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

在美国和全世界，癌症的发病率和死亡率在2005年急剧增加。 发达国家和发展中国家。癌症导致约13%的人类死亡，760万人死亡 2007年，癌症。在美国，死于肺癌的人比死于乳腺癌、结肠癌、肾癌和前列腺癌的人要多。 加起来最近的研究表明，退伍军人患肺癌的可能性比普通人高25%至75%。 没有在军队服役的人。癌症免疫治疗的进展正在导致以下方面的突破： 治疗表达嵌合抗原受体（CAR-T）的T细胞的连续转移导致持久的缓解， B细胞恶性肿瘤的诊断。用针对PD-1、PD-L1和CTLA-4的抗体阻断检查点导致 在患有各种恶性肿瘤的患者中部分和完全缓解。然而，存在着重大的局限性。与 除了黑色素瘤患者外，只有少数患者对检查点阻断有反应。共同 诸如前列腺癌和结肠直肠癌的癌症通常没有反应。因此，其他方法是 实现癌症免疫疗法的全部潜力。 用表达Vγ 2 VS 2 TCR的γδ Τ细胞治疗是一种这样的方法。与αβ T细胞不同， Vγ 2 V δ2 T细胞的增殖不受MHC限制，而是需要IG超家族蛋白，亲丁酸蛋白3A 1， 感测外来微生物类异戊二烯代谢物HMBPP和自身代谢物IPP。这种感知允许 肿瘤细胞被Vγ 2 VS 2 T细胞识别和杀死，而不依赖于它们的突变负荷。Vγ2Vδ2 T 在许多感染过程中，细胞安全地扩增到非常高的数量（高达1/2的循环T细胞），在那里它们杀死 感染的细胞并分泌炎性Th 1细胞因子、趋化因子和生长因子。 两种方法被用于用Vγ 2 VS 2 T细胞治疗癌症。第一种是用刺激免疫- 如HMBPP的溴代醇类似物或氨基二膦酸盐唑来膦酸与低剂量IL- 2.虽然治疗已导致部分缓解，这些疫苗最终导致无反应性和删除 Vγ 2 V δ2 T细胞。第二种是过继转移Vγ 2 VS 2 T细胞。这种方法是安全的，而且... 在3例实体瘤患者中诱导完全缓解，并诱导部分缓解或稳定的疾病， 在别人身上放松。然而，为了广泛采用，Vγ 2 VS 2 T细胞疗法需要更有效。 活细菌疫苗已被用于预防肺结核、伤寒和兔热病。细菌 产生激活先天免疫的化合物和刺激αβ T细胞的抗原，为免疫系统提供帮助。 Vγ 2 VS 2 T细胞，因为它们扩增。我们现在已经鉴定出一种减毒李斯特菌菌株， pands Vγ 2 V δ2 T细胞。李斯特菌优先聚集在肿瘤中，这应该允许特定的焦油- 获得过继转移的Vγ 2 V δ2 T细胞至肿瘤以及肿瘤部位的TCR刺激 关于HMBPP我们还发现，Vγ 2 V δ2 T细胞快速表达检查点受体，如CTLA-4、PD-1、CTLA-2、CTLA-3、CTLA-4和CTLA-4。 1、TIM-3和LAG-3，并且添加PD-1检查点阻断显著增强了 在小鼠模型中通过Vγ 2 VS 2 T细胞的前列腺肿瘤免疫。我们还发现了一本小说 双膦酸盐前药。为了实现这些目标，我们将： 疫苗和评估剂量和时间的免疫接种，评估李斯特菌的能力，以目标和活动， 过继性转移Vγ 2 V δ2 T细胞以控制肿瘤，并评估结合检查点的有效性。 阻断和新的PTA双膦酸盐前药与过继转移的Vγ 2 V δ2 T细胞。 我们拥有一支优秀的团队，拥有出色的业绩记录，并拥有丰富的工作经验， γδ T细胞和类异戊二烯代谢。我们建立了体内模型和技术。一个有效的列表- RIA疫苗已被鉴定。制造更多疫苗的分子方法已经发展得很好。与此同时- 结论：Vγ 2 V δ2 T细胞的细菌疫苗免疫治疗具有广泛的应用前景 用于通过直接活化和通过增强过继转移来治疗许多不同的肿瘤。

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. Advances in cancer immunotherapy are leading to breakthroughs in treatment. Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) results in durable remis- sions for B cell malignancies. Checkpoint blockade with antibodies against PD-1, PD-L1, and CTLA-4 results in partial and complete responses in patients with a variety of malignancies. Yet, significant limitations exist. With the exception of patients with melanoma, only a minority of patients respond to checkpoint blockage. Common cancers such as prostate and colorectal cancer generally do not respond. Thus, additional approaches are needed to realize the full potential of cancer immunotherapy. Treatment with γδ T cells expressing Vγ2Vδ2 TCRs is one such approach. Unlike αβ T cells, the response of Vγ2Vδ2 T cells is not MHC restricted but instead requires the Ig superfamily protein, butyrophilin 3A1, to sense the foreign-microbial isoprenoid metabolite, HMBPP, and the self-metabolite, IPP. This sensing allows tumor cells to be recognized and killed by Vγ2Vδ2 T cells independent of their mutational burden. Vγ2Vδ2 T cells safely expand to very high numbers during many infections (up to 1 in 2 circulating T cells) where they kill infected cells and secrete inflammatory Th1 cytokines, chemokines, and growth factors. Two approaches are being used to treat cancer with Vγ2Vδ2 T cells. The first is to immunize with stimula- tors such as the bromohydrin analog of HMBPP or the aminobisphosphonate zoledronic acid with low-dose IL- 2. Although treatment has resulted in partial remissions, these vaccines eventually cause anergy and deletion of the Vγ2Vδ2 T cells. The second is to adoptively transfer Vγ2Vδ2 T cells. This approach is safe and has in- duced complete remissions in three patients with solid tumors, and induced partial remissions or stable dis- ease in others. However, for widespread adoption, Vγ2Vδ2 T cell therapy needs to be more effective. Live bacterial vaccines have been used to prevent tuberculosis, typhoid fever, and tularemia. The bacteria produce compounds that activate innate immunity and antigens that stimulate αβ T cells to provide help to the Vγ2Vδ2 T cells as they expand. We have now identified an attenuated Listeria strain that consistently ex- pands Vγ2Vδ2 T cells. Listeria preferentially accumulate in tumors which should allow the specific tar- geting of adoptively-transferred Vγ2Vδ2 T cells to tumors as well as TCR stimulation at the tumor site by HMBPP. We also find that Vγ2Vδ2 T cells rapidly express checkpoint receptors such as CTLA-4, PD- 1, TIM-3, and LAG-3 upon stimulation and that adding PD-1 checkpoint blockade markedly enhances prostate tumor immunity by Vγ2Vδ2 T cells in a mouse model. We have also identified a novel bisphosphonate prodrug. To accomplish these goals, we will: delete inlB in ΔactA prfA* (G155S) Listeria vaccine and assess dosing and timing of immunization, assess the ability of Listeria bacteria to target and acti- vate adoptively transferred Vγ2Vδ2 T cells to control tumors, and assess effectiveness of combining checkpoint blockade and a new PTA bisphosphonate prodrug with adoptively transferred Vγ2Vδ2 T cells. We have an outstanding team with an excellent track record and have extensive experience working with γδ T cells and isoprenoid metabolism. We have established in vivo models and techniques. An effective Liste- ria vaccine have been identified. The molecular methods to create more vaccines are well developed. In con- clusion, immunotherapy using bacterial vaccines for Vγ2Vδ2 T cells has the potential to be broadly applicable for the treatment of many different tumors both by direct activation and through potentiating adoptive transfer.