Hybridoma Library Research Articles

Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy

In adenovirus-derived gene therapy, one of the problems is the difficulty in specific targeting. We have recently demonstrated that monoclonal antibody (mAb) libraries screened by fiber-modified adenovirus vector (Adv-FZ33), which is capable of binding to immunoglobulin-G (IgG), provide a powerful approach for the identification of suitable target antigens for prostate cancer therapy. Hybridoma libraries from mice immunized with androgen-dependent prostate cancer cell line LNCaP were screened and mAb were selected. Through this screening, we obtained one mAb, designated LNI-29, that recognizes a glycoprotein with an apparent molecular mass of 100 kD. It was identified as neural cell adhesion molecule 2 (NCAM2). Some prostate and breast cancer cell lines highly expressed NCAM2 whereas normal prostate cell lines expressed NCAM2 at low levels. In contrast to the low efficiency of gene transduction by Adv-FZ33 with a control antibody, LNI-29-mediated Adv-FZ33 infection induces high rates of gene delivery in NCAM2-positive cancers. NCAM2-mediated therapeutic gene transduction of uracil phosphoribosyltransferase (UPRT) had a highly effective cytotoxic effect on NCAM2-positive cancer cells, whereas it had less of an effect in cases with a control antibody. In conclusion, NCAM2 should be a novel gene therapy target for the treatment of prostate and breast cancer.

Modulation of Lysyl Oxidase-like 2 Enzymatic Activity by an Allosteric Antibody Inhibitor

In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

The Wuerzburg Hybridoma Library against Drosophila Brain

: This review describes the present state of a project to identify and characterize novel nervous system proteins by using monoclonal antibodies (mAbs) against the Drosophila brain. Some 1,000 hybridoma clones were generated by injection of homogenized Drosophila brains or heads into mice and fusion of their spleen cells with myeloma cells. Testing the mAbs secreted by these clones identified a library of about 200 mAbs, which selectively stain specific structures of the Drosophila brain. Using the approach “from antibody to gene”, several genes coding for novel proteins of the presynaptic terminal were cloned and characterized. These include the “cysteine string protein” gene (Csp, mAb ab49), the “synapse-associated protein of 47 kDa” gene (Sap47, mAbs nc46 and nb200), and the “Bruchpilot” gene (brp, mAb nc82). By a “candidate” approach, mAb nb33 was shown to recognize the pigment dispersing factor precursor protein. mAbs 3C11 and pok13 were raised against bacterially expressed Drosophila synapsin and calbindin-32, respectively, after the corresponding cDNAs had been isolated from an expression library by using antisera against mammalian proteins. Recently, it was shown that mAb aa2 binds the Drosophila homolog of “epidermal growth factor receptor pathway substrate clone 15” (Eps15). Identification of the targets of mAbs na21, ab52, and nb181 is presently attempted. Here, we review the available information on the function of these proteins and present staining patterns in the Drosophila brain for classes of mAbs that either bind differentially in the eye, in neuropil, in the cell-body layer, or in small subsets of neurons. The prospects of identifying the corresponding antigens by various approaches, including protein purification and mass spectrometry, are discussed.

897. Na,K-ATPase β1-Mediated Selective Gene Transfer into Neurons

Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. Selective gene transfer requires high efficiency as well as tissue specificity of the target molecule employed. Selection of an optimal target molecule is critical. Here, we contrived the hybridoma library screening to identify optimal target molecules for neuron selective gene transfer using fiber mutant adenoviruses as targeting tools. We generated genetically modified adenoviruses incorporating Z33, an IgG Fc-binding motif from Staphylococcus protein A, at the fiber (Adv-FZ33 and Adv-FdZ).

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine.

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine.

Isolation and characterization of a monoclonal anti-quadruplex DNA antibody from autoimmune "viable motheaten" mice.

A cell line that produces an autoantibody specific for DNA quadruplex structures has been isolated and cloned from a hybridoma library derived from 3-month-old nonimmunized autoimmune, immunodeficient "viable motheaten" mice. This antibody has been tested extensively in vitro and found to bind specifically to DNA quadruplex structures formed by two biologically relevant sequence motifs. Scatchard and nonlinear regression analyses using both one- and two-site models were used to derive association constants for the antibody-DNA binding reactions. In both cases, quadruplexes had higher association constants than triplex and duplex molecules. The anti-quadruplex antibody binds to the quadruplex formed by the promoter-region-derived oligonucleotide d(CGCG4GCG) (Ka = 3.3 x 10(6) M-1), and has enhanced affinity for telomere-derived quadruplexes formed by the oligonucleotides d(TG4) and d(T2G4T2G4T2G4T2G4) (Ka = 5.38 x 10(6) and 1.66 x 10(7) M-1, respectively). The antibody binds both types of quadruplexes but has preferential affinity for the parallel four-stranded structure. In vitro radioimmunofilter binding experiments demonstrated that purified anti-DNA quadruplex antibodies from anti-quadruplex antibody-producing tissue culture supernatants have at least 10-fold higher affinity for quadruplexes than for triplex and duplex DNA structures of similar base composition and length. The antibody binds intramolecular DNA triplexes formed by d(G4T3G4T3C4) and d(C4T3G4T3G4), and the duplex d(CGCGCGCGCG)2 with an affinities of 6. 76 x 10(5), 5.59 x 10(5), and 8.26 x 10(5) M-1, respectively. Competition experiments showed that melted quadruplexes are not effective competitors for antibody binding when compared to native structures, confirming that the quadruplex is bound structure-specifically. To our knowledge, this is the first immunological reagent known to specifically recognize quadruplex structures. Subsequent sequence analysis demonstrates homologies between the antibody complementarity determining regions and sequences from Myb family telomere binding proteins, which are hypothesized to control cell aging via telomeric DNA interactions. The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.

Organic Artificial Proteinase with Active Site Comprising Three Salicylate Residues

The first organic artificial proteinase based on synthetic materials is obtained by building active sites on the backbone of poly(ethylenimine) (PEI). The active site comprising three salicylate residues is prepared by preassemblage of three molecules of a salicylate derivative with Fe(III) ion followed by cross-linkage of the salicylates with PEI and demetalation of the resulting polymer. Proteinase activity of the artificial enzyme is demonstrated with hydrolytic cleavage of γ-globulin (Gbn). Both the heavy (50 kDa) and the light (25 kDa) chains of Gbn are effectively cleaved into peptides smaller than 5 kDa. The optimum activity of the artificial proteinase is manifested at pH 5−7. The half-life for cleavage of the two chains of Gbn by the artificial proteinase is ∼1 h at pH 7 and 50 °C when the concentration of the artificial active site is 0.4−1 mM. The activity (at pH 7) of the artificial proteinase prepared with PEI is comparable to that (at pH 9) of a catalytic antibody elicited by a joint hybridoma and combinatorial antibody library approach and to that (at pH < 3) achieved by the intramolecular carboxyl group in the hydrolysis of N-methyl maleamic acid. Thus, the approach reported here is useful for designing active sites of artificial enzymes.

Direct Selection for Catalysis from Combinatorial Antibody Libraries Using a Boronic Acid Probe: Primary Amide Bond Hydrolysis

This report describes a joint hybridoma and combinatorial antibody library approach to elicit catalysts for primary amide bond hydrolysis. By immunization with a trigonal boronic acid hapten 3a and construction of a Fab (antigen-binding fragment) library, a diastereoselective catalyst for hydrolysis of the tripeptide primary amide substrate 1a was selected. In contrast, no antibody catalyst was isolated by standard hybridoma methods of monoclonal antibody production following immunizations with hapten 3a. The active Fab, BL25, obeys Michaelis−Menten kinetic behavior (kcat/kuncat ca. 4 × 104, Km = 150 μM) and is competitively inhibited by a boronic acid hapten analog 3b (Ki = 9 μM). Kinetic and binding studies both point to Fab selection of a hydrated tetrahedral anionic form of the boronic acid hapten 3a which serves to mimic the putative transition state 4 for catalysis of water addition to the primary amide bond. Fab-BL25 exhibits exquisite substrate selectivity, as a methyl ester analog of 1a is not ac...

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the fresh-water planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians.

The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.

Proteins expressed specifically in neurons and transported to synaptic terminals are likely to constitute important molecular elements of nervous system function. In an effort to characterize synapse-associated proteins (SAPs) of Drosophila, we have isolated from a hybridoma library several monoclonal antibodies (MABs) that selectively stain synaptic terminals in immunohistochemical preparations. MAB nc46 binds to most but not all synaptic terminals of the Drosophila nervous system, it also recognizes a protein with homologous distribution in other dipteran flies and binds to large parts of fish CNS. In Western blots the antibody labels a Drosophila brain protein of 47 kDa and cross-reacts with brain proteins from several species including insects, fish, mouse and man. From these data we conclude that the corresponding gene has been conserved in evolution at least among diptera. Using MAB nc46 and expression cloning we have identified the 'sap47' gene coding for the 'synapse-associated protein of 47 kDa' of Drosophila melanogaster. Sequence analysis of genomic and cDNA clones reveals the intron-exon structure of the gene and characterizes the complete open reading frames of two alternatively spliced transcripts. The sap47 gene is located in 89A8-B3 on chromosome 3R and codes for two almost identical inferred polypeptides of 347 and 351 amino acids with no significant sequence homology to known proteins.

Biochemical properties of a novel cell wall protein associated with elongation growth in higher plants

A monoclonal antibody of IgM-type (TIM-11B2) was screened from a hybridoma library. The antibody recognizes a 40 kDa glycoprotein, p40, with high specificity. This protein was detected in all plant species examined so far and was found to be located both solubly and ionically-bound within the primary cell wall. The strongest immunobiochemical signals of p40 were found in tissues undergoing elongation growth, whereas in other tissues only a faint signal could be detected. Those included the non-elongating parts of different seedlings, such as the apical part of monocot primary leaves or the leaves of dicots grown in light. Inhibition of pea epicotyl growth by white light irradiation resulted in a strong decrease of the immunostain signal

Labeled antigen capture assay: A method for detecting monoclonal antibodies to cloned gene products

We report here a relatively easy and highly sensitive assay for detecting monoclonal antibodies to the product of virtually and cloned gene. The protocol, termed labeled antigen capture assay (LACA), is a solid-phase type radioimmunoassay which uses a bacteriophage T7 expression system to generate exclusively radiolabeled antigen. Thus, to generate radiolabeled antigen for screening, the gene encoding the protein of interest need only be subcloned downstream of a T7 promoter, and the new construct transformed into an Escherichia coli strain harboring a compatible plasmid which encodes a thermal inducible copy of the T7 RNA polymerase. Expression of the T7-promoted gene in the presence of rifampicin and [ 35S]cysteine (or methionine) yields labeled antigen, which is then “captured” by specific monoclonal antibody and detected by autoradiography. Our results indicate that as little as 30 ng of specific monoclonal antibody can be detected using the LACA protocol. The protocol is applicable to the product of any cloned gene but is particularly useful in the case where the biochemical properties of the gene product of interest are unavailable. In this report we use the LACA protocol to screen a hybridoma library for monoclonal antibodies to the STb heat-stable enterotoxin (STb) of E. coli. Mice were immunized with a genetically constructed, affinity-purified Protein A-STb hybrid protein, and following spleen cell fusion and HAT selection, hybridomas were screened by the described LACA protocol for production of STb-specific monoclonal antibody. Of over 1500 hybridomas tested 138 were positive, by primary LACA screening, for STb-specific IgG monoclonal antibodies.

A Cysteine-String Protein is Expressed in Retina and Brain ofDrosophila

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

Cloning and expression of the cDNA for the murine interferon gamma receptor.

A murine interferon gamma (IFN-gamma) receptor cDNA was isolated by screening a murine T-cell hybridoma library prepared in lambda gt10 with probes prepared from a human IFN-gamma receptor cDNA. The 2.1-kilobase (kb) cDNA encoded a serine-rich polypeptide of 477 amino acids that was 52% identical to the human protein. Southern and Northern (RNA) blot analyses indicated the presence of a single receptor gene and a single predominant 2.3-kb receptor transcript. Human embryonic kidney fibroblasts, stably transfected with the murine IFN-gamma receptor cDNA, expressed murine IFN-gamma receptors as detected by flow cytometry with either ligand or a receptor-specific monoclonal antibody. Nontransfected cells bound neither ligand nor antibody. Radioligand-binding analysis demonstrated that the transfectants expressed 530,000 murine IFN-gamma receptors per cell and bound murine IFN-gamma with a Ka of 1 x 10(9) M-1. However, despite high-level expression of murine IFN-gamma receptors, the transfected human cells responded only to human and not to murine IFN-gamma as detected by enhancement of major histocompatibility class I antigen expression and induction of antiviral activity. These results thus document the isolation and expression of a full-length murine IFN-gamma receptor cDNA and suggest that additional species-specific components may be necessary to form a biologically active IFN-gamma receptor.

Monoclonal antibodies as tools in membrane biochemistry. Identification and partial characterization of the dicarboxylate transporter from pea leaf mitochondria.

Monoclonal antibodies specific for the dicarboxylate transporter of pea mitochondria were prepared and used to identify this substrate carrier. The hybridoma library was derived from mice that had been immunized with total mitochondrial membranes. The monoclonal antibodies specific for the dicarboxylate transporter were selected by screening hybridoma supernatants for their ability to inhibit malate- and succinate-dependent oxalacetate reduction by osmotically shocked pea leaf mitochondria. Three monoclonal antibodies were shown to be specific for the dicarboxylate transporter by their ability to 1) inhibit malate and succinate metabolism without affecting alpha-ketoglutarate, citrate, pyruvate, glycine, glutamate, or aspartate metabolism by mitochondria and 2) inhibit malate uptake by a partially purified transporter fraction reconstituted into asolectin vesicles. The dicarboxylate transporter was identified by Western blotting and immunoprecipitation and had an apparent molecular mass of 26,000 Da. The techniques described should prove useful for identifying a number of membrane proteins that can be assayed in situ but are difficult to assay following dissolution of the membrane.

Use of hybridoma libraries in the study of the genetics and development of Drosophila.

Use of hybridoma libraries in the study of the genetics and development of Drosophila.