CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils.

The CD66 Ag is a neutrophil-specific "activation Ag" in that it is detected in low density on resting cells but its surface expression is up-regulated by stimulation (with the chemotactic peptide FMLP, the calcium ionophore A23187, and 12-O-tetradeconoyl-phorbol-13-acetate). Phosphorylation is an important mechanism of regulation of protein function. Although most studies of protein phosphorylation have focused on intracellular reactions, recent studies have provided evidence for the existence of ectoprotein kinase activity on the surface of several types of cells including human neutrophils. The role of ectoprotein kinase activity in cell function is unknown and little is known about the endogenous substrates of this enzyme system. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed that CD66 mAb specifically recognize a approximately 180-kDa phosphoprotein on the surface of human neutrophils. This protein was one of the major endogenous substrates for human neutrophil ectoprotein kinase activity. Phosphoamino acid analysis of the 180-kDa protein revealed that it contained predominantly phosphotyrosine. Preclearing studies demonstrated that this protein was also recognized by CD15 mAb, and by polyclonal anticarcinoembryonic Ag antiserum. In addition, the CD66 mAb reacted with purified carcinoembryonic Ag, biliary glycoprotein, and "nonspecific cross-reacting Ag." Thus, the neutrophil protein recognized by CD66 mAb appears to be a approximately 180-kDa form of the classical "nonspecific cross-reacting Ag" on human neutrophils.     

Human monoclonal antibodies specific for capsular polysaccharides of Klebsiella recognize clusters of multiple serotypes

We report the generation and the characterization of a set of human monoclonal antibodies (HmAb) specific for Gram-negative bacteria of Klebsiella pneumoniae. The eight human hybridomas secrete either IgM kappa, IgA1 kappa, or IgA2 kappa antibodies. One HmAb binds bacteria of only one serotype. Five HmAb recognize non-overlapping clusters of 2, 3, or 10 different serotypes. The remaining two HmAb both recognize three serotypes. Two serotypes are recognized by both HmAb, and in addition both HmAb bind one more nonidentical serotype. These results suggest that in man, epitopes are immunodominant, different from serotype-specific determinants detected by conventional rabbit antisera. Screening of clinical isolates revealed that the HmAb recognize not only representative typing strains but also most isolates of the corresponding serotype. In addition, most of the isolates that were non-typable by polyclonal antisera were recognized by one of the HmAb. Fine specificity analyses revealed that all HmAb are highly specific for the isolated capsular polysaccharides (CPS) of bacteria within the corresponding cluster of serotypes. However, the avidity of a HmAb for the different CPS can differ significantly. Taken together, our results suggest that the unequivocal interactions between HmAb and CPS offer the basis for an alternative, better defined classification system, and that passive immunization with a limited number of HmAb may provide a feasible strategy for the protection against the majority of fatal, nosocomial infections with multidrug-resistant strains of K. pneumoniae.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection.

Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.     

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.     

Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies

Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.     

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.     

Characterization of monoclonal antibodies raised toStreptococcus mutans

Monoclonal antibodies (MAb) to whole cells ofStreptococcus mutants P-4 (serotype e) were generated. Of the four MAb used in this investigation, three were found to cross-react with mouse heart tissue (MAb-HT) in the solid-phase ELISA, while the remaining MAb was found to be specific forS. mutants only (MAb-SM). Serotypic characterization has shown that MAb-SM as well as MAb-HT recognize strains ofS. mutans representing all of the eight currently known serological groups. In addition, MAb-HT showed reactivity withStreptococcus pyogenes, a Lancefield group-A streptococcus frequently implicated in rheumatic fever. Western blot analysis has shown that MAb-SM recognizes a 14,000-dalton component present in a partially purified membrane fraction recovered following glass-bead breakage ofS. mutans P-4 cells. Two of the MAb-HT were found to react with a 35,000-dalton component from this same preparation.     

Identification of a human natural killer cell target cell antigen with monoclonal antibodies

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.     

Study of Yersinia pseudotuberculosis surface antigen epitopes using monoclonal antibodies

A study of the influence of exogenous factors on the immunochemical activity of the bacterium Yersinia pseudotuberculosis and lipopolysaccharide preparations isolated from bacteria was performed using monoclonal antibodies. It was shown that the hybridomas that were obtained in this work produce antibodies against different and, most likely, species-specific epitopes associated with lipopolysaccharide O-side chains. The concentration of these epitopes increased with a decrease in the temperature, at which the bacteria were cultivated. An inhibitory effect of proteinase K, pepsin, and trypsin on the immunochemical activity of bacterial cells, determined using a solid-phase enzyme immunoassay, was demonstrated. Treatment with sodium periodate showed no uniform effect on the reactions between monoclonal antibodies and antigens (lipopolysaccharides and microbial cells), as adjudged by an immunoassay, which is most likely a consequence of the different localization of lipopolysaccharide epitopes recognized by the antibodies from four hybridomas.     

Identification of mutans streptococci with monoclonal antibodies

Mutans streptococci have been correlated with dental caries. The identification of the species within this group is still a problem. The characterization of a monoclonal antibody (Mab) OMVU10 against S. sobrinus as well as the isolation and characterization of Mabs against S. mutans (OMVU30 and OMVU31), S. cricetus (OMVU40) and mutans streptococci (OMVU2) is described. The epitope specificity for OMVU10 and OMVU31 was cell-wall antigen B in both cases although both Mabs recognized different species-specific epitopes. OMVU40 was cross reactive with Steptococcus sanguis taxon 3. All other Mabs were specific for one species. Using these Mabs, a key to the identification of mutans streptococci is developed. This key was tested for 85 wild type isolates of mutans streptococci and proved to be highly reliable and easy to perform.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

Comparison of cell surface antigen extracts from two serotypes of Pasteurella haemolytica

Cells of Pasteurella haemolytica serotypes A1 and A6 were extracted with sodium salicylate and the chemical and antigenic composition of both extracts determined. The extracts were concentrated by ultrafiltration and the serotype antigen, measured by the indirect haemagglutination test, was estimated to have a molecular weight between 100 000 and 300 000. The chemical composition of sodium salicylate extracts (SSEs) from both serotypes was similar, having protein, carbohydrate, fatty acid and phosphorus present in the ratio 10:1:0.5:0.1. SDS-PAGE of both SSEs gave similar profiles with at least 48 bands present. These results suggest that sodium salicylate removes the outer membrane of P. haemolytica. Crossed immunoelectrophoresis indicated that a major serotype-specific antigen was present in SSEs of both strains. This antigen was extracted from the SSE with hot phenol/water and analysed by gas chromatography. The sugar composition of A1 and A6 phenol/water extract (PWE) was qualitatively identical although some differences in proportions were observed. A1 and A6 PWE antigens protected mice against homologous serotype challenge and A6 PWE protected against heterologous (A1) challenge.     

Analysis of epithelial cell surface polarity with monoclonal antibodies

The hybridoma technique of K?hler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.     

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.     

Contribution of cell surface protein antigen PAc of Streptococcus mutans to bacteremia

Streptococcus mutans, a major cariogenic bacterium, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. Mutant strains of S. mutans MT8148, defective in the major surface proteins glucosyltransferase (GTF) B-, C-, and D-, and protein antigen c (PAc), were constructed by insertional inactivation of each respective gene with an antibiotic resistant cassette. Susceptibility to phagocytosis was determined by analyses of interactions of the bacteria with human polymorphonuclear leukocytes, and the PAc-defective mutant strain (PD) showed the lowest rate of phagocytosis. Further, when PD and MT8148 were separately injected into the jugular veins of Sprague-Dawley rats, PD was recovered in significantly larger numbers and for a longer duration, and caused more severe systemic inflammation than MT8148, indicating that S. mutans PAc is associated with its systemic virulence in blood. Next, 100 S. mutans clinical isolates from 100 Japanese children and adolescents were analyzed by Western blotting using antisera raised against recombinant PAc, generated based on the pac sequence of MT8148. Four of the 100 strains showed no positive band and each exhibited a significantly lower phagocytosis rate than that of 25 randomly selected clinical strains (P < 0.01). In addition, three of the 100 strains possessed a lower molecular weight PAc and a significantly lower rate of phagocytosis than the 25 reference strains (P < 0.05). These results suggest that S. mutans PAc may be associated with phagocytosis susceptibility to human polymorphonuclear leukocytes, with approximately 7% of S. mutans clinical isolates possible high-risk strains for the development of bacteremia.     

Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries

We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schiff's reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pI of 5.8.     

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, K_D = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.     

High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, K_D = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.