Ragupathi Govind, Damani Payal, Srivastava Geeta, Srivastava Om, Sucheck Steven J, Ichikawa Yoshi, Livingston Philip O
Laboratory of Tumor Vaccinology, Melanoma and Sarcoma Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Cancer Immunol Immunother. 2009 Sep;58(9):1397-405. doi: 10.1007/s00262-008-0654-7. Epub 2009 Feb 4.
Sialyl Lewis(a) (sLe(a)), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe(a) is known to be the ligand for endothelial cell selectins suggesting a role for sLe(a) in cancer metastases and adhesion. For these reasons, sLe(a) may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe(a) is structurally similar to sLe(x) and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe(a) will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe(a) hexasaccharide and its conjugation to KLH to construct a sLe(a)-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe(a)-HSA by ELISA and against sLe(a) positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe(a) plus GPI-0100 or unconjugated sLe(a) mixed with KLH plus GPI-0100 failed to produce antibodies against sLe(a). However, mice immunized with sLe(a)-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe(a)-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe(a) by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe(a) positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le(y), Le(x), and sLe(x) by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe(a) positive cancer in clinical settings.
唾液酸化路易斯抗原（a）（sLe(a)），也被称为CA19-9抗原，可被鼠源单克隆抗体mAb19-9识别，在癌细胞表面以糖脂和O-连接糖蛋白的形式表达。它在包括结肠癌和胰腺癌在内的多种胃肠道上皮恶性肿瘤、乳腺癌和小细胞肺癌中高表达，但在正常组织中表达有限。已知sLe(a)是内皮细胞选择素的配体，提示sLe(a)在癌症转移和黏附中发挥作用。基于这些原因，sLe(a)可能是抗体介导的免疫治疗（包括单克隆抗体和肿瘤疫苗）的良好靶点。然而，sLe(a)在结构上与sLe(x)以及其他与血型相关的碳水化合物相似，这些碳水化合物在多形核白细胞和其他循环细胞上广泛表达，这引发了人们对针对sLe(a)进行免疫接种会诱导产生与这些更广泛表达的自身抗原发生反应的抗体的担忧。我们之前在小鼠和患者中均已表明，将多种碳水化合物癌症抗原与钥孔戚血蓝蛋白（KLH）偶联，并将这种偶联物与皂苷佐剂QS-21或GPI-0100混合给药，是诱导针对这些癌症抗原的抗体的最有效方法。我们在此首次描述了sLe(a)六糖戊烯基糖苷的全合成及其与KLH的偶联，以构建sLe(a)-KLH偶联物。将五组小鼠在6周内皮下接种四次。通过ELISA检测血清对sLe(a)-人血清白蛋白（HSA）的反应，并通过荧光激活细胞分选术（FACS）检测血清对sLe(a)阳性人细胞系腺癌SW626和小细胞肺癌（SCLC）DMS79的反应。正如预期的那样，用未偶联的sLe(a)加GPI-0100或未偶联的sLe(a)与KLH加GPI-0100免疫的小鼠未能产生针对sLe(a)的抗体。然而，用不含GPI-0100的sLe(a)-KLH偶联物免疫的小鼠产生了低水平的抗体，而用sLe(a)-KLH加GPI-0100免疫的小鼠通过ELISA产生了针对sLe(a)的显著更高滴度的IgG和IgM抗体。通过FACS检测，这些抗体具有高反应性，并介导了对sLe(a)阳性SW626和DMS79细胞的强效补体介导的细胞毒性。通过斑点印迹免疫染色，它们对一系列其他与血型相关的抗原，包括Le(y)、Le(x)和sLe(x)，未显示出可检测到的交叉反应性。这种疫苗已准备好在临床环境中作为治疗sLe(a)阳性癌症的主动免疫疗法进行测试。
