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

Gamma Delta T 细胞用于癌症免疫治疗的细菌代谢工程

• 批准号: 8598011
• 负责人: CRAIG T MORITA
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598011
• 关键词: Accounting; Age; Antigens; BCG Live; Bacteria; Binding; Biochemical Reaction; Blood; Blood Cells; CD8B1 gene; CD94 Antigen; Cancer Etiology; Cancer Vaccine Related Development; Cancer Vaccines; Cells; Cessation of life; Colon Carcinoma; Developed Countries; Developing Countries; Diphosphates; Effectiveness; Engineering; Environmental Hazards; FCGR3B gene; Fc Receptor; Generations; Goals; Growth; Growth Factor; Gulf War; Haplotypes; Heating; Human; Immune system; Immunization; Immunoglobulins; Immunotherapy; In Vitro; Incidence; Infection; Interleukin-2; Koreans; Lead; Listeria; Listeria monocytogenes; Lymphoma; Macaca mulatta; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Metabolism; Military Personnel; Monkeys; Mus; Natural Immunity; Partial Remission; Pathway interactions; Patients; Peptides; Peripheral; Persian Gulf; Production; Proliferating; Recurrence; Renal carcinoma; Salmonella; Salmonella enterica; Serratia; Site; Solid Neoplasm; Stable Disease; Streptococcus; Surface; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Tissues; Tumor Antibodies; Tumor Immunity; Tumor Specific Peptide; United States; Vaccines; Veterans; Vietnam; Virulence; adaptive immunity; anergy; antibody-dependent cell cytotoxicity; bisphosphonate; cancer immunotherapy; cancer type; chemical synthesis; cytokine; drug synthesis; field study; high risk; in vivo; innovation; isoprenoid; killings; malignant breast neoplasm; metabolic engineering; mevalonate; microbial; mortality; neoplastic cell; novel vaccines; prenyl; prevent; repaired; rituximab; sarcoma; tumor

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.

描述（由申请人提供）： 在美国和世界各地，发达国家和发展中国家的癌症发病率和死亡率都急剧增加。癌症约占人类死亡的 13%，2007 年有 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多。最近的研究表明，退伍军人患肺癌的可能性比未参军的人高 25% 至 75%；然而，肺癌和其他实体瘤的治疗方法仍然有限。早在 1900 年代，Coley 就成功地使用热灭活的链球菌和沙雷氏菌的混合物来治疗多种肉瘤和其他癌症。如今使用活卡介苗进行类似治疗有助于预防膀胱癌复发。这些疗法的有效性证明可以通过细菌激活免疫系统来治疗恶性肿瘤。目前正在尝试多种方法，通过使用肿瘤特异性肽抗原的 12 T 细胞来刺激肿瘤免疫。然而，由于 CD4 和 CD8 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 细胞的免疫治疗需要针对每种 MHC 单倍型进行个体化。 相比之下，34 个 T 细胞代表了 T 细胞的独特子集，通过以 MHC 不受限制的方式识别非肽抗原，连接先天性免疫和适应性免疫。人类 34 T 细胞的主要亚群使用其 V32V42 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。 V32V42 T 细胞在多种感染的情况下在血液中扩增，然后在外周部位积聚。 HMBPP 的识别会激活 34 个 T 细胞产生 Th1 细胞因子，杀死受感染的细胞，并产生生长因子来修复粘膜表面。人类 34 T 细胞还有助于调节恶性肿瘤。一旦激活，V32V42 T 细胞就会利用其 TCR 和 NK 受体识别并杀死多种肿瘤细胞，无论其组织来源、MHC 表达或 MHC 单倍型如何。由于 V32V42 T 细胞也表达 CD16 免疫球蛋白 Fc 受体，因此它们可以杀死抗肿瘤抗体致敏的肿瘤细胞。近年来，单独使用异戊二烯焦磷酸盐或双磷酸盐和 IL-2 刺激 V32V42 T 细胞或与利妥昔单抗联合治疗淋巴瘤的免疫疗法已显示出前景，因为这些治疗使淋巴瘤和转移性肾癌或前列腺癌患者的病情完全和部分缓解或稳定。然而，重复免疫会导致 V32V42 T 细胞无反应和缺失。 细菌的代谢工程是一个新的研究领域，迄今为止，其重点是改变细菌以进行药物或化学合成或替代燃料的生产。通过修改特定的生化反应或引入新的生化反应来实现细菌代谢的定向变化。没有任何组织尝试通过代谢改造细菌来生产 34 种疫苗。我们现在建议通过代谢改造沙门氏菌和李斯特菌以过量产生 HMBPP，开发用于 V32V42 T 细胞癌症免疫治疗的新疫苗。这两个物种均已用于癌症疫苗，但存在显着差异，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同时使用 MEP 和甲羟戊酸途径并静脉注射。这些根本差异可能会导致 V32V42 刺激质量不同，因此两者都会被追求。这些疫苗将激活 V32V42 T 细胞，通过 TCR 和 NKR 识别或通过与 CD16 结合的抗肿瘤单克隆抗体的抗体依赖性细胞毒性来杀死肿瘤细胞。 为了实现我们的目标，我们将：(1) 对疫苗细菌进行代谢工程改造以过量产生 HMBPP，(2) 在体外测试工程细菌的 HMBPP 水平、生长速率、毒力以及在人类细胞中感染和增殖的能力，(3) 在猴子体内测试工程细菌，以及 (4) 评估代谢工程刺激的 V32V42 T 细胞的能力 细菌来控制肿瘤。