A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both ¹²⁵I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of ³H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen     

Deficiency of neutrophil membrane antigen detected by monoclonal antibody in rheumatoid arthritis

Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.     

Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64

The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 cDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the anigen it recognizes.     

Recombinant CD63/ME491/neuroglandular/NKI/C-3 antigen inhibits growth of established tumors in transgenic mice

Attempts to vaccinate against tumors can be hindered by the induction of immunological tolerance to the target Ag as a result of Ag expression on normal tissues. In this study, we find that transgenic mice expressing the melanoma-associated Ag CD63/ME491/neuroglandular/NKI/C-3 on their normal tissues do, in fact, exhibit immunological tolerance to the Ag, recapitulating the conditions in cancer patients. In these mice, growth of murine melanoma cells expressing the Ag after gene transfer was inhibited by immunization with Ag-expressing recombinant vaccinia virus combined with IL-2, but not by immunization with the protein alone, anti-idiotypic Abs, or irradiated tumor cells. The effect of the recombinant virus was demonstrated both for nonestablished and established tumors. Infiltration with both CD4(+) and CD8(+) T lymphocytes was significantly more extensive in tumors from experimental mice than in tumors from control mice. MHC class I-positive, but not class I-negative, tumors were inhibited by the vaccine, suggesting that MHC class I-restricted T lymphocytes play a role in the antitumor effects. Abs did not appear to be involved in the vaccine effects. CD63 was immunogenic in 2 of 13 melanoma patients, pointing to the potential of this Ag, combined with IL-2, as a vaccine for melanoma patients.     

Sialosyllactotetraosylceramide, a novel ganglioside antigen detected in human carcinomas by a monoclonal antibody

A novel ganglioside was detected in a small cell lung carcinoma by TLC-immunostaining of gangliosides with a monoclonal antibody, the C-50 MAb. Structural characterization showed this ganglioside to be IV3NeuAc-LcOse4Cer, a hitherto unknown ganglioside. This ganglioside has also been detected as a minor component in many different carcinomas using the C-50 MAb. The normally dominant CA-50 ganglioside antigen is IV3NeuAc, III4Fuc-LcOse4Cer. Based upon solid-phase binding to IV3NeuAc, III4-LcOse4Cer and IV3NeuAc-LcOse4Cer it is concluded that the C-50 MAb recognizes an epitope present in sialylated type I carbohydrate chains.     

A rat oocyte differentiation antigen (OA-1) detected by a monoclonal antibody

Cell surface antigenic changes associated with differentiation of the rat oocyte and early embryo have been demonstrated with a monoclonal antibody (anti-OA-1). Antigen is first detectable coincident with initiation of oocyte growth, is a constant feature of all growing oocytes and displays a redistribution during meiotic maturation. Following fertilization, antigen is detectable on the surface of the embryo through the four-cell stage. This first monospecific marker for the rat oocyte and embryo should prove useful in probing structure/function relationships in oocyte growth, meiotic maturation fertilization, and/or early embryonic development.     

Novel isoglobo-neolacto-series hybrid glycolipid detected by a monoclonal antibody is a rat colon tumor-associated antigen

Isoglobotetraosylceramide (GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)Glc (beta 1-1)Cer), the major glycolipid species in dimethylhydrazine-induced rat tumors of colorectal origin, was not detected in epithelial cells of normal colon but was present in the non-epithelial stroma and could be extracted from each of nine tumors studied. Monoclonal antibodies produced against isoglobotetraosylceramide detected this and another novel rat tumor-associated glycolipid not present in epithelial cells nor in non-epithelial stroma of normal rat colon (Brodin, T., Thurin, J., Str?mberg, N., Karlsson, K.-A. and Sj?gren, H.O. (1985) Eur. J. Immunol. 16, 951-956). This novel glycolipid was present in 8/9 of the studied tumors and was also present in two in vitro cell clones. These were originally obtained from a W49/T4 colon tumor isograft. The novel glycolipid was characterized by mass spectrometry, 1H-NMR, and methylation analysis as a hybrid between the isoglobo- and neolacto-series, with the structure GalNAc(beta 1-3)Gal(alpha 1-3)Gal(beta 1-4)GlcNA(beta 1-3)Gal (beta 1-4)Glc(beta 1-1)Cer.     

Altered cell surface antigen expression in bladder carcinoma detected by a new hemagglutinating monoclonal antibody

A hemagglutinating monoclonal IgM antibody (MoAb145) was produced against a high incidence red blood cell membrane antigen. By the specific red cell adherence test, the antibody also reacted with human bladder epithelium; in addition, expression of the MoAb145 antigen was lost in some cases of transitional cell carcinoma of the bladder, in a manner similar to the ABH blood group. Hemagglutination studies with a panel of erythrocytes lacking specific high incidence red blood cell membrane antigens indicated that MoAb145 did not recognize ABH specificity but rather a determinant absent from rare MN variant erythrocytes, including En(a-) erythrocytes, which lack glycophorin-alpha. Failure of MoAb145 to stain, by indirect immunofluorescence, the erythroleukemia cell line K562, which expresses glycophorin-alpha and the MN blood group, and failure to inhibit MoAb145 hemagglutination with an erythrocyte sialoglycoprotein fraction that contained MN blood group activity suggests that MoAb145 does not recognize either glycophorin-alpha or the MN blood group, but rather another membrane determinant, which is altered in En(a-) erythrocytes. This study demonstrates a new epitope detected by MoAb145 that is shared between human erythrocyte membranes and bladder epithelia, and is affected by neoplastic transformation in transitional cell carcinoma of the bladder.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Immunopurification, characterization, and nature of membrane association of human melanoma-associated oncofetal antigen gp87 defined by monoclonal antibody 140.240

A melanoma-associated oncofetal antigen, gp87 (a p97-like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two-step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde-insolubilized MoAb 140.240 (ascites fluid) resulted in a 13-fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr-activated Sepharose 4B the bound material from the first step was further purified resulting in a 330-fold purification with 90% recovery. SDS-polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalanine in gp87. We have also compared gp87 with two well defined antigens, HLA-A,B,C (integral membrane protein) and "94K" melanoma/carcinoma-associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate-buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP-40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA-A,B,C antigens, could only be extracted with NP-40, strongly suggesting that gp87 is an integral melanoma cell component.     

Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon

To develop monoclonal antibodies (mAb) recognizing human melanoma-associated antigens (MAA) susceptible to modulation by immune interferon (IFN-gamma), hybridomas were constructed with splenocytes from a BALB/c mouse immunized with IFN-gamma-treated melanoma cells Colo 38. Screening of supernatants with control and IFN-gamma-treated melanoma cells showed that the mAb CL203 and CL207 display preferential reactivity with IFN-gamma-treated melanoma cells. The two mAb recognize the same (or spatially close) determinant on a 96,000 MAA which has a density of 0.36 X 10(6) antigenic sites/cell on untreated melanoma cells Colo 38 and of 1.39 X 10(6) and 1.54 X 10(6) on melanoma cells Colo 38 treated with IFN-gamma (final concentration, 200 U/ml) for 24 and 48 hr, respectively. The effect of IFN-gamma on the 96,000 MAA is dose- and time-dependent, reversible, and blocked by inhibitors of RNA and protein synthesis. Furthermore, the effect of IFN-gamma on the induction of the 96,000 MAA appears to be specific, inasmuch as IFN-alpha and IFN-beta do not induce the expression of the 96,000 MAA. The latter is also induced by IFN-gamma in a variety of carcinoma cell lines, but its level is markedly lower than on melanoma cells. Furthermore, the apparent m.w. of the antigen synthesized by the carcinoma cell lines in the presence of IFN-gamma ranges between 93,000 and 96,000. This molecular heterogeneity appears to reflect differences in the degree of glycosylation of the polypeptide moiety because the antigen synthesized by a variety of cell lines in the presence of tunicamycin has an apparent m.w. of 51,000.     

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.     

Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody

The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the “UIC2 shift”. Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r = 0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.     

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Selective in vitro toxicity of purothionin conjugated to the monoclonal antibody 225.28S to a human high-molecular-weight melanoma-associated antigen

The toxic agent purothionin was conjugated to the monoclonal antibody 225.28S to a human high-molecular-weight melanoma-accociated antigen. The toxic conjugate displayed in vitro toxicity to cultured human Colo 38 melanoma cells as indicated by reduced uptake of 3H-thymidine following a 24-h incubation and loss of cell viability following a 7-day incubation. The effect is dose-dependent and is specific since addition of the toxic conjugate to a cultured Raji B lymphoid cells did not affect their 3H-thymidine uptake or their viability.     

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.     

Isolation of the melanoma-associated antigen p23 using antibody phage display

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.     

Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.