Biomedical subjects

M Neumaier

Publications and source records attributed to M Neumaier.

49 records · Page 3Linked to original sources

PubMed1994

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction.

PURPOSE: To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas. PATIENTS AND METHODS: A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay. RESULTS: In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method. CONCLUSION: Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.

Base Sequence↗

PubMed1993

Biliary glycoprotein, a potential human cell adhesion molecule, is down-regulated in colorectal carcinomas.

Biliary glycoprotein (BGP) is the human homologue of a cell adhesion molecule (CAM) of the rat designated Cell-CAM. The BGP gene is a member of the carcinoembryonic antigen gene family, which belongs to the immunoglobulin superfamily. BGP is expressed in cells of epithelial and myeloid origin. In granulocytes, BGP is a main antigen of the CD66 cluster of differentiation antigens that mediate the binding to endothelial E-selectin. Since BGP is a major human CAM, the expression of BGP was studied in 21 colorectal carcinoma tissue specimens and in the respective adjacent normal mucosae. As an internal control for epithelial mRNA, the expression of cytokeratin 18 was evaluated in parallel. In addition, the expression of carcinoembryonic antigen and nonspecific crossreacting antigen, which are highly homologous to BGP, was investigated. Two BGP mRNAs of 3.9 and 1.5 kilobases were detected in the normal colonic mucosa samples. The median of the tumor-to-normal ratios of mRNA expression was 0.2 for both BGP mRNAs. In contrast, the median was 1.2 for cytokeratin, 1.0 for carcinoembryonic antigen, and 1.4 for nonspecific crossreacting antigen. Relative to cytokeratin 18 expression, the expression of BGP was reduced to < or = 0.1 in half of the tumors and to < or = 0.4 in > 80% of the tumors. These findings indicate that the loss or reduced expression of the adhesion molecule BGP is a major event in colorectal carcinogenesis.

Antigens, CD↗

PubMed1993

Molecular characterization of a cloned idiotypic cascade containing a network antigenic determinant specific for the human carcinoembryonic antigen.

The monoclonal anti-idiotypic antibody (maId) 6G6.C4, directed against the carcinoembryonic antigen (CEA)-specific T84.66 immunoglobulin, was recently shown to act as a surrogate antigen for CEA in experimental animals. In this report, we have extended our studies. 1) The kinetics of complex formation in this CEA-specific idiotypic cascade were investigated using Biosensor technology. 2) Bacterial expression studies show that the mimicked epitope can be delimited to the A3 domain of CEA. 3) We cloned and characterized the genes coding for maId 6G6.C4. 4) Comparison of this epitope-bearing domain with the hypervariable region sequences of 6G6.C4 yields substantial amino acid similarity. Sequence homology between maId 6G6.C4 and anti-CEA antibodies binding to the T84.66 epitope led us to investigate the interaction of maId 6G6.C4 and CEA. Surprisingly, 6G6.C4 specifically binds to CEA but not CEA-related antigens in Western blots. 6G6.C4 and T84.66 recognize different epitopes on CEA. Our results suggest that the T84.66 epitope functionally mimicked by maId 6G6.C4 may be involved in the homophilic binding between CEA molecules, and that heterophilic interactions in the CEA-family are mediated by different binding sites. A model for the intermolecular adhesion of CEA is presented.

Amino Acid Sequence↗

PubMed1993

Monoclonal, anti-domain and anti-peptide antibodies assign the molecular weight 160,000 granulocyte membrane antigen of the CD66 cluster to a mRNA species encoded by the biliary glycoprotein gene, a member of the carcinoembryonic antigen gene family.

mAb directed against the CD66 cluster of granulocyte differentiation Ag recognize Ag of the carcinoembryonic Ag family. A major Ag in extracts from granulocyte membranes bound by CD66 antibodies exhibits a relative molecular mass of 160,000. According to recent data, this Ag may be a product of the biliary glycoprotein (BGP) gene that belongs to the CEA gene family. As a result of alternative splicing, the BGP gene is transcribed into at least seven distinct mRNA species. To identify splice variants of BGP, antisera were raised against the A2 domain expressed in bacteria and to a peptide comprising the C-terminal 23 amino acids encoded by the 3' exon of the BGP gene. The antisera and an mAb specific for members of the BGP family were used to identify potential BGP splice variants in granulocyte membranes. For comparison, the binding of antibodies to Ag purified from human bile was investigated. In the membrane preparation from granulocytes, the only Ag identified by the mAb, the domain antiserum and the peptide antiserum, was the Ag of M(r) 160,000 recognized by a CD66 antibody. These results indicate that the M(r) 160,000 granulocyte membrane Ag of the CD66 cluster is the product of the BGP-specific mRNA containing all coding sequences of the BGP gene. Among two major biliary glycoproteins present in human bile, the M(r) 115,000 Ag contains the A2 domain, whereas the domain is lacking in the "classical" biliary glycoprotein of M(r) 85,000. None of the bile Ag bound the peptide antiserum.

Antibodies, Monoclonal↗

PubMed1992

A monoclonal anti-idiotypic antibody bearing the image of an epitope specific to the human carcinoembryonic antigen.

One concept for immune therapy of patients bearing carcinomas involves monoclonal anti-idiotypic antibodies (Malds) to trigger the immune system of the host into a response against the tumor cells. Current theory states that so-called internal image Malds bearing epitopes specific to a given tumor-associated antigen would be most suited for that purpose. We report here the generation of syngeneic Malds generated against the murine monoclonal immunoglobulin T84.66 (Ab1), which defines a single epitope on the protein moiety of the carcinoembryonic antigen (CEA). This antigenic determinant is unique to CEA, as it is absent in other members of the CEA gene family that are expressed in a variety of normal human tissues, including granulocytes. The Mald 6G6.C4 (Ab2) exhibits the immunochemical features of an internal image antibody mimicking the epitope recognized by the idiotype T84.66. In enzyme immunoassays the binding of Ab1 to Ab2 is completely inhibited by CEA. In addition, Mald 6G6.C4 only binds to native but not to the denatured or reduced idiotype. Immunization of rabbits with F(ab')2-fragments of 6G6.C4 results in antisera (Ab3) that show specificity to CEA in both binding and inhibition enzyme immunoassays as well as in Western blots. Finally, Ab3 did not detect NCA, a major CEA-related glycoprotein in Western blots, either in a purified form or in a crude tumor extract, indicating a high specificity of the anti-anti-idiotypic response. In summary, these immunochemical data show that the monoclonal anti-idiotype 6G6.C4 can functionally mimic a CEA-specific epitope in rabbits and may do so in humans. Therefore, this antibody may have a clinical potential as a network antigen for active immune therapy in patients suffering from CEA-positive carcinomas.

Animals↗

PubMed1990

Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells.

Carcinoembryonic antigen (CEA) is one of the best characterized tumor-associated antigens and is extensively used in the in vitro immunodiagnosis of human colon adenocarcinomas. Among a number of anti-CEA monoclonal antibodies, the murine monoclonal antibody T84.66 shows the highest specificity and affinity for CEA and has been used successfully for in vivo tumor imaging in mice and humans. We report here the cloning and sequencing of the genes coding for monoclonal antibody T84.66 and the amino acid sequence of the variable regions for the heavy and light chains. We also report the construction of mouse/human chimeric IgG1 antibody genes using T84.66 variable region genes and human constant region genes. The resulting chimeric gene constructs were transfected into murine myeloma cells (Sp2/0) by electroporation and into Chinese hamster ovary cells by lipofection. The chimeric antibodies obtained exhibited the same specificity and affinity for CEA as that of the T84.66 immunoglobulin produced by the murine hybridoma cell line. Antibody concentrations in culture medium supernatants were clonally variable but similar (15-480 ng/ml) for both Sp2/0 and Chinese hamster ovary transfectants; the average production by Chinese hamster ovary transfectants was only 3-5-fold less than Sp2/0 transfectants. Ascites production of Sp2/0 transfectants is sufficiently high (900 micrograms/ml) for initial in vivo studies with humanized T84.66.

Amino Acid Sequence↗

PubMed1988

Characterization of a cDNA clone for the nonspecific cross-reacting antigen (NCA) and a comparison of NCA and carcinoembryonic antigen.

NCA (nonspecific cross-reacting antigen), a glycoprotein found in normal lung and spleen, is immunologically related to carcinoembryonic antigen (CEA), which is found in over 95% of colon adenocarcinomas. From a human genomic library, we previously cloned part of an NCA gene and showed that the amino-terminal region has extensive sequence homology to CEA (Thompson, J. A., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, Ch. W., Riggs, A. D., and Shively, J.E. (1987) Proc. Natl. Acad. Sci. U. S.A. 84, 2965-2969). We now present the nucleotide sequence of a cDNA clone, containing the entire coding region of NCA (clone 9). The clone was obtained from a lambda gt 10 library made from the colon carcinoma cell line SW 403; the clone contains a 34-amino acid leader sequence, 310 amino acids for the mature protein, and 1.4 kilobases of 3'-untranslated region of the NCA gene. A comparison of the NCA sequence to the CEA sequence (Oikawa, S., Nakazato, H., and Kosaki, G. (1987) Biochem. Biophys. Res. Commun. 142, 511-518; Zimmerman, W., Ortlieb, B., Friedrich, R., and von Kleist, S. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2690-2694) shows that both proteins contain doublets of an immunoglobulin-like domain, of which there are one copy in NCA and three copies in CEA, a 108-amino acid amino-terminal domain with no cysteine residues, and a carboxyl-terminal hydrophobic domain of sufficient length to anchor the glycoproteins in the cell membrane. Overall, the corresponding coding regions possess 85% sequence homology at the amino acid level and 90% homology at the nucleotide level. Forty nucleotides 3' of their stop codons, the CEA and NCA cDNAs become dissimilar. The 108-amino acid amino-terminal region together with part of the leader peptide sequence corresponds exactly to a single exon described in our previous work. The data presented here further demonstrate the likelihood that CEA recently evolved from NCA by gene duplication, including two duplications of the immunoglobulin-like domain doublet of NCA.

Amino Acid Sequence↗

PubMed1988

Molecular cloning of a cDNA coding biliary glycoprotein I: primary structure of a glycoprotein immunologically crossreactive with carcinoembryonic antigen.

We have isolated and sequenced four overlapping cDNA clones from a normal adult human colon library, which together gave the entire nucleotide sequence for biliary glycoprotein I (BGP I). BGP I is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily in the immunoglobulin gene superfamily. The deduced amino acid sequence of the combined clones for BGP I revealed a 34-residue leader sequence followed by a 108-residue N-terminal domain, a 178-residue immunoglobulin-like domain, a 108-residue region specific to BGP I, a 24-residue transmembrane domain, and a 35-residue cytoplasmic domain. The nucleotide sequence of BGP I exhibited greater than 80% identity with CEA and nonspecific crossreacting antigen (NCA) in the leader peptide, N-terminal domain, and immunoglobulin-like domain. The BGP I-specific domain, designated A', was 56.7% and 55.8% identical at the nucleotide level and 42.6% and 39.6% identical at the amino acid level to the immunoglobulin-like domain of NCA and the first immunoglobulin-like domain of CEA, respectively. Beyond nucleotide position 1375 the 3' region of the BGP I cDNA was found to be specific for BGP I. Hybridization of a probe from this region to electrophoretic blots of RNAs from different human tissues showed a predominant 2.8-kilobase (kb) message accompanied by weaker bands 4.1 and 2.1 kb in size. The same probe gave a single band in Southern blot analysis of restricted total human DNA. Using a coding region probe from the BGP I domain A', we observed 4.1- and 2.1-kb messages. Lack of the 2.8-kb band suggested that different forms of BGP I may be generated by posttranscriptional modification of the same gene. We propose that BGP I diverged from NCA by acquiring an immunoglobulin-like domain substantially different from the domains found in NCA or CEA and also a new cytoplasmic domain. The latter feature should result in a substantially different membrane anchorage mechanism of BGP I compared to CEA, which lacks the cytoplasmic domain and is anchored via a phosphatidylinositol-glycan structure. Protein structural analysis of BGP I isolated from human bile revealed a blocked N terminus, 129 amino acids of internal sequence that are in agreement with the translated cDNA sequence, and five glycosylation sites in the peptides sequenced.

Amino Acid Sequence↗

PubMed1987

Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences.

Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.

Amino Acid Sequence↗

PubMed1986

Transblot studies with biotin-labeled proteins: electrophoretic mobilities and detection limits.

Molecular weight standard proteins, mouse IgG as well as several antigens cross-reacting with the carcinoembryonic antigen (CEA), were biotin labeled, submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The bound proteins were revealed by the use of avidin-peroxidase conjugates and a suitable substrate. The ratio of N-hydrosuccinimido biotin (NHSB) to protein yielding the lowest detection limit was determined. At an optimal NHSB/protein ratio, 0.33 ng of IgG heavy chains and 0.17 ng of IgG light chains could be visualized. With the exception of human albumin and ovalbumin, the increase in apparent molecular weight after biotin labeling was less than 10% for the proteins tested. The method has proven to be a valuable addition to Western blots performed with CEA-related antigens and monoclonal anti-CEA antibodies.

Animals↗

PubMed1985

Delineation of four carcinoembryonic antigen (CEA) related antigens in normal plasma by transblot studies using monoclonal anti-CEA antibodies with different epitope specificities.

Antigens related to the carcinoembryonic antigen (CEA) were isolated from normal human plasma by perchloric acid extraction, gel permeation chromatography and immunoaffinity chromatography using a monoclonal antibody with broad specificity and high affinity. The antigens were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The binding of five monoclonal anti-CEA antibodies with different epitope specificities to the immobilized antigens was analyzed. Two antigens with mol. wts of greater than 200,000 and 177,000 bound all five antibodies, and two antigens with mol. wts of 114,000 and 85,000 bound three of the five antibodies. The findings reported indicate that even monoclonal antibodies with high specificity for colonic cancer CEA detect CEA-related antigens in normal human plasma.

Antibodies, Monoclonal↗

PubMed1985

Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography.

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.

Antibodies, Monoclonal↗

PubMedDate not supplied

A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.

We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.

Animals↗