Genetic Cancer Vaccines

鉴于是否有可能对独特型进行免疫的一般问题已经通过已完成的II期试验得到了回答，并且已经设计了随机对照的III期临床试验来回答临床疗效问题，第三个主要研究目标是简化这些个体化疫苗的生产，使这种疗法更加实用。因此，我们已经利用新技术和疫苗递送系统来设计用于将独特型（否则为非免疫原性抗原）配制成免疫原性疫苗的替代方法，并在临床前同基因小鼠淋巴瘤模型中对其进行测试。 任何不需要蛋白质表达的递送系统对于简化疫苗生产的目标具有巨大的潜力。在我们看来，DNA疫苗接种最吸引人的方面是它的简单性和疫苗生成的容易性，尽管有限的效力可能是一个限制。通过DNA疫苗接种体内表达编码肿瘤抗原的外源基因仅需要将基因在真核或病毒元件的调节元件下克隆到表达盒中，然后通过肌内或皮内施用途径将其注射到溶液中或通过DNA包被的金颗粒的颗粒介导的轰击（基因枪）递送到表皮中。特别地，抗体工程的进展使得容易地鉴定和克隆鼠和人IG可变区基因，包括来自B细胞恶性肿瘤的特异性V基因成为可能。一旦V基因被克隆，它们可以组合成单链Fv（sFv）形式，编码仅由用短的15个氨基酸接头框内连接在一起的VH和VL基因组成的单一多肽。 趋化性细胞因子（Chemotactic cytokines）被认为是调节专职APC（包括DC）选择性地通过外周淋巴结到达淋巴结的关键效应分子之一。趋化因子是一组小的分泌性蛋白质（7-15 kDa），其通过协调血液传播的白细胞的选择性迁移、渗出和活化来诱导炎症反应。已有研究表明，单核细胞趋化蛋白（MCP-3）、巨噬细胞炎性蛋白（MIP）-1 α、巨噬细胞衍生趋化因子（MDC）和基质细胞衍生因子（SDF）-1等趋化因子对DC具有趋化作用。趋化因子通过与特定的细胞表面七螺旋G蛋白偶联受体结合而起作用，该受体在与配体结合后内化。DC表达多种趋化因子受体，包括CCR 1、CCR 2、CCR 5、CXCR 1和CXCR 3。 我们探索了一种新的假设，即通过编码由淋巴瘤独特型（sFv）融合到促炎趋化因子部分（作为KLH的替代品）组成的融合蛋白，可以大大提高体内DNA疫苗接种的效率。假设抗肿瘤免疫可以通过用由趋化因子和肿瘤抗原组成的融合蛋白在体内靶向APC来触发（Nat Biotech 17：253-258，1999）。具体地，该想法是表达的sFv-趋化因子蛋白将靶向APC，用于趋化因子受体介导的sFv抗原的结合、摄取和加工，用于随后呈递给CD 4+和/或CD 8 + T细胞。 该策略已在两种不同的B细胞淋巴瘤38 C-13和A20上进行了测试，它们分别表达表面IgM和IgG 2a。通过RT/PCR将相应的sFv克隆为与作为原型的促炎趋化因子基因MCP-3和IP-10的融合物。趋化因子融合体保留了生物学功能，并能结合趋化因子受体，在体外和体内诱导趋化性。这些融合物已经在两种肿瘤模型中作为蛋白质和DNA疫苗进行了测试。具体地，用编码IP-10-或MCP-3-sFv融合物的质粒（而不是单独的sFv）通过基因枪免疫的小鼠诱导针对大的肿瘤攻击（20倍于最小致死剂量）的保护性抗肿瘤免疫。此外，T细胞亚群耗竭实验表明，MCP-3-sFv融合诱导效应CD 4+和CD 8 + T细胞，这是保护所需的。此外，在两种肿瘤模型中的保护水平与原型Id-KLH蛋白的保护水平相当，并且在A20模型中可能上级。 后两个特征将这些sFv-趋化因子融合物与其他报道的DNA Id疫苗区分开来。特别是，CD 8 + T细胞免疫的产生将这些融合与先前报道的淋巴瘤Id与GM-CSF的融合区分开来，后者仅引起抗体应答。另一项最近的研究报道，用编码与破伤风毒素片段C融合的sFv的DNA接种疫苗引发了CD 4 + T细胞介导的保护性免疫应答。然而，没有发现诱导CD 8 + T细胞的证据。

Given that the general question of whether it is possible to immunize against idiotype has been answered by the completed Phase II trial and that a randomized, controlled Phase III clinical trial has been designed to answer the question of clinical efficacy, a third major research objective is to streamline the production of these individualized vaccines to make this therapy more practical. Accordingly, we have taken advantage of novel technologies and vaccine delivery systems to design alternative methods for formulating idiotype, an otherwise nonimmunogenic antigen, into an immunogenic vaccine and tested them in preclinical, syngeneic murine lymphoma models. Any delivery system which does not require protein expression holds tremendous potential for the goal of streamlining vaccine production. In our view, the most appealing aspect of DNA vaccination is its simplicity and ease of vaccine generation, although limited potency may be a limitation. In vivo expression of foreign genes encoding the tumor antigen by DNA vaccination requires only that the gene is cloned under regulatory elements of eukaryotic or viral elements into an expression cassette, which is then either injected in solution via intramuscular or intradermal routes of administration or delivered into the epidermis by particle mediated bombardment of DNA-coated gold particles (gene gun). In particular, advances in antibody engineering have made it possible to readily identify and clone both murine and human Ig variable region genes, including specific V-genes from B-cell malignancies. Once V-genes are cloned, they can be combined into a single chain Fv (sFv) format, encoding a single polypeptide consisting solely of VH and VL genes linked together inframe with a short, 15 amino acid linker. Chemotactic cytokines, termed chemokines, are thought to be among the key effector molecules regulating the trafficking of professional APC, including DC, selectively through peripheral tisues to reach lymph nodes. Chemokines are a group of small secreted proteins (7-15 kDa) that induce inflammatory responses by orchestrating the selective migration, diapedesis and activation of bloodborne leukocytes. Several chemokines, such as monocyte chemotactic protein (MCP-3), macrophage inflammatory protein (MIP)-1 alpha, macrophage-derived chemokine (MDC) and stromal cell-derived factor (SDF)-1 have been reported to be chemotactic for DC. Chemokines act by binding to a specific cell-surface heptahelical G-protein-coupled receptor, which is internalized after binding with the ligand. DC express a variety of chemokine receptors, including CCR1, CCR2, CCR5, CXCR1, and CXCR3. We explored a novel hypothesis, that the efficiency of DNA vaccination in vivo could be greatly increased by encoding a fusion protein consisting of lymphoma idiotype (sFv) fused to a proinflammatory chemokine moiety (as a substitute for KLH). The hypothesis is that anti-tumor immunity can be triggered by targeting APC in vivo with a fusion protein consisting of chemokine and tumor antigen (Nat Biotech 17:253-258, 1999). Specifically, the idea is that the expressed sFv-chemokine protein will be targeted to APC for chemokine receptor-mediated binding, uptake, and processing of sFv antigen for subsequent presentation to CD4+ and/or CD8+ T cells. The strategy has been tested on two different B-cell lymphomas, 38C-13 and A20, which express surface IgM and IgG2a, respectively. The respective sFv were cloned by RT/PCR as fusions to pro-inflammatory chemokine genes MCP-3 and IP-10, as prototypes. Chemokine fusions retained biologic function and could bind chemokine receptors and induce chemotaxis both in vitro and in vivo. These fusions have been tested as protein and DNA vaccines in both tumor models. Specifically, mice immunized by gene gun with plasids encoding IP-10- or MCP-3-sFv fusions, but not sFv alone, induced protective anti-tumor immunity against a large tumor challenge (20 times the minimum lethal dose). Moreover, T-cell subset depletion experiments revealed that MCP-3-sFv fusions induced effector CD4+ and CD8+ T cells, which were required for the protection. Furthermore, the level of protection was equivalent to that of the prototype Id-KLH protein in both tumor models, and may have been superior in the A20 model. These latter two features distinguish these sFv-chemokine fusions from other reported DNA Id vaccines. In particular, the generation of CD8+ T-cell immunity distinguishes these fusions from previously reported fusions of lymphoma Id with GM-CSF which elicited exclusively antibody responses. Another recent study reported that vaccination with DNA encoding sFv fused with fragment C of tetanus toxin elicited a CD4+ T-cell-mediated protective immune response. However, evidence for the induction of CD8+ T cells could not be found.