Metabolic Engineering of Bacteria for Cancer Immunotherapy by Gamma Delta T Cells

描述（由申请人提供）： 在美国和全世界，癌症的发病率和死亡率在发达国家和发展中国家都急剧增加。2007年，癌症导致约13%的人类死亡，760万人死于癌症。在美国，死于肺癌的人数比乳腺癌、结肠癌、肾癌和前列腺癌的总和还要多。最近的研究表明，退伍军人患肺癌的可能性比未在军队服役的人高25%至75%;然而，肺癌和其他实体瘤的治疗仍然有限。早在20世纪初，Coley就成功地使用热灭活链球菌和沙雷氏菌的混合物来治疗各种肉瘤和其他癌症。今天使用活BCG的类似治疗有助于预防膀胱癌复发。这些疗法的有效性提供了证据，证明有可能通过用细菌激活免疫系统来治疗恶性肿瘤。目前正在尝试各种各样的方法来使用肿瘤特异性肽抗原通过12 T细胞刺激肿瘤免疫。然而，由于CD 4和CD 8 12 T细胞以MHC限制的方式识别这些与MHC分子结合的肽，因此使用12 T细胞的免疫疗法需要针对每个MHC单倍型进行个体化。 相比之下，34 T细胞代表了一个独特的T细胞亚群，通过以MHC不受限制的方式识别非肽抗原来桥接先天性免疫和获得性免疫。人34 T细胞的主要亚群使用其V32 V42 T细胞受体来识别外来微生物类异戊二烯代谢物HMBPP和自身代谢物IPP。V32 V42 T细胞在各种感染的血液中扩增，然后在外周部位积累。HMBPP的识别激活34 T细胞产生Th 1细胞因子，杀死感染的细胞，并产生生长因子来修复粘膜表面。人类34 T细胞也有助于调节恶性肿瘤。一旦激活，V32 V42 T细胞使用其TCR和NK受体识别并杀死各种肿瘤细胞，而不管其组织来源、MHC表达或MHC单倍型。由于V32 V42 T细胞也表达CD 16免疫球蛋白Fc受体，它们杀死由抗肿瘤抗体致敏的肿瘤细胞。近年来，用异戊烯焦磷酸盐或双膦酸盐和IL-2单独或与利妥昔单抗联合刺激V32 V42 T细胞用于淋巴瘤的免疫疗法已显示出前景，因为这些治疗导致淋巴瘤和转移性肾癌或前列腺癌患者的完全和部分缓解或稳定的疾病。然而，重复免疫导致无反应性和V32 V42 T细胞的缺失。 细菌的代谢工程是一个新的研究领域，到目前为止，一直专注于改变细菌用于药物或化学合成或用于替代燃料的产生。细菌代谢的定向变化是通过改变特定的生化反应或引入新的生化反应来实现的。没有一个研究小组试图通过代谢工程改造细菌来获得34种疫苗。我们现在提出通过代谢工程沙门氏菌和李斯特菌细菌过量生产HMBPP来开发用于V32 V42 T细胞癌免疫治疗的新疫苗。这两个物种都已用于癌症疫苗，但差异显著，因为革兰氏阴性沙门氏菌使用MEP途径并口服给药，而革兰氏阳性李斯特菌使用MEP和甲羟戊酸途径并静脉内给药。这些基本差异可能导致V32 V42刺激的性质不同，因此将同时进行这两种刺激。这些疫苗将通过TCR和NKR识别或通过与CD 16结合的抗肿瘤mAb的抗体依赖性细胞毒性激活V32 V42 T细胞以杀死肿瘤细胞。 为了实现我们的目标，我们将：（1）代谢工程化疫苗细菌以过量产生HMBPP，（2）在体外测试工程化细菌的HMBPP水平、生长速率、毒力以及在人细胞中感染和增殖的能力，（3）在猴体内测试工程化细菌，以及（4）评估由代谢工程化细菌刺激的V32 V42 T细胞控制肿瘤的能力。

DESCRIPTION (provided by applicant): In the United States and throughout the world, cancer incidence and mortality has increased dramatically in both developed and developing nations. Cancer causes ~13% of human deaths with 7.6 million people dying from cancer in 2007. More people in the US die of lung cancer than breast, colon, kidney, and prostate cancers combined. Recent studies show that veterans are 25 to 75 percent more likely to develop lung cancer than people who did not serve in the military; yet therapies for lung cancer and other solid tumors are still limited. Early in the 1900's, Coley successfully used a mix of heat-killed Streptococcus and Serratia bacteria to treat a variety of sarcomas and other cancers. Similar treatments today with live BCG help prevent recurrence of bladder cancer. The effectiveness of these therapies provides evidence that it is possible to treat malignancies by activating the immune system with bacteria. A wide variety of approaches are now being tried to stimulate tumor immunity by 12 T cells using tumor-specific peptide antigens. However, since CD4 and CD8 12 T cells recognize these peptides bound to MHC molecules in an MHC-restricted fashion, immunotherapy with 12 T cells needs to be individualized for each MHC haplotype. In contrast, 34 T cells represent a unique subset of T cells that bridge innate and adaptive immunity by recognizing nonpeptide antigens in an MHC-unrestricted manner. The major subset of human 34 T cells use their V32V42 T cell receptors to recognize the foreign-microbial isoprenoid metabolite, HMBPP and the self- metabolite, IPP. V32V42 T cells expand in the blood with a variety of infections and then accumulate at peripheral sites. Recognition of HMBPP activates 34 T cells to produce Th1 cytokines, kill infected cells, and produce growth factors to repair mucosal surfaces. Human 34 T cells also help to regulate malignancies. Once activated, V32V42 T cells use their TCR and NK receptors to recognize and kill a wide variety of tumor cells irrespective of their tissue origin, MHC expression, or MHC-haplotype. Since V32V42 T cells also express the CD16 immunoglobulin Fc receptor, they kill tumor cells sensitized by anti-tumor antibodies. In recent years, immunotherapy with prenyl pyrophosphates or bisphosphonates and IL-2 to stimulate V32V42 T cells alone or in conjunction with rituximab for lymphoma has shown promise since these treatments resulted in complete and partial remissions or stable disease in patients with lymphoma and metastatic renal or prostate cancer. However, repeat immunizations lead to anergy and deletion of V32V42 T cells. Metabolic engineering of bacteria is a new field of study that has, up to now, been focused on altering bacteria for drug or chemical synthesis or for the generation of alternative fuels. Directed changes in bacterial metabolism are made by modifying specific biochemical reactions or by introducing new ones. No group has attempted to metabolically engineer bacteria to derive 34 vaccines. We now propose to develop new vaccines for V32V42 T cell cancer immunotherapy by metabolic engineering Salmonella and Listeria bacteria to overproduce HMBPP. Both species have been used for cancer vaccines but differ significantly since the Gram negative Salmonella uses the MEP pathway and is given orally whereas the Gram positive Listeria uses both the MEP and mevalonate pathways and is given intravenously. These fundamental differences may result in qualitatively different V32V42 stimulation so both will be pursued. These vaccines will activate V32V42 T cells to kill tumor cells by TCR and NKR recognition or by antibody-dependent cellular cytotoxicity through anti-tumor mAbs bound to CD16. To accomplish our goals, we will: (1) metabolically engineer vaccine bacteria to overproduce HMBPP, (2) test engineered bacteria in vitro for HMBPP levels, growth rates, virulence, and ability to infect and proliferate in human cells, (3) test engineered bacteria in vivo in monkeys, and (4) assess the ability of V32V42 T cells stimulated by metabolically engineered bacteria to control tumors.