Image guided immunotherapy and targeted radionuclide therapy of metastatic melanoma

摘要 黑色素瘤是最致命的皮肤癌。如果早期发现，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选择提高了生存率，但个体反应不均匀，可能 部分原因是受治疗干预影响的保护性肿瘤浸润淋巴细胞（TIL）的质量。 迫切需要：1）识别具有侵袭性表型的患者; 2）改进治疗以增加 这类患者的总体生存率;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，我们提出了一个多方面的方法，成像和治疗转移性乳腺癌。 黑色素瘤，将在原发性和转移性疾病的小鼠模型中进行测试。有很大的兴趣， 靶向极晚期抗原-4（VLA-4;也称为整合素α4β1），用于癌症成像和多种癌症的治疗。 骨髓瘤和黑色素瘤，其中它通过以下方式在肿瘤生长、血管生成和转移中起促进作用： 促进癌细胞的粘附和迁移。在人类中，黑色素瘤中VLA-4的表达增加 与转移的发展相关。我们先前表明，高亲和力肽模拟物VLA-4 用68 Ga（用于正电子发射断层扫描（PET）; T1/2 = 68 min）和177 Lu（用于 放射性核素治疗; T1/2 = 6.7天）被小鼠中的B16 F10黑素瘤肿瘤强烈吸收。一个最近 已发表的研究表明，将外部光束照射（XRT）与 在B16 F10荷瘤小鼠中靶向CTLA-4和PD 1/PD-L1治疗的联合免疫治疗剂。 由于XRT不是治疗广泛播散或微转移性疾病的最佳选择，我们的目标是改善这一点 通过研究用177 Lu-DOTA-PEG 4-LLP 2A进行的VLA-4靶向全身放疗的重要发现 （177 Lu-LLP 2A），与抗CTLA-4和抗PD-1免疫疗法组合。我们还将开发PET 靶向PD-L1和CD 8的成像示踪剂，以提供实时、非侵入性的肿瘤细胞、髓样细胞、 衍生细胞和T细胞对治疗的应答。我们假设用177 Lu-LLP 2A进行靶向放射治疗， 与双重抗CTLA-4和抗PD-1免疫疗法相结合， PD-1和CD 8 + T细胞的成像将允许监测早期“临床”肿瘤。 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68 Ga的吸收- 和177 Lu标记的LLP 2A在黑色素瘤小鼠模型（BP 20）中的作用，其中肿瘤具有共同的 BRAFV 600 E突变，以及来源于患者黑素瘤转移的人肿瘤中的BRAFV 600 E突变; 2）确定BRAFV 600 E突变的人肿瘤中的BRAFV 600 E突变; 比较177 Lu-LLP 2A和XRT联合双重免疫疗法的最佳治疗策略 B16 F10皮下和播散性肿瘤，以及具有BRAFV 600 E突变的BP 20肿瘤;和3） 开发和验证抗小鼠PD-L1微抗体和抗小鼠CD 8单域抗体（sdAb）PET 分别用64 Cu（T1/2 = 12.7h）和68 Ga（T1/2 = 68 min）标记的显像剂。组合疗法 并进行早期反应的PET成像。如果成功，我们将证明， 靶向放射性核素治疗和免疫治疗可有效治疗晚期黑色素瘤， 同时识别一组PET示踪剂用于治疗功效的非侵入性监测。

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.