STRATEGIES OF VACCINATION AGAINST MUC-1 ANTIGEN

本修订申请的总体目标是测试针对MUC-1的疫苗接种的新概念的免疫原和策略。MUC-1是一种高度糖基化的粘蛋白，表达于腺上皮的腔侧，由20个氨基酸的30-90个串联重复序列组成，其中存在免疫显性表位。在恶性转化期间，粘蛋白过度表达，并且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，使得肿瘤细胞对免疫效应机制不太敏感，并且有利于T细胞的凋亡。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合力的抗体可引起MUC-1抗原在肿瘤细胞表面的重新分布（通过交联和加帽），因此恢复宿主针对表达MUC-1的肿瘤细胞的全部免疫潜力;（2）具有细胞毒性活性的T细胞，其杀死表达粘蛋白的肿瘤细胞，特别是微转移。本文提出的疫苗接种实验是基于实验室中开发的方法和原理，并设计为通过在抗体的高变环中表达MUC-1寡肽来最大化MUC-1寡肽的免疫原性，即，具有有限柔性的结构锚定到保守的免疫球蛋白折叠中。这种方法允许它们作为构象约束单元表达，这是诱导生物活性B细胞应答的必要先决条件。在抗体的高变环中表达的异源表位也是优先细胞内切割的位点，使得容易产生用于活化T细胞的肽。与这种前蛋白工程方法的优点相结合，将使用新开发的基于DNA的疫苗接种方法（体细胞转基因免疫），通过该方法，编码MUC-1表位的抗体的基因本身在体内用作疫苗以诱导抗体和T细胞。研究将在正常C57 B16小鼠中进行，在人MUC-1转基因小鼠中进行以评估由自身耐受性提出的限制（如果有的话），并在敲除α（1->3）半乳糖基转移酶的小鼠中进行以确定天然存在的抗体（其存在于这些KO小鼠中）是否限制疫苗接种可诱导的特异性细胞应答的程度。将在正常小鼠和转基因小鼠的肿瘤保护（预防或下调生长）实验中评价此类疫苗接种的效力。

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.