CN105992590B - 靶定癌症的il-12免疫疗法 - Google Patents
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Abstract
本发明涉及癌症免疫疗法。本发明具体涉及先天性或适应性抗肿瘤免疫的诱导，其通过优选将靶向IL‑12分子与IL‑2和/或IL‑7联合给予癌症患者来引发，所述癌症患者受肌肉、骨、神经、软骨、腱、血管等的癌症所累，优选受肉瘤所累。本发明具体涉及使用呈特异性免疫球蛋白细胞因子融合蛋白形式的IL‑12(称为NHS‑IL12)，优选与对治疗所述癌症疾病表现出延长的药代动力学的IL‑2和/或IL‑7形式组合使用。
Description
发明领域：
本发明涉及癌症免疫疗法。本发明具体涉及先天性或适应性抗肿瘤免疫的诱导，其通过优选将靶向IL-12分子与IL-2和/或IL-7联合给予癌症患者来引发，所述癌症患者受肌肉、骨、神经、软骨、腱、血管等的癌症所累，优选受肉瘤所累。
本发明具体涉及使用呈特异性免疫球蛋白细胞因子融合蛋白形式的IL-12 (称为NHS-IL12)，优选与对治疗所述癌症疾病(具体而言肉瘤)表现出延长的药代动力学的IL-2和/或IL-7形式组合使用。
发明背景
癌症免疫疗法包括多种不同的治疗途径，包括‘被动’给予肿瘤特异性单克隆抗体及其它免疫系统组分、‘主动’免疫以引发或增强针对肿瘤细胞的特异性T细胞介导的免疫反应、过继转移离体修饰的T细胞以及使用免疫调节剂非特异性增强免疫反应性。免疫疗法已对广泛范围的癌症管理具有重要影响，但其主要限于使用单克隆抗体的被动免疫疗法。癌症免疫疗法的领域很复杂且迅速发展。免疫疗法在其作用机制以及产生的反应类型上与常规化学疗法不同，并且常规反应标准不能提供对免疫治疗剂的改善疾病活性的可靠评价。许多主动免疫疗法掺入多种组分(例如抗原、辅剂和递送载体)。此外，日渐认识到的是，稳健的治疗活性需要将免疫与其它免疫调节策略组合，所述免疫调节策略针对增强全身免疫反应性并克服肿瘤通过其促成致耐受性环境的免疫抑制机制。
横纹肌肉瘤(RMS)为在儿童中最常见的软组织肿瘤并且在晚期伴随非常不良的预后(Oberlin等，2008，J. Clin. Oncol. 26:2384-2389)。手术切除、化疗和放疗常常因肿瘤位于棘手的解剖部位并倾向于扩散而失败。因此，存在对于备选治疗策略的高度未满足的需求。
近期数据建议将同种异体干细胞移植(alloSCT)作为在包括RMS在内的晚期实体瘤中的可行策略(Koscielniak等，2005，J. Clin. Oncol. 23:242-244)。然而，在此情况下肿瘤控制的机制并未充分阐明。尽管可在体外产生针对RMS的细胞毒性T细胞和自然杀伤(NK)细胞应答(van den Broeke等，2006，Cancer Res. 66:1818-1823)，但免疫效应细胞的抗肿瘤活性具有许多面。除杀伤之外，通过干扰素-γ (IFN-γ)以及肿瘤坏死因子受体1(TNFR1)-依赖性途径的组合信号转导可导致肿瘤休眠(Muller-Hermelink等，2008，Cancer Cell 13:507-518)，因此，诱导分泌这些细胞因子可以是局部控制肿瘤生长的重要方式。事实上，数个研究已显示在alloSCT之后患者的存活率改善，伴有产IFN-γ T细胞的频率增加(Wiegering等，2011，Cancer Immunol. Immunother. 60:693-703)。因此，CD4+ T细胞可与CD8+ CTL同等重要。
此网状系统中的主要参与者IL-12可诱导肿瘤消退并影响先天性和适应性免疫(例如Trinchieri，G. 2003，Nat. Rev. Immunol. 3:133-146)。除其在引发T细胞上的作用之外，IL-12亦使TH17细胞复原成TH1表型，恢复M1巨噬细胞功能并介导DC-NK相互作用。
白介素-12 (IL-12)为一种多效促炎细胞因子，其通过免疫系统的多种细胞对感染响应而产生，所述细胞包括吞噬细胞、B细胞和激活的树突状细胞(Colombo和Trinchieri(2002)，Cytokine & Growth Factor Reviews，13: 155–168)。IL-12在介导先天性和适应性免疫系统武器（arms）的相互作用、作用于T细胞和自然杀伤(NK)细胞、增强细胞毒性淋巴细胞的增殖和活性以及其它炎性细胞因子尤其是干扰素-γ的产生上起重要作用。IL-12为由α-链(p35亚基，IL-12p35)和β-链(p40亚基，IL-12p40)组成的异二聚体分子，所述链通过二硫键共价连接以形成生物学上活性的74 kDa异二聚体。
已显示内源性IL-12的存在对于免疫抵抗广泛种类（broad array）的病原体以及移植和化学诱导的肿瘤而言为必需的(Gateley等，(1998)，Annu. Rev. Immunol.，16: 495-521)。基于诱导IFN-γ以及激活诸如CD8+ T细胞和NK细胞等效应细胞，IL-12已被证实具有潜在的抗肿瘤活性(Brunda等(1993)，J. Exp. Med.，178: 1223-30)。高水平的IFN-γ通过T细胞和NK细胞对IL-12的响应而产生[Kobayashi等，1989，J Exp Med；170:827-45]，导致通过旁分泌增量调节I类和II类MHC表达而增强的抗原呈递[Wallach等，1982 Nature 1982；299:833-69]。因为其经证明的抗肿瘤活性，IL-12已作为用于治疗广泛种类的癌症的免疫治疗剂(Atkins等(1997)，Clin. Cancer Res.，3: 409-17；Gollob等(2000)，Clin. Cancer Res.，6: 1678-92；和Hurteau等(2001)，Gynecol. Oncol.，82: 7-10)以及作为用于癌症疫苗的辅剂(Lee等(2001)，J. Clin. Oncol. 19: 3836-47)在人类临床试验中测试，所述广泛种类的癌症包括肾癌、结肠癌、卵巢癌、黑素瘤和T细胞淋巴瘤。
全身给予IL-12主要显示针对一些实体瘤的功效，但因其剂量限制性毒性所致，其在治疗上的用途为有限的(Gollob等，2000，Clin. Cancer Res. 6:1678-1692)。在早期临床开发十多年之后，严重毒性以及对rIL-12的普遍较低的响应率的备案病例迄今为止仍阻止其临床开发。
因此，将IL-12靶向递送至肿瘤微环境代表用于肿瘤免疫疗法的非常有前景的途径，因为其可使细胞因子为更有效且毒性更低。因此，提供一种有效且尤其是可行的治疗性癌症免疫治疗途径，为本发明的主题，其通过使用该药物的有效抗肿瘤免疫而进行。
发明概述
用于改善促炎细胞因子(例如IL-2和IL-12)的安全性的策略为经由与靶向肿瘤的抗体融合将其递送导向至肿瘤。所述抗体-细胞因子融合蛋白或“免疫细胞因子”，先前已证实其在临床前模型中增强抗肿瘤免疫的能力(Gillies SD. In Lustgarten J，Cui Y，Li S，编辑Targeted Cancer Immune Therapy(靶向癌症免疫疗法). New York，New York， USA: Springer；2009:241-256)。为了使免疫细胞因子耐受性达到最大，选为载体的抗体必须与仅在肿瘤中存在的抗原特异性结合。针对坏死相关抗原的抗体提供引人注目的递送途径，所述抗原在肿瘤中大量存在但在正常组织中不存在(例如Epstein等，1988，Cancer Res 1988；48:5842-48)。
DNA/组蛋白结合抗体选择性靶定肿瘤坏死区域的能力已在临床前和临床上充分研究。利用此理论，通过将2个人IL-12异二聚体与NHS76抗体重链的C端融合，工程改造出靶定坏死的IL-12免疫细胞因子(称为NHS-IL12)(WO 2000/001822)。NHS76为全人源噬菌体展示来源的IgG1抗体，其因结合DNA/组蛋白的特殊能力而被选中并从而在体内靶定肿瘤[Sharifi等，2001，Hybrid Hybridomics；20:305-12]。
发现的是，通过给予上述靶向IL-12药物的IL-12驱动的人免疫系统不仅杀伤癌细胞，而且还使用备选机制减弱肿瘤生长，即通过在癌细胞中诱导衰老和/或分化，从而导致将癌细胞或癌组织恢复成原始细胞或组织。提出的靶向IL 12疗法特异性导致癌细胞的TH1-诱导的生长停滞和分化。如果将所述靶向IL-12与免疫调节剂(例如白介素，如IL-2和IL-7)联合或组合给予，效果尤其增加。
根据本发明，IL-12以绑定且靶向的形式（a bound an targeted form）使用，例如IL12-融合蛋白，优选由免疫球蛋白和IL-12组成的融合蛋白，所述免疫球蛋白能够靶定患者组织表达的抗原或特异性受体分子，其中优选所述免疫球蛋白的C端与所述免疫细胞因子的N端连接。根据本发明，所述靶定肿瘤的IL-12为与IL-12连接的治疗上有效的单克隆抗体或其生物学活性部分(例如Fab片段或scFv)。
在本发明的一个优选实施方案中，所述治疗上有效的抗体或其靶向部分针对肿瘤坏死中暴露的DNA-组蛋白H1复合物。在又一个具体实施方案中，所述靶向IL-12分子为由IL-12和上下文详述的已知的全人源IgG1抗体NHS76组成的各自的融合蛋白（respectivefusion protein）。
根据本发明，所述靶向IL-12可用于癌症免疫疗法。根据本发明，所述靶向IL-12可用于癌症免疫疗法，其中所述癌症涉及肌肉、骨、神经、软骨、腱、血管等，优选肉瘤，以及更优选横纹肌肉瘤(RMS)。根据本发明，所述靶向IL-12可用于单一疗法或者与免疫调节剂组合使用，所述免疫调节剂例如白介素、干扰素、CpGs、趋化因子或葡聚糖。
在本发明的一个具体实施方案中，所述免疫调节剂为IL-2和/或IL-7，优选IL-2。在又一个具体和优选的实施方案中，所述免疫调节剂为IL-2。所述免疫调节剂优选与较大的蛋白质或免疫球蛋白或其片段(例如Fab、scFv、Fc)共价结合或融合，或者为与免疫球蛋白的复合物。在本发明的一个实施方案中，IL-2与抗体的Fc部分共价融合。在本发明的又一个实施方案中，IL-2以复合物形式与抗-IL2抗体结合。在本发明的又另一个实施方案中，IL-7与抗体的Fc部分共价融合。
因此，本发明涉及用于癌症治疗的靶定肿瘤的IL-12，其中所述药物在患者中诱导和/或刺激针对癌症疾病的免疫反应，所述癌症疾病例如肉瘤以及其它实体瘤，其中所述免疫反应的诱导或刺激引发癌细胞的衰老，和/或出人意料地使癌细胞或癌组织恢复成原始细胞或组织，其中所述靶定肿瘤的IL-12为针对在肿瘤坏死中暴露的人DNA-组蛋白H1复合物的，并且优选经由其C端与IL-12(优选与IL-12的p35亚基)融合的治疗上有效的单克隆抗体或其生物学活性部分。
本发明进一步涉及用于与免疫调节剂和/或免疫补充剂(例如白介素、干扰素或趋化因子)的组合治疗的靶定肿瘤的IL-12，其用于在受癌症疾病所累的患者中诱导和/或刺激针对所述癌症疾病的免疫反应，其中所述诱导或刺激导致癌细胞衰老，以及使癌细胞或癌组织恢复成原始细胞或组织，其中所述靶定肿瘤的IL-12为针对在肿瘤坏死中暴露的人DNA-组蛋白H1复合物、并且经由其C端与IL-12融合的治疗上有效的单克隆抗体或其生物学活性部分。
根据本发明，所述靶定肿瘤的IL-12疗法对于导致癌细胞衰老有用，所述衰老优选通过在对患者的免疫系统进行所述刺激或诱导之后增加内源IFNy和/或TNF的产生来发生，所述刺激或诱导通过所述靶向IL-12疗法触发。发现的是，通过根据本发明的所述治疗，癌细胞的衰老导致稳定的生长停滞。此外发现的是，癌细胞的衰老和组织恢复不依赖于直接的免疫特异性细胞毒性效应，例如产生和激活NK细胞和巨噬细胞，尽管这些细胞通过IL-12触发。
根据本发明，所述靶定肿瘤的IL-12疗法在癌症免疫治疗中为有用的，其中所述癌症疾病涉及实体瘤，或者肌肉、骨、神经、软骨、腱、血管以及脂肪组织或纤维组织的肿瘤。在本发明的一个具体实施方案中，所述疗法用于治疗肉瘤，以及优选横纹肌肉瘤(RMS)。
发现的是，所提出的癌症免疫疗法，其中组合使用如上下文所述的NHS-IL12和IL-2或IL-7，不仅引起癌细胞的衰老，而且额外引发生肌分化的诱导以及癌细胞恢复成原始组织细胞，表明IL-12驱动的人免疫系统不仅杀伤癌细胞，但也使用备选机制以减弱肿瘤生长。靶定肿瘤的IL-12至少在使用全人源化同种异体免疫系统重组的NSG小鼠中显著消除肿瘤生长。
根据本发明，单独的IL-2和IL-7既不会使肿瘤生长停止，亦不会诱导有效的抗肿瘤免疫反应。需要使用IL-12刺激同种异体人免疫系统以有效地控制人源化小鼠中的肿瘤生长。在此情况下更有效的为NHS-IL12和IL-2或IL-7的组合，尤其当IL-2或IL-7为其在身体中的半寿期增加的形式时。在一个实施方案中，将IL-2或IL-7与免疫球蛋白耦合或结合，优选将IL-2或IL-7与免疫球蛋白的Fc片段的C端融合。在又另一个实施方案中，将IL-2与抗-IL2抗体(例如MAb602)复合。
NHS-IL12/ILMAB602组合显示关于总存活率和减小肿瘤体积的显著优势。组织学表明的是，NK细胞或CD68+噬菌体浸润癌症（cancer infiltration）并不需要IL-2。对比鲜明的是，需要根据本发明的IL-12构建体来驱使巨噬细胞进入表达II类MHC的M1-型中，其有效地产生效应子分子和炎性细胞因子(TNF、IL-12、IL-6、IL-1β)。
根据本发明的数据可得到以下提示，IL-12至少在所使用的这个特殊系统中促进同种异体免疫系统杀伤癌细胞，例如肌肉、骨、神经、软骨、腱、血管以及脂肪组织或纤维组织的癌细胞，优选RMS细胞。有趣的是，免疫组织学未显示癌细胞杀伤、凋亡或坏死的证据，表明优选以结合或复合形式与IL-2或IL-7组合的靶向IL-12癌症免疫疗法，驱动不依赖于杀伤的癌症控制的关键机制。然而最重要的是，根据本发明通过给予靶向IL12疗法的肿瘤生长停滞，开始重新以立体横纹构型表达结蛋白，其为一种肌肉特异性蛋白并且为心脏、骨骼和平滑肌中的中间丝状体的重要亚基。尤其是呈条纹形式的结蛋白的存在表明肌细胞中的高度分化(van,d.，V，Schaart等，1992，Cell Tissue Res. 270:189-1981)。
总之，本发明人提供的数据为癌细胞的TH1-诱导的生长停滞和分化的第一份体内证据。这可通过靶向递送IL-12 (优选与IL-2或IL-7组合)到肿瘤微环境并随后激活p16INK4a途径来实现。
发明详述
根据本发明，术语“衰老”意指限制癌细胞的寿命并阻止无限细胞增殖的生长停滞程序。
根据本发明，术语“直接的免疫特异性细胞毒性效应”意指在免疫反应期间免疫特异性细胞毒性细胞(例如NK细胞、巨噬细胞、T-细胞毒性细胞或树突状细胞)的主动参与和作用。
根据本发明，术语“癌症免疫疗法”意指的这样的治疗性疗法，其通过诱导、增强或阻抑免疫反应来刺激或恢复免疫系统对抗癌症的能力。癌症免疫疗法导致针对疾病特异性抗原的靶定免疫活性，其通过增加靶标的免疫细胞识别或通过减少疾病相关的免疫阻抑来进行。
根据本发明，优选根据本发明将靶向IL-12 (例如NHS-IL12)的给予与IL-2或IL-7组合，优选与呈结合、融合或复合形式的IL-2和IL-7组合，如上下文详述所述。所述共同给予可遵循具体的剂量和时间方案同时或序贯进行，其中在后者情况下，所述免疫调节剂可在给予所述靶向IL-12分子(如NHS-IL12)之前或之后数小时或数天给予。
根据本发明，原则上可能将靶向IL-12 (如NHS-IL12)的给予与放疗和/或化疗组合。
根据本发明，与上述靶向IL-12融合分子组合使用的化学治疗剂可以是例如甲氨蝶呤、长春新碱、阿霉素、顺铂、含氯乙基亚硝基脲类的非糖物、5-氟尿嘧啶、丝裂霉素C、博来霉素、多柔比星、达卡巴嗪、泰素、fragyline、Meglamine GLA、戊柔比星、卡莫司汀（carmustaine）、UFT (替加氟/尿嘧啶)、ZD 9331、多西紫杉醇/紫杉特尔、氟尿嘧啶(5-FU)、长春碱以及来自此类的其它良好的化合物。根据本发明，施用化疗达至少两个周期，优选2-8个周期，更优选2-5个周期。一个周期介于21-35天，优选介于21-28天。所述化学治疗剂的剂量方案取决于多个可能的患者相关和药物相关的情况和性质。
根据本发明，通过标准辐射实施放疗，其中施用总共40-120 Gy，优选至少50 Gy，更优选介于50-75 Gy。所述放射疗法常常分成几部分进行，其中施用1.5-3.5 Gy/天达至少四天，优选依次5-7天。根据本发明，在21-35天之内施用总的放射剂量，优选在28天之内施用。在有需要或有利时，可在放射开始时或在中间间隔施用3.5-15 Gy、优选5-10 Gy的加强剂量。根据本发明，可在给予所述靶向IL-12分子和任选本发明的免疫调节剂之前或之后实施放疗，或同时实施。
化-放疗治疗可通过给予能够调节免疫系统的剂来完成。通过例如施用介于100-400 mg/m2优选250 mg/m2的相对低剂量的环磷酰胺，患者的免疫系统可得到激活或增强。通常，在开始疫苗接种之前(通常1-5天，优选2-5天)的单剂量应足以为有效的。
根据本发明，如上所述优选的靶向IL-12为NHS-IL12。如上所述，IL-12为由通过二硫键共价连接的α-链(p35亚基，IL-12p35)和β-链(p40亚基，IL-12p40)组成的异二聚体分子。在本发明的融合蛋白中，p35亚基与(二聚)抗体NHS76的两条重链的每一条的C端连接。p40亚基通过共价结合与p35亚基连接。在实际中，所述分子通过重组方法制备，其使用将重链和p35亚基表达为融合蛋白并单独表达p40亚基的DNA构建体进行，所述p40亚基原位与如所述表达的NHS-p35亚基融合物结合。
SEQ ID NO.1描述成熟的(野生型)人IL-12的α链，即人p35亚基的成熟氨基酸序列：
SEQ ID NO. 2描述成熟的(野生型)人IL-12的β链，即人p40亚基的成熟氨基酸序列：
SEQ ID NO. 3描述如根据本发明所使用和修饰的Mab NHS的λ轻链的氨基酸序列，其包含信号序列(斜体，前19个aa)、可变结构域(用下划线表示)和去免疫性L103V突变(粗体)。所述信号序列并非成熟多肽链的一部分：
SEQ ID NO. 4描述重链/人p35融合物的氨基酸序列，其包含信号序列(斜体，前19个aa)、可变结构域(用下划线表示)以及hu p35的第一个氨基酸的R→A取代(粗体)、hu p35(用下划线和斜体表示)。所述信号序列并非成熟多肽链的一部分：
SEQ ID NO. 5描述带有信号序列(用斜体字表示)和以粗体显示的变异序列(KSKREKKDRV突变成KDNTEGRV)的人p40的氨基酸序列。所述信号序列并非成熟多肽链的一部分：
在NSG小鼠中建立全功能免疫系统
为了在体内测试所述NHS-IL12构建体在人类系统中的毒性和潜在治疗功效，对NSG小鼠移植huCD34+干细胞和FcIL-7刺激植入物（stimulated engraftment with FcIL-7）(图1A)。移植>99.9%纯的huCD34+细胞到NSG小鼠中在12周内建立全部造血谱系。此外，在骨髓、胸腺以及脾和胸腺等同物中发现复合的T-细胞受体(TCR)所有组分（repertoire）。同时，数据显示，移植小鼠形成了非常类似于人体内情况的人T-细胞免疫系统。所述小鼠亦产生正常NK细胞，如通过NKp受体和杀伤细胞免疫球蛋白样受体(KIR)所显示，其反映供体NK所有组分。
肿瘤接种
在干细胞移植之后12周皮下接种1x106个同种异体A204细胞，在3周内在所有小鼠中得到侵略性生长的肿瘤。与患有RMS的患者的干细胞移植(SCT)一致（in line withstem-cell transplantation(SCT) of patients with RMS），免疫系统未能抵制同种异体A204。肿瘤迅速生长，尽管可靠的表达I类和II类表面HLA、MICA/B、粘连蛋白-2 (CD112)和脊髓灰质炎病毒受体(PVR，CD155)但完全缺乏UL16结合蛋白(ULBP) 1-4。
使用靶定组蛋白的IL-12融合蛋白的肉瘤治疗
在肿瘤接种之后，实体A204 RMS肿瘤在3周后建立。随后，使用单独的FcIL-7 (对照)、NHS-IL12/FcIL-7或NHS-IL12/IL-2MAB602每周处理小鼠达5周(图1A)。静脉内注射所述构建体未导致可见的全身毒性，既无急性毒性也无随时间显现出的毒性(图1B：4只小鼠/组，100天)。在仅用FcIL-7处理的小鼠中，肉瘤显示指数生长。4/7小鼠在第5周之前死亡，以及3只小鼠在第52天因肉瘤负担所致而到达终点标准(图1C)。在Fc-IL7组，从第25天到第52天观察到6.5倍肿瘤生长，而在NHS-IL12/IL-2MAB602中，所述生长减小为1.8倍(P<0.05，单尾t-检验)。因此，肉瘤在接受NHS-IL12/IL-2MAB602的小鼠中保持显著生长停滞(n=11)(图1C)，导致所有小鼠(miceved)存活(图1B和1D)。NHS-IL12/FcIL-7仅对2/11小鼠保护较短时间(图1B和1C)，因为其分别在第43天和第49天死亡。为了从全部3组得到分析，除了每个NHS-IL12处理组的4只小鼠之外，在第52天(短期处理)处死小鼠，所述4只小鼠保持存活并接受治疗直至第100天(长期处理)。NHS-IL12/FcIL-7长期处理在1/4小鼠中成功阻止肿瘤生长，在2/4小鼠中延迟肿瘤生长以及在1只小鼠中清除肿瘤。NHS-IL12/IL-2MAB602长期处理在3/4小鼠中清除肿瘤并在剩余小鼠中阻止肿瘤生长(图1D)。
NHS-IL12构建体的生物分布
所述单克隆抗体NHS76识别坏死癌症区域中的胞内抗原。因此对NHS-IL12是否优先与肉瘤部位结合进行分析。使用123I-标记NHS-IL12的SPECT/CT生物分布研究显示NHS-IL12在肉瘤微环境内部的显著体内富集(图2A)。123I-标记NHS-IL12的定量显示与对侧肌肉相比在肿瘤中的4-6倍放射性核富集。123I计数在静脉内NHS-IL12施用之后26 h时在肿瘤区达到峰值，而在正常肌肉组织中123I计数随时间保持稳定(图2B)，证实NHS-IL12优先与人肉瘤结合。
肿瘤特异性免疫反应
为了了解不同处理方案的治疗功效潜在的差异，本发明人进行了对浸润（infiltrating）A204肉瘤的人免疫细胞的组织学、免疫组织化学(IHC)以及广泛的分子和功能表征。
引人注目的是，FcIL-7-处理小鼠的肉瘤只有很少浸润，其专有地包含巨噬细胞(CD68+)和NK细胞(CD56+)。对比鲜明的是，使用任一NHS-IL12方案处理小鼠的肉瘤显示含有NK细胞、巨噬细胞和大量CD4+和CD8+ T细胞的密集的单核细胞浸润。所有处理组的NK细胞均表达NKG2D mRNA和DNAM-1 (图3A)，其为肉瘤相关的表面分子粘连蛋白-2 (CD112)和PVR(CD155)的配体。操纵NK-细胞分化和激活的表面分子的mRNA表达严格地需要NHS-IL12构建体。然后FcIL-7或IL-2MAB602调节NHS-IL12构建体对浸润的NK-细胞群体的作用。NKG2E、NKp44和NKp46仅在用NHS-IL12/FcIL-7处理小鼠的肿瘤中发现，而NKp30表达限于NHS-IL12/IL-2MAB602-处理小鼠和NHS-IL12/FcIL-7长期处理小鼠的肉瘤(图3A)。FcIL-7处理小鼠的肉瘤强表达CD161 (图3B)和TH17-主导转录因子RORC (图4A)，其表征产IL-17的表型(Billerbeck等(2010) Proc. Natl. Acad. Sci. USA. 107:3006-11)。对比鲜明的是，NHS-IL12/FcIL-7-处理小鼠亦具有强的CD161表达(图4B)，但显著更低的RORC表达(图4A)，其表征分泌高水平的IFN-γ和TNF但缺乏溶胞活性的效应子和中心记忆T细胞(Takahashi等(2006) J. Immunol 176:211-216)。使用NHS-IL12/IL-2MAB602处理的小鼠缺乏CD161mRNA (图3B)和RORC mRNA (图4A)，有力地表明产IL-17的NK细胞或T细胞表型在这些小鼠中受到阻抑(Laurence等(2007) Immunity 26:371-381；Ghoreschi等(2010) Nature 467:967-971)。
因为KIR分子即使在缺乏MHC-I的环境中亦损害NK-细胞功能(27)，对FcIL-7-或NHS-IL12/FcIL-7-处理小鼠的肉瘤中的KIR表达进行分析。整个肉瘤的qRT-PCR在两组中显示相似水平。正如预期，正常小鼠肌肉组织寻靶NK细胞和人肉瘤异种移植物的浸润肿瘤的淋巴细胞的KIR-表达不同，小鼠肌肉显示KIR2DL3和KIR2DL4，而人肉瘤显示额外的KIR2DL1和KIR3DL1。尽管表达不同的KIR，但NK细胞仍为有功能的，因为从肉瘤组织新鲜分离的NK细胞在使用NHS-IL12体外刺激之后释放IFN-γ。除NK细胞之外，表征TH17先天淋巴细胞群体，如表达TCRVα24的iNKT细胞、NKp46+ NK或γδ T细胞的mRNA表达几乎专有地存在于经NHS-IL12/FcIL-7处理小鼠的肉瘤中（Besides NK cells, mRNA expression was foundcharacterizing TH17 innate lymphocyte populations, like TCRVα24-expressingiNKT cells, NKp46+ NK or γδ T cells almost exclusively in sarcomas of micetreated with NHS-IL12/FcIL-7）(图3A、3C、3D)。
除先天淋巴细胞群体之外，所有NHS-IL12-处理组的肿瘤还显现了广谱的CD3+ T细胞(图4B和4C)。这些在用FcIL-7处理小鼠的肉瘤中为不存在的，所述肉瘤在Vβ谱型分析中几乎不显示信号(图4B和4C)以及无浸润性CD8+ T细胞。NHS-IL12-处理肉瘤显示广泛的TCR所有组分，其通过在多个Vβ-家族内的寡克隆峰或单克隆峰证实(图4C)，因为其发生在优先扩增限制性T-细胞克隆期间。CDR3区的克隆和测序证实，在两个处理组中，所述峰包含有限数量的具有强扩增的TRBV片段的不同T-细胞克隆，例如在NHS-IL12/FcIL-7组的所有个体中的TRBV29-1，或者在NHS-IL12/IL-2MAB602组中的TRBV5-5和TRBV18 (图4B和4C)。分别调节IFN-γ和IL-17的转录因子T-bet和RORC的相对表达，反映各组浸润肿瘤的淋巴细胞中TH1偏倚的程度。T-bet/RORC比率在仅FcIL-7的组中为<0.05，而其在接受含Fc-IL7或IL-2MAB602的NHS-IL12的小鼠中为19倍(0.8)和44倍(2.2) (图4D)。与T-bet/RORC表达一致，与仅FcIL-7的组相比，Foxp3在两个NHS-IL12组中均为约1/10 (图4A)。因此，低的Foxp3表达与T-细胞激活标志物CD40L的强表达负相关(图4A和4D)。
NHS-IL-12处理肉瘤中的衰老诱导
NHS-IL12构建体在所有处理小鼠中强烈阻抑肉瘤形成(图1)。出人意料的是，仅NHS-IL12/IL-2MAB602-组的肉瘤含有大量的穿孔蛋白和粒酶K mRNA (图4A)，而用NHS-IL12/FcIL-7处理的小鼠近乎(virtually)不含穿孔蛋白并且表达低水平的粒酶K mRNA(图4A)。这有力地表明控制肉瘤的免疫反应包含不同于细胞溶解的机制。此外，肉瘤并未包含数量足以通过杀伤或凋亡解释癌症控制的CD4+或CD8+ T细胞。
因此分析了免疫反应对肉瘤细胞增殖的作用，如通过增殖标志物增殖细胞核抗原(PCNA)和Ki67所确定。在迅速生长的FcIL-7-对照肿瘤中，50%的肉瘤细胞对PCNA或Ki67染色阳性，显示大部分细胞正在增殖(图5A和5B)。在NHS-IL12-处理组中，PCNA-染色(图5A)和Ki67-染色的肉瘤细胞均显著低于FcIL-7-对照中的染色肉瘤细胞(图5A和5B)。为了确定所述免疫反应是否仅使细胞周期停滞或者其是否诱导如在细胞因子诱导的衰老中所见到的稳定的生长停滞，将肉瘤针对一种增殖标志物和衰老相关的磷酸化异染色质蛋白1 (p-HP1γ或p16INK4a，亦称为细胞周期调节周期蛋白依赖性激酶抑制物2A (CDKN2A))进行复染。仅FcIL-7-处理的肉瘤显示高PCNA/Ki67表达以及同时非常低的p16INK4a/核p-HP-1γ表达，证实这些肉瘤细胞正在迅速增殖(图5A和5B)。对比鲜明的是，高达70%的来自经NHS-IL12/IL-2MAB602或NHS-IL12/FcIL-7处理小鼠的肉瘤细胞表达衰老标志物p-HP1γ或p16INK4a (图5A和5B)，而不含PCNA (PCNA-/p-HP1γ+) (图5A，上排插图)和Ki67 (Ki67-/p16INK4a) (图5A，下排插图)。
数据清楚显示的是，IL-12驱动的人免疫系统的刺激可使用癌细胞的衰老作为约束肿瘤生长的重要机制。
鉴于IFN-γ和TNF为IL-12驱动的TH1-免疫的两个主要的效应细胞因子并且因为这两个细胞因子可诱导衰老，将非常早期代数的多个患者来源的人RMS细胞系与渐增剂量的IFN-γ和TNF一起孵育。单独的任一种细胞因子并未导致生长抑制或者仅导致轻微生长抑制。然而，其在组合时在2/3肉瘤中导致永久的衰老界定（senescence-defining）的生长停滞(图5C)。重要的是，抗衰老肉瘤不表达细胞周期调节子p16INK4a (未显示)，证实IFN-γ和TNF-诱导的衰老严格需要p16INK4a的激活。
A204横纹肌肉瘤中的分化诱导
鉴于IFN-γ-和TNF-控制的免疫反应导致RMS中永久的细胞周期停滞，而骨骼肌分化取决于成肌细胞较早退出细胞周期以允许肌肉特异性基因的表达和细胞融合成多核肌管，所以存在这样一个问题：是否此生长停滞亦可能影响A204肉瘤的分化。结蛋白为用于成横纹肌细胞分化的可靠标志物，其在未分化或低分化的RMS中为不存在的。因此，A204肉瘤在移植之前即不显示表征肌细胞的横纹，亦不表达结蛋白(未显示)。在移植到人源化小鼠中之后，经FcIL-7-处理的增殖肉瘤保持为低分化的，其具有弥散分布在肿瘤内的少量单个结蛋白+细胞但没有横纹(图6A和6B)。对比鲜明的是，NHS-IL12处理不仅在A204肉瘤中抑制肿瘤生长并诱导衰老，而且还逐渐恢复成肌结构和标志物表达。NHS-IL12/FcIL-7诱导随机分布的线性区域的成熟RMS细胞，其明显地表达结蛋白和横纹区。朝功能性肌肉组织的这种分化在来自经NHS-IL12/IL-2MAB602处理小鼠的肉瘤中甚至更显著。所述生长停滞的肉瘤显示绳状/绳样增殖的分化区，其具有广泛穿透肿瘤的筛状/管样结构(图6A和6B)。因此，肉瘤的生长停滞在体内诱导衰老，并且与肌细胞(A204 RMS的来源)的细胞命运特异性标志物的恢复相关。
在体外，单独施用组合的IFN-γ/TNF使A204肉瘤细胞不可挽回地生长停滞，并立即增加周期蛋白依赖性激酶抑制子p21CIP1/WAF1的mRNA(在未处理对照的第+1天为7倍，第5天为10倍)，其在骨骼肌中内在地使细胞退出周期（cycling）(图7A和7B)。引人注目的是，在5天之内IFN-γ/TNF亦在肉瘤培养物中大量诱导双核和多核细胞(>80%的多核细胞) (图7C和7D)，其具有如在后期末见到的近端核，表明p16INK4a-介导的胞质分裂的不可逆阻断。此外，肉瘤细胞重新表达另外两种肌细胞分化调节标志物(Myf-6和肌球蛋白重链-II) (图7E)，并变形成SA-β-半乳糖苷酶+伸长的多核细胞，形成成肌巨细胞(图7C和7D)，其为晚期肌细胞分化中的步骤。单独给予IFN-γ/TNF在体外亦使RMS细胞系RH30和患者来源的RMS细胞系SRH (第14代)不可逆地生长停滞(图8)，但是不会使ZCRH (p16缺陷型，第9代)不可逆生长停滞。
在代表三胚层组织的一组肿瘤细胞系中的分化诱导
为了测试通过IFN-γ/TNF诱导的生长停滞和分化是否可能具有更大的相关性，在体外对代表三胚层组织的一组肿瘤细胞系测试了两个TH1细胞因子组合IFN-y/TNF。3/3成胶质细胞瘤细胞系(T98G、Ln229、A172)、2/2成神经细胞瘤细胞系(LS和LAN-1)以及癌细胞系MCF-7 (乳腺癌)、HCT-116 (结肠直肠癌)和Hep3b (肝细胞癌)停止增殖，一致表达SA-β-Gal并显示退变类型的细胞异型和核异型(图8)。组织病理学特征包括大型深染多形核（large hyperchromatic pleomorph nuclei）以及类似于骨骼肌中由细胞损伤导致的活性/非瘤的多核成肌巨细胞的形态。在成胶质细胞瘤中，核异型类似于在周围神经的良性“古老神经鞘瘤”中所见的类型(图8)。多核巨细胞的形成已在许多器官中报道并且与感染、损伤、自身免疫和肿瘤有关。
药物组合物
可将根据本发明的靶向IL-12融合蛋白掺入适于给予的药物组合物中。所述组合物通常包含抗体可变区和药学上可接受的载体。意图本文所用的术语“药学上可接受的载体”包括与药物给予相容的任何和所有溶剂、分散介质、包被物、抗菌和抗真菌剂、等渗和吸收延迟剂等。所述介质和物质用于药学上活性物质的用途为本领域众所周知的。
将本发明的药物组合物进行配制以与其预期给予途径相容。给予途径的实例包括胃肠外(例如静脉内、皮内、皮下)、口服(例如吸入)、透皮(局部)、透粘膜和直肠给予。用于胃肠外、皮内或皮下施用的溶液剂或混悬剂可包含以下组分：无菌稀释剂，例如注射用水、盐水溶液、不挥发性油、聚乙二醇、甘油、丙二醇或其它合成溶剂；抗菌剂，例如苯甲醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸；缓冲剂，例如醋酸盐、柠檬酸盐或磷酸盐以及用于调节张力的物质，如氯化钠或葡萄糖。pH可使用酸或碱调节，例如盐酸或氢氧化钠。所述胃肠外制剂可封装在安瓿、一次性注射器或者由玻璃或塑料制成的多剂量瓶中。
包含本发明的靶向IL-12融合蛋白和IL-7/IL-2分子的药剂可具有0.01-100% (w/w)的浓度，但所述量根据药剂的剂型而不同。
给予优选为每两周一次或每月一次，但可根据给定个体中的药代动力学情况而频率更高或更低。用于约70公斤成人的如本申请中所述的抗体融合蛋白的剂量为每次剂量在约50-1000毫克的范围内，每次剂量优选范围为约100-500毫克。用于70 kg成人每月治疗一次的的最优选剂量为约200-400毫克。
附图简述：
图1
研究设计、RMS激发和治疗之后的肿瘤生长和存活。(A)对4-6周龄NSG小鼠进行亚致死辐照并使用CD34+ CD3-移植物人源化。在第12周用1×106个A204细胞接种完全植入的小鼠。18天之后当肿瘤体积为50–200 mm3时开始免疫治疗。在5周处理之后处死小鼠，此时FcIL-7组的肿瘤达到体重的20%。使NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602组的四只小鼠继续存活和处理至少到肿瘤接种之后的第95天。(B) FcIL-7、NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602对存活的影响。使用时序检验(log-rank test)比较存活率曲线。与FcIL-7组相比，NHS-IL12组的存活非常显著地更好。在FcIL-7对照组中，4只动物在第52天之前死亡以及3只在第52天因过度的肿瘤生长而被处死。在NHS-IL12/FcIL-7处理组中，2只小鼠在第52天之前死亡，在长期治疗组中，1只在第56天死亡以及1只在第74天死亡。(C) FcIL-7、NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602对肿瘤生长的影响。使用FcIL-7 (20 µg) (圆形)、NHS-IL12 (20 µg)/FcIL-7 (20 µg) (三角形)或者使用NHS-IL12 (20 µg)和与MAB602 (15 µg)复合的IL-2 (1.5 µg) (正方形)每周静脉内处理含人RMS A204的小鼠。肿瘤大小(mm3)表示为短期处理(5周)的各组7只小鼠的平均值±SD。(D)长期处理(14周，>95天)期间各NHS-IL12组的4只小鼠的单个肿瘤的大小(NHS-IL12/FcIL-7：正方形；NHS-IL12/IL-2MAB602：圆形)。
图2：
123I-标记的NHS-IL12在人A204肿瘤异种移植物病损中积累。(A)在注射治疗剂量(30 µg)的123I-标记的NHS-IL12之后2、26和46 h之后进行的体内SPECT扫描，显示与肌肉组织(虚线圆)相比NHS-IL12在肿瘤(实线圆)中的特异性积累。(B)在给予之后26 h，123I-NHS-IL12的吸收在肿瘤病损中达到其最大值，而在肌肉中，在整个扫描时间内未能检测到特异性信号。对计数进行衰减校正以针对123I在测量时间点之间的放射性衰减进行调整(n=2)。*P≤ 0.05。
图3
FcIL-7、NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602对先天免疫的影响。(A)使用基于RT-PCR的片段长度分析对各组中个体的肿瘤匀浆检测主要的触发受体NKG2C、-D和-E、DNAM-1以及自然杀伤细胞受体NKp30、-44和-46。注意组内的高度一致性。(B) CD161在肿瘤匀浆和肌肉匀浆中的表达。量以平均荧光密度给出。各点代表一个独立的肿瘤。** P≤0.01，*** P≤ 0.001，使用单尾学生t检验评价。(C)第52天显示iNKT细胞(不变Vα24和Vβ11)、Vδ1和-2链以及NKp46的TCR转录物。T=肿瘤组织，M=肌组织，LT=第100天的长期处理的小鼠，S=单峰，G=Vα24链表达的高斯分布。(D)检测为单峰或呈高斯分布的A204肿瘤中的TCRVα24 mRNA表达。在人中，iNKT TCRVα24优先与TCRVß11链相关联。
图4
FcIL-7、NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602影响下的αβ T细胞的克隆能力分析。(A)基于实时PCR检测肿瘤匀浆中的多种免疫标志物。(B)将靶基因的表达针对人CD45的表达标准化。通过CDR3-大小谱型分析确定25个TRBV片段的表达。填充的方格表示高达12个片段的表达。每个垂直列代表一只小鼠，深色方格表示选择用于CDR3序列分析的TRBV片段。(C)所选TRBV片段的CDR3区蛋白质序列，粗体的氨基酸代码标示同源序列。(SEQ ID NO.6：LDKVGQETQYF；SEQ ID NO. 7：VDRTGEPFYGYTF；SEQ ID NO. 8：STRTGSYNYPLHF；SEQ IDNO. 9：LAEGVTEAFF；SEQ ID NO.10：LAKGVTEAFF；SEQ ID NO. 11：SLNLVLPGGAEAFF；SEQ IDNO. 12：PRFSMNTEAFF；SEQ ID NO. 13：SRGSFESYNEAVLRAQ；SEQ ID NO. 14：SRGSFESYNEQFF；SEQ ID NO. 15：SRGSFESYNEQVLR；SEQ ID NO. 16：RTVANGFSPLHF)。(D)肿瘤匀浆中T-bet/RORC-和Foxp3/CD40L-表达比率(n=4)。* P≤ 0.05，** P≤ 0.01，*** P≤0.001。
图5
通过NHS-IL12/FcIL-7和NHS-IL12/IL-2MAB602处理诱导衰老标志物和抗增殖效应。(A)通过针对核p-HP1γ组合以PCNA (上排)或者p16INK4a组合以Ki67 (下排)的免疫荧光复染确定肿瘤切片内的细胞衰老和增殖(1:100)。插图显示更高放大倍数的图片(1:300)以显示p-HP1γ或p16INK4a染色的核点。(B)使用FcIL-7、NHS-IL12/FcIL-7或NHS-IL12/IL-2MAB602处理之后p-HP1γ-阳性细胞(即具有多于5个核点的细胞)、p16INK4a–阳性细胞(如通过更高放大倍数(1:300)所确定)或Ki67-阳性细胞的平均百分数(n=3)。** P≤ 0.01，***P≤ 0.001，使用单尾学生检验评价。(C)在原发性人RMS癌细胞制备物中的细胞因子诱导的生长停滞。以2×104个细胞/9.6 cm2的密度接种CCA细胞(eRMS，第7代)、SRH (eRMS，第8代)或ZCRH细胞(aRMS，代数> 9)。第3天和第4天，使用100 ng/ml IFN-γ和10 ng/ml TNF或单独的培养基(对照)处理细胞。第7天，去除细胞因子，将细胞用胰蛋白酶消化、计数并以2×104个细胞/9.6 cm2重新接种。在孵育又4天(ZCRH 和SRH)或10天(CCA)之后，对活细胞计数。在不存在(Co.)或存在IFN-γ加TNF时应答细胞CCA和SRH以及非应答细胞ZCRH的生长曲线。显示平均细胞数± SEM (n=3)。
图6
作为A204 RMS中生肌分化标志物的结蛋白的体内表达和组织。(A)将来自所有组(经FcIL-7、NHS-IL12/FcIL-7或NHS-IL12/IL-2MAB602处理)的肿瘤(n=3/组)的组织学载玻片针对结蛋白染色以及(B)通过设盲病理学家（blinded pathologist）进行分析。LT：长期处理；ST：短期处理。
图7
在未处理和经细胞因子处理的A204细胞中多核的衰老A204细胞以及p21和成肌标志物的表达。(A)在使用IFN-γ和TNF (++)或作为对照的培养基(-)处理前后p21的相对表达，通过定量RT-PCR测量(n=3)。(B) IFN-γ和TNF处理(倒三角形)终止癌细胞增殖但不会杀伤肉瘤细胞(n=3)。用于比较，正常细胞培养物(圆形)显示未受阻碍的增殖(n=3)。(C)上排：经细胞因子处理的A204癌细胞为衰老的(黑色箭头：灰色染色)和多核的(白色箭头，DAPI染色)。下排：经培养基处理作为阴性对照的A204肉瘤细胞对于SA-β-Gal和单核细胞为阴性的(DAPI-染色)。(D)与标准培养的A204细胞(左)相比的在A204细胞中经细胞因子诱导的伸长的多核形态和合胞体形态(中和右)，通过透射显微镜描述。(E)成肌转录因子在A204和RH30肉瘤细胞系中的相对表达。3天内与培养基(-)相比通过100 ng/ml IFN-γ和10 ng/ml TNF (++)在A204细胞中诱导MHCII和MYF-6 mRNA的表达(n=3)。
图8
代表中胚层、内胚层和外胚层来源的细胞系在使用100 ng/ml IFN-γ组合以10ng/ml TNF的单剂量处理之后与培养基对照(未处理)相比显示多核表型和生长停滞(第1天)。成胶质细胞瘤的图片在处理之后第37天获得，其它图片均在处理之后第6天获得。所有处理细胞系的细胞在第43天均存活，所述时间为最终检验细胞的时间点。RMS：横纹肌肉瘤，GBM：成胶质细胞瘤，BC：乳腺癌，CRC：结肠直肠癌，HCC：肝细胞癌，NB：成神经细胞瘤
实施例：
(1) NSG小鼠的人源化。HuCD34+干细胞来源于来自亲代供体的剩余的G-CSF动员的外周血干细胞，其已通过CD34+选择耗尽T-细胞(CliniMACS，Miltenyi，德国)。用20%DMSO/80% 5%-HAS溶液使细胞悬浮并随后使用Sylab方冰装置（icecube device）并以受控的冷冻速率冻存。融化之后，将细胞用台盼蓝染色并用Neubauer细胞计数室计数。关于科学使用剩余细胞的知情同意书依照Helsinki宣言从所有供者获得。在融化之后通过第二轮CD3+消耗将CD34+群体的纯度进一步增加到>99.99% (LS MACS，Miltenyi，德国)。干细胞供体对于RMS A204细胞系而言均为HLA-错配的。在经亚致死辐照(250 cGy)的NSG小鼠的尾静脉中注射含1×106个huCD34+细胞的100 µl预热PBS。通过每周施用20 µg FcIL-7 (Merck，德国)支持植入。在各NHS-IL12处理组中，4只动物接受具有Fc-IL7或IL-2MAB602的长期NHS-IL12细胞因子处理达最多15周(100天)。
(2)肿瘤植入和测量。胎儿RMS A204细胞获自ATCC (HTB 82)，使之融化，用RPMI1640+10% FBS传代培养（sub-culture）1代，用无菌盐水清洗三次，使用PCR支原体检测试剂盒AppliChem (德国)检测支原体，并植入经移植的NSG小鼠中。每只小鼠，通过皮下注射在右侧腹中移植1×106个肿瘤细胞。肿瘤体积用以下方程确定：VT=a×b×d×π/6，此处a、b和d描述肿瘤的长、宽和高。在肿瘤接种之后52天处死小鼠，所述时间为FcIL-7组中动物的肿瘤达到≥体重的20%的时间点。使每个NHS-IL12组的四只小鼠继续存活用于长期处理。
(3)抗人组蛋白/人IL-12融合蛋白和IL-2/抗IL-2抗体复合物的给予。在肿瘤已生长至≥150µl之后，在经植入的人源化NSG小鼠中经由穿刺尾静脉每周一次给予20 µg的NHS-IL12融合蛋白连同20 µg FcIL-7 (均来自Merck，德国)。如上所述，使另一组小鼠每周接受1.5 µg IL-2加15 µg抗IL-2 mAB MAB602的混合物。在注射之前使重组人IL-2(PROLEUKIN，Aldesleukin，Chiron，USA)和MAB602 (抗hIL-2 mABCD122，克隆5355，R&DSystems)在室温下共同孵育15 min。
(4)体内SPECT/CT成像。使用Inveon多模态SPECT/CT (Siemens Healthcare，Knoxville，TN，USA)进行接种小鼠的体内成像。无载体碘化钠(123I)购自GE Healthcare并使用Pierce®碘化试剂(Thermo Scientific)进行NHS-IL12的放射性碘标记。经由尾静脉给小鼠注射30 μg用18 MBq 123I标记的NHS-IL12，并在示踪物给予之后2、26和46小时获得体内SPECT/CT图像。在注射和测量期间，使用含1.5%异氟烷的氧气(0.5 l/min)将小鼠麻醉。基于关于用以涵盖整个肿瘤的数个切片的CT信息在重建的SPECT图像上勾勒出兴趣区(ROI)的轮廓。将相同大小的ROI置于相同动物左后足的未受影响的肌肉组织上以用于参照。使用衰减校正的计数以用于评价123I–NHS-IL12吸收。
(5)六色流式细胞术。在移植之后10-12周对数个个体评价免疫重建，并使用以下与鼠科动物无交叉反应但对人表位而言为特异性的鼠mAb分析外周血、脾、骨髓和胸腺，所述鼠mAb：CD62L(Dreg56)-FITC、CD25(2A3)-APC、CD3(SK7)-PerCP、CD8(SK1)-PerCP、CD8(SK1)-APC-H7、CD4(SK3)-FITC、CD4(SK3)-PerCP、CD14(M5E2)-PE、CD56(My31)-PE、HLA-DR(L243)-PerCp、NKp30(P30-15)-PE、NKp44(P44-8)-APC、NKp46(9E2)-PE及其相应的IgG同种型(均来自BD Pharmingen，德国)。CD45(MEM-28)-Pacific Blue、CD19(HIB19)-PerCP、CD3(MEM-57)-Alexa Fluor 700、CD4(MEM-241)-Alexa Fluor 700 (Exbio，捷克斯洛伐克)。具有各自同种型IgG的CD45(HI30)-PE (Biolegend，德国)。在移植之后12-14周通过眶后出血和FACS染色常规检查植入。使用ULBP-1(Z-9)、ULBP-2(2F9)、ULBP-3(F16)、ULBP-4(6E6)(均来自Santa Cruz，USA)、MICA/B(6D4)APC CD112(R2.525)PE、CD155(SKII.4)PE (均来自Biolegend，德国)、HLA-ABC(W6/32)PE (DAKO Cytomation，德国)、第二抗体RAM-PE(X56)(BD Pharmingen，德国)和同种型对照(Beckman Coulter和R&D Systems，德国)表征A204细胞系。使用Diva©软件用LSR II (BD Biosciences)进行流式细胞术。
(6)免疫组织学染色。将冷冻的组织载玻片(5 µm 厚度)在4%缓冲的福尔马林中孵育(2 min)，用蒸馏水清洗，在压力室中用pH 6.0柠檬酸盐缓冲液煮沸(4 min)，并用Tris-NaCl-Tween清洗。将样品载玻片转移至湿室并用Zytochem-Plus AP聚合物试剂盒(ZytomedSystems，德国)染色。第一抗体为：CD3 (SP7，1:50；DCS Innovative Diagnostic SystemeGmBH，德国)、单克隆兔抗人CD4 (SP35，1:50，Zytomed Systems)、CD8 (C8/144B，1:100)、CD56 (123C3-D5，1:20)、CD68 (PG-M1，1:150)、HLA-DR-α (TAL.1B5，1:200)、结蛋白(D33，1:100，均来自DAKO，德国)、穿孔蛋白(5810，1:200，Novocastra/Leica，德国)。使用Permanent AP Red试剂盒(Zytomed Systems，德国)进行最终染色。进行单盲法用于分析免疫组织学载玻片。
(7)免疫荧光。如前所述将新鲜冷冻的人异种移植A204肿瘤的冷冻切片染色(Zhang和Adams (2007) Cell Cycle 6:784-789)。简单地说，使用高碘酸盐-赖氨酸-多聚甲醛固定冷冻切片并使用驴血清(1:20)于室温(RT)封闭30 min。然后使载玻片与兔抗p-HP1γ (1:80；Abcam，UK)、鼠抗PCNA (1:50；Cell Signaling Technology，USA)、鼠抗p16INK4a (1:50；Santa Cruz Biotechnology，德国)或兔抗Ki67 (1:100；Abcam，USA)一起于RT孵育1h。3次清洗之后，使切片与Cy3-或Cy5-缀合的驴抗兔抗体和Cy3-或Cy5-缀合的驴抗鼠抗体(均来自Dianova，德国)一起孵育。在使用Mowiol (Hoechst，德国)封固载玻片之前，使用Yopro (1:2000；Invitrogen，德国)将核染色(5 min)，并用Leica TCS-Sp/Leica DMRB共聚焦激光扫描显微镜(Leica Microsystems，德国)分析切片。使用Leica共聚焦软件LCS (Version 2.61)处理图像。
(8)使用TNF和IFN-γ处理患者来源的原发性RMS和癌细胞系。使用早前精确描述的程序确定不同癌细胞系(横纹肌肉瘤RH30、SRH；成胶质细胞瘤T98G、Ln229、A172；乳腺癌MCF-7；结肠直肠癌HCT-116；肝细胞癌Hep3b和成神经细胞瘤LAN-1、LS)在细胞因子处理之后的永久生长停滞(Braumuller等，2013，Nature494:361-365)。通过以较低数量(4,000或8,000个细胞/cm2)接种肿瘤细胞并与含或不含10 ng/ml TNF + 100 ng/ml IFN-γ的培养基一起孵育来完成用以研究RMS中的早期分化事件的短期培养。
(8) KIR表达分析。如前所述进行表达分析(77)。使用5`FAM标记的反向引物，在终点PCR中用特异性引物确定NKp30、-44、-46、DNAM-1、CD161转录物。在具有用于长度标准的Gene Scan-600 LIZ和GeneMapper软件(二者均来自Applied Biosystems，德国)的ABI测序仪中对PCR产物分析片段长度；使用MFI进行半定量分析。
(9) T-bet、RORC、Foxp3、CD40L、粒酶B、粒酶K的表达。使用cDNA特异性引物(所有序列可根据需要获得)和iQ SYBR Green Supermix BioRad (Munich，德国)，用实时PCR使用BioRad C1000热循环仪/CFX96实时系统(Munich，德国)确定瘤内基因表达。将表达针对人CD45表达标准化。使用特异性引物组(Search LC，德国)和FastStart DNA SYBR Green I(Roche，德国)检测RORC表达。
(10) Vα-24链鉴定。对此，使用Han等(Han等，1999，J. Immunol. 163:301-31)公布的方案。
(11) Vβ谱型分析。使用较小修饰根据Gorski等(Gorsk等，1994，J. Immunol.152:5109-511)分析TCR Vβ-链表达的多样性和TCR所有组分的复杂性。使用5`FAM-标记的Cβ-引物，以及具有Gene Scan-600 LIZ和GeneMapper软件的ABI测序仪用于检测扩增子(二者均来自Applied Biosystems，德国)。
(12)γδ免疫观测（immunoscope）。如先前所公布来确定免疫观测(Dechanet等，J.，1999，J. Clin. Invest103:1437-1449)。
(13) TCR-CDR3区的鉴定。使用标准程序将Vβ PCR产物克隆到pGEMTeasy(Promega，德国)中并在XL1-Blue感受态细胞(Stratagene，USA)中扩增。将插入片段阳性克隆直接转移至再扩增Vβ PCR。在2.5%琼脂糖凝胶上分析5µl PCR等分，并将相关长度的PCR产物送至Seqlab (德国)用于序列分析。使用EMBOSS Transeq free软件实施将cDNA翻译成蛋白质序列。
Claims (12)
1.靶定肿瘤的IL-12和IL-7或其衍生物的组合在制备药物中的用途，所述药物用于在受癌症疾病所累的患者中诱导和/或刺激针对所述癌症疾病的免疫反应，其中所述诱导或刺激引起通过所述药物导致的
(i)癌细胞的衰老或生长停滞，和/或
(ii)使癌细胞或癌组织恢复成原始细胞或组织，
和其中所述靶定肿瘤的IL-12为一种治疗上有效的单克隆抗体或其生物学活性部分，其针对暴露在肿瘤坏死中的人DNA-组蛋白H1复合物，并经由其C端与IL12融合。
2.根据权利要求1所述的用途，其中以无修饰形式（naked form）、以共价结合形式或复合形式使用IL-7或其衍生物。
3.根据权利要求2所述的用途，其中所述IL-7或其衍生物与免疫球蛋白重链或其一部分共价融合，或者与抗IL2抗体形成复合物。
4.根据权利要求3所述的用途，其中所述免疫球蛋白重链或其一部分为Fc部分。
5.根据权利要求1-3中任一项所述的用途，其中所述衰老是导致稳定的生长停滞的衰老。
6.根据权利要求1-3中任一项所述的用途，其中所述衰老是不依赖于直接的免疫特异性细胞毒性效应的衰老。
7.根据权利要求1-3中任一项所述的用途，其中所述癌症疾病涉及实体瘤，或者肌肉、骨、神经、软骨、腱、血管以及脂肪组织或纤维组织的肿瘤。
8.根据权利要求7所述的用途，其中所述癌症为肉瘤。
9.根据权利要求1-3中任一项所述的用途，其中IL-12经由其p40和/或p30亚基的N端与抗体或其生物学有效部分的C端融合。
10.根据权利要求1-3中任一项所述的用途，其中所述抗体为全人NHS76抗体并且命名为NHS-IL12。
11.一种药物试剂盒，其包含
(i)第一包装，所述包装包含全人单克隆抗体NHS76，其针对暴露在肿瘤坏死中的人DNA-组蛋白H1复合物并经由其C端与IL12的p40和/或p30亚基的N端融合，和
(ii)第二包装，所述包装包含用于诱导和/或刺激针对癌症疾病的免疫反应的IL-7或其衍生物。
12.根据权利要求11所述的药物试剂盒，其中所述IL-7或其衍生物呈无修饰形式、呈共价结合形式或复合形式。
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| TN2020000015A1 (en) | 2017-08-03 | 2021-10-04 | Amgen Inc | Interleukin-21 muteins and methods of treatment |
| AR112797A1 (es) | 2017-09-08 | 2019-12-11 | Amgen Inc | Inhibidores de kras g12c y métodos para utilizarlos |
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| JP5070200B2 (ja) * | 2005-04-22 | 2012-11-07 | イーライ リリー アンド カンパニー | Ｔｇｆベータ１に特異的な抗体 |
| PL1966238T3 (pl) * | 2005-12-30 | 2012-09-28 | Merck Patent Gmbh | Warianty interleukiny-12p40 o polepszonej stabilności |
| CA2933705A1 (en) * | 2013-12-16 | 2015-06-25 | Merck Patent Gmbh | Survivin-directed cancer vaccine therapy |
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| WO2010046097A1 (en) * | 2008-10-21 | 2010-04-29 | Merck Patent Gmbh | Cancer treatments with radiation and immunocytokines |
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| RU2016136990A3 (zh) | 2018-10-10 |
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| CN105992590A (zh) | 2016-10-05 |
| EP3107573A1 (en) | 2016-12-28 |
| WO2015124297A1 (en) | 2015-08-27 |
| ES2703826T3 (es) | 2019-03-12 |
| RU2689160C2 (ru) | 2019-05-24 |
| JP2017507937A (ja) | 2017-03-23 |
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