Ando Hiroshi, Matsushita Takefumi, Wakitani Masako, Sato Takashi, Kodama-Nishida Sachiko, Shibata Kenji, Shitara Kenya, Ohta So
Antibody Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Machida, Tokyo, Japan.
Biol Pharm Bull. 2008 Sep;31(9):1739-44. doi: 10.1248/bpb.31.1739.
Tn-antigen (alpha-N-acetyl-galactosamine(GalNAc)-Ser/Thr) is a cancer-associated carbohydrate antigen expressed in various epithelial and hematological cancers, and although a number of anti-Tn IgG and IgM antibodies have been generated, they have not been fully validated for cancer immunotherapy. In this study, we generated a novel murine anti-Tn IgG1 monoclonal antibody, KM3413, by immunization of mucins purified from a culture supernatant of LS180: a human colon cancer cell line. The binding of KM3413 was detected against consecutive Tn-antigens (Tn3 and Tn2), but not against monovalent antigens (Tn1). The affinity (K(D)) of KM3413 was determined to be about 10(-7) M with BIAcore. Cross-reactivity against type-A blood antigen, which shares a sugar residue, alpha-linked GalNAc, with Tn-antigen, was not detected. Next, we generated mouse-human chimeric IgG1 of KM3413 (cKM3413) and evaluated its anti-tumor activities against Jurkat: a human T-lymphoid leukemia cell line. In vitro assay revealed that cKM3413 induced antibody-dependent cellular cytotoxicity (ADCC) and direct killing activity with cross-link antibody. Furthermore, treatment of cKM3413 (1 or 10 mg/kg) showed significantly better survival of Jurkat-inoculated C.B-17/lcr-scid Jcl mice compared with controls using PBS treatment (p<0.001). These results suggest that humanized antibody against clustered Tn-antigens is a promising therapeutic antibody against Tn-positive cancers.
Tn抗原（α-N-乙酰半乳糖胺（GalNAc）-丝氨酸/苏氨酸）是一种与癌症相关的碳水化合物抗原，在各种上皮癌和血液系统癌症中表达。尽管已经产生了多种抗Tn IgG和IgM抗体，但它们尚未在癌症免疫治疗中得到充分验证。在本研究中，我们通过免疫从人结肠癌细胞系LS180的培养上清液中纯化的粘蛋白，产生了一种新型鼠抗Tn IgG1单克隆抗体KM3413。检测到KM3413与连续的Tn抗原（Tn3和Tn2）结合，但不与单价抗原（Tn1）结合。用BIAcore测定KM3413的亲和力（K(D)）约为10(-7) M。未检测到与A型血抗原的交叉反应性，A型血抗原与Tn抗原共享一个糖残基，即α-连接的GalNAc。接下来，我们产生了KM3413的鼠-人嵌合IgG1（cKM3413），并评估了其对人T淋巴细胞白血病细胞系Jurkat的抗肿瘤活性。体外试验表明，cKM3413通过交联抗体诱导抗体依赖性细胞毒性（ADCC）和直接杀伤活性。此外，与使用PBS处理的对照组相比，用cKM3413（1或10 mg/kg）处理的接种Jurkat的C.B-17/lcr-scid Jcl小鼠的存活率显著提高（p<0.001）。这些结果表明，针对聚集Tn抗原的人源化抗体是一种有前途的抗Tn阳性癌症的治疗性抗体。
