HT-29, and Caco2) were cultured in the presence of PDMP in order to deplete them of GSLs. The cells were then subjected to GSL analysis or used to test bacterial adherence and cytokine production. The globo-GSLs were identified by thin-layer chromatography. Bacterial adherence was quantitated by microscopy, and interleukin-6 secretion was quantitated by the B9 bioassay. The interaction of bacteria with the globo-GSLs was studied by using E. coli strains and recombinant clones expressing P fimbriae. E. coli strains expressing type 1 fimbriae binding to mannose-containing glycoproteins were used as controls. PDMP treatment was found to reduce the content of the globo-GSLs in mammalian cells and the adherence of P-fimbriated E. coli to these cells. In contrast, PDMP treatment had no effect on the adherence of type 1-fimbriated E. coli or their activation of cytokine production by A498 cells. P-fimbriated E. coli elicited an interleukin-6 response in the A498 cells; this response was reduced after treatment with PDMP. The results emphasize the role of GSLs as receptors for P-fimbriated E. coli and for the cytokine response elicited by attaching bacteria.

Fresh human A1 erythrocytes, washed and pretreated in phosphate buffer with or without papain, were incubated at 37 degrees C with blood group-degrading enzymes from the human fecal Ruminococcus torques strain IX-70. The effects were assayed as changes in hemagglutination patterns, and blood group activities of alkali stable glycolipid extracts from the enzyme-treated cells using Dolichos biflorus anti-A1 lectin, Ulex europaeus type 1 anti-H lectin, and various monoclonal anti-A antibodies. Hemolysis was negligible (less than or equal to 1% after 6 h), and the osmotic fragility increased slightly only after papain treatment. The papain-untreated A1 erythrocytes lost D. biflorus agglutinability within minutes at room temperature with the unfractionated bacterial enzyme mixture IX-70 (42 mU 1,3-alpha-N-acetylgalactosaminidase (alpha-GalNAc'ase)/ml), but remained A active by strong agglutination with BioClone anti-A antibody even after 6 h of incubation. Thin layer chromatographic (TLC) immunostaining of extracted lipids showed hydrolysis of D. biflorus binding glycosphingolipids with more than six monosaccharides after 1 h, i.e., at a slower rate than the loss of D. biflorus agglutinability. Disappearance of these glycosphingolipids after 1 h paralleled the appearance of U. europaeus agglutinability and the strong binding of this lectin to glycolipid extracts in TLC immunoassays. A partly purified 1,3-alpha-GalNAc'ase (XI-117) (100 mU/ml) and a 1,2-alpha-fucosidase fraction (XI-50) containing alpha-GalNAc'ase (10 mU/ml) did not degrade blood group A active glycosphingolipids but completely abolished the D. biflorus agglutinability within 6 h. Papain pretreatment exposed U. europaeus receptors on the cell surface without changing the A1 hemagglutination pattern. It also facilitated a complete degradation of D. biflorus and U. europaeus reactive glycolipids with the IX-70 enzyme mixture within 6 h. The D. biflorus lectin was a good discriminator of A1/A2 subjects using erythrocyte lipid extracts but had a low affinity for the blood group A type 3 and type 4 glycosphingolipids in the TLC-overlay technique. In conclusion this study shows that (i) loss of D. biflorus A1 hemagglutination does not correlate with a loss of D. biflorus binding glycosphingolipids and (ii) loss of D. biflorus binding glycosphingolipids does not correlate with a loss of D. biflorus agglutinability. The results indicate that the serological D. biflorus agglutinability of A1 erythrocytes is not dependent on medium-sized glycosphingolipids (hexa- to dodecaglycosylceramides).

Escherichia coli strains which colonize the human urinary tract express lectins specific for different members of the globoseries of glycolipids, e.g., globotetraosylceramide and globo-A. This study investigated the importance of globo-A expression for attachment to human uroepithelial cells, colonization of the urinary tract, and severity of urinary tract infection. The expression of receptor-active glycolipids by erythrocytes and epithelial cells was analyzed by thin-layer chromatography and bacterial overlay as well as by bacterial binding to those cells. The epithelial expression of the globo-A receptor was restricted to individuals of blood group A with a positive secretor state. Consequently, globo-A binding E. coli strains attached only to epithelial cells from these individuals. In contrast, globoside-recognizing strains attached in similar numbers to uroepithelial cells regardless of the ABH blood group and secretor state of the donor. The role of host receptor expression for infection with globo-A-specific E. coli was analyzed in 1,473 children with urinary tract infections. All those infected with strains exclusively expressing globo-A-specific adhesins were found to be of blood group A, compared with 45% in the population at large (P less than 0.006). The inflammatory response (fever, C-reactive protein, erythrocyte sedimentation rate) of individuals infected with these strains was lower than that in individuals with infections caused by globoside binding strains. The results demonstrate the importance of fitness between host receptors and bacterial adhesins for infection and suggest that minor receptor epitope differences have profound effects on the disease process.