IMMUNOTOXIN THERAPY OF OLID TUMORS--PRECLINICAL STUDIES

我们通过融合Fv片段生产重组免疫毒素 一种名为PE38的假单胞菌外毒素A突变形式的抗体。为 免疫毒素疗法或其他癌症靶向疗法 如果成功，就有必要检测实体瘤上的抗原靶。 我们为发现新的抗原性靶标所做的努力概述如下： (1)卵巢癌和间皮瘤表达分化抗原 名为间甲硫磷。我们已经使用噬菌体展示技术来识别新的 抗间硫蛋白的单链抗体。其中一个与高密度脂蛋白结合 亲和力(Kd约为10 nm)。一种含有这种FV的免疫毒素非常 对含有间充质蛋白的细胞具有细胞毒性。进一步的临床前评估 正在进行中。(2)胶质母细胞瘤：用噬菌体展示技术分离 Fv(MR1)通常与EGF受体的突变形式结合 出现在胶质母细胞瘤和一些癌症上。MR1(FV)PE38仅 对表达突变受体的细胞具有细胞毒性。诱变 正在使用程序来增加MR1的亲和力。(3)前列腺 癌症：我们构建了常规和单链免疫毒素 单抗E4，被认为是前列腺特异性的。免疫毒素是 对前列腺癌细胞株和其他几种细胞的细胞毒作用 上皮性癌症。不幸的是，最近的研究表明，单抗E4可能 与正常肠道反应。(4)细胞内突变蛋白：许多 作为癌基因的突变蛋白位于细胞内。自.以来 由这些蛋白质衍生的突变多肽位于细胞上。 表面上应该有可能产生识别这些细胞的抗体 并利用这些化合物将细胞毒剂靶向细胞。至 确定这样的方法是否可行，我们已经制作了一个重组 具有特异性识别MHC多肽的Fab的免疫毒素 复杂，但不只是MHC或多肽。这个重组体 免疫毒素仅对表达该特定复合体的细胞具有细胞毒性作用。 这些研究表明，细胞毒剂可以被设计成识别 细胞内蛋白质以MHC-肽复合体的形式出现在细胞内 也表明识别MHC-肽的抗体 癌细胞上的复合体可能对癌症治疗有用。我们是 试图分离出这样的抗体。 研制了一种二硫键稳定的免疫毒素(E23(DsFv)PE38)。 与乳腺癌和几个 其他类型的癌症。该代理产生完全回归的 异种移植物在裸鼠体内生长，灵长类动物对其耐受性良好。一个 该免疫毒素的临床批次已经制备完毕，将进行IND 于1997年底提交申请。 由于免疫毒素是如此强大和特定的细胞毒剂，它们 除了癌症治疗外，还有其他用途。我们已经开发出一种方法 使用免疫毒素(抗Tac(Fv)PE38)建立新的疾病模型 治疗表达人白介素2受体的转基因小鼠 组织特异性方式。在最近的一项实验中，患有外周疾病的小鼠 已经产生了自主神经病变。

We produce recombinant immunotoxins by fusing the Fv fragments of antibodies to a mutant form of Pseudomoans exotoxin A termed PE38. For immunotoxin therapy or other targeted therapies of cancer to be successful, it is necessary to detect antigenic targets on solid tumors. Our efforts to detect new antigenic targets are summarized as follows: (1) Ovarian cancers and mesotheliomas express a differentiation antigen termed mesothelin. We have used phage display techniques to identify new single chain antibodies to mesothelin. One of these binds with high affinity (Kd approximate 10 nm). An immunotoxin containing this Fv is very cytotoxic to mesothelin containing cells. Further preclinical evaluation is in progress. (2) Glioblastomas: Phage display was used to isolate an Fv (MR1) that binds to a mutant form of the EGF receptor commonly present on glioblastomas and some carcinomas. MR1(Fv)PE38 is only cytotoxic to cells expressing the mutant receptor. Mutagenesis procedures are being used to increase the affinity of MR1. (3) Prostate cancer: We constructed conventional and single chain immunotoxins with MAb E4 which was thought to be prostate specific. The immunotoxins were cytotoxic to prostate cancer cell lines and also to several other epithelial cancers. Unfortunately recent studies show that MAb E4 may react with normal intestine. (4) Intracellular mutant proteins: Many mutant proteins that act as oncogenes are located within the cell. Since mutant peptides derived from these proteins are located on the cell surface it should be possible to produce antibodies that recognize these complexes and use these to target cytotoxic agents to the cell. To determine if such an approach is feasible, we have made a recombinant immunotoxin with a Fab that specifically recognizes a MHC-peptide complex but not the MHC or the peptide alone. This recombinant immunotoxin was only cytotoxic to cells expressing the specific complex. These studies show that cytotoxic agents can be designed that recognize intracellular proteins displayed as MHC- peptide complexes on the cell surface, and also indicate that antibodies which recognize MHC-peptide complexes on cancer cells could be useful for cancer therapy. We are trying to isolate such antibodies. A disulfide stabilized immunotoxin (e23(dsFv)PE38) has been made that binds to the erb B2 oncoprotein present on breast cancers and several other types of cancer. The agent produces complete regressions of xenografts growing in nude mice and is well tolerated by primates. A clinical batch of the immunotoxin has been prepared and an IND will be filed in late 1997. Because immunotoxins are such potent and specific cytotoxic agents, they have other uses besides cancer therapy. We have developed an approach to create new disease models by using an immunotoxin (anti-Tac(Fv)PE38) to treat transgenic mice which express the human IL2 receptor in a tissue specific manner. In a recent experiment, mice with peripheral autonomic neuropathy have been produced.