Adoptive Immunotherapy of Cancer with IL-12 Secreting Tumor-Targeted T cells

描述（由申请人提供）：通过逆转录病毒插入编码嵌合抗原受体（CAR）的基因，可以对T细胞进行遗传修饰，以靶向肿瘤细胞上表达的抗原。CAR通常由与CD 28共刺激受体跨膜和胞质信号传导结构域融合的鼠抗体衍生的肿瘤靶向单链片段长度抗体（scFv）组成，所述CD 28共刺激受体跨膜和胞质信号传导结构域与CD 36链的胞质信号传导结构域融合。表达所得CAR基因产物的T细胞随后识别并裂解表达靶向抗原的正常细胞以及肿瘤细胞。19- 28 z是对在正常B细胞以及大多数B细胞肿瘤上表达的CD 19抗原特异性的CAR。表达19- 28 z CAR的人T细胞在体外和体内在SCID-米色小鼠中裂解CD 19+肿瘤细胞系。尽管这些数据与遗传操作克服肿瘤细胞逃避免疫检测的能力一致，但这些研究未能解决这种基于基因的免疫方法的其他潜在局限性。具体地，先前的研究已经证明，虽然遗传靶向的T细胞易受肿瘤微环境中存在的其他因子的抑制，包括CD 4 + CD 25 hi调节性T细胞（TGF 2）以及抑制性细胞因子TGF 2。进一步修饰以表达IL-12细胞因子的T细胞变得对这些抑制因子具有抗性。最近的研究表明，人效应T细胞对培养的自体TGF 2或外源性TGF 2的抑制具有显著的抗性。利用转基因C57 BL 6鼠CD 19敲除、hCD 19敲入和hCD 19基因敲除，（mCD 19-/-hCD 19 +/-）小鼠的免疫抑制作用，以1）更好地定义免疫抑制微环境的作用，2）评估分泌IL-12的遗传靶向T细胞在调节该微环境中的作用，3）研究安全性以及开发和测试自杀基因方法以在完全肿瘤根除后选择性地删除修饰的T细胞。本提案的目的1将检验以下假设：分泌IL-12的靶向T细胞对肿瘤微环境中存在的显著抑制因子具有抗性，包括TGF 3、抑制性细胞因子TGF 2和IL-10，以及肿瘤微环境中表达的PD-L1对T细胞的PD-1活化。该提议的目的2将检验以下假设：靶向T细胞的局部IL-12分泌将募集或激活其他宿主免疫元件，包括自然杀伤细胞以及无反应性TIL，从而拓宽抗肿瘤免疫库。该方案的目的3将研究IL-12分泌靶向T细胞在免疫活性宿主中的潜在副作用，并进一步测试一种新的自杀基因方法，该方法旨在完全根除肿瘤后根除过继转移的T细胞，从而增强这种治疗方法的安全性。这些研究产生的数据最终将为在未来的血液系统和实体瘤恶性肿瘤患者过继免疫治疗临床试验中使用分泌IL-12的肿瘤靶向T细胞提供依据。 公共卫生相关性：通过使用基因治疗方法，癌症患者自身的免疫细胞可以被修饰，使得它们随后可以识别并杀死癌细胞。我们已经在使用这项技术来测试血癌（白血病）患者的治疗方法。该项目的目标是改善这些细胞的功能，使其更适合治疗化疗后持续存在的癌症患者。

DESCRIPTION (provided by applicant): T cells may be genetically modified to target antigens expressed on tumor cells through the retroviral insertion of a gene encoding a chimeric antigen receptor (CAR). A CAR is typically composed of a murine antibody- derived tumor-targeted single chain fragment length antibody (scFv) fused to the CD28 co-stimulatory receptor transmembrane and cytoplasmic signaling domains, fused to the cytoplasmic signaling domain of the CD3 6 chain. T cells expressing the resulting CAR gene product subsequently recognize and lyse normal as well as tumor cells which express the targeted antigen. 19-28z is a CAR specific to the CD19 antigen expressed on normal B cells as well as most B cell tumors. Human T cells expressing the 19-28z CAR lyse CD19+ tumor cell lines both in vitro, and in vivo in SCID-Beige mice. Although these data are consistent with the ability of genetic manipulation to overcome tumor cell escape from immune detection, these studies fail to address other potential limitations of this gene-based immune approach. Specifically, prior studies have demonstrated that while genetically targeted T cells are susceptible to inhibition by other factors present in the tumor microenvironment including CD4+ CD25hi regulatory T cells (Tregs) as well as the inhibitory cytokine TGF2. T cells further modified to express the IL-12 cytokine become resistant to these inhibitory factors. Recent studies have demonstrated a remarkable resistance of human effector T cells to inhibition by either cultured autologous Tregs or exogenous TGF2. A clinically relevant syngeneic and immune competent hCD19+ tumor model has been developed in the laboratory utilizing transgeneic C57BL6 murine CD19 knockout, hCD19 knockin (mCD19-/- hCD19+/-) mice to 1) better define the role of the immunosuppressive microenvironment, 2) assess the role of IL-12 secreting genetically targeted T cells in modulating this microenvironment, and 3) study the safety as well as develop and test suicide gene approaches to selectively delete modified T cells following complete tumor eradication. Aim1 of this proposal will test the hypothesis that IL-12 secreting targeted T cells are resistant to prominent suppressive factors present in the tumor microenvironment including Tregs, the inhibitory cytokines TGF2 and IL-10, and PD-1 activation on T cells by PD-L1 expressed within the tumor microenvironment. Aim 2 of this proposal will test the hypothesis that localized IL-12 secretion by targeted T cells will recruit or activate other host immune elements, including natural killer cells as well as anergic TILs, thereby broadening the anti-tumor immune repertoire. Aim 3 of the protocol will study the potential side effects of IL-12 secreting targeted T cells in the immune competent host, and further test a novel suicide gene approach designed to eradicate adoptively transferred T cells following complete tumor eradication thereby enhancing the safety profile of this therapeutic approach. Data generated from these studies will ultimately serve to provide the rationale of utilizing IL-12 secreting tumor targeted T cells in future clinical trials of adoptive immunotherapy in patients with both hematologic and solid tumor malignancies. PUBLIC HEALTH RELEVANCE: By using gene therapy approaches, a cancer patient's own immune cells can be modified in such a way that they can subsequently recognize and kill cancer cells. We are already using this technology to test this treatment approach in patients with blood cancers (leukemias). The goal of this project is to improve the function of these cells to make them better suited to treat patients with persistent cancer after chemotherapy.