Production and characterization of highly tumor-specific rat monoclonal antibodies recognizing the extracellular domain of human L-type amino-acid transporter 1.
L-type large amino acid transporter (LAT) 1, the first light chain (lc) of cluster of differentiation 98 (CD98) to be identified, is associated with the heavy chain (hc) of CD98 and expressed on the surface of various tumor cells irrespective of their origin. Because LAT1 is a 12-pass membrane protein and its possible immunogenic extracellular region is very small, specific monoclonal antibodies (mAb) had not been developed. We report the successful preparation and characterization of mAb recognizing the extracellular domain of human LAT1 protein. Two mAb were selected from hybridoma clones established by fusing mouse myeloma cells and spleen cells from rats immunized against RH7777 rat hepatoma cells expressing recombinant green fluorescent protein fused to human LAT1 protein. Designated SOL22 and SOL69, these mAb specifically reacted with the extracellular domain of LAT1 on cells transfected with cDNA of LAT1, but not with cells transfected with cDNA of other CD98 lc, namely, LAT2, y(+)LAT1, y(+)LAT2, and xCT amino acid transporters. These mAb immunoprecipitated 35- and 90-kDa proteins under reducing conditions in extracts prepared from human HeLa tumor cells, indicating the existence of intermolecular disulfide bonds between cysteine residues in the 90-kDa hc and 35-kDa lc (LAT1). SOL22 and SOL69 mAb reacted with a wide variety of living unfixed human tumor cell lines, but were only weakly reactive with HEK293F human embryonic kidney cells and human peripheral blood cells. Comparative immunohistochemical analyses of normal human tissues with anti-CD98 hc and anti-LAT1 revealed LAT1 to be an excellent molecular target for antibody therapy, possibly even superior to CD98 hc.
Ohno
Yoshiya
Y
Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502, Japan.
Suda
Kentaro
K
Masuko
Kazue
K
Yagi
Hideki
H
Hashimoto
Yoshiyuki
Y
Masuko
Takashi
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000276:immunology*; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011916:Rats, Inbred F344; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D027283:Large Neutral Amino Acid-Transporter 1; D009363:Neoplasm Proteins; D049452:Green Fluorescent Proteins