Development, characterization, and use of monoclonal antibodies made to antigens expressed on the surface of fetal nucleated red blood cells.

BACKGROUND: Current methods for obtaining fetal cells for prenatal diagnosis are invasive and carry a small (0.5-1.0%) but definite risk of miscarriage. An attractive alternative would be isolation of fetal cells from peripheral maternal blood using antibodies with high specificity and avidity. METHODS: To generate antibodies, we purified nucleated red blood cells (NRBCs) from fetal livers and used them as the immunogen to generate monoclonal antibodies (mAbs) directed against surface antigens. RESULTS: The four antibodies recognized at least two conformationally sensitive epitopes of the transferrin receptor. Isolation of NRBCs from 252 maternal blood samples using these antibodies in magnetic activated cell sorting after an initial density gradient centrifugation yielded 0-419 NRBCs per 25 mL of maternal blood. One antibody, 2B7.4, not only isolated the highest number of NRBCs (>10 in 90% of the samples) but also isolated these NRBCs in 78 consecutive maternal samples. CONCLUSION: Antibody 2B7.4 shows promise for the isolation of NRBCs from maternal blood and should allow studies concerning the source of these cells, fetal vs maternal, and the factors controlling their prevalence.

Identification of cross-reactive antibodies with low opsonophagocytic activity for Streptococcus pneumoniae.

Opsonophagocytic capacity and concentrations of antibodies to the capsular polysaccharide (PS) of Streptococcus pneumoniae serotypes 6A, 6B, 19F, and 19A were determined for sera from adults immunized with 14- and 23-valent S. pneumoniae capsular PS vaccines. The concentration of anticapsular PS antibodies strongly correlated (r = .72 to r = .91) with the opsonophagocytic activities. However, 10%-20% of serum samples display strikingly less opsonophagocytic activity for the cross-reactive serotypes than expected on the basis of their antibody concentrations to the cross-reactive serotypes. From 1 poorly opsonic serum sample, kappa and lambda fractions of anti-6B antibodies were purified by affinity chromatography. Kappa (but not lambda) type antibody was found to have less opsonophagocytic capacity than expected. Thus, ELISA measurements of antibody responses to pneumococcal capsular PSs may not be reflective of opsonophagocytic function of the antibody.

The repertoire of human antibodies to the carbohydrate capsule of Streptococcus pneumoniae 6B.

Antibodies to Streptococcus pneumoniae 6B capsular polysaccharide (PS) induced with a 23-valent PS vaccine among 25 adults were examined. The magnitude of antibody responses among different subjects was highly correlated with the amount of anti-6B antibodies expressing IgG (r = 0.98) and lambda (r = 0.93) isotypes. Most individuals produced one or two dominant IgG antibody clones as identified by their isoelectric points. Two antibody clones with unique amino acid sequences could be readily purified, and the sequences of their light chains match those of A1/A17 V kappa and hslv2046 V lambda genes. Anti-6B antibodies isolated from different subjects used various VL genes and differed in their cross-reactivity with 6A PS. An isoelectric focusing study suggests that some IgG antibodies induced with 6B PS bind 6A PS with lower avidity.

A modified Farr assay is more specific than ELISA for measuring antibodies to Streptococcus pneumoniae capsular polysaccharides.

In an attempt to develop an assay specific for antibody to capsular polysaccharide (PS) of Streptococcus pneumoniae, the ability of ELISA and Farr (radioactive antigen-binding) assay for antibodies to 6B and 19A PS to be affected by antibodies to C-polysaccharide (C-PS) was compared. Preabsorption with C-PS reduced values obtained by ELISA for anti-6B antibody by > 3-fold in 5 of 10 preimmune and 7 of 26 postimmune sera. In contrast, absorption reduced values by > 3-fold in 0 of 36 samples studied with the Farr assay. Similar results were observed when the absorption was done with CSR-SCS2 S. pneumoniae. Furthermore, when anti-19A antibody levels were examined, preabsorption with R36a S. pneumoniae reduced ELISA values by 3-fold in 7 of 22 samples, whereas no samples had 3-fold reduction by Farr assay. Thus, the Farr assay for capsular PS is less affected than the ELISA by anti-C-PS antibody.

Specific binding of vascular permeability factor to endothelial cells.

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor, has recently been purified from guinea pig, human, and bovine sources. We show that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF. Two classes of sites with KDs of approximately 10 pM and 1 nM were detected for all endothelial cell types examined. Guinea pig [125I]VPF binding to endothelial cells was inhibited by human VPF (ID50 = 0.8 ng/ml) and by suramin (ID50 = 75 micrograms/ml) but not by heparin. Cross-linking experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes migrated very slowing in SDS-PAGE, indicating that they were of very high molecular weight and probably highly cross-linked. A portion of the molecules migrated as 270 kDa complexes, indicating that the molecular weight of the endothelial cell VPF receptor is about 230 kDa.

Vascular permeability factor: a tumor-derived polypeptide that induces endothelial cell and monocyte procoagulant activity, and promotes monocyte migration.

Systemic infusion of low concentrations of tumor necrosis factor/cachectin (TNF) into mice that bear TNF-sensitive tumors leads to activation of coagulation, fibrin formation, and occlusive thrombosis exclusively within the tumor vascular bed. To identify mechanisms underlying the localization of this vascular procoagulant response, a tumor-derived polypeptide has been purified to homogeneity from supernatants of murine methylcholanthrene A-induced fibrosarcomas that induces endothelial tissue factor synthesis and expression (half-maximal response at approximately 300 pM), and augments the procoagulant response to TNF in a synergistic fashion. This tumor-derived polypeptide was identified as the murine homologue of vascular permeability factor (VPF) based on similar mobility on SDS-PAGE, an homologous NH2-terminal amino acid sequence, and recognition by a monospecific antibody to guinea pig VPF. In addition, VPF was shown to induce monocyte activation, as evidenced by expression of tissue factor. Finally, VPF was shown to induce monocyte chemotaxis across collagen membranes and endothelial cell monolayers. Taken together, these results indicate that VPF can modulate the coagulant properties of endothelium and monocytes, and can promote monocyte migration into the tumor bed. This suggests one mechanism through which tumor-derived mediators can alter properties of the vessel wall.

Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis.

Human vascular permeability factor. Isolation from U937 cells.

Human vascular permeability factor (hVPF) is a glycoprotein that promotes fluid and protein leakage from blood vessels. The function of hVPF is at present unknown, but the potent bioactivities of this protein suggest that it could act during inflammation, wound healing, and tumor angiogenesis. hVPF was purified from serum-free conditioned medium of the human histiocytic lymphoma cell line U937 as a disulfide-linked dimeric 40-kDa protein that promoted dermal blood vessel leakage in guinea pigs at a dose of 20 ng (3 x 10(-9) M) and promoted in vitro endothelial cell growth at concentrations as low as 50 PM. Multiple forms of hVPF with apparent pI values greater than 7.5 were resolved using pH gradient electrophoresis. Antibodies against guinea pig vascular permeability factor were found to cross-react with hVPF. The N-terminal amino acid sequence of hVPF was similar to, but not identical with, the N-terminal sequence of guinea pig vascular permeability factor.

Fibrin-enhanced endothelial cell organization.

The formation of cloned bovine endothelial cells into capillary-like tubes is accelerated from 3-7 days to 2-18 h in the presence of fibrin. Indirect immunofluorescence showed the presence of both fibrin and fibronectin in the strands along which the cells organized. Electronmicroscopy revealed the same type of cell structures as form in the absence of fibrin; it also revealed a gradual decrease with time of the fibrin within the putative lumen. Fibrin and fibronectin are commonly present during angiogenesis in vivo, thus these in vitro observations may well have relevance to the in vivo process.

The formation of capillary-like tubes by calf aortic endothelial cells grown in vitro.

Cloned, large vessel endothelial cells derived from fetal bovine and bovine calf aortas formed three-dimensional structures in vitro without tumor-conditioned medium or special substrata. Transmission electron microscopy showed the structures to be hollow tubes composed of typical endothelial cells with overlapping and interdigitating cytoplasmic processes typical of those seen in in vivo capillaries. The putative lumen of these tubes generally contained abundant electron-dense fibrous material, which by ruthenium red and indirect immunofluorescent staining appeared to be extracellular matrix. This suggests that the endothelial cell orientation in the tubes is the reverse of that normally found in in vivo vessels.

An assay measuring the stimulation of several types of bovine endothelial cells by growth factor(s) derived from cultured human tumor cells.

Endothelial cell growth factor(s) from several previously untested human tumor cell lines (i.e., SK-HEP-1, MG63, A375, TE671-C1, RD) were detected using a low cell inoculum growth assay. The final cell density in the 2-cm2 wells was determined by a highly sensitive DNA content measurement performed directly in the tissue culture plates. The sensitivity of the assay to human tumor cell growth factors depended critically on the low cell inocula, 2,000 to 5,000 cells/well. Most of the bovine endothelial cells used were cloned from primary cultures; all the cell lines obtained from various fetal and nonfetal sources responded to the growth factor(s) (up to a 16x stimulation) as well as to endothelial cell growth supplement. Dose response curves showing the cell specific response of bovine endothelial cells were obtained. The growth stimulatory activity and the in vivo chick embryo chorioallantoic membrane assay responses correlated sufficiently to imply that the assay is detecting tumor angiogenesis factor or some closely related activity. This in vitro assay should prove useful in the identification and purification of tumor-derived factors and in the elucidation of the role of these factors in the events comprising angiogenesis.

Amphotericin B plus 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU-NSC no. 409962) in advanced cancer. Phase I and preliminary phase II results.

Twenty-one patients with advanced metastatic cancer received amphotericin B (AmB) plus BNUC in a Phase I chemotherapy trial. Of 11 patients with measurable metastases from bronchogenic carcinoma, five had partial antitumor responses lasting 1.5 to 12+ months, and one had objective improvement. Only two of six patients with other types of tumors had objective improvement of short duration. No consistent evidence of immunologic stimulation was observed in eight patients studied. These results suggest that amphotericin B may increase the therapeutic ratio of BCNU, and further trials of this new concept in chemotherapy of advanced tumors are in progress. The dose-limiting toxicity was myelosuppression, usually thrombocytopenia. No enhancement of BCNU toxicity by the addition of AmB was observed. The recommended dose for future studies is: AmB, 7.5 mg/m2 on day 1, 15 mg/m2 on day 1, 30 mg/m2 on days 3 and 4; plus BCNU, 250 mg/m3 on day 4. The regimen is repeated every 6 to 8 weeks.