STRATEGIES OF VACCINATION AGAINST MUC-1 ANTIGEN

这一修订申请的总体目标是测试针对MUC-1的疫苗接种新概念的免疫原和策略。MUC-1是一种高度糖基化的粘蛋白，表达于腺上皮细胞的管腔侧，由20个氨基酸组成的30-90个串联重复序列组成，其中存在一个免疫优势表位。在恶性转化过程中，粘蛋白过度表达，产生粘蛋白的细胞失去管腔极性，粘蛋白在细胞表面表达，使肿瘤细胞不太容易受到免疫效应机制的影响，有利于T细胞的凋亡。针对MUC-1的疫苗旨在产生两种效果：(1)具有足够特异性和亲和力的抗体可导致MUC-1抗原在肿瘤细胞表面的重新分布(通过交联和封盖)，从而恢复宿主对表达MUC-1的肿瘤细胞的全部免疫学潜力；(2)具有细胞毒活性的T细胞可杀死表达粘蛋白的肿瘤细胞，尤其是微转移细胞。本文提出的疫苗接种实验是基于实验室开发的方法和原理，旨在通过在抗体的高变量环中表达MUC-1寡肽来最大化它们的免疫原性，即固定在保守的免疫球蛋白折叠中的具有有限灵活性的结构。这种方法允许它们以构象受限单位的形式表达，这是诱导生物活性B细胞反应的必要前提。在抗体的高变环中表达的异源表位也是细胞内优先切割的部位，因此很容易产生激活T细胞的多肽。结合这种亲蛋白质工程方法的优点，将使用一种新开发的基于DNA的疫苗接种方法(体细胞转基因免疫)，通过这种方法，编码MUC-1表位的抗体基因本身在体内被用作疫苗来诱导抗体和T细胞。研究将在正常C57B16小鼠、人类MUC-1转基因小鼠中进行，以评估自我耐受提出的限制(如果有的话)，并在α(1-gt；3)半乳糖基转移酶敲除小鼠中进行研究，以确定自然产生的抗体(存在于这些KO小鼠中)是否限制了疫苗诱导特定细胞反应的程度。这种疫苗的效果将在正常和转基因小鼠的肿瘤保护(防止或抑制生长)实验中进行评估。

The overall goal of this revised application is to test immunogens and strategies of new conception for vaccination against MUC-1. MUC-1, a highly glycosylated mucin expressed on the luminal side of glandular epithelial, consists of 30-90 tandem repeats of 20 amino acids within which there exists an immunodominant epitope. During malignant transformation mucin is over-expressed, and mucin-producing cells lose luminal polarity with mucin becoming expressed all around the cell surface rendering tumor cells less susceptible to immune effector mechanisms and favoring apoptosis of T cells. Vaccination against MUC-1 is intended at generating two effects: (1) antibodies of sufficient specificity and avidity could cause redistribution (via cross-linking and capping) of the MUC-1 antigen at the surface of tumor cells hence reinstating the full immunological potential of the host against tumor cells expressing MUC-1; (2) T cells with cytotoxic activity that kill mucin-expressing tumor cells, particularly micro-metastasis. The vaccination experiments proposed herein are based on methods and principled developed in the laboratory and designed at maximizing the immunogenicity of MUC-1 oligopeptides by expressing them in the hyper-variable loops of antibodies, i.e., structures with limited flexibility anchored into the conserved immunoglobulin fold. This approach allows for their expression as conformationally-constrained units, a necessary prerequisite for the induction of biologically active B cell responses. Heterologous epitopes expressed in hyper-variable loops of antibodies are also site of preferential intracellular cleavage so that peptides for the activation of T cells are easily generated. Combined with the advantages of this pro protein engineering approach will be the use of a newly-developed method of DNA-based vaccination (Somatic Transgene Immunization), a method through which the genes of antibodies coding for MUC-1 epitopes are themselves utilized in vivo as vaccines to induce both antibodies and T cells. Studies will be done in normal C57B16 mice, in human MUC-1 transgenic mice to evaluate the limitations (if any) proposed by self tolerance, and in mice knock-out for the alpha (1->3) galactosyl transferase to determine if naturally-occurring antibodies (which are present in these KO mice) limit the extent to which specific cellular responses can be induced by vaccination. The efficacy of this type of vaccination will be evaluated in experiments of tumor protection (prevention or down-regulation of growth) in normal as well as transgenic mice.