The use of multiple endpoints to assess cellular responses to environmental contaminants in the interstitial marine ciliate Euplotes crassus.

This paper presents the results of investigations on the suitability of Euplotes crassus, an interstitial marine ciliate, to be used as model organism in ecotoxicology and thereafter to evaluate the toxicity of estuarine and coastal sediments upon laboratory exposure. Nowadays, anthropogenic activities have resulted in accumulation of metals and organic pollutants in the environment as well as in the food chain hence leading to serious ecological and human health problems. This may pose a risk to benthic and epibenthic organisms and it is crucial to discover toxicity tests that will identify adverse effects of sediment-associated chemicals on benthic organisms. Due to their nature as a eukaryotic cell/organism and their position in the food web, ciliated protozoa are suitable models for evaluating the effects of pollution on aquatic communities. Lethal and sublethal effects of exposure to inorganic and organic pollutants were tested on the cell mortality, replication rate, lysosomal membrane stability and endocytosis rate of E. crassus. Increasing nominal concentrations of individual and mixtures of mercury, copper, and benzo(a)pyrene were investigated in this study as they might be bioavailable in naturally occurring polluted sites. A significant decrease in the mean replication rate (p<0.05) was found after 24h exposures to m/µM concentrations of all tested pollutants. At the same time, significant decreases of lysosomal membrane stability (p<0.05) were observed for Cu (5 µM), Hg (10 nM), and B(a)P (200 nM). Among the entire suite of tests, endocytosis rate test demonstrated the highest sensitivity. Exposures to binary mixtures of all studied pollutants were performed showing both inorganic-organic and inorganic-inorganic additive and/or antagonist effects. Moreover, medium salinity was also varied to mimic estuarine-like environmental conditions linking biological response to ionic strengths. Under these conditions significant increases of both endocytosis rate and lysosomal membrane stability were observed and related to the increment of some Hg- and Cu-related toxic complexes. The studied biomarkers were always able to discriminate between the effects of organic and inorganic pollutants. Together with the short time and simplicity of the test procedures, results obtained in this study indicate that E. crassus is a promising and convenient bioindicator for evaluating the toxicity of different environmental matrixes like pore water, sediments and wastewaters--polluted by metals and organic pollutants.

Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression.

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.

Functional effect of point mutations in the alpha-folate receptor gene of CABA I ovarian carcinoma cells.

The alpha-folate receptor (alpha FR) is overexpressed in 90% of nonmucinous ovarian carcinomas. In addition to the known role of alpha FR binding and mediating the internalization of folates, functional interaction of alpha FR with signaling molecules was recently shown. To identify a model to study the role of alpha FR in ovarian carcinoma, we characterized the alpha FR gene in the ovarian carcinoma cell line CABA I in comparison to a reference line, IGROV1. In CABA I cells, Northern blot analysis revealed an alpha FR transcript of the expected length and FACS analysis indicated receptor expression on the cell membrane; however, RNase protection assay revealed no specific signals. Southern blot and genomic PCR analysis suggested the presence of a rearrangement(s) involving the 5' region of the gene in CABA I cells as compared to IGROV1 cells. Cloning and sequencing of CABA I alpha FR cDNA revealed several point mutations. The partitioning of alpha FR in membrane microdomains from CABA I cells and its association with regulatory molecules was comparable to that of IGROV1 cells. By contrast, the alpha FR expressed on the CABA I cell membrane bound folic acid with lower affinity, and ectopic expression of the corresponding cDNA in CHO cells confirmed impaired folic acid binding. Thus, CABA I cells may provide a tool to delineate functional domains of the alpha FR.

HIV-1 Tat protein mimicry of chemokines.

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24-51) encompassing the "chemokine-like" region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to beta-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of beta-chemokines from the beta-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24-51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic beta-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.

Targeting of saporin to Hodgkin's lymphoma cells by anti-CD30 and anti-CD25 bispecific antibodies.

CD25 and CD30 represent suitable target molecules for bispecific antibody (bimAb)-driven toxin delivery to lymphoid tumour cells. We describe two new anti-CD30/anti-saporin bimAbs (termed CD30 x sap1 and CD30 x sap2), produced by hybrid hybridomas, which react against non-cross-reactive epitopes of the saporin molecule, and compared their effect with a bimAb reacting with saporin and with CD25 (CD25 x sap1). In a protein synthesis inhibition assay these bimAbs were able to enhance saporin toxicity (IC50 = 8.5 x 10(-9) M in the absence of mAbs) with a similar activity: in the presence of 10(-9) M CD30 x sap1 bimAb the IC50 was 2.75 x 10(-11) M, whereas with CD30 x sap2 bimAb the IC50 was 6.5 x 10(-11) M and CD25 x sap1 bimAb displayed an IC50 of 3 x 10(-11) M (as saporin). The combined use of the two anti-CD30 bimAbs further increased cytotoxicity by 100-fold, resulting in an IC50 of 1.9 x 10(-13) M. A slightly less efficient improvement was obtained by combining the CD25 x sap1 bimAb with the CD30 x sap2 bimAb directed against a different toxin epitope (saporin IC50 to 7 x 10(-13) M). In contrast, no synergistic effect was observed using the combination of the anti-CD25 bimAb with the anti-CD30 bimAb reacting with the same epitope of saporin (IC50 = 4.5 x 10(-11) M). Analysis of FITC-saporin binding to L540 cells by flow cytometry demonstrated that the appropriate combinations of the two anti-CD30/anti-saporin bimAbs or of the anti-CD30/anti-saporin and anti-CD25/anti-saporin bimAbs had a cooperative effect on the binding of the ribosome-inactivating protein (RIP) to the cells, when compared with single bimAbs.

Expression of two interleukin-15 mRNA isoforms in human tumors does not correlate with secretion: role of different signal peptides.

Interleukin (IL)-15 is a four-helix bundle cytokine sharing several biological properties with IL-2. By reverse transcriptase-polymerase chain reaction analysis, human cancer cell lines of different histotypes are shown to express two IL-15 amplification products: a 524-bp band corresponding to the IL-15 mRNA found in macrophages, and another of 643 bp corresponding to an alternatively spliced mRNA including a 119-bp alternative exon. IL-15 was undetectable in the supernatant of tumor cell lines expressing either one or both of the mRNA isoforms as evaluated by a bioassay or by ELISA, indicating that IL-15 is not secreted. However, IL-15 could be detected intracellularly in some tumor cells by confocal microscopy analysis. Since the pre-proteins encoded by the two mRNA isoforms differ in the signal peptide sequence, we have analyzed the characteristics of these signal peptides and their possible role in controlling secretion. The two IL-15 cDNA isoforms, expressed in COS-7 cells, induced very low levels of IL-15 secretion. However, substitution of the sequence encoding natural signal peptide(s) with the one from IgV kappa chain in the IL-15 cDNA results in a significantly higher secretion of biologically active IL-15 (15-30-fold) upon cDNA transfection. A poor efficiency of natural signal peptides may represent one of the mechanisms involved in the control of IL-15 secretion.

Analysis of IL-2 receptor expression and of the biological effects of IL-2 gene transfection in small-cell lung cancer.

We have analysed the expression of interleukin-2 receptor (IL-2R) on a panel of small-cell lung cancer (SCLC) cell lines. None of the 11 SCLC cell lines studied expressed detectable surface IL-2R alpha or beta chains by indirect immunofluorescence. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses indicated that only one out of 11 cell lines expressed detectable IL-2R beta mRNA while two expressed a weak positivity for IL-2R gamma. Five SCLC cell lines were transfected with the plasmid vector RSV.5 neo containing IL-2 cDNA coding sequence. Stable transfectants secreted biologically active IL-2 (ranging from 25 to 100 U ml-1 in the culture supernatant). IL-2 transfection did not produce significant modifications in the expression of surface molecules such as IL-2R alpha and beta chains, intercellular adhesion molecule-1 (ICAM-1), CD44, HLA class I and II or in IL-2R beta or gamma mRNA. More importantly, IL-2-transfected N592 and NCI H69 cell lines completely lost their tumorigenic potential in nude mice after subcutaneous injection, whereas experimental controls transfected with RSV.5 neo vector only, displayed an in vivo growth pattern identical to that of untransfected cells. In addition, in the N592 model, IL-2-producing N592 inhibited the growth of wild-type N592 injected at the same site, while injection of parental cells on the opposite side did not significantly affect the growth of wild-type tumour cells. Histopathological analysis of the rejection process of IL-2-transfected cells demonstrated the presence of MAC-1+, MAC-3+ macrophages and of RB68C5+ granulocytes, whereas T cells were undetectable and NK cells were scarcely represented. In addition, a reduction of the tumour blood vessels was observed. The possible relevance of these data for the development of vaccination strategies using cytokine-engineered tumour cells in SCLC is discussed.

Effect of a new model of hemodialysis potassium removal on the control of ventricular arrhythmias.

The primary aim of this multicenter, prospective, randomized cross-over study was to clarify whether a new model of hemodialysis (HD) potassium (K) removal using a decreasing intra-HD dialysate K concentration and a constant plasma-dialysate K gradient (treatment B) is capable of reducing the arrhythmogenic effect of standard HD, which has a constant dialysate K concentration and decreasing plasma-dialysate K gradient (treatment A). The secondary aim was to verify whether this new model is clinically safe. In treatment B, the initial dialysate K concentration had to be 1.5 mEq/liter less than the plasma K concentration, and exponentially decrease to 2.5 mEq/liter at the end of HD. Forty-two chronic HD patients with an increase in premature ventricular complexes (PVC) during dialysis were enrolled from 18 participating centers, and randomly assigned to either sequence 1 (ABA) or sequence 2 (BAB). A pool of 333 of 378 expected ECG Holter recordings were checked for signal quality; 269 (71%) from 36 patients (86%) had a satisfactory signal quality and 108 were selected for analysis (1 per patient per period). There was a difference in the natural logarithm of the increase in PVC/hr and PVC couplets/hr during HD between treatments A and B (1.70 +/- 1.59 vs. 1.09 +/- 1.76 and 0.94 +/- 0.86 vs. 0.64 +/- 1.01, a reduction of 36% and 32%, P = 0.011 and 0.047, respectively) without any carry over effect (P = 0.61 and 0.24, respectively). The fact that this decrease of one third is due to a lower plasma-dialysate K gradient is supported by the observation that it was more evident during the first than the last two hours of HD (a reduction in the natural logarithm of the increase in PVC/hr and PVC couplets/hr of 60% and 60%, P 0.002 and 0.009, vs. 26% and 17%, P = 0.098 and 0.332, respectively): the initial plasma-dialysate K gradient was 2.3 times lower during treatment B than during treatment A, without adversely affecting pre-HD plasma K levels. These results could have a considerably clinical impact not only because of the possibility of physiologically decreasing the arrhythmogenic effect of HD, but also because this effect can be considered a "marker" of the electrophysiological derangement induced by the administration of standard HD three times a week for years ("electric disequilibrium syndrome").

The natural killer-related receptor for HLA-C expressed on T cells from CD3+ lymphoproliferative disease of granular lymphocytes displays either inhibitory or stimulatory function.

Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related "p58" receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T-cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb-triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2-cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.

In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).

Targeting of type 1 ribosome-inactivating proteins to CD30+ or CD25+ hematologic neoplasias by bispecific antibodies.

In this study, we compared the ability of different bispecific monoclonal antibodies (BsmAb) and immunotoxins to deliver the type 1 ribosome-inactivating proteins (RIP) saporin and gelonin through the CD25 or CD30 target molecules to Hodgkin's lymphoma cells. An anti-CD25/antisaporin and an anti-CD30/antisaporin BsmAb enhanced the toxicity of the relevant RIP against the CD25+CD30+ L540 Hodgkin's lymphoma cell line, although targeting by anti-CD30 BsmAb appeared eight times more efficient. Two anti-CD30/antigelonin BsmAb, reacting with different epitopes of the gelonin molecule, were able to enhance gelonin toxicity against L540 cells and had a synergistic effect when used in combination. Among CD25-CD30+ Hodgkin's lymphoma lines, which were resistant to targeting by anti-CD25/saporin BsmAb, one (L428) was sensitive to both gelonin and saporin delivered by anti-CD30 BsmAb. Another CD25-CD30+ cell line (COLE) was completely resistant to the toxic effect of gelonin targeted by the two synergistic BsmAb, as well as to an anti-CD30/gelonin immunotoxin. However, these cells were partially sensitive to saporin delivered by an anti-CD30/anti-saporin BsmAb, and they were efficiently killed by an anti-CD30/saporin immunotoxin. These results indicate that heterogeneity in the sensitivity to certain RIP, such as gelonin, exists among tumor cells of the same histotype.

Differential sensitivity of CD30+ neoplastic cells to gelonin delivered by anti-CD30/anti-gelonin bispecific antibodies.

Lymphocyte activation antigens, such as CD30, represent suitable target molecules for antibody-driven drug delivery in haemopoietic malignancies. A ribosome-inactivating protein (RIP) type 1 of potential interest for mAb targeting is gelonin, which displays a lower toxicity, as compared to other RIPs. In this study, two anti-CD30/antigelonin bispecific monoclonal antibodies (bimAbs), secreted by hybrid hybridomas, were used to deliver this RIP to CD30+ tumour cells. The two bimAbs, termed D4 and A18, were produced using the same anti-CD30 mAb and two anti-gelonin mAbs, directed to unrelated epitopes of the gelonin molecule. These bimAbs enhanced gelonin toxicity (IC50 5 x 10(-8) M, in the absence of mAbs) against the CD30+ L540 Hodgkin's lymphoma cell line in a protein synthesis inhibition assay. Thus, in the presence of 10(-9) M D4 bimAb, protein synthesis was inhibited with an IC50 of 5 x 10(-10) M as gelonin, whereas with A18 bimAb the IC50 was 8 x 10(-11) M. More interestingly, the combined use of the two bimAbs had a synergistic effect, since the IC50 of gelonin reached 6 x 10(-12) M. Among CD30 tumour cell lines, the Hodgkin's lymphoma L428 was also sensitive to gelonin delivered by bimAbs (IC50 6 x 10(-11) M), whereas the COLE Hodgkin's cell line and the T-ALL Jurkat were completely resistant to the toxic effect of gelonin and bimAbs. COLE and Jurkat cells were also resistant to a gelonin/anti-CD30 conventional immunotoxin, whereas they were sensitive to a saporin/anti-CD30 immunotoxin. This suggests that the resistance to gelonin is not related to a lack of internalization through the CD30 molecule but is associated with some property of the RIP.

Insulin-like growth factor-I (IGF-I) and IGF-binding proteins blood serum levels in women with early- and late-stage breast cancer: mutual relationship and possible correlations with patients' hormonal status.

The pathogenesis and progression of breast cancer involve complex interactions between hormones and polypeptide growth factors such as insulin-like growth factor-I (IGF-I). IGF-I has been found in stromal fibroblasts derived from malignant and benign breast tissue and it is a mitogen for several breast cancer cell lines. It circulates bound to specific high-affinity binding proteins, which could act as either positive or negative modulators of tumorigenesis. This study has been addressed to characterize IGF-I and its binding proteins in the serum of 85 unselected patients with early breast cancer. The IGF-I concentration was assessed by radioimmunoassay of 69 out of 85 samples before and after dissociation of the IGF-I and IGF-binding protein (IGF-BP) complex whereas IGF-BP of all 85 sera were analyzed by Western ligand blotting; estradiol and progesterone were measured by radioimmunoassay in native serum samples. In our study no differences in IGF-I serum levels between pre- and post-menopausal patients were observed. Patients with higher estradiol and progesterone serum levels did not present different IGF-I concentrations compared to patients with lower serum levels. Furthermore, IGF-I median values were not found to depend on estrogen receptor (ER) status. A heterogeneous quali-quantitative molecular pattern of binding proteins was detected: IGF-BP3 and IGF-BP1 were the most and the least expressed respectively. No correlations between ER status, or parameters related to the hormonal status, and IGF-I or binding proteins expression were observed. No significant differences in IGF-I concentration and IGF-BP expression were observed between cancer patients and a control group matched for age and menopausal status. Finally, preliminary collection of 20 sera derived from patients with late breast cancer was analyzed for IGF-I and its binding proteins content.

T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function.

A new anti-p58 monoclonal antibody (mAb), termed CH-L, has been used to characterize a minor subset of T lymphocytes co-expressing p58 and CD3 molecules. In two-color immunofluorescence analysis, CH-L+CD3+ cells represented 0.5 to 6% of the peripheral blood lymphocytes (in 20 healthy donors). Clonal analysis showed that most CD3+CH-L+ T cell clones expressed the CD8+4- T cell receptor (TcR) alpha/beta+ phenotype, while only a few were CD8-4+ TcR alpha/beta, CD8-4- TcR alpha/beta+ or CD8-4- TcR gamma/delta+. Western blot analysis indicated that the CH-L mAb identifies the same 56-58-kDa diffuse band in both T and natural killer cell (NK) clones. A minority of T cell clones also expressed other NK-related markers such as CD16, CD56 and CD94 and two clones also reacted with the anti-p58 mAb EB6. Interestingly, most clones displayed cytolytic activity in an anti-CD3 mAb-triggered redirected killing assay against the Fc gamma receptor+ P815 target cells and NK-like activity against K562 and Raji cells. In contrast, the IGROV-1 ovarian carcinoma cell line was resistant to cytolysis by all of these clones. Since p58 molecules have previously been shown to exert regulatory functions on NK-mediated lysis, we investigated whether anti-p58 mAb could also influence cytotoxicity mediated by CD3+p58+ T lymphocytes. Lysis of P815 target cells, triggered by anti-CD3 mAb, could be inhibited by anti-p58 mAb in 8 out of 12 cytolytic clones tested, while 4 clones were not inhibited. In addition, anti-p58 mAb enhanced the cytolytic activity of 3 clones against IGROV-1 and of 4 other clones against Raji target cells. Taken together, these data indicate that p58+ T cells express heterogeneous phenotypes and different forms of TcR and, in most instances, display cytolytic functions. Perhaps more importantly, the p58 molecule appears to modulate the cytolytic activity triggered via the CD3/TcR complex.

Characterization of a cyclosporin A-sensitive activation pathway in cultured T and natural killer cells.

Previously, the authors have described a molecule, identified by the LD6 monoclonal antibody (MoAb), present at the cell surface of long-term cultured T and Natural Killer (NK) cells which is involved in cell triggering. In the study described here the authors used biotin surface labelling and immunoprecipitation to show that LD6 MoAb recognizes a surface protein of approximately 65 kDa. In combination with submitogenic concentrations of phorbol esters (PMA); LD6 MoAb was able to induce accumulation of mRNA specific for GM-CSF, gamma-IFN and TNF-alpha and release of these cytokines by LD6+ T-cell lines. Both lymphokine production and lymphokine-specific mRNA accumulation induced by the LD6 MoAb were blocked totally by Cyclosporin A (CsA). To investigate the mechanism(s) of signal transduction through this activatory pathway, the authors performed Ca++ mobilization experiments. The results of these experiments suggested a role for Ca++ in signal transduction. The Ca++ mobilization induced by LD6 MoAb cross-linking could be inhibited totally by the use of pertussis toxin, indicating a possible role for G proteins in signalling through the LD6 MoAb-reactive molecule. Western blot analysis performed with an anti-phosphotyrosine antibody did not suggest that tyrosine kinase activation has a role.

The LFA-1/ICAM cell adhesion pathway is involved in tumor-cell lysis mediated by bispecific monoclonal-antibody-targeted T lymphocytes.

We investigated the role of the LFA-1/ICAM, VLA-4/VCAM-1 and CD2/LFA-3 adhesion pathways in the cytolysis of tumor cells mediated by an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb). The biMAb induced efficient lysis of EGF-R+ tumor cells (A431, HT-29, IGROV-1 and MDA-MB468) by cytotoxic T lymphocytes (CTL) cultured in IL-2. Pretreatment of effector cells by anti-LFA-1 alpha (CD11a) and anti-LFA-1 beta (CD18) MAbs significantly inhibited cytolysis of all types of EGF-R+ tumor cells, while anti-CD2 and anti-VLA-4 MAbs were virtually ineffective. We investigated the expression of adhesion-molecule counter-receptors on tumor target cells by indirect immunofluorescence. HT-29, A431 and MDA-MB 468 tumor cells expressed an ICAM-1+2-3- VCAM-1- LFA-3+ phenotype, while IGROV-1 was ICAM-1-2+3- VCAM-1- LFA-3+. Pre-treatment of A431, HT-29 and MDA-MB468 with anti-ICAM-1 MAb inhibited cytolysis, further supporting the functional involvement of the LFA-1/ICAM adhesion pathway in biMAb-targeted tumor-cell lysis. In addition, treatment of target cells with TNF alpha or IFN gamma for 24 hr increased the expression of ICAM-1 in HT-29, A431 and MDA-MB468 (ICAM-2 was induced on IGROV-1) and also enhanced the sensitivity of these target cells to biMAb-targeted cytotoxicity. These data suggest that up-regulation of ICAM-molecule expression by inflammatory cytokines may increase susceptibility of tumor cells to biMAb-targeted lysis. Anti-LFA-1 MAbs did not significantly inhibit the formation of conjugates between biMAb-coated T lymphocytes and tumor cells. Co-aggregation of LFA-1 molecules with biMAb-bound CD3 molecules resulted in a more sustained and prolonged increase in the intracellular concentration of free Ca++ in CD8+ cultured CTL lines. These findings indicate that in T cells targeted by anti-CD3/anti-TAA biMAb LFA-1 may act as a co-receptor molecule which enhances signal transduction through the CD3/TCR complex.

Targeting of T lymphocytes against EGF-receptor+ tumor cells by bispecific monoclonal antibodies: requirement of CD3 molecule cross-linking for T-cell activation.

Targeting of T lymphocytes against epidermal growth-factor-receptor (EGF-R)+ tumor cells was achieved by constructing a hybrid hybridoma which secretes an anti-EGF-R/anti-CD3 bispecific monoclonal antibody (biMAb) of hybrid isotype (IgG1/IgG2a). Purification of biMAb molecules from parental anti-EGF-R and anti-CD3 MAbs was performed by protein-A chromatography. The purified biMAb was able to trigger the lysis of EGF-R+ tumor cell lines (A431, IGROV-1, MDA-468 and U-87) and of NIH-3T3 transfectants expressing human EGF-R by cytolytic T lymphocytes, but it was ineffective in the case of EGF-R-negative tumor targets. Normal EGF-R+ cells (keratinocytes and endometrial cells) were also susceptible to biMAb-targeted cytolysis. However, the amount of biMAb required to induce half-maximal cytolysis of tumor cells over-expressing the EGF-R molecule (A431) was considerably lower than that required to induce lysis of EGF-R+ tumor or normal cells which express EGF-R at considerably lower density. The ability of such biMAbs to deliver activation signals to T cells was evaluated by Ca++ mobilization and lymphokine production experiments. The soluble anti-EGF-R/anti-CD3 biMAb failed to induce intracellular Ca++ increases, which occurred only after cross-linking induced by an anti-mouse IgG antibody. Secretion of lymphokines (IFN-gamma, TNF-alpha and GM-CSF) was induced by contact of the biMAb-coated effector cells with the relevant tumor target, whereas the soluble biMAb was virtually ineffective. In addition, biMAb-coated effector cells retained the ability to recognize and to lyse EGF-R+ tumor cells for a prolonged period of time. Our data indicate that activation of effector cells targeted by biMAbs can only occur at the tumor site, where cross-linking of surface CD3 molecules is induced by contact with the tumor cells.

Targeting of saporin to CD25-positive normal and neoplastic lymphocytes by an anti-saporin/anti-CD25 bispecific monoclonal antibody: in vitro evaluation.

This study has been designed to verify the specific toxicity of saporin, a type 1 ribosome-inactivating protein (RIP), with the same activity as ricin A chain, targeted by a bispecific monoclonal antibody (bimAb) recognising both the CD25 antigen and the RIP. The CD25 antigen is expressed by lymphoid populations upon activation and by leukaemias and lymphomas with an activated membrane phenotype (Hodgkin's lymphoma, anaplastic large cell lymphoma, adult T cell leukaemia). The bimAb-saporin mixture was tested on CD25+ targets at different bimAb and saporin concentrations. Saporin, in the presence of a bimAb concentration of 10(-9) M, inhibited protein synthesis by CD25+ neoplastic lymphocytes (L540 and MT2 cell lines) with IC50S (concentrations giving 50% of inhibition) ranging from 8 x 10(-12) M to 3 x 10(-11) M. The saporin-bimAb mixture was also effective in blocking the phytohaemagglutinin-driven proliferation of normal lymphocytes, whereas it displayed the same level of toxicity exerted by saporin alone on an irrelevant CD25-negative cell line (EBV-infected B lymphoblastoid cell line). From these results it is possible to envisage a clinical use of this bimAb as a cytotoxic agent for CD25+ leukaemias and lymphomas, as well as an immunosuppressive agent for severe immune disorders such as graft-vs-host disease (GVHD) and transplanted organ rejection.

Use of anti-CD3 and anti-CD16 bispecific monoclonal antibodies for the targeting of T and NK cells against tumor cells.

To target T lymphocytes against EGF-R+ tumors, we constructed anti-CD3/anti-EGF-R bimAbs either by the generation of a hybrid hybridoma (quadroma) or by a chemical cross-linking method. Analysis of the in vitro functional activity of these two different constructs indicated that the quadroma-secreted bimAb was more efficient in targeting the CD3+8+ clones against EGF-R+ target cells with respect to the bimAb produced by chemical method. In addition, the quadroma-produced bimAb is able to induce cytolysis of EGF-R+ tumor cell lines of PHA-induced lymphoblasts that had been expanded in IL-2-containing medium, whereas tumor cells lacking expression of EGF-R were not lysed. Resting PBL targeted by the bimAb did not display significant cytotoxicity against the relevant tumor. An anti-CD16 hybridoma (IgG1) was fused with an anti-folate-binding protein hybrid (IgG2a) to construct bimAbs to target NK cells against NK-resistant ovarian carcinomas. The hybrid IgG1/IgG2a bimAb triggered the specific lysis of relevant target cells by resting NK cells, but it was ineffective when CD8+TCR alpha/beta+ cultured cell populations were used as effectors. Only marginal increases of cytolytic activity could be induced by the bimAb when IL-2-activated PBL (i.e., LAK cells) were used as effectors due to the high cytolytic activity of these cells against the relevant tumors in the absence of bimAb. The possible use of anti-CD16 or anti-CD3 bimAbs for the development of different cellular immunotherapy strategies against cancer is discussed.