Project 1: Combinatorial adjuvants promote uniform and selective intratumoral CTL infiltration in colorectal cancer

项目1：摘要 肿瘤微环境（TME）的CTL浸润预测结直肠癌患者的长期生存 癌症（CRC）。它还区分了具有微卫星不稳定性的患者的小子集（<5%）- 高[MSI-H] CRC，显示高水平的肿瘤内CTL，并对PD-1阻断有反应， 没有反应的CRC患者。我们的初步数据表明a）基于TLR 3的佐剂诱导 选择性地在肿瘤间质中而不是在周围非肿瘤组织中的CTL吸引趋化因子; B） TLR 3配体（如林他托莫德）与IFNα的组合协同诱导高水平的CTL-1， 在所有肿瘤病变中均匀地包含C 0X 2阻断剂;以及c）包含C 0X 2阻断剂增强了CKM在肿瘤中的特异性。 促进CTL吸引但抑制Treg吸引。三组分趋化因子调节剂 方案（CKM rintatolimod，IFNα celecoxib）增强了肿瘤内CTL的积累，延长了生存期， 与PD 1和PD-L1阻断剂协同诱导腹膜内MC 38肿瘤小鼠的治愈（> 150天存活）， 对单独的PD-1阻断有抗性。我们完成了对肝硬化患者全身（i.v）CKM的I期评价， 转移性CRC（NCT 01545141），观察到其非常好的耐受性和改善的CTL与Treg的比率 TME中的标志物（与我们仅接受标准治疗的患者相比）。我们建议： 目标1.评估i. v. -管理CKM，以促进当地 IIa期试验NCT 03403634中肝转移性CRC病变中的CTL蓄积。比较治疗前与 12例肝转移性CRC患者的治疗后肿瘤活检，我们将检测系统性CKM是否会 消除TME异质性并均匀地增加TME中的CTL数量，但不增加周围组织中的CTL数量。 目标二。评价连续与周期性应用CKM的免疫和抗肿瘤作用， PD 1阻断和额外免疫的优势，以获得持久的抗肿瘤益处。在 在临床前研究中，我们将测试CKM吸引的DC、NK细胞和T细胞将a）促进 局部和全身肿瘤特异性免疫，和B）将扩增CKM启动的CTL的肿瘤内产生 IFNγ和TNFα依赖性机制中的引诱剂，导致TME持续条件化， PD 1阻断的持续抗肿瘤活性，即使在没有额外接种的情况下。 目标3：进行I/II期试验，以测试CKM联合PD-1阻滞剂在以下患者中的临床活性： 微卫星稳定（MSS）CRC患者。在I/II期试验中，我们将评估临床疗效（iORR; iRESIST）CKM/抗PD-1治疗在19例传统上对免疫治疗耐药的MSS-CRC患者中的应用。 项目作用：项目1的独特作用是评估有效性、均匀性和肿瘤- 选择性的全身应用CKM和开发CKM为基础的治疗与持续的抗癌效果。 它的成功将为我们提供一种工具，将免疫疗法的治疗益处扩展到特别大的 目前对检查点阻断无反应的MSS-CRC和其他癌症患者组。

PROJECT 1: ABSTRACT CTL infiltration of tumor microenvironments (TME) predicts prolonged survival of patients with colorectal cancer (CRC). It also differentiates between the small subset of patients (<5%) with microsatellite instability- high [MSI-H] CRC, who show high levels of intratumoral CTLs and respond to PD-1 blockade from the rest of CRC patients who do not respond. Our preliminary data demonstrate that a) TLR3-based adjuvants induce CTL-attracting chemokines selectively in tumor stroma, but not surrounding non-tumor tissues; b) that combination of TLR3 ligands (such as rintatolimod) with IFNα synergistically induce high levels of CTL- attractants uniformly in all tumor lesions; and c) that inclusion of COX2 blockers enhances specificity of CKM in promoting CTL attraction but suppressing Treg attraction. The three-component chemokine-modulatory regimen (CKM rintatolimod, IFNα celecoxib) enhanced intratumoral CTL accumulation, prolonged survival and synergized with PD1- and PD-L1 blockers in inducing cures (> 150 day survival) in mice with i.p. MC38 tumors, resistant to PD-1 blockade alone. We completed phase I evaluation of systemic (i.v) CKM in patients with liver- metastatic CRC (NCT01545141), observing its very good tolerability and improved ratios of CTL-to-Treg markers in TME (compared to our patients receiving standard care only). We propose to: Aim 1. Evaluate the in-patient immunologic effectiveness of i.v.- administered CKM to promote local CTL accumulation in liver-metastatic CRC lesions in phase IIa trial NCT03403634. Comparing pre- versus post-treatment tumor biopsies of 12 patients with liver-metastatic CRC, we will test if systemic CKM will abrogate the TME heterogeneity and uniformly increase CTL numbers in TMEs, but not in surrounding tissues. Aim 2. Evaluate the immune and antitumor effects of sequential versus cyclic application of CKM and PD1 blockade and the advantage of additional immunization for long-lasting anti-tumor benefit. In preclinical studies, we will test the hypotheses that the CKM-attracted DCs, NK cells and T cells will a) promote local and systemic tumor-specific immunity and b) will amplify the CKM-initiated intratumoral production of CTL attractants in an IFNγ and TNFα-dependent mechanism, resulting in sustained conditioning of the TME for continued antitumor activity of PD1 blockade, even in the absence of additional vaccination. Aim 3. Perform a phase I/II trial to test the clinical activity of CKM combined with PD-1 blockade in patients with microsatellite-stable (MSS) CRC. In phase I/II trial, we will evaluate the clinical efficacy (iORR; iRESIST) of CKM/anti-PD-1treatment in 19 patients with MSS-CRC, traditionally resistant to immunotherapy. Programmatic Role: The unique role of Project 1 is to evaluate the effectiveness, uniformity and tumor- selectivity of systemically-applied CKM and develop CKM-based treatments with sustained anticancer effect. Its success will provide us with a tool to extend the therapeutic benefit of immunotherapy to a particularly large group of patients with MSS-CRC and other cancers currently non-responsive to checkpoint blockade.