STRATEGIES OF VACCINATION AGAINST MUC-1 ANTIGEN

本修订后的应用程序的总体目标是测试免疫原和策略的新概念接种针对MUC-1。muc1是一种高度糖基化的粘蛋白，表达于腺上皮的管腔侧，由30-90个20个氨基酸的串联重复序列组成，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腔极性，粘蛋白在细胞表面周围表达，使肿瘤细胞对免疫效应机制不敏感，有利于T细胞凋亡。针对MUC-1的疫苗接种旨在产生两种效果：(1)具有足够特异性和亲和力的抗体可以引起肿瘤细胞表面MUC-1抗原的重新分布（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潜能；(2)具有细胞毒活性的T细胞，能够杀死表达黏液蛋白的肿瘤细胞，特别是微转移细胞。本文提出的疫苗接种实验基于实验室开发的方法和原则，旨在通过在抗体的超可变环中表达MUC-1寡肽来最大化其免疫原性，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。这种方法允许它们作为构象约束单位表达，这是诱导具有生物活性的B细胞反应的必要先决条件。在抗体的高可变环中表达的异源表位也是细胞内优先切割的位点，因此很容易产生用于激活T细胞的肽。结合这种前蛋白工程方法的优势，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），该方法通过编码MUC-1表位的抗体基因本身在体内作为疫苗来诱导抗体和T细胞。研究将在正常C57B16小鼠中进行，在人类MUC-1转基因小鼠中进行，以评估自我耐受性提出的局限性（如果有的话），并在敲除α(1->3)半乳糖转移酶的小鼠中进行，以确定自然产生的抗体（存在于这些KO小鼠中）是否限制了接种疫苗诱导特异性细胞反应的程度。这类疫苗的功效将在正常小鼠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.