Ohno Yoshiya, Suda Kentaro, Masuko Kazue, Yagi Hideki, Hashimoto Yoshiyuki, Masuko Takashi
Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502, Japan.
Cancer Sci. 2008 May;99(5):1000-7. doi: 10.1111/j.1349-7006.2008.00770.x. Epub 2008 Feb 24.
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.
L型大氨基酸转运体（LAT）1是分化簇98（CD98）的首个被鉴定的轻链（lc），它与CD98的重链（hc）相关联，并表达于各种肿瘤细胞表面，无论其来源如何。由于LAT1是一种12次跨膜蛋白，其可能具有免疫原性的细胞外区域非常小，因此尚未开发出特异性单克隆抗体（mAb）。我们报告了识别人类LAT1蛋白细胞外结构域的单克隆抗体的成功制备及其特性。从通过将小鼠骨髓瘤细胞与用表达与人LAT1蛋白融合的重组绿色荧光蛋白的RH7777大鼠肝癌细胞免疫的大鼠脾细胞融合而建立的杂交瘤克隆中筛选出两种单克隆抗体。命名为SOL22和SOL69，这些单克隆抗体与用LAT1 cDNA转染的细胞上的LAT1细胞外结构域特异性反应，但不与用其他CD98 lc的cDNA转染的细胞反应，即LAT2、γ(+)LAT1、γ(+)LAT2和xCT氨基酸转运体。在从人HeLa肿瘤细胞制备的提取物中，这些单克隆抗体在还原条件下免疫沉淀了35 kDa和90 kDa的蛋白质，表明在90 kDa的重链（hc）和35 kDa的轻链（LAT1）中的半胱氨酸残基之间存在分子间二硫键。SOL22和SOL69单克隆抗体与多种未固定的活人类肿瘤细胞系反应，但与HEK293F人胚肾细胞和人外周血细胞的反应较弱。用抗CD98重链和抗LAT1对正常人体组织进行的比较免疫组织化学分析表明，LAT1是抗体治疗的极佳分子靶点，甚至可能优于CD98重链。
