Image guided immunotherapy and targeted radionuclide therapy of metastatic melanoma

摘要 黑色素瘤是最致命的皮肤癌。如果及早发现，通常可以通过手术治愈；然而， 转移性黑色素瘤的治疗选择有限，5年存活率仅为15%-20%。虽然 免疫疗法作为一线治疗方案提高了存活率，个体反应可能参差不齐 部分原因是受治疗干预影响的保护性肿瘤浸润性淋巴细胞(TIL)的质量。 迫切需要：1)识别具有攻击性表型的患者；2)改进治疗以增加 此类患者的总体存活率；以及3)开发改进的监测手段，以辨别患者是否 对治疗有反应。在这里，我们提出了一种多方面的转移性肿瘤的成像和治疗方法。 黑色素瘤，在原发和转移性疾病的小鼠模型中进行测试。人们对……很感兴趣 靶向甚晚抗原-4(Vla-4；又称整合素α4-β1)用于多发性骨髓瘤的肿瘤成像和治疗 骨髓瘤和黑色素瘤，在肿瘤生长、血管生成和转移中发挥促进作用 促进癌细胞的黏附和迁移。在人类黑色素瘤中，VLA-4的表达增加 与肿瘤转移的发生密切相关。我们之前已经证明了高亲和力的模拟肽VLA-4 标记有68Ga(正电子发射断层扫描，T1/2=68分钟)和177Lu(用于正电子发射断层扫描)的靶向试剂 放射性核素治疗；T1/2=6.7d)被小鼠B16F10黑色素瘤肿瘤贪婪地吸收。A最近 已发表的研究表明，将外照射(XRT)与 针对CTLA-4和PD1/PD-L1治疗B16F10荷瘤小鼠的联合免疫治疗药物。 由于xrt不是治疗广泛播散或微转移疾病的最佳选择，我们的目标是改进这一点。 用177Lu-DOTA-PEG4-LLP2A研究VLA-4靶向全身放射治疗的重要发现 (177 Lu-LLP2A)，联合抗CTLA-4和抗PD-1免疫治疗。我们还将开发PET 针对PD-L1和CD8的成像示踪剂可提供对肿瘤细胞、髓系细胞和 衍生细胞和T细胞对治疗的反应。我们假设177Lu-LLP2A靶向放射治疗， 联合抗CTLA-4和抗PD-1双重免疫治疗将是治疗黑色素瘤的高效方法 荷瘤小鼠，PD-1和CD8+T细胞成像将允许监测早期的“临床” 对治疗的反应。为了解决我们的假设，我们提出了以下目标：1)验证68Ga- 和177Lu标记的LLP2A在小鼠黑色素瘤模型(BP20)中，其中肿瘤具有共同的 BRAFV600E突变，以及来自患者黑色素瘤转移的人类肿瘤；2)确定 177Lu-LLP2A和XRT联合双重免疫治疗的最佳治疗策略比较 B16F10皮下和播散性肿瘤，以及具有BRAFV600E突变的BP20肿瘤；以及3) 制备并验证抗鼠PD-L1微体和抗鼠CD8单域抗体(SdAb)PET 显像剂分别标记64Cu(t1/2=12.7h)和68Ga(t1/2=68min)。综合疗法 并将进行早期反应的PET成像。如果成功，我们将展示这种结合 靶向放射性核素治疗和免疫治疗可以有效地治疗晚期黑色素瘤，而 同时识别一组PET示踪剂，以非侵入性监测治疗效果。

ABSTRACT Melanoma is the most lethal form of skin cancer. If discovered early, it is usually cured with surgery; however, treatment options are limited for metastatic melanoma, with a 5-year survival of only 15-20%. Although immunotherapy as a first-line treatment option has improved survival, individual response is uneven, possibly due in part to the quality of protective tumor infiltrating lymphocytes (TILs) impacted by treatment intervention. There is critical need to: 1) identify patients with an aggressive phenotype; 2) improve therapies to increase overall survival of such patients; and 3) develop improved monitoring means to discern whether patients are responding to therapy. Here we propose a multi-faceted approach for imaging and therapy of metastatic melanoma, to be tested in mouse models of primary and metastatic disease. There is great interest in targeting very late antigen-4 (VLA-4; also called integrin α4β1) for cancer imaging and therapy for multiple myeloma and melanoma, where it plays a facilitating role in tumor growth, angiogenesis and metastasis by promoting adhesion and migration of cancer cells. In humans, increased expression of VLA-4 in melanoma correlates with development of metastasis. We previously showed that high-affinity peptidomimetic VLA-4 targeted agents labeled with 68Ga (for Positron Emission Tomography (PET); T1/2 = 68 min) and 177Lu (for radionuclide therapy; T1/2 = 6.7 d) are avidly taken up by B16F10 melanoma tumors in mice. A recently published study showed dramatic efficacy from combining external beam irradiation (XRT) with the combination immunotherapy agents targeting CTLA-4 and PD1/PD-L1 therapy in B16F10 tumor-bearing mice. As XRT is not optimal for treating widely disseminated or micrometastatic disease, we aim to improve upon this important finding by investigating VLA-4 targeted, systemic, radiotherapy with 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A), in combination with anti-CTLA-4 and anti-PD-1 immunotherapy. We will also develop PET imaging tracers targeting PD-L1 and CD8 to provide real-time, non-invasive monitoring of tumor cells, myeloid- derived cells and T cells in response to therapy. We hypothesize that targeted radiotherapy with 177Lu-LLP2A, combined with dual anti-CTLA-4 and anti-PD-1 immunotherapy, will be highly effective in treating melanoma tumor-bearing mice, and that imaging of PD-1 and CD8+ T-cells will allow the monitoring of early “clinical” responses to therapy. To address our hypotheses, we propose the following aims: 1) validate uptake of 68Ga- and 177Lu-labeled LLP2A in a mouse model of melanoma (BP20) where the tumors have the common BRAFV600E mutation, and in human tumors derived from patient melanoma metastases; 2) determine the optimal treatment strategy comparing 177Lu-LLP2A and XRT in combination with dual immunotherapy in B16F10 subcutaneous and disseminated tumors, and in BP20 tumors with the BRAFV600E mutation; and 3) develop and validate anti-mouse PD-L1 minibodies, and anti-mouse CD8 single domain antibody (sdAb) PET imaging agents labeled with 64Cu (T1/2 = 12.7 h) and 68Ga (T1/2 = 68 min), respectively. The combination therapy and PET imaging of early response will be performed. If successful, we will demonstrate that combining targeted radionuclide therapy and immunotherapy can effectively treat advanced-stage melanoma, while simultaneously identifying a panel of PET tracers for non-invasive monitoring of treatment efficacy.