Xenograft-like rejection of tumors in a-gal glycolipids

描述(申请人提供)：治疗难治性病变是实体瘤患者死亡的主要原因。尽管肿瘤细胞表达各种肿瘤相关抗原(TAA)，但由于肿瘤内的免疫抑制微环境、调节性T细胞(Treg)的活性以及抗原提呈细胞(APC)缺乏适当的TAA递呈，这些细胞通常对免疫系统“隐藏”。我们提出了一种新的免疫疗法，通过瘤内注射携带α-Gal表位的神经鞘糖脂胶束(Gala1-3Galb1-4GlcNAc-R)(命名为GSL？Gal)来破坏这种病变并将其转化为内源性肿瘤疫苗。由于天然的抗Gal抗体(人类免疫球蛋白的1%)与GSL？Gal的α-Gal表位结合，该注射可引起肿瘤内强烈的炎症反应。抗-Gal与非灵长类哺乳动物(如猪)细胞上自然表达的α-Gal表位结合可诱导人和猴子对异种移植的快速排斥反应。我们假设瘤内注射GSL？Gal将以类似于异种移植排斥反应的机制导致病变的破坏，并将诱导针对微转移的保护性免疫反应，其效力足以克服Treg细胞的抑制作用。GSL？Gal可能通过以下机制发挥作用：1.通过抗Gal/GSL？Gal相互作用激活补体，产生趋化因子。2.在异种移植排斥反应中，GSL？Gal插入细胞膜，导致抗Gal介导的肿瘤细胞破坏。3.抗Gal对肿瘤细胞的调理作用，靶向它们被炎症征集到肿瘤中的APC有效摄取，从而将肿瘤转化为内源性疫苗。我们对缺乏α-Gal表位并产生抗Gal的基因敲除小鼠的初步研究表明，在很大比例的小鼠中，治疗后的黑色素瘤病变被破坏并转化为疫苗。1.研究GSL-Gal诱导B16黑色素瘤皮损内炎症反应的机制。2.探讨Treg细胞在预防GSL？Gal治疗无效的小鼠诱导局部和全身保护性抗肿瘤免疫反应中的作用。3.在自发性乳腺癌模型中，观察GSL-Gal瘤内注射对原发灶形成的保护作用，以及在诱导保护性免疫反应防止其他病变和转移方面的作用。这些研究的成功将使我们能够建立GSL？GAL在有化疗难治性皮损的癌症患者中的临床试验。 我们的目标是开发一种新的方法来治疗复发的化疗难治性实体肿瘤患者，通过向病变内注射一种名为糖鞘糖脂的物质，该物质含有一种名为a-半乳糖表位的碳水化合物结构。这种物质被称为GSL？Gal。这种疗法利用了这样一个事实，即所有人类都有一种名为抗Gal的天然抗体，含量非常高。该抗体与GSL？Gal表面的α-Gal表位相互作用，引起局部炎症。瘤内注射GSL？Gal可在抗Gal抗体的辅助下破坏病变，并将治疗后的病变转化为自身疫苗。这种疫苗“教育”免疫系统将微转移中的肿瘤细胞(不同器官中的小肿瘤细胞群)识别为必须被摧毁的细胞。我们将在一种独特的小鼠模型中研究这种治疗方法，该模型模拟相关的人类免疫学参数。老鼠模型研究的成功将使我们能够将这种治疗方法应用于癌症患者。

DESCRIPTION (provided by applicant): Therapy refractory lesions are the main cause of death in solid tumor patients. Although the tumor cells in such lesions express various tumor associated antigens (TAA), they are usually "concealed" from the immune system because of immunosuppressive microenvironment within the tumor, activity of regulatory T (Treg) cells and lack of appropriate presentation of TAA by antigen presenting cells (APC). We propose a novel immunotherapy to destroy such lesions and convert them into endogenous tumor vaccines by intratumoral injection of micelles of glycosphingolipids bearing a-gal epitopes (Gala1-3Galb1-4GlcNAc-R) (designated GSL?gal). This injection induces a strong intratumoral inflammatory reaction due to binding of the natural anti- Gal antibody (1% of immunoglobulins in humans) to a-gal epitopes of GSL?gal. Anti-Gal binding to a-gal epitopes naturally expressed on cells of non-primate mammals (e.g. pigs) induces rapid rejection of xenografts in humans and monkeys. We hypothesize that intratumoral injection of GSL?gal will result in destruction of lesions in a mechanism similar to xenograft rejection and will induce a protective immune response against micrometastases, potent enough to overcome the suppressive effect of Treg cells. The effects of GSL?gal will be mediated by the following mechanisms: 1. Chemotactic factors generation due to complement activation by Anti-Gal/GSL?gal interaction. 2. Insertion of GSL?gal into cell membranes, resulting in anti-Gal mediated destruction of tumor cells as in xenograft rejection. 3. Opsonization of tumor cell by anti-Gal, targeting them for effective uptake by APC recruited into the tumor by the inflammation, thereby converting tumors into endogenous vaccines. Our preliminary studies in knockout mice lacking a-gal epitopes and producing anti-Gal, demonstrated destruction of treated melanoma lesions and their conversion into vaccine in a large proportion of mice. Our specific aims are: 1. To study the mechanisms by which GSL?gal induces inflammation within of B16 melanoma lesions. 2. To evaluate the role of Treg cells in preventing induction of a local and systemic protective anti-tumor immune response in the ~30% of mice that do not respond to GSL?gal treatment. 3. To determine in a model of spontaneous mammary carcinoma the protective effect of intratumoral GSL?gal injection in the development of the primary lesion and on induction of a protective immune response preventing additional lesions and metastases. Success in these studies will enable us to establish clinical trials with GSL?gal in cancer patients with chemotherapy refractory lesions. Our objective is to develop a novel method for treating cancer patients with recurrent chemotherapy refractory solid tumors, by injection into the lesion a substance called glycosphingolipids containing a carbohydrate structure called the a-gal epitope. This substance is referred to as GSL?gal. The treatment exploits the fact that all humans have a natural antibody called anti-Gal in very large amounts. This antibody interacts with a-gal epitopes on GSL?gal and induces local inflammation. Injection of GSL?gal into tumor lesions destroys the lesions with the assistance of anti-Gal and convert treated lesions into a self vaccine. This vaccine "educates" the immune system to recognize tumor cells in micrometastases (small groups of tumor cells in various organs) as cells that have to be destroyed. We will study this treatment in a unique mouse model that simulates the pertinent human immunological parameters. Success in the studies in the mouse model will enable us to apply this treatment to cancer patients.