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Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.

Carcinoembryonic antigen (CEA) is a member of the immunoglobulin gene superfamily with one predicted variable domain-like region (N domain; 108 amino acids) and three sets of constant domain-like regions (A1B1, A2B2, and A3B3; 92 amino acids for A domains and 86 amino acids for B domains). In addition, CEA possesses two signal peptides, one at the amino terminus and one at the carboxyl terminus. Both are removed during posttranslational processing, with the one at the carboxyl terminus being replaced by a glycosylphosphatidylinositol (GPI) moiety. We have previously expressed the full length complementary DNA clone for CEA in Chinese hamster ovary cells and murine L cells, demonstrating proper processing of nascent polypeptide chains to mature, fully glycosylated CEA including the GPI anchor. Using the same full length CEA complementary DNA clone and the polymerase chain reaction, we have now constructed expression clones for secreted versions of the N domain, the A3B3 domain, and the A3 and B3 subdomains. The clones were expressed in HeLa cells using the beta-actin promoter. A stop codon was introduced at the end of the A3B3 and the A3 and B3 domains to allow secretion instead of retention on plasma membranes with the GPI anchor. Expressed products were purified to homogeneity by affinity chromatography using monoclonal antibodies specific for each domain and by reversed phase high pressure liquid chromatography. Purified domains were characterized by Western blotting, antibody binding and inhibition studies, amino-terminal sequence and amino acid analyses, and laser desorption/time of flight mass spectrometry. These analyses revealed that the monomeric N domain is of size 15,990, with a glycosylation mass of about 4100, in good agreement with two N-linked glycosyl units of about mass 2100. There is some evidence that the N domain forms dimers. The N domain reacted with antibodies specific for this domain with an affinity similar to that of intact CEA. The A3B3 domain had a mass of 34,462, with a glycosylation mass of 14,900, in good agreement with seven N-linked glycosylation sites of average mass 2100. The A3B3 domain reacted only with antibodies specific for this domain, with a slightly lower affinity than that of native CEA. The amino-terminal sequences of the N domain and A3B3 domain proteins demonstrated proper processing of the signal peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Five members of the human CEA gene family [human pregnancy-specific beta 1-glycoprotein (PS beta G); hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family.