Generation of monoclonal antibodies directed against organ-specific endothelial cell surface determinants.

Organ-specific determinants expressed on the luminal surface of vascular endothelia are often unstable when cells are removed from their normal tissue environment and grown in culture. Unspecific endothelial cells of large vessel origin [e.g., bovine aorta (BAEC)] can be modulated to express and preserve such determinants when they are grown on the extracellular matrix of the desired organ. Lung matrix-modulated BAEC were used here to generate MAb against lung-specific vascular endothelia. Immunization was accomplished with outside-out membrane vesicles obtained by incubating BAEC monolayers grown on lung matrix with a low-strength paraformaldehyde solution. In four of the six fusions performed, this active immunization was preceded by passive immunization with mouse antiserum directed against membrane vesicles from BAEC grown on plastic. Among the growing hybrids, 7.6% secreted MAb that bound efficiently to both BAEC grown on lung-derived matrix and BAEC grown on plastic, while 3.5% (50) secreted MAb that bound primarily to BAEC grown on lung matrix. The fusion data show that only a passive/active immunization protocol yielded MAb directed against lung-specific endothelia. For example, MAb 6D3 and 5F5 selectively recognized endothelia from small- and medium-sized venules of bovine lungs, but failed to react with endothelial cells in other organs and tissues.     

Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.     

A monoclonal antibody detecting unusual Thy-1 determinants

20-10-5S is a monoclonal antibody produced by the fusion of C3H anti-C3H.SW splenocytes with the SP2/0 cell line. The antibody appears to react with Thy-1 determinants by several criteria including cytotoxicity patterns, functional assays, genetic analyses, and competitive binding experiments. However, the antibody and the determinants it detects are unusual in that: 1) 20-10-5S is autoreactive; 2) the antibody shows allospecificity for Thy-1.2 vs Thy-1.1 antigens only on peripheral lymphocytes and not on thymocytes; and 3) the antibody reacts only with determinants on murine T cells and not with antigens on brain tissue or on rat thymocytes. It therefore seems that 20-10-5S reacts with murine T cell-specific Thy-1 determinants that are lost or modified during maturation of the cells on which they are expressed.     

Activation of C1 by monoclonal antibodies directed against C1q

Eleven monoclonal antibodies directed against the subcomponent C1q of the first component of human complement, C1, were prepared and tested for binding to intact C1q and to the collagenous portion, the C1q stalks. All of the monoclonals bound well to the intact C1q. Eight out of the eleven exhibited strong binding to the collagenous stalks, while three bound very weakly, if at all, to the stalks and, thus, were presumed to bind to the pepsin-sensitive region which includes the C1q heads. For one of the latter monoclonals, this was confirmed by electron microscopy. Five of the monoclonals were purified by C1q affinity chromatography. When tested with C1 reassembled from its subunits, two of these purified monoclonal antibodies markedly enhanced the rate of spontaneous activation.     

Species-specific recognition patterns of monoclonal antibodies directed against vimentin.

Two commercially available monoclonal antibodies raised against the intermediate filament protein vimentin were characterized concerning their species-specific reaction pattern on vertebrate cells. The antibody V9 exhibited extensive reactivity with vimentin of all mammalian species tested, but specifically did not detect vimentin in mouse cells and chicken fibroblasts. The antibody VIM 3B4 recognized vimentin in cells of chicken and most mammalian species, except for rodent species. Characterization of the binding site of VIM 3B4 on human vimentin by limited proteolysis and immunoblotting as well as by sequence comparison strongly suggested that the epitope is located in the coil 2 part of the vimentin rod domain. Site-directed mutagenesis of a mouse vimentin cDNA clone followed by in vivo expression showed that VIM 3B4 could detect rodent vimentin containing a single amino acid substitution (valine for leucine) at position 353 of the mouse vimentin sequence. Practical application for this finding was demonstrated by the unequivocal identification of a modified murine vimentin protein, distinct from the endogenous vimentin, in a cytoplasmic intermediate filament network in mouse skin fibroblasts transfected with a recombinant plasmid expression vector.     

Monoclonal antibodies directed against different antigenic determinants of rotavirus

We have developed a series of monoclonal antibodies against the calf strain RIT 4237 (subgroup 1) and the human strain 82-561 (subgroup 3) of rotavirus, both grown in tissue culture, and also against the human rotavirus 81-2162 (subgroup 2), extracted from a fecal specimen. A variety of different specificities was observed among these antibodies, namely, antibodies against group and subgroup determinants, as well as neutralizing antibodies. By using monoclonal antibodies against the subgroup antigen in an enzyme-linked immunoassay system, the constant predominance of subgroup 2 viruses in humans was confirmed in 74 stools collected from children in Brussels who suffered a diarrheal illness between July 1981 and June 1983. The availability of these antibodies also made it possible to improve the sensitivity and the specificity of the test system.     

Characterization of monoclonal antibodies directed against the rat neurotensin receptor NTS1

G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to a variety of ligands and represent major drug targets. Despite their medical importance, detailed structural information is limited because only one GPCR has been crystallized and its structure determined. To develop tools to aid in the formation of well-ordered crystals, we generated monoclonal antibodies with high affinity to the rat neurotensin receptor. All antibodies bound to the C-terminus of the receptor, which may reflect the selection strategy used to identify high-affinity binders. Further characterization revealed that some antibodies bound to the receptor in a sodium chloride sensitive manner, but others did not. Epitope mapping revealed distinct antigenic regions within the receptor C-terminus. Tight binding of Fab fragments to the receptor was verified by size exclusion chromatography.     

Sharing of Ia antigens between species. IV. Interspecies cross-reactivity of monoclonal antibodies directed against polymorphic mouse Ia determinants

The specificity of interspecies Ia cross-reactions has been analyzed by testing a panel of monoclonal antibodies (mAb) to mouse I-E and I-A antigens for reactivity with pig Ia antigens. Our earlier studies showed that mouse anti-I-E alloantisera recognized common determinants on Ia antigens of other species, whereas anti-I-A alloantisera showed much more limited cross-reactivity. These results were confirmed using a panel of 17 anti-I-E mAb, 10 of which were cytotoxic to pig cells. 2D gel electrophoretic analyses of precipitates with these mAb of 35S-labeled, NP40 solubilized pig cells revealed a limited set of protein spots that appeared to be identical to the subset of pig Ia antigens precipitated by A.TH anti-A.TL alloantiserum. Because the cross-reactive mouse sera were produced in mouse strains that do not express an I-E molecule (H-2b and H-2s), it was anticipated that the cross-reacting antibodies would be reactive with the monomorphic determinant of the I-E molecule, Ia.7. However, comparison of the reactivity of these mAb with pig cells and mouse cells revealed that the cross-reactivity on pig cells correlated not with Ia.7 but rather with detection of epitope(s) of the I-E molecule associated with inter-strain polymorphism. Anti-I-A cross-reactions were also detected, but were weaker and more limited. These findings may have implications for the evolution of Ia antigens in mammalian species.     

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.     

Monoclonal antibodies directed against human FcRn and their applications

The MHC class I-like Fc receptor (FcRn) is an intracellular trafficking Fc receptor that is uniquely responsible for the extended serum half-life of antibodies of the IgG subclass and their ability to transport across cellular barriers. By performing these functions, FcRn affects numerous facets of antibody biology and pathobiology. Its critical role in controlling IgG pharmacokinetics has been leveraged for the design of therapeutic antibodies and related biologics. FcRn also traffics serum albumin and is responsible for the enhanced pharmacokinetic properties of albumin-conjugated therapeutics. The understanding of FcRn and its therapeutic applications has been limited by a paucity of reliable serological reagents against human FcRn. Here, we describe the properties of a new panel of highly specific monoclonal antibodies (mAbs) directed against human FcRn with diverse epitope specificities. We show that this antibody panel can be used to study the tissue expression pattern of human FcRn, to selectively block IgG and serum albumin binding to human FcRn in vitro and to inhibit FcRn function in vivo. This mAb panel provides a powerful resource for probing the biology of human FcRn and for the evaluation of therapeutic FcRn blockade strategies.Key words: FcRn, IgG, monoclonal antibody, albumin, therapy     

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

Specificity and function of monoclonal antibodies directed against Ewing sarcoma cells

A selection of 16 monoclonal antibodies has been produced against a fresh Ewing's sarcoma (ES) tumor mixed with a permanent ES cell line. The majority of antibodies identify an 80-kDa molecule, which is not detected on healthy tissues except on certain cultured monocytes. One antibody recognize the CD2 ligand MIC2 and 2 antibodies (numbers 13 and 16) define a higher-molecular-mass antigen. Antibody 16 is also expressed on mesenchymal fibroblasts of bone marrow or fetal origin. Tumorspecific antigen expression is potentially linked to the chromosome 22 abnormality decribed in Ewing's sarcoma.     

Characterization of monoclonal antibodies directed against trail or trail receptors

A subset of tumour necrosis factor receptor family members is involved in death transducing signals and is, therefore, referred as the "death receptors." Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumour cells but only rarely in normal cells. Five distinct receptors have been described for TRAIL: TRAIL R1 (DR4), TRAIL R2 (DR5, TRICK), TRAIL R3 (TRID, DcR1), TRAIL R4 (TRUNDD, DcR2), and osteoprotegerin. In the Eighth International Workshop on Human Leukocyte Differentiation Antigens, 10 monoclonal antibodies (mAbs) reported to be specific for TRAIL or for TRAIL receptors were submitted. In the present study, the mAb specificity was determined by ELISA. Using these mAbs, investigation on the expression of TRAIL and TRAIL receptors was performed. Some of them were able to modulate TRAIL induced programmed cell death.     

Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.     

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen.

Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.     

Characterization of murine monoclonal antibodies directed against basic fibroblast growth factor

Monoclonal antibodies (McAbs) were developed that identify the complete (1-146 aa) and the NH2-terminal truncated (des 1-15) form of bovine basic fibroblast growth factor (bFGF). Four McAbs, designated McAbs 6, 8, 38, and 42, bind the complete form of bFGF found in bovine pituitary, brain, and adrenal gland. One of these McAbs, McAbs 42, also binds to the des 1-15 aa form of bFGF found in bovine adrenal gland, kidney, and corpus luteum. None of the McAbs binds bovine-brain-derived acidic FGF (aFGF). McAbs 6, 8, and 38 recognized the same epitope located within the first ten residues of the NH2-terminal of complete bFGF. McAb 42 recognizes a "core" epitope found on both the complete and des 1-15 aa bFGFs. The McAbs are murine IgGs with affinity constants of 10(7)-10(8) liter/M for bovine-pituitary-derived bFGF. McAbs 8 and 42 have been used in a two-site ELISA to detect the complete form of bFGF. The ELISA is sensitive to 38.5 fmole/well of bFGF and is not affected by the presence of calf serum or bovine-brain-derived aFGF. These McAbs should be useful in distinguishing the native and des 1-15 aa forms of bFGF from each other, and from aFGF and other growth factors.     

Bone marrow transplantation across major histocompatability barriers in mice. III. Treatment of donor grafts with monoclonal antibodies directed against Lyt determinants

We studied the effect of eliminating T cells from donor grafts of mice in a system in which bone marrow was transplanted across major histocompatibility barriers. BALB/c bone marrow (added as a source of hematopoietic stem cells) combined with equal volumes of spleen cells (added as a source of GVHD-promoting cells) was pretreated in vitro with monoclonal anti-Lyt-1.2 or Lyt-2.2 plus absorbed rabbit complement before injection into C57BL/6 total-body-irradiated recipients. Functional activity of anti-Lyt monoclonal antibodies was determined in CML assay. Treatment with anti Lyt-1.2 plus C did not have any anti-stem cell activity, as measured by CFU-S assay, and protected recipients from the onset of lethal GVHD. Treatment with Lyt-2.2 plus C also did not reduce CFU-S; however, mice receiving treated marrow did develop GVHD and were all dead by 2 mo, as were untreated control mice. Surviving "anti-Lyt-1.2 + C chimeras" demonstrated a high percentage of donor mononuclear cells in their peripheral blood. Similar results were obtained when C3H/HeN donor BMS was treated with monoclonal anti-Lyt-1.1 plus C and injected into C57BL/6 recipients. These findings show that monoclonal antibodies directed against determinants unrelated to Thy-1 can eliminate T cells in the presence of C and successfully protect transplanted mice from lethal GVHD. They also suggest that these anti-Lyt antibodies may be useful tools in determining subpopulations of T cells that contribute to the development of GVHD.     

Characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2.

Monoclonal antibodies (MAbs) raised against the core proteins of human immunodeficiency virus type 1 (HIV-1; laboratory strain HTLV-IIIB) and HIV-2 (strain ROD) were investigated in a variety of tests, e.g., enzyme-linked immunosorbent assay (ELISA), immunostaining of Western immunoblots, immunofluorescence, immunoprecipitation, and alkaline phosphatase anti-alkaline phosphatase assay. The MAbs were grouped according to their cross-reactions. Seven HIV-1-specific MAbs reacted exclusively with HIV-1, and five showed cross-reactivity with HIV-2 and simian immunodeficiency virus of macaques in ELISA. Four of the 15 MAbs against HIV-2 reacted only with the HIV-2 protein p26. Six showed cross-reactivity with HIV-1, and five showed a broad reaction with all three viruses. Overlapping 30-amino-acid-long peptides derived from the p24 protein sequence of HIV-1 were used in an epitope-mapping system. Three different immunogenic regions (A, B, and C) could be defined. Specific regions where anti-HIV-1 and -HIV-2 MAbs cross-reacted were mapped with shorter oligopeptides.