Molecular cloning of cDNA for the carcinoma-associated antigen GA733-2.

Abstract

Defined by monoclonal antibody GA733, the GA733-2 antigen is a cell surface 40-kDa glycoprotein associated with human carcinomas of various origins. Molecular clones for the GA733-2 antigen were isolated from a colorectal carcinoma cell line cDNA library using the high-efficiency COS cell expression system. A 1.4-kilobase cDNA species was enriched by immunoselection with monoclonal antibody. The authenticity of individual clones was established by immunologic and sequence criteria. At the amino acid sequence level, GA733-2 was found to be >99% identical to the previously described KSA antigen defined by monoclonal antibody KS1/4. The amino acid sequence derived from the previously described GA733-related gene, GA733-1, was found to be 49% identical to GA733-2. The positions of 12 cysteine residues in the extracellular domains of the two GA733 antigens are conserved, as is the overall distribution of hydrophobic and hydrophilic residues. A 1.45-kilobase transcript of the GA733-2/KSA gene was found to be expressed in cell lines derived from colorectal and pancreatic carcinoma.in a colorectal carcinoma cell line. Both GA733-2 and GA733-1 sequences have significant homology to an exon-encoded repeat unit in thyroglobulin and the HLA-DR-associated invariant chain.Here we report the isolation of cDNA clones for the GA733-2 antigen using the high-efficiency COS cell expression system (7-9) and the sequence of the GA733-2 cDNA.t Molecular clones for the GA733-2 antigen were enriched from a colorectal carcinoma cell line cDNA library by iterating cycles of expression of the plasmid library in COS cells, immunoselection of antigen-positive cells by "panning" with the mAb GA733, and transfer of plasmid DNA from COS cells into Escherichia coli. The sequence of GA733-2 was compared to that of the carcinoma-associated antigen KSA (10, 11) and to the related GA733-1 antigen (6). A group of related mAbs (1, 12, 13) were assayed for reactivity to the antigen expressed by the GA733-2 clone. The size and expression of the GA733-2 mRNA in tumor cell lines were also studied.Monoclonal antibodies (mAbs) defining tumor-associated cell surface antigens are currently being evaluated for use in the diagnosis and therapy ofhuman cancer. Initial studies of mAbs C017-1A and GA733 (1), demonstrating cytotoxic activity in vitro and tumoricidal activity in experimental animals (for review, see ref.2), led to an evaluation of their immunotherapeutic potential in cancer patients. Idiotypic cascades have been demonstrated in cancer patients treated with mAb C017-1A, providing support to the hypothesis that antigen-specific anti-anti-idiotypic antibody (Ab3) responses may explain some of the observed delayed clinical responses (3).The independently derived mAbs GA733 and C017-1A define the same 40-kDa cell surface glycoprotein (4), hereafter referred to as the GA733-2 antigen. It is expressed by carcinomas of several histologic types and to some extent by normal tissues (5). The molecular cloning of cDNA for the tumor-associated GA733-2 an...

Metastasis-association of the rat ortholog of the human epithelial glycoprotein antigen EGP314

“…Within the extracellular part there is an EGF-like domain (AA 26 ± 48) and a thyroglobin-like repeat (AA 60 ± 122). Corresponding to the human and the mouse molecule (Bergsagel et al, 1992;Bjork et al, 1993;Szala et al, 1990b). D5.7A has 12 conserved cystein residues two consensus sequences for phosphorylation and two potential N-glycosylation sites.…”

“…Like EGP314 (Bergsagel et al, 1992;Bjork et al, 1993;Linnenbach et al, 1993;Perez and Walker, 1989;Strnad et al, 1989;Szala et al, 1990b) it is a type 1 transmembrane molecule with 12 conserved cystein residues and two N-glycosylation sites. Furthermore, an EGF-like domain and a thyroglobin-like repeat are conserved (Fornaro et al, 1995;Kiefer et al, 1991;Malthiery and Lissitzky, 1987).…”

“…C4.4A is a phophatidyl-inositol anchored molecule with partial sequence homology to uPAR (RoÈ sel et al, 1998). Here we report on the cloning and characterization of a molecule, named D5.7A, the ortholog to the murine and human panepithelial antigen EGP314, also known as EGP40, GA733-2, ESA, KSA, 17-1A antigen and Ep-CAM (Bergsagel et al, 1992;Bjork et al, 1993;Bumol et al, 1988;Edwards et al, 1986;Fernsten et al, 1990;GoÈ ttlinger et al, 1986;Lando et al, 1993;Larson et al, 1988;de Leij et al, 1994;Momburg et al, 1987;Nelson et al, 1996;Perez and Walker, 1989;Ross et al, 1984;Simon et al, 1990;Spurr et al, 1986;Stein et al, 1994;Strnad et al, 1989;Szala et al, 1990b). As revealed by transfection of non-metastasizing tumor cells, D5.7A appears to be a cell-cell adhesion molecule which may facilitate tumor progression by its growth promoting capacity upon ligand interaction.…”

Inhibition of tumor growth by recombinant vaccinia virus expressing GA733/CO17-1A/EpCAM/KSA/KS1-4 antigen in mice

“…Clones were tested for tumorigenicity in Balb / c mice. A tumorigenic clone was transfected with the human GA733 -2 cDNA 22 and selected with G418 (Gibco BRL, Grand Island, NY ). FACS analysis revealed H -2k d expression in $98% of the transfectants and GA733 Ag expression in $86% of the cells in vitro (not shown ).…”

A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood

“…MAbs directed against EGP-2, such as C017-lA, KS1/4 and MOC3 1, have been studied extensively in diagnostic and therapeutic approaches in cancer (Lobuglio et al, 1986;Elias et al, 1990; Kroesen et al, 1993;Herlyn et al, 1994;Kosterink et al, 1995). The human EGP2 cDNA was independently cloned by different research groups (Bumol et al, 1988;Strnad et al, 1989;Szala et al, 1990) and, more recently, its gene structure on chromosome 4q has been partly determined. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons interspersed by introns of variable length (Linnenbach et al, 1993).…”