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

描述（由申请人提供）：T细胞可以通过逆转录病毒插入编码嵌合抗原受体（CAR）的基因进行基因修饰，以靶向肿瘤细胞上表达的抗原。CAR通常由小鼠抗体衍生的肿瘤靶向单链片段长度抗体（scFv）组成，融合到CD28共刺激受体跨膜和细胞质信号域，融合到cd36链的细胞质信号域。表达产生的CAR基因产物的T细胞随后识别并裂解表达目标抗原的正常细胞和肿瘤细胞。19-28z是一种针对CD19抗原的CAR，在正常B细胞和大多数B细胞肿瘤上表达。在SCID-Beige小鼠体内和体外，表达19-28z CAR的人T细胞裂解CD19+肿瘤细胞系。尽管这些数据与基因操作克服肿瘤细胞逃避免疫检测的能力是一致的，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。具体而言，先前的研究表明，虽然遗传靶向T细胞容易受到肿瘤微环境中存在的其他因素的抑制，包括CD4+ CD25hi调节性T细胞（Tregs）和抑制性细胞因子TGF2。进一步修饰表达IL-12细胞因子的T细胞对这些抑制因子产生抗性。最近的研究表明，人效应T细胞对培养的自体Tregs或外源性TGF2的抑制具有显著的抗性。利用转基因C57BL6小鼠CD19敲除、hCD19敲除蛋白（mCD19-/- hCD19+/-）小鼠，在实验室建立了具有临床意义的同基因和免疫能力的hCD19+肿瘤模型，以1)更好地确定免疫抑制微环境的作用，2)评估IL-12分泌的基因靶向T细胞在调节这种微环境中的作用。3)研究安全性，并开发和测试自杀基因方法，选择性地删除肿瘤完全根除后修饰的T细胞。本课题的目的1将验证IL-12分泌的靶向T细胞对肿瘤微环境中存在的Tregs、抑制性细胞因子TGF2和IL-10等重要抑制因子以及肿瘤微环境中表达的PD-L1对T细胞的激活PD-1具有抗性的假设。本提案的目的2将验证靶向T细胞局部分泌IL-12将招募或激活其他宿主免疫元件，包括自然杀伤细胞和无能TILs，从而扩大抗肿瘤免疫库的假设。该方案的目标3将研究IL-12在免疫能力强的宿主中分泌靶向T细胞的潜在副作用，并进一步测试一种新的自杀基因方法，旨在根除肿瘤完全根除后的过继性转移T细胞，从而提高这种治疗方法的安全性。从这些研究中产生的数据将最终为在血液学和实体瘤恶性肿瘤患者的过继免疫治疗的未来临床试验中使用IL-12分泌肿瘤靶向T细胞提供理论依据。公共卫生相关性：通过使用基因治疗方法，可以对癌症患者自身的免疫细胞进行修饰，使其随后能够识别并杀死癌细胞。我们已经用这项技术在血癌（白血病）患者身上测试了这种治疗方法。该项目的目标是改善这些细胞的功能，使它们更适合治疗化疗后持续性癌症患者。

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.