CN113045635A - 一种特异性结合hla-a2分型的多肽组合物及应用 - Google Patents
一种特异性结合hla-a2分型的多肽组合物及应用 Download PDFInfo
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Abstract
本发明属于医学免疫学领域,具体涉及一种特异性结合HLA‑A2分型的多肽组合物及应用,该多肽组合物选自一种或多种衍生于抗原蛋白Survivin、Her2、CEA、hTERT、MAGE‑A3、EGFR、gp100或p53的多肽,衍生于各抗原蛋白的多肽含有各抗原蛋白的抗原表位或者抗原表位的变体。该多肽组合物通过负载抗原呈递细胞后呈递并激活特异性的CD8+细胞毒性T淋巴细胞而达到对肿瘤细胞的靶向毒性作用。本发明的多肽组合物及其衍生的肿瘤疫苗、DC疫苗、药物组合物能够显著激活免疫效应细胞,特别是T细胞,明显提高其激活相关的细胞因子的分泌水平和对肿瘤细胞的杀伤水平,具有潜在的临床价值。
Description
技术领域
本发明属于医学免疫学领域,具体地涉及一种多肽组合物及应用。
背景技术
近年来,通过外科手术结合放化疗治疗癌症取得了一些进展,提高了患者生存率,尤其是乳腺、肺、前列腺和肾脏扩散性癌症病人的生存率得以提高。然而,大多数这类治疗都具有显著的毒副作用,易伤害到正常的细胞。
肿瘤在机体内能引发体液免疫应答和细胞免疫应答。肿瘤抗原在细胞内加工成肽段后与细胞表面的主要组织相容性复合体I类分子结合并呈递给CD8+细胞毒性淋巴细胞,或先从肿瘤细胞上脱落,再由抗原提呈细胞摄取、加工成肽段后与表面主要组织相容性复合体II类分子结合并呈递给CD4+辅助性淋巴细胞,进而诱发机体的抗肿瘤细胞免疫应答。对抗肿瘤免疫及恶性肿瘤进展过程中遗传改变的认识的加深,使得人类能够开发更有选择性的安全治疗方法,采用通过激活免疫系统的方法以攻击正在发生的肿瘤,即肿瘤疫苗。根据肿瘤疫苗的具体用途,可以分为预防性疫苗和治疗性疫苗两类。预防性疫苗的主要功能为控制肿瘤的发生;治疗性疫苗以肿瘤相关抗原为基础,主要用于化疗后的辅助治疗。肿瘤疫苗中的一种为基于树突状细胞(DC)的疫苗。由于DC大量表达共刺激分子,并且具有既可有效地致敏CD4+辅助T细胞(T helper,Th)又可致敏CD8+细胞毒性T细胞(Cytotoxic TLymphocyte,CTL)的能力,故DC细胞与B淋巴细胞与巨噬细胞不同。DC通过负载肿瘤抗原并诱导为成熟的DC,产生特异性抗肿瘤免疫应答。基于此,已经用DC开发出多种抗肿瘤疫苗,包括肿瘤抗原肽负载DC、肿瘤全细胞抗原负载DC、肿瘤细胞RNA负载DC、肿瘤细胞DNA负载DC、外泌体(exosome)负载DC、细胞因子、趋化因子基因修饰DC。目前DC疫苗已经在恶性黑色素瘤、前列腺癌、肾癌等癌种当中进行了尝试,并部分取得了成功。各种形式的DC疫苗已在肿瘤的免疫治疗中开始尝试并在初步临床试用中显示出了良好的疗效。其中由美国Dendreon公司生产的DC疫苗Provenge在2010年被美国国家食品药品监督管理局批准上市,用于晚期尤其是对激素疗法失效的前列腺癌病人,疗效显示与安慰剂相比,其能够延长患者的生存时间超过4个月(Nature Medicine,2010,16(6):615.)。
当前大多数临床试验中仍然采用自体全肿瘤裂解液来负载DC,具体操作是将病人自身的肿瘤组织通过多次循环冻融进行裂解,以裂解液对DC细胞进行刺激(CancerImmunol Immunother,2006,55:819;medical oncology,2006,23:273.)。冻融循环会诱导产生肿瘤细胞坏死,但冻融诱导的肿瘤细胞坏死并不具有免疫源性,甚至会抑制Toll样受体(TLR)诱导的DC细胞成熟及正常功能(Hatfeld P,Merrick AE,West E,O’Donnell D,Selby P,Vile R,et al.Optimization of dendritic cell loading with tumor celllysates for cancer immunotherapy.J Immunother(2008)31(7):620–632),并且病人的肿瘤组织并非总是容易获得。肿瘤细胞裂解液,纯化的肿瘤相关抗原和肿瘤衍生mRNA也被证明可以用作负载DC的抗原来源。肿瘤细胞裂解液能够提供多种抗原供DC加载,并能诱导CD4+和CD8+T细胞应答和赋予DC不同的损伤相关分子模式(Damage-Associated MolecularPatterns,DAMP)以确保DC的成熟,但也会向DC提供免疫调节型细胞因子,诱导DC细胞发生耐受转化(Guida M,Pisconte S,Colucci G.Metastatic melanoma:the new era oftargeted therapy.Expert Opin Ther Targets 2012;16Suppl 2:S61-70);纯化的肿瘤相关抗原负载DC能够激活抗原特异性T细胞应答,并诱导CD4+和CD8+T细胞应答,但单次使用的不同的抗原种类数量有限。肿瘤衍生mRNA可以转入肿瘤相关抗原和共刺激分子,保证I型MHC的抗原呈递,并且不需要交叉呈递(Robbins PF,Morgan RA,Feldman SA,Yang JC,Sherry RM,Dudley ME,Wunderlich JR,Nahvi AV,Helman LJ,Mackall CL,et al.Tumorregression in patients with metastatic synovial cell sarcoma and melanomausing genetically engineered lymphocytesreactive with NY-ESO-1.J Clin Oncol2011;29:917-24),但不能诱导DC细胞成熟,也不能诱导有效的CD4+免疫应答,同时单次使用的不同的抗原种类数量也有限。
当使用肿瘤相关抗原中的短肽片段作为负载DC的抗原时,可以有效地增加单次使用时能够涉及到的抗原种类的数量,提高CD4+和CD8+T细胞的免疫应答水平,但需要先测定受试者的HLA单倍型,并在选定的肿瘤相关抗原中选取适当的肽段验证其是否能够与该HLA单倍型结合。HLA等位基因在不同种族人群中具有高度的多态性。根据世界卫生组织统计,截至2018年4月,I型HLA等位基因的数量已超过13000个,其中HLA-A等位基因4200个,HLA-B等位基因5091个,HLA-C等位基因3854个(http://www.hla.alleles.org/nomenclature/stats.html)。其中,亚洲人群常见的HLA分型多为HLA-A2、A3和A24(Experimental andTherapeutic Medicine,2011,2:109-117.)。HLA-A2、A11和A24三种分型能够覆盖超过中国人口的90%(Immunol Today,1996;17:261.)。HLA-A2属于HLA-A2超型,在中国人群中频率最高为45.9%,HLA-A11属于HLA-A3超型,在白种人(Caucasian)中频率最低,为37.5%,在中国人中频率最高,达52.7%;HLA-A24属于HLA-A24超型,在白种人中频率最低,为23.9%,在中国人中频率为40.1%,在日本人中频率最高,为58.6%(Curr Opinion In Immunol,1998,10:478-482;Immunogenetics,1999,50(3-4):201-212.)。目前针对各HLA分型都缺少包含能够有效被抗原呈递细胞呈递的免疫源性多肽组合物及包含这类多肽组合物的肿瘤疫苗。
发明内容
在肿瘤免疫中,肿瘤相关抗原的抗原表位的多肽片段与抗原呈递细胞如DC表面的HLA结合,形成HLA-肿瘤抗原表位肽复合物被TCR识别,进而被呈递给T细胞,使能够识别相应肿瘤抗原表位的T细胞被特异性激活并扩增。扩增的T细胞成为特异性靶向该肿瘤相关抗原的细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL),对表达该肿瘤相关抗原的肿瘤细胞产生细胞介导的免疫杀伤作用。
衰老细胞可以激活先天免疫反应和适应性免疫反应,维持组织稳态。此外,新发现表明程序性诱导细胞衰老可能对调节生殖过程很重要,部分原因是免疫清除。抗原p16、p53及p21与衰老细胞及肿瘤的发生具有显著的联系。目前被广泛认可的衰老细胞的生物标志物(如β-半乳糖苷酶,p16INK4A)即包括p16,p53和p21在细胞周期的调控中具有与p16相似的作用,且在多种肿瘤细胞中均可见p16或p53的调控突变或缺失。
为提高细胞介导的肿瘤免疫杀伤作用,本发明基于肿瘤相关抗原和细胞衰老相关蛋白筛选特异性结合HLA分型(A2)的免疫源性多肽组合物,及基于该免疫源性多肽组合物获得的负载该免疫源性多肽组合物的细胞、激活的免疫效应细胞和引发体内免疫应答反应的方法。肿瘤相关抗原有Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100,细胞衰老相关蛋白有p53。本发明的技术方案如下:
多肽组合物
本发明提供一种多肽组合物,选自一种或多种衍生于抗原蛋白Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100或p53的多肽,为各相应抗原蛋白的一部分,其长度等于或短于相应全长抗原蛋白;优选地,衍生于各抗原蛋白的多肽含有(1)各抗原蛋白的抗原表位,抗原表位具有免疫源性;或者含有经氨基酸替换、添加或删除而获得的同(1)中抗原表位活性相同的抗原表位,优选与(1)中抗原表位的氨基酸序列具有75%以上同源性,再优选与(1)中抗原表位的氨基酸序列具有85%以上同源性,更优选与(1)中抗原表位的氨基酸序列具有95%以上同源性,即为与(1)中抗原表位具有等同功能的变体,具有改变的氨基酸序列,如在(1)中抗原表位氨基酸序列上存在一个或多个氨基酸替换,或有一个或多个氨基酸被添加在(1)中抗原表位氨基酸序列上,或有一个或多个氨基酸从(1)中抗原表位氨基酸序列上被删除,而不影响包含(1)中抗原表位的多肽及包含(1)中抗原表位的多肽的多肽组合物的功能。作为本发明一个实施方案,有1-5个氨基酸,优选有1-3个氨基酸被添加到(1)中抗原表位氨基酸序列的N端和/或C端。多肽组合物中衍生于各抗原蛋白的多肽间两两的质量比为1~5:1,优选为1~3:1,更优选为1:1。
作为本发明一个实施方案,多肽组合物选自任意两种或三种衍生于抗原蛋白Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100或p53的多肽,Her2优选含有SEQ ID NO:6所示的氨基酸序列;具体如衍生于抗原蛋白Survivin、CEA和Her2的多肽,Her2优选含有SEQID NO:6所示的氨基酸序列,或衍生于Her2、hTERT和MAGE-A3的多肽,或衍生于P53、EGFR和gp100的多肽,或衍生于Survivin、P53和Her2的多肽,Her2优选含有SEQ ID NO:6所示的氨基酸序列,或衍生于Her2的两种不同多肽(如含有SEQ ID NO:2和SEQ ID NO:6所示的氨基酸序列)和衍生于hTERT的多肽;优选地,衍生于各抗原蛋白的多肽含有(1)各抗原蛋白的抗原表位,抗原表位具有免疫源性;或者含有经氨基酸替换、添加或删除而获得的同(1)中抗原表位活性相同的抗原表位,优选与(1)中抗原表位的氨基酸序列具有75%以上同源性,再优选与(1)中抗原表位的氨基酸序列具有85%以上同源性,更优选与(1)中抗原表位的氨基酸序列具有95%以上同源性。多肽组合物中任意两种衍生于各抗原蛋白的多肽的质量比为1~5:1,优选为1~3:1,更优选为1:1。多肽组合物中任意三种衍生于各抗原蛋白的多肽的质量比为1~5:1~5:1,优选为1~3:1~3:1,更优选为1:1:1。
作为本发明一个实施方案,多肽组合物选自衍生于抗原蛋白Survivin、CEA及选自Her2、hTERT、MAGE-A3、EGFR、gp100或p53中任意一种的多肽,Her2优选含有SEQ ID NO:6所示的氨基酸序列;具体如衍生于Survivin、CEA和Her2的多肽,Her2优选含有SEQ ID NO:6所示的氨基酸序列,或衍生于Survivin、CEA和hTERT的多肽,或衍生于Survivin、CEA和hTERT的多肽,或衍生于Survivin、CEA和MAGE-A3的多肽,或衍生于Survivin、CEA和EGFR的多肽,或衍生于Survivin、CEA和gp100的多肽,或衍生于Survivin、CEA和p53的多肽;优选地,衍生于各抗原蛋白的多肽含有(1)各抗原蛋白的抗原表位,抗原表位具有免疫源性;或者含有经氨基酸替换、添加或删除而获得的同(1)中抗原表位活性相同的抗原表位,优选与(1)中抗原表位的氨基酸序列具有75%以上同源性,再优选与(1)中抗原表位的氨基酸序列具有85%以上同源性,更优选与(1)中抗原表位的氨基酸序列具有95%以上同源性。Survivin、CEA及选自Her2、hTERT、MAGE-A3、EGFR、gp100或p53中任意一种多肽间的质量比为1~5:1~5:1,优选为1~3:1~3:1,更优选为1:1:1。
多肽组合物中衍生于各抗原蛋白的各多肽的长度在50个氨基酸以内;优选在30个氨基酸以内。具体地,衍生于各抗原蛋白的多肽的长度可以是5-30个氨基酸、8-25个氨基酸、8-15个氨基酸或9-12个氨基酸。作为本发明一个实施方案,衍生于各抗原蛋白的各多肽的长度为9个氨基酸。
抗原蛋白Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100或p53具有本领域所周知的含义和序列,包括各抗原蛋白的氨基酸野生型序列和各种已知的或可能存在的变体序列。
所述衍生于抗原蛋白Survivin的多肽含有SEQ ID NO:1所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:1所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:1所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:1所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白Her2的多肽含有SEQ ID NO:2所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:2所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:2所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:2所示的氨基酸序列具有95%以上同源性;或SEQ ID NO:6所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:6所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:6所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:6所示的氨基酸序列具有95%以上同源性。所述衍生于抗原蛋白Her2的多肽优选含有SEQ ID NO:6所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列。
所述衍生于抗原蛋白CEA的多肽含有SEQ ID NO:3所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:3所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:3所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:3所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白hTERT的多肽含有SEQ ID NO:4所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:4所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:4所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:4所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白MAGE-A3的多肽含有SEQ ID NO:5所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:5所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:5所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:5所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白P53的多肽含有SEQ ID NO:7所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:7所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:7所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:7所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白EGFR的多肽含有SEQ ID NO:8所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:8所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:8所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:8所示的氨基酸序列具有95%以上同源性。
所述衍生于抗原蛋白gp100的多肽含有SEQ ID NO:9所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:9所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:9所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:9所示的氨基酸序列具有95%以上同源性。
本发明多肽组合物可以通过本领域众所周知的常规方法制备得到,包括但不限于通过固相合成的化学合成后经HPLC将合成产物与副产品分离,以及在活细胞内表达编码包含本发明多肽组合物中抗原片段的多肽的核酸或通过体外的无细胞翻译系统翻译上述编码核酸后通过纯化,获得本发明多肽组合物中的抗原肽片段。此外,本发明所述多肽组合物中所包含的不需要的小分子可通过充分透析被去除,所得产物冻干后可添加其他载体或赋形剂以形成需要的制剂。还应当理解,本发明多肽组合物中可能附带的一些在疫苗组分中产生的氨基酸、突变体、化学修饰等基本不会干扰抗体或TCR识别抗原表位序列。
本发明多肽组合物与抗原呈递细胞如DC表面的HLA(尤其是HLA-A2亚型)结合,形成HLA-肿瘤抗原表位肽复合物被TCR识别,进而被呈递给T细胞,使能够识别相应肿瘤抗原表位的T细胞被特异性激活并扩增,扩增的T细胞成为特异性靶向该肿瘤相关抗原的细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL),对表达该肿瘤相关抗原的肿瘤细胞产生细胞介导的免疫杀伤作用。即本发明多肽组合物可刺激体内细胞介导的免疫应答和/或体液免疫应答,产生预防和/或治疗肿瘤的效果,可用于制备检测、和/或预防、和/或治疗肿瘤的药物、或试剂、或试剂盒,该药物、或试剂、或试剂盒包括但不限于生物制品。
负载多肽组合物的细胞
本发明还提供负载上述多肽组合物的细胞,所述细胞为抗原呈递细胞(antigenpresenting cell,APC),抗原呈递细胞的具有与被负载的多肽相匹配的HLA分型(A2亚型)。所述抗原呈递细胞为专职抗原呈递细胞(professional antigen presenting cell)或非专职抗原呈递细胞(non-professional antigen presenting cell)。所述专职抗原呈递细胞为淋巴细胞(B细胞)、树突状细胞DC、巨噬细胞、内皮细胞或干细胞,优选地为树突状细胞、巨噬细胞或淋巴细胞,更优选为树突状细胞。DC可以是自体来源或异体来源,DC也可以是分离自活性个体或人工构建的与天然DC具有相似生物学特性的DC细胞系,人工构建的DC细胞系的形态和/或基因表型与天然DC类似,如CN108546679A所记载的转导有表达Tax基因的慢病毒载体的DC细胞,其CD3表达为阴性,CD70、CD80、CD83、CD86、CCR7和HLA-DR等DC标志分子均有表达;又如US20050272151A1所记载的GEN2.2细胞系,其为浆细胞样DC细胞系,具有CD4+,HLA-DR+,CD123+,CD45RA+,CD11c-,CD13-的表型;DC还可以分化自DC前体细胞系,如分化自PBMC的单核细胞或MUTZ-3细胞系,MUTZ-3细胞系为表达单核细胞标志分子单核细胞特异性酯酶和CD14的细胞系(Santegoets SJ,van den Eertwegh AJ,van deLoosdrecht AA,Scheper RJ,de Gruijl TD.Human dendritic cell line models for DCdifferentiation and clinical DC vaccination studies.J Leukoc Biol.2008Dec;84(6):1364-73.)。所述非专职抗原呈递细胞为表达I型HLA的抗原呈递细胞。
本领域技术人员知晓并理解,将抗原呈递细胞(APC)以暴露于抗原的方式被“脉冲”或加载上述的多肽组合物,暴露的时间足够长,使得上述的多肽组合物被呈递到所述APC的表面,获得负载多肽组合物的抗原呈递细胞;“暴露”方式实现抗原呈递细胞与上述多肽组合物的接触,如共孵育培养等。具体地,APC被暴露于以多条短多肽片段形式存在的抗原中,即暴露于抗原肽下,抗原肽被直接加载到APC表面。除短多肽片段外,APC可以与衍生于抗原蛋白的大片段、完整的抗原全蛋白或包含抗原蛋白的颗粒一起孵育,抗原蛋白的大片段、完整的抗原全蛋白或包含抗原蛋白的颗粒可以被APC通过胞吞等方式吞入包内,而后被溶酶体或蛋白酶体加工为短多肽片段并最终被运载并呈递到APC的表面,与APC表面的HLA结合形成抗原呈递复合物。
负载多肽组合物的抗原呈递细胞可以是在体外(in vitro)或体内(in vivo)使APC接触上述多肽组合物制得。当APC在体外加载上述多肽组合物的抗原肽时,APC可以在培养皿或孔板上铺板生长,然后再暴露于足够量的包含抗原肽的多肽组合物,并与之接触足够长的时间使抗原肽与APC相结合。抗原肽结合APC所需要使用的量和结合时间可以通过本领域所周知的检测方法进行确定。本领域技术人员所知晓的其他方法,如免疫检测或结合检测,可用于检测暴露于包含抗原肽的多肽组合物后的APC上是否负载有抗原肽。
本发明提供一种负载上述多肽组合物的细胞的制备方法,步骤包括将上述多肽组合物与抗原呈递细胞接触,多肽组合物负载于抗原呈递细胞上,获得负载多肽组合物的抗原呈递细胞。
上述多肽组合物与抗原呈递细胞的接触为共同孵育,共同孵育所用培养基为本领域任何常规的适合培养免疫细胞的培养基,如AIM-V、DMEM或RPMI-1640中任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为1-2天,优选为2天,以本发明多肽组合物中抗原肽能够结合到抗原呈递细胞表面HLA(A11亚型)形成HLA-抗原肽复合物为标准;共同孵育的温度为室温~37℃;共同孵育过程中上述多肽组合物中各多肽的浓度为10-100μg/mL,优选为20-80μg/mL,更优选为40μg/mL。
抗原呈递细胞与多肽组合物接触,对抗原呈递细胞的成熟有一定的促进作用,但仍需加入其他促成熟因子与负载多肽组合物的抗原呈递细胞接触,进一步促进抗原呈递细胞的成熟化。当负载多肽组合物的抗原呈递细胞仍处于未成熟状态时,还包括将负载多肽组合物的抗原呈递细胞与促成熟因子接触,诱导抗原呈递细胞成熟,获得负载多肽组合物的处于成熟状态的抗原呈递细胞。
负载多肽组合物的抗原呈递细胞与促成熟因子的接触为共同孵育,共同孵育所用培养基为本领域任何常规的适合培养免疫细胞的培养基,如AIM-V、DMEM或RPMI-1640中任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为8-72小时,优选为24-48小时,更优选为24小时;共同孵育的温度为室温~37℃;促成熟因子选自TNF-α、IL-1β、IL-6、PGE2、IFN-γ、poly(I:C)、R848或ATP中一种或多种,优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为IFN-γ、poly(I:C)和R848,或者优选为IFN-γ、poly(I:C)、R848和ATP,IFN-γ的工作浓度为10-1000IU/mL,优选为100-300IU/mL,更优选为100IU/mL;poly(I:C)的工作浓度为1-200μg/mL,优选为20-40μg/mL,更优选为30μg/mL;R848的工作浓度为0.1-50μg/mL,优选为1-10μg/mL,更优选为5μg/mL;ATP的工作浓度为0.1-10mM,优选地为0.1-5mM,更优选为1mM;优选为IFN-γ、poly(I:C)、R848和ATP,IFN-γ的工作浓度为100IU/mL,poly(I:C)的工作浓度为30μg/mL,R848的工作浓度为5μg/mL。
所述抗原呈递细胞包括专职抗原呈递细胞或非专职抗原呈递细胞。所述专职抗原呈递细胞为淋巴细胞(B细胞)、树突状细胞DC、巨噬细胞、内皮细胞或干细胞等,优选地为树突状细胞、巨噬细胞或淋巴细胞,更优选为树突状细胞。所述非专职抗原呈递细胞为表达I型HLA的抗原呈递细胞。
所述抗原呈递细胞可由抗原呈递细胞的前体细胞分化而成,其制备包括:将抗原呈递细胞的前体细胞与细胞因子接触,诱导分化成抗原呈递细胞。
作为本发明一个实施方案,负载上述多肽组合物的细胞的制备方法包括:(1)将上述多肽组合物与未成熟树突状细胞接触,多肽组合物负载于未成熟树突状细胞上,获得负载多肽组合物的未成熟树突状细胞;
(2)将负载多肽组合物的未成熟树突状细胞与树突状细胞促成熟因子接触,诱导未成熟树突状细胞成熟,获得负载多肽组合物的成熟树突状细胞。
步骤(1)中接触为共同孵育,共同孵育所用培养基为本领域任何常规的适合培养免疫细胞的培养基,如AIM-V、DMEM或RPMI-1640中任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为1-2天,优选为2天,以本发明多肽组合物中抗原肽能够结合到抗原呈递细胞表面HLA(A2亚型)形成HLA-抗原肽复合物为标准;共同孵育的温度为室温~37℃;共同孵育过程中上述多肽组合物中各多肽的浓度为10-100μg/mL,优选为20-80μg/mL,更优选为40μg/mL。
步骤(2)中接触为共同孵育,共同孵育所用培养基为本领域任何常规的适合培养免疫细胞的培养基,如AIM-V、DMEM或RPMI-1640中的任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为8-72小时,优选为24-48小时,更优选为24小时;共同孵育的温度为室温~37℃;树突状细胞促成熟因子选自TNF-α、IL-1β、IL-6、PGE2、IFN-γ、poly(I:C)、R848或ATP中一种或多种,优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为IFN-γ、poly(I:C)和R848,或者优选为IFN-γ、poly(I:C)、R848和ATP,IFN-γ的工作浓度为10-1000IU/mL,优选为100-300IU/mL,更优选为100IU/mL;poly(I:C)的工作浓度为1-200μg/mL,优选为20-40μg/mL,更优选为30μg/mL;R848的工作浓度为0.1-50μg/mL,优选为1-10μg/mL,更优选为5μg/mL;ATP的工作浓度为0.1-10mM,优选地为0.1-5mM,更优选为1mM;优选为IFN-γ、poly(I:C)、R848和ATP,IFN-γ的工作浓度为100IU/mL,poly(I:C)的工作浓度为30μg/mL,R848的工作浓度为5μg/mL。
步骤(2)中,成熟树突状细胞的验证方法是检测本领域所周知的成熟DC表面所表达的分子标志和/或成熟DC所分泌的细胞因子,包括但不限于CD80、CD83、CD86、CCR7、HLA-ABC、HLA-DR或IL-6。所述验证方法所采用的检测手段可以是本领域任何能够检测上述分子标志和/或细胞因子的检测手段,包括但不限于ELISA、Western杂交或流式细胞仪检测。
步骤(1)中,未成熟树突状细胞由树突状细胞的前体细胞分化而成,步骤包括:将树突状细胞的前体细胞与细胞因子接触,诱导分化成未成熟树突状细胞。
树突状细胞的前体细胞与细胞因子的接触为共同孵育,共同孵育所用培养基为本领域任何常规的适合培养免疫细胞的培养基,如AIM-V、DMEM和RPMI-1640中的任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的温度为室温~37℃;共同孵育的时长为2-6天,优选为3-5天,更优选为5天。
所述树突状细胞的前体细胞为CD14+DC前体细胞,优选为血液中PBMC的单核细胞;所述细胞因子包括GM-CSF与IL-4,GM-CSF的工作浓度为50-500ng/mL,优选为50-100ng/mL,更优选为100ng/mL;IL-4的工作浓度为5-100ng/mL,优选为10-50ng/mL,更优选为50ng/mL。
所述单核细胞通过分离个体的PBMC后进行培养而获得。所述PBMC的分离方法可以为本领域所周知的方法,例如抽取个体的血液,通过密度梯度离心法分离。分离的PBMC经过培养后,贴壁细胞即基本为单核细胞。培养PBMC的时间优选为2-8小时,更优选为8小时。
所述树突状细胞的前体细胞还可以为造血干细胞谱系来源的CD34+DC前体细胞,所述细胞因子包括GM-CSF与IL-4,所述CD34+DC前体细胞分离自脐带血,经过大量扩增后再与细胞因子GM-CSF和IL-4相接触而被诱导分化为未成熟DC。CD34+DC前体细胞大量扩增的方法可以为本领域所知晓的方法,具体可见WO2010055900A1中所公开的方法。
未成熟DC的前体细胞还可以为永生化的DC前体细胞系,如上述MUTZ-3细胞系,其为表达单核细胞标志分子单核细胞特异性酯酶和CD14的细胞系。
本发明上述负载多肽组合物的细胞表面形成HLA-肿瘤抗原表位肽复合物,通过TCR识别被呈递给T细胞,使能够识别相应肿瘤抗原表位的T细胞被特异性激活并扩增,扩增的T细胞成为特异性靶向该肿瘤相关抗原的细胞毒性T淋巴细胞(Cytotoxic TLymphocyte,CTL),对表达该肿瘤相关抗原的肿瘤细胞产生细胞介导的免疫杀伤作用。即本发明负载多肽组合物的细胞可刺激体内细胞介导的免疫应答和/或体液免疫应答,产生预防和/或治疗肿瘤的效果,可用于制备检测、和/或预防、和/或治疗肿瘤的药物、或试剂、或试剂盒,该药物、或试剂、或试剂盒包括但不限于生物制品。
激活的免疫效应细胞
本发明还提供一种激活的免疫效应细胞,由以下方法制备而成:将上述负载本发明多肽组合物的细胞与未激活的免疫效应细胞接触而得。本发明所提供的负载多肽组合物的细胞如DC,在其表面表达某一亚型的HLA如A2,与该某一特定亚型的HLA相匹配的抗原肽如肿瘤抗原肽与之结合后形成HLA-抗原肽复合物,进而被T细胞表面特异性识别该复合物的受体TCR所识别并结合,表达该TCR的T细胞因此受到刺激并开始增殖。
所述免疫效应细胞选自T细胞或NK细胞,优选地为T细胞。所述负载本发明多肽组合物的细胞选自淋巴细胞(B细胞)、树突状细胞DC、巨噬细胞、内皮细胞或干细胞等,优选为负载本发明多肽组合物的淋巴细胞、巨噬细胞或树突状细胞,更优选为负载本发明多肽组合物的树突状细胞。所述免疫效应细胞与负载本发明多肽组合物的细胞来自相同个体或不同个体,优选来自相同个体。所述负载多肽组合物的细胞与免疫效应细胞的数量比为本领域所周知的任何比例,只要所述负载多肽组合物的细胞能够有效激活识别其表面HLA-抗原肽复合物的免疫效应细胞即可,负载多肽组合物的细胞与免疫效应细胞的数量比优选为1︰10-50,再优选为1︰10-30,更优选为1︰10。
所述负载多肽组合物的细胞与免疫效应细胞的接触为共同孵育。共同孵育的时长为2-48小时,优选为24-48小时,更优选为24小时。共同孵育在培养基中进行,所述培养基为AIM-V、DMEM或RPMI1640,优选为AIM-V培养基,更优选为含有2%v/vFBS AIM-V培养基,作为优选实施方案中,所述AIM-V培养基中还包含IL-2,IL-2的工作浓度为10-100U/mL,优选为100U/mL。
本发明上述激活的免疫效应细胞为特异性靶向肿瘤相关抗原的细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL),对表达肿瘤相关抗原的肿瘤细胞产生细胞介导的免疫杀伤作用。即本发明激活的免疫效应细胞可刺激体内细胞介导的免疫应答和/或体液免疫应答,产生预防和/或治疗肿瘤的效果,可用于制备检测、和/或预防、和/或治疗肿瘤的药物、或试剂、或试剂盒,该药物、或试剂、或试剂盒包括但不限于生物制品。
药物、试剂或试剂盒
该药物、试剂或试剂盒具有检测、和/或预防、和或治疗肿瘤的活性,包括但不限于生物制品,含有本发明上述的多肽组合物、和/或负载多肽组合物的细胞、和/或激活的免疫效应细胞。
该药物、试剂或试剂盒还含有佐剂和/或免疫调节剂。佐剂或免疫调节剂均为本领域所周知的能够提升免疫应答效应和/或降低免疫毒副作用的化合物小分子、生物大分子、组合物、复合物或提取物。佐剂或免疫调节剂可先于本发明多肽组合物、或可与本发明多肽组合物一起、或可晚于本发明多肽组合物递送至目标细胞或目标机体,增强细胞或机体对抗原的免疫应答或改变免疫应答类型,降低免疫应答毒副作用。
所述佐剂或免疫调节剂选自铝佐剂(例如氢氧化铝、铝盐)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、前列腺素E2、α干扰素、短小棒状杆菌、脂多糖、细胞因子、水包油乳液、油包水乳液、纳米乳液、微粒递送系统、脂质体、微球、生物可降解微球、斑块病毒体、蛋白脂质体、蛋白酶体、免疫刺激复合体(ISCOMs、ISCOMATRIX)、微颗粒、纳米颗粒、生物可降解的纳米颗粒、硅纳米颗粒、聚合微米/纳米颗粒、聚合薄片状底物颗粒(PLSP)、微颗粒树脂、纳米脂质体聚合凝胶(nanolipogel)、合成的/生物可降解的及生物相容性半合成或天然聚合物或树枝状聚合物(如PLG、PLGA、PLA、聚己酸内酯、硅聚合物、聚酯、聚二甲基硅氧烷、聚苯乙烯磺酸钠、聚苯乙烯苄基三甲基氯化铵、聚苯乙烯二乙烯基苯树脂、聚磷腈、聚-[二-(羧基乙酰苯氧基)磷腈(PCPP)、聚-(甲基丙烯酸甲酯)、葡聚糖、聚乙烯吡咯烷酮、透明质酸及衍生物、壳聚糖及其衍生物、多糖、δ菊粉多糖、糖脂(合成的或天然的)、脂多糖、一种或多种聚阳离子化合物(如聚氨基酸、聚-(γ-谷氨酸))、聚-精氨酸-HCl、聚-L-赖氨酸、多肽、生物高聚物)、阳离子二甲基二(十八烷基)铵(DDA)、α-半乳糖苷神经酰胺及其衍生物、古细菌脂质及衍生物、内酰胺、gallen、甘油酯、磷脂和螺旋体中一种或多种。
该药物、试剂或试剂盒还含有医药学上可接受的载体和/或赋形剂,该类载体或赋形剂不对多肽组合物、和/或负载多肽组合物的细胞、和/或激活的免疫效应细胞的性质及生物学功能等产生任何实质性影响。
该药物、试剂或试剂盒可制备成医药学可接受的具有检测、和/或预防、和/或治疗作用的任意剂型,如冻干粉末或者液体制剂,液体制剂中包含灭菌注射用水或有机溶剂,如DMSO。
含有多肽组合物的药物或试剂可以被注射到受试者体内,通过负载到受试者体内的抗原呈递细胞(如DC)上与识别所述肿瘤疫苗中抗原肽的相应亚型的HLA相结合后形成HLA-抗原肽复合物,所述HLA-抗原肽复合物与相应的特异性TCR结合,激活表达该特异性TCR的T细胞。
也可以是,自体血液中分离获得的抗原呈递细胞的前体细胞(如DC前体细胞)分化而成的抗原呈递细胞,如来源于脐带血的CD34+的造血前体细胞或来源于外周血CD14+的单核细胞分化而来的DC,与本发明多肽组合物体外共同孵育后,获得含有本发明多肽组合物和负载多肽组合物的抗原呈递细胞(尤其是负载多肽组合物并处于成熟状态的抗原呈递细胞),再输入体内,通过抗原呈递细胞呈递其负载的抗原肽进而激活特异性T细胞应答;在输入体内的同时施用降低免疫排斥反应的试剂,如抑制内源TCR表达的抑制剂。
该药物或试剂的施用可以在外科去除肿瘤之前或之后发生,或在放化疗治疗肿瘤之前或之后发生。该药物或试剂可以与其他组合物或药物产品一起或联合向患者施用。应理解,本发明该药物除了可以施用于已经罹患肿瘤的个体外,也可以施用于没有患肿瘤但具有患肿瘤风险的个体。
该药物可通过节内注射被施用于腹股沟节,也可经皮下或皮内施用于接受治疗的患癌症病人的手足,或其他施用路径,例如肌肉内注射或血液注射。
该药物施能够治疗和/或预防的肿瘤包括但不限于肺癌、非小细胞肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、间皮瘤、大肠癌、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤、骨癌、胰腺癌、肾细胞癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、胆管癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、尿路上皮癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童实体瘤、淋巴细胞性淋巴瘤、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、霍奇金淋巴瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症,包括石棉诱发的癌症和各类白血病与淋巴癌以及各类癌前病变。
引发体内免疫应答反应的方法
为达到预防和/或治疗肿瘤的效果,本发明还提供一种引发体内免疫应答的方法,向体内输入有效剂量的多肽组合物、负载多肽组合物的细胞和激活的免疫效应细胞中一种或多种。
为达到预防目的,本发明多肽组合物、负载多肽组合物的细胞和激活的免疫效应细胞中一种或多种在肿瘤或肿瘤病变发生前对受试者个体施用。在某些情况下,该药物在上述一种或多种肿瘤发病后对受试者个体施用,旨在预防出现更进一步的症状或已经发生的症状进一步恶化。本发明多肽组合物、负载多肽组合物的细胞和激活的免疫效应细胞中一种或多种的预防性施用旨在预防或减轻任何后续的症状。为达到治疗目的,本发明多肽组合物、负载多肽组合物的细胞和激活的免疫效应细胞中一种或多种在癌症发生时或癌症发生后对受试者个体施用,旨在减轻已经产生的癌症的症状。
针对任何特定治疗应用的有效剂量取决于不同的因素,如癌症的种类、癌症发病的程度、受试者个体的状况如年龄、性别、体重、身体各项指标的水平等以及所施用的具体药剂的组分以及施用的具体方式,例如,可以通过包括但不限于静脉内、肌肉内、皮内、经皮、动脉内、腹膜内、创伤内、颅内、关节内、前列腺内、胸膜内、气管内、鞘内、鼻腔内、阴道内、直肠内、不经肠胃地、全身地、局部地、肿瘤内、经腹膜、脑室内、经皮下、经结膜下、经粘膜、透粘膜地、心包内、脐带内、眼眶内、经口、透皮地、经肺内、经吸入、经注射、经植入、经回输、经连续回输、经局部灌注、经导管、经灌洗、用乳剂和用脂质体组合物中一种或多种手段对受试者个体施用。对于所施用的包含本发明多肽组合物、负载多肽组合物的细胞和激活的免疫效应细胞中一种或多种的药物的有效剂量,本领域技术人员可以按照药物中包含的具体组分根据经验决定,而无需进行另外不必要的试验。
本发明所取得的积极效果在于:本发明提供的多肽组合物通过负载DC后呈递并激活特异性的CD8+细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)而达到对肿瘤细胞的靶向毒性作用。本发明的多肽组合物及其衍生的肿瘤疫苗、DC疫苗、药物组合物能够显著激活免疫效应细胞,特别是T细胞,明显提高其激活相关的细胞因子的分泌水平和对肿瘤细胞的杀伤水平,具有潜在的临床价值。
下面对本发明涉及的部分术语进行解释。非如下另行定义,否则本文中的术语按照本领域通常所用的方式使用。
在本发明中,术语“多肽”指由通过酰胺键(也称为肽键)线性连接的单体(氨基酸)组成的分子。术语“多肽”是指具有两个或更多个氨基酸的任何一个或多个链,并且不是指产物的具体长度。因此,肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指具有两个或更多个氨基酸的一个或多个链的任何其他术语包含在“多肽”的定义中,并且术语“多肽”可以代替这些术语中的任何一个、或可与其互换使用。术语“多肽”还旨在指多肽在表达后修饰的产物,包括而不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知保护/阻断基团来进行的衍生、蛋白水解裂解或通过非标准的氨基酸来进行的修饰。多肽可来自天然生物来源或通过重组技术来产生,但不是必然从一个指定的核酸序列翻译而来。它可以按任何方式、包括通过化学合成来产生。
术语“衍生于……的多肽”表示蛋白或多肽的全长、片段、修饰物或突变体。术语“片段”在涉及多肽时可为保留全长蛋白或多肽的抗原性的任意长度的多肽。在一个或多个实施方案中,所述片段是全长蛋白的抗原表位。在一个或多个实施方案中,所述片段包含50、40、30个以内的氨基酸。例如,所述片段的长度可以是5-30个氨基酸、8-25个氨基酸、8-15个氨基酸或9-12个氨基酸,或有5上述端点任意组合形成的范围,如9-15个氨基酸。在一个或多个实施方案中,所述片段的长度可以是9个氨基酸。
术语“变体”或“突变体”是指与参照序列相比,通过一个或多个氨基酸的插入、缺失或取代(替换),氨基酸序列发生变化但保留至少一种生物活性的肽或多肽。在一个或多个实施方案中,所述变体或突变体包括与参照序列(如本文所述SEQ ID NO:1、2、3、4、5、6、7、8、9各片段)具有至少75%,优选至少80%,优选至少85%,优选至少90%,优选至少95%,优选至少97%的序列相同性并保留参照序列的生物学活性(如作为抗原表位)的氨基酸序列。所述取代可以是非保守氨基酸取代或保守氨基酸取代。保守氨基酸取代指取代的氨基酸与野生型多肽中对应的氨基酸具有类似的结构和化学性质,性能相近或相似,不改变蛋白质或多肽的功能。例如,保守氨基酸取代包括的,具有相似侧链的氨基酸残基的家族,这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此,在本发明多肽中用来自同一侧链类的另一氨基酸残基替换一个或几个位点,将不会在实质上影响其活性。
术语“野生型”具有其在此项技术中所理解的含义,其是指具有如在自然界中发现的呈“正常”(如与突变体、患病者、改变者等形成对比)状态或情形的结构和/或活性的实体。所属领域的技术人员将了解,野生型基因和多肽通常以多种不同形式(例如等位基因)存在。
术语“肿瘤相关抗原”或“TAA”,是指被肿瘤细胞特异性表达或被肿瘤细胞以与相同组织种类的非肿瘤细胞相比较高频率或密度表达的抗原。肿瘤相关抗原可以是正常不被宿主表达的抗原;它们可经突变、截短、错误折叠、或正常被宿主表达的分子的其他方式异常显现;它们可与正常表达的分子相同但以异常高水平表达;或它们可以在异常的情况或环境中表达。肿瘤相关抗原可以是,例如,蛋白或蛋白片段、复杂碳水化合物、神经节苷脂、半抗原、核酸、或这些或其他生物分子的组合。
术语“疫苗”是指用于施用至哺乳动物的免疫原性组合物,其用于在哺乳动物中引发针对特定抗原的免疫反应。疫苗典型地包含相似于或衍生自该免疫反应的目标的试剂(已知为“抗原”或“免疫原”),该目标诸如造成疾病的微生物或肿瘤细胞。意图用于肿瘤诸如癌症的治疗的疫苗,典型地包含衍生自目标肿瘤上所发现的肿瘤相关抗原且能够引发对目标肿瘤上的肿瘤相关抗原的免疫原性的抗原。
术语“Survivin”是指Survivin的表达产物凋亡抑制蛋白,可以是人凋亡抑制蛋白或非人的凋亡抑制蛋白。
术语“Her2”是指Her2基因的表达产物人表皮生长因子受体2。
术语“CEA”是指CEA基因的表达产物癌坯抗原蛋白。
术语“hTERT”是指hTERT基因的表达产物转染端粒酶逆转录酶蛋白。
术语“MAGE-A3”是指MAGE-A3基因的表达产物黑色素瘤抗原蛋白。
术语“EGFR”是指EGFR基因的表达产物表皮生长因子蛋白。
术语“gp100”是指gp100基因的表达产物gp100蛋白,可以是人gp100蛋白或非人的哺乳动物gp100蛋白。
术语“p53”是指p53基因的表达产物p53蛋白,可以是人p53蛋白或非人的哺乳动物p53蛋白。
术语“癌症”、“肿瘤”和“恶性”指或描述在哺乳动物中典型地是特征为不受控制的细胞生长的生理病症。癌症的实例包括但不限于上皮癌,包括腺癌、淋巴瘤、胚细胞瘤、黑色素瘤、肉瘤、和白血病。此类癌症的更具体的实例包括鳞状细胞癌、小细胞肺癌、非小细胞肺癌、胃肠癌、霍奇金氏淋巴瘤和非霍奇金氏淋巴瘤、胰腺癌、成胶质细胞瘤、神经胶质瘤、宫颈癌、卵巢癌、肝癌(诸如肝癌和肝细胞瘤)、膀胱癌、乳腺癌(包括激素介导的乳腺癌)、结肠癌、结直肠癌、子宫内膜癌、骨髓瘤(诸如多发性骨髓瘤)、唾液腺癌、肾癌(诸如肾细胞癌和维尔姆斯氏瘤)、基底细胞癌、黑色素瘤、前列腺癌、外阴癌、甲状腺癌、睾丸癌、食道癌、血癌(包括但不限于急性髓细胞性白血病(AML)和多发性骨髓瘤(MM))、各种类型的头颈癌(包括但不限于鳞状细胞癌)、以及粘液性起源的癌症(诸如粘液性卵巢癌)、胆管癌(肝)以及肾乳头状癌。在某些实施例中,该血癌选自下组,该组由以下各项组成:霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤、多发性骨髓瘤、急性成淋巴细胞性白血病、急性骨髓性白血病、慢性淋巴细胞性白血病、以及慢性骨髓性白血病。
术语“免疫调节剂”,意指一种物质,当它与免疫原混合时能够比免疫原单独存在时诱发更强的免疫应答,和/或降低免疫毒副作用。例如,一种免疫增强剂能够提高免疫源性,并提供优异的免疫应答。又例如,一种免疫增强剂能够通过提高巨噬细胞和其他抗原呈递细胞上的共刺激因子的表达来起作用。
术语“佐剂”是指非特异性免疫增强剂,当其与抗原一起或预先递送入机体时,其可增强机体对抗原的免疫应答或改变免疫应答类型,和/或降低免疫毒副作用。佐剂有很多种,包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
术语“DC促成熟因子”是指任何能够促进未成熟DC向成熟DC转化的蛋白质、核酸、多肽、复合物、提取物、分离物或以上的组合物,其可以通过与未成熟DC接触而使未成熟DC向成熟DC转化。成熟DC的验证方法可以是检测本领域所周知的成熟DC表面所表达的分子标志和/或成熟DC所分泌的细胞因子,包括但不限于CD80、CD83、CD86、CCR7、HLA-ABC、HLA-DR和IL-6。
术语“分离的”是指通过人工的方法将天然存在的生物大分子如蛋白质、多肽、核酸、抗体,或其形成的复合物等从体内的天然状态中分离、提纯或在体外被从头合成后所形成的状态。
术语“工作浓度”是指某种试剂或某种有效成分在溶液体系中发挥功效时的实际的浓度。通常,某种试剂或某种有效成分会在使用前配置成为浓度较高的母液或储存液储存,在使用时再按一定比例加入到最终反应体系中被稀释,通常其稀释后获得的最终浓度即为工作浓度。
术语“负载”是指蛋白质、多肽或核酸分子直接与细胞表面上的受体相结合,形成细胞与蛋白质、多肽或核酸分子的复合体。所述结合作用可以是共价结合,也可以是非共价结合,包括受体与配体的相互结合。例如,负载抗原肽的DC即为抗原肽与DC表面表达的与所述抗原肽的HLA分型相匹配的HLA分子相结合而形成的DC-抗原肽复合体。
术语“DC-细胞毒性T淋巴细胞”或“DC-CTL”是指被负载抗原肽的成熟DC所激活的细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte,CTL),其能够特异性结合表达含有所述成熟DC所负载的抗原肽的抗原,并对表达该抗原的细胞产生细胞杀伤作用,是通过DC介导的细胞免疫的主要执行者。
术语“抗原呈递细胞”(APC)是指能够表达主要组织相容性复合物(MHC)I型或II型的一类细胞,能够通过MHC结合抗原肽形成MHC-抗原肽复合物并进一步结合T细胞表面的受体进而激活T细胞的一类细胞,包括但不限于树突状细胞(dendritic cell,DC)、单核细胞/巨噬细胞、B细胞、Langerhans细胞。
术语“负载抗原的抗原呈递细胞”包括已暴露于抗原并被抗原活化的APC。例如,APC可以在体外(例如在有抗原存在的培养期间)负载抗原。APC也可以通过暴露于抗原而在体内被负载。“抗原负载的APC”通常以两种方式之一制备:(1)被称为抗原肽的小片段直接“脉冲”到APC的外部与MHC分子结合;(2)APC与多肽大片段、完全蛋白质或蛋白质颗粒孵育,然后多肽大片段、完全蛋白质或蛋白质颗粒由APC摄取。这些多肽大片段或蛋白质分子通过APC被消化成小的肽段,并且最终被运输并呈递在APC表面上。此外,还可以通过将编码抗原的多核苷酸导入细胞中来产生负载抗原的APC。
附图说明
图1为实施例1经细胞因子诱导培养的负载不同多肽组合物的各DC表面成熟标志物的比例。负载不同多肽组合物的各DC组横向从左至右依次为成熟标志物CD80、CD83、CD86比例示意图。对照组指未负载任何多肽的DC细胞。
图2为实施例2流式检测负载不同多肽组合物的DC-CTL表面标志物的比例。CD3/CD4、CD3/CD8、CD25、CD69、CD107a、CD137各活化标志物检测组中从左至右依次为对照组、负载多肽组合物1、负载多肽组合物2和负载多肽组合物3的检测结果。
图3为实施例2负载不同多肽组合物的DC-CTL表面标志物CD3+/CD137的比例。
图4为实施例2负载不同多肽组合物的DC-CTL表面标志物CD3+/CD107α的比例。
图5为实施例2负载不同多肽组合物的DC-CTL胞内IFN-γ分泌水平。
图6为实施例2负载不同多肽组合物的DC-CTL胞内IFN-γ分泌水平流式检测图。
图7为实施例3负载不同多肽组合物的DC-CTL对胰腺癌肿瘤细胞PANC-1的杀伤效果。
图8为实施例3负载不同多肽组合物的DC-CTL对胰腺癌肿瘤细胞PANC-1的杀伤后上清TNF-α分泌水平。
图9为实施例3负载不同多肽组合物的DC-CTL对胰腺癌肿瘤细胞PANC-1的杀伤后上清IFN-γ分泌水平。
图10为对比例DC负载HLA-A2结合型多肽组合物1(优选肽组合)、肽替换组1和肽替换组2的DC-CTL对肿瘤细胞系PANC-1的杀伤曲线。
图11为对比例DC负载HLA-A2结合型多肽组合物1、肽替换组1和肽替换组2的DC-CTL对肿瘤细胞系PANC-1的杀伤后上清TNF-α分泌水平。
图12为对比例DC负载HLA-A2结合型多肽组合物1、肽替换组1和肽替换组2的DC-CTL对肿瘤细胞系PANC-1的杀伤后上清IFN-γ分泌水平。
以上附图中标注的“对照组或T细胞组”除有特别说明外,均指用未负载任何多肽的DC刺激的T细胞实验组。
以上附图中标注的“肽组合1组、组合1、负载多肽组合1、肽组合1”均指负载多肽组合物1的实验组;“肽组合2组、组合2、负载多肽组合2、肽组合2”均指负载多肽组合物2的实验组;“肽组合3组、组合3、负载多肽组合3、肽组合3”均指负载多肽组合物3的实验组。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例中所述“室温”是指进行试验的操作间的温度,一般为25℃。
实施例中所述“过夜”指超过8小时。
以下实施例针对中国人群中比例较高的HLA分型-A2型,提供了多种包含多条肿瘤抗原肽的多肽组合物,每种多肽组合物中包括了针对HLA-A2分型的至少3条肿瘤相关抗原表位多肽。以下实施例所用到的多肽如下表1所示:
表1
以下实施例所用到的多肽组合物如下表2所示:
表2
以下实施例中的DC前体细胞与T细胞从供体受试者的血液PBMC中分离获得,具体步骤如下:HLA-A2型供体受试者的血液与等体积生理盐水混合,总体积为35mL,沿管壁缓慢加入含15mLFicoll的离心管中,可见血液与Ficoll明显分层,800g离心20min,离心后吸取白色细胞层,转至另一离心管中,加生理盐水,1500rpm,10min,离心清洗,弃去废液,再加生理盐水离心清洗一次。清洗后的细胞转入培养瓶中培养贴壁过夜,收集悬浮细胞即T细胞,计数,冻存,剩余贴壁细胞为单核细胞(DC前体细胞),可诱导分化为DC。
以下实施例中若无特别说明,所实用的多肽组合物中的每种多肽的工作浓度均为40μg/mL。
实施例1不同HLA-A2结合型多肽组合物诱导对DC成熟的影响
操作步骤:
(1)未成熟DC细胞的培养:DC前体细胞加入不含血清的AIM-V培养基(购自Gibco),添加IL-4与GM-CSF至工作浓度分别为50ng/mL与100ng/mL后继续培养。培养至第三天半量换液,补加IL-4与GM-CSF至各自工作浓度分别为50ng/mL与100ng/mL。
(2)未成熟DC细胞的多肽负载:第五天后,DC前体细胞分为4个组,分别为对照组,组1,组2,组3。在组1中加入多肽组合物1混合物,在组2中加入多肽组合物2混合物,在组3中加入多肽组合物3混合物。各肽工作浓度均为40μg/mL。对照组中细胞不添加任何抗原肽。
(3)DC成熟与检测:培养至第七天,按标示用量加入IFN-γ、poly(I:C)和R848,IFN-γ的工作浓度为100IU/mL,poly(I:C)的工作浓度为30μg/mL,R848的工作浓度为5μg/mL,继续培养24h,用以进一步刺激活化DC。使用CD80,CD83,CD86流式抗体对培养的DC细胞进行检测。
结果:
如图1所示,经细胞因子刺激后的DC细胞,包括对照组、多肽组合物1组、多肽组合物2组和多肽组合物3组的DC细胞,CD80,CD83,CD86阳性率均较高(均大于99%),显示诱导后的DC成熟度好。对照组与负载不同多肽组合物的实验组间无显著差异。
实施例2不同多肽组合物诱导DC激活的T细胞的细胞活化表型检测
操作步骤:
(1)负载抗原肽的DC与T细胞共孵育:取实施例1中负载不同抗原肽的DC按照1:10的比例与对应donor的T细胞进行共孵育,孵育时间72h形成DC-CTLs。培养条件为AIM-V培养基中添加2%FBS,100U/mL的IL-2因子,其中对照组为未负载抗原的DC。
(2)流式表型:使用CD3、CD4和CD8抗体对DC-CTLs进行标记,通过流式检测分析不同实验组中CD3CD4+和CD3CD8+细胞比例。通过CD3和CD137,CD107a,CD25,CD69,IFN-γ抗体标记DC-CTLs,分析相应的细胞表型比例。
结果:
流式检测结果图2显示,使用不同的多肽组合物1,多肽组合物2,多肽组合物3共孵育后,DC-CTLs与对照组T细胞相比,CD3+CD4T细胞和CD3+CD8T细胞占比均未发生明显变化。然而一些活化相关marker检测结果显示,除CD69中无显著差异外,其他的活化指标CD25,CD137(图3)和CD107α(图4)中,负载多肽组合物组比未负载多肽组合物组均有很大程度的提升,且多肽组合物1均高于其他两组负载多肽组合物实验组。进一步流式检测CD3+细胞胞内IFN-γ表达,负载多肽组合物的实验组CD3+胞内IFN-γ高于对照组(图5,图6),而负载多肽组合物1的实验组胞内IFN-γ也高于负载多肽组合物2组和负载多肽组合物3组(图5,图6)。
上述结果显示,负载多肽组合物后获得DC-CTLs,其对CD4+、CD8+比例影响较小,但一些T细胞活化标志物显著上调,表明DC负载多肽组合物后对T细胞的功能活化有重要作用。更高的胞内细胞因子IFN-γ,提示活化的细胞可能有更好的肿瘤杀伤能力。
实施例3不同多肽组合物诱导DC激活的T细胞对胰腺癌细胞PANC-1的特异性杀伤作用影响
操作步骤:
(1)肿瘤细胞系杀伤实验:
取实施例2中不同实验组的DC-CTLs,使用实时无标记细胞功能分析仪(RTCA)检测其对胰腺癌肿瘤细胞PANC-1杀伤效果。胰腺癌肿瘤细胞PANC-1为HLA-A2型,与患者HLA分型、使用的肿瘤抗原肽HLA分型相同。具体的步骤:
(a)调零:每孔加入50μL DMEM培养液,放入仪器中,选择step 1,调零;
(b)靶细胞铺板:胰腺癌细胞PANC-1(购买于美国菌种保藏中心ATCC)按每孔104个细胞/50μl铺在包含检测电极的板中,放置数分钟,待细胞稳定后,再放入仪器中,开始step2,培养细胞;
(c)加入效应细胞:靶细胞培养24h后,暂停step 2。加入按实施例2方法用多肽组合物1及多肽组合物2,多肽组合物3获得的DC-CTLs,每孔50μl,效靶比分别设置为10:1,以采用未负载任何多肽组合物的来自同一供体受试者的T细胞作为对照,开始step 3,继续共培养24h后,观察仪器记录的细胞功能曲线。
(2)杀伤后的上清细胞因子检测
使用细胞因子试剂盒对杀伤后的上清分别进行IL-2,IL-4,IL-6,IL-10,TNF-a,IFN-γ细胞因子含量检测。
结果:
如图7所示,使用多肽组合物1,多肽组合物2和多肽组合物3分别负载DC后处理T细胞获得的DC-CTL对靶细胞PANC-1的杀伤曲线。与未负载多肽组合物的DC-CTL组相比,负载多肽组合物的各实验组在加入效应细胞24h后有更强的杀伤效果,表明负载的HLA-A2抗原肽可以有效呈递给T细胞并增强T细胞的杀伤能力。进一步分析显示,负载多肽组合物1的杀伤曲线低于多肽组合物2和多肽组合物3组,表现出对PANC-1细胞更优的杀伤能力。
流式检测PANC-1杀伤后细胞上清中TNF-α细胞因子浓度如图8显示,负载肽组合物1的实验组上清中TNF-α细胞因子浓度最高,为314.55pg/mL,其次是负载肽组合物3的实验组和肽组合物2的实验组,分别为252.84pg/mL和206.90pg/mL。
流式检测PANC-1杀伤后细胞上清中IFN-γ因子浓度中(图9),负载肽组合物1的实验组上清中IFN-γ细胞因子浓度最高,为1999.53pg/mL,其次是负载肽组合物3的实验组和肽组合物2的实验组,分别为1471.55pg/mL和1339.53pg/mL,与TNF-α因子检测结果相近。
上述结果表明在三组HLA-A2抗原肽中,负载抗原肽组合1的DC-CTL对胰腺癌细胞PANC-1有更强的杀伤能力。
对比例1肽替换对多肽组合物1诱导DC激活的T细胞对胰腺癌细胞PANC-1的特异性杀伤作用影响
操作步骤:
未成熟DC细胞的培养:DC前体细胞加入不含血清的AIM-V培养基(购自Gibco),添加IL-4与GM-CSF至工作浓度分别为50ng/mL与100ng/mL后继续培养,培养至第三天半量换液。
(1)未成熟DC细胞的多肽负载:第五天后,DC前提细胞分为4个组,分别为对照组,多肽组合物1组,肽替换组1,肽替换组2。在多肽组合物1组中加入多肽组合物1混合物;将多肽组合物1中的肽3替换为肽7,其余不变,为肽替换组1;将多肽组合物1中的肽1替换为肽2,肽3替换为肽4,其余不变,为肽替换组2;各肽工作浓度均为40μg/mL。
(2)培养至第七天,按标示用量加入IFN-γ、poly(I:C)和R848,IFN-γ的工作浓度为100IU/mL,poly(I:C)的工作浓度为30μg/mL,R848的工作浓度为5μg/mL,继续培养24h,用以进一步刺激活化DC。
负载抗原肽的DC与T细胞共孵育:取实施例1中负载不同抗原肽的DC按照1:10的比例与对应donor的T细胞进行共孵育,孵育时间72h形成DC-CTLs。培养条件为AIM-V培养基中添加2%FBS,100U/mL的IL-2因子,其中对照组为未负载抗原的DC。
肿瘤细胞系PANC-1细胞杀伤实验:取不同实验组的DC-CTLs,使用实时无标记细胞功能分析仪(RTCA)检测其对胰腺癌肿瘤细胞PANC-1杀伤效果。具体的步骤:
(a)调零:每孔加入50μL DMEM培养液,放入仪器中,选择step 1,调零;
(b)靶细胞铺板:胰腺癌细胞PANC-1(购买于美国菌种保藏中心ATCC)按每孔104个细胞/50μl铺在包含检测电极的板中,放置数分钟,待细胞稳定后,再放入仪器中,开始step2,培养细胞;
(c)加入效应细胞:靶细胞培养24h后,暂停step 2。加入按实施例2方法用多肽组合物1及多肽组合物2,多肽组合物3获得的DC-CTLs,每孔50μl,效靶比分别设置为10:1,以采用未负载任何多肽组合物的来自同一供体受试者的T细胞作为对照,开始step3,继续共培养24h后,观察仪器记录的细胞功能曲线。
杀伤后的上清细胞因子检测:使用细胞因子试剂盒对杀伤后的上清分别进行IL-2,IL-4,IL-6,IL-10,TNF-a,IFN-γ细胞因子含量检测。
结果:
如图10所示,使用多肽组合物1,肽替换组1和肽替换组2分别负载DC后处理T细胞获得的DC-CTL对靶细胞PANC-1的杀伤曲线。与负载多肽组合物1的DC-CTL组相比,抗原肽3、抗原肽1被其他抗原替换后,肽替换组2和肽替换组1的DC-CTL对胰腺癌细胞PANC-1的杀伤能力逐渐减弱,表明抗原肽3和抗原肽1在多肽组合物1中有重要作用。
流式检测PANC-1杀伤后细胞上清中TNF-α细胞因子浓度如图11显示,多肽组合物1中TNF-α分泌量最高,其次是肽替换组1和肽替换组2,各组的TNF-α含量分别为385.42pg/mL,266.55pg/mL,205.42pg/mL。流式检测PANC-1杀伤后细胞上清中IFN-γ因子浓度如图12显示,多肽组合物1有更优的细胞因子分泌量,为1882.77pg/mL,高于肽替换组1和肽替换组2的1413.85pg/mL和1244.90pg/mL。
如图10-12显示,多肽组合物1(优选肽组合)相比肽替换组1和肽替换组2,其针对胰腺癌PANC-1细胞杀伤效果更强,包括TNF-α和IFN-γ的细胞因子也有更高的分泌量。另外,无论是针对胰腺癌PANC-1细胞的杀伤效果还是相应的细胞因子分泌量,肽替换组1都优于肽替换组2。
上述结果表明在针对胰腺癌PANC-1细胞的杀伤中,抗原肽1和抗原肽3在优选的HLA-A2型抗原肽组合中有关键作用,具有很强的不可替换性。
本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
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Claims (17)
1.一种特异性结合HLA-A2分型的多肽组合物,其特征在于,选自一种或多种衍生于抗原蛋白Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100或p53的多肽,优选自任意两种或三种衍生于抗原蛋白Survivin、Her2、CEA、hTERT、MAGE-A3、EGFR、gp100或p53的多肽,更优选自衍生于抗原蛋白Survivin、CEA及选自Her2、hTERT、MAGE-A3、EGFR、gp100或p53中任意一种的多肽;优选衍生于各抗原蛋白的多肽间两两的质量比为1~5:1,优选为1~3:1,更优选为1:1。
2.根据权利要求1所述的多肽组合物,其特征在于,衍生于各抗原蛋白的多肽含有(1)各抗原蛋白的抗原表位,或者含有经氨基酸替换、添加或删除而获得的同(1)中抗原表位活性相同的抗原表位,优选与(1)中抗原表位的氨基酸序列具有75%以上同源性,再优选与(1)中抗原表位的氨基酸序列具有85%以上同源性,更优选与(1)中抗原表位的氨基酸序列具有95%以上同源性。
3.根据权利要求1所述的多肽组合物,其特征在于,所述衍生于抗原蛋白Survivin的多肽含有SEQ ID NO:1所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:1所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:1所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:1所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白Her2的多肽含有SEQ ID NO:2所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:2所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:2所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:2所示的氨基酸序列具有95%以上同源性;或SEQ ID NO:6所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ IDNO:6所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:6所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:6所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白CEA的多肽含有SEQ ID NO:3所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:3所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:3所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:3所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白hTERT的多肽含有SEQ ID NO:4所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:4所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:4所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:4所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白MAGE-A3的多肽含有SEQ ID NO:5所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:5所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:5所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:5所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白P53的多肽含有SEQ ID NO:7所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:7所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:7所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:7所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白EGFR的多肽含有SEQ ID NO:8所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:8所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:8所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:8所示的氨基酸序列具有95%以上同源性;
所述衍生于抗原蛋白gp100的多肽含有SEQ ID NO:9所示的氨基酸序列或其经氨基酸替换、添加或删除而获得的具有相同活性的氨基酸序列,优选与SEQ ID NO:9所示的氨基酸序列具有75%以上同源性,再优选与SEQ ID NO:9所示的氨基酸序列具有85%以上同源性,更优选与SEQ ID NO:9所示的氨基酸序列具有95%以上同源性。
4.一种负载权利要求1-3任一项所述多肽组合物的细胞。
5.根据权利要求4所述的细胞,其特征在于,所述细胞为抗原呈递细胞,包括专职抗原呈递细胞或非专职抗原呈递细胞,专职抗原呈递细胞包括淋巴细胞、树突状细胞DC、巨噬细胞、内皮细胞或干细胞,优选为树突状细胞、巨噬细胞或淋巴细胞,更优选为树突状细胞;非专职抗原呈递细胞为表达I型HLA的抗原呈递细胞。
6.权利要求4或5所述细胞的制备方法,其特征在于,步骤包括:将权利要求1-3中任一项所述多肽组合物与抗原呈递细胞接触,多肽组合物负载于抗原呈递细胞上,获得负载多肽组合物的抗原呈递细胞。
7.根据权利要求6所述的制备方法,其特征在于,所述接触为共同孵育,共同孵育所用培养基为AIM-V、DMEM或RPMI-1640中任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为1-2天,优选为2天;共同孵育的温度为室温~37℃;共同孵育过程中权利要求1-3中任一项所述多肽组合物中各多肽的浓度为10-100μg/mL,优选为20-80μg/mL,更优选为40μg/mL。
8.根据权利要求6所述的制备方法,其特征在于,还包括将负载多肽组合物的抗原呈递细胞与促成熟因子接触,诱导抗原呈递细胞成熟,获得负载多肽组合物的处于成熟状态的抗原呈递细胞。
9.根据权利要求8所述的制备方法,其特征在于,所述接触为共同孵育,共同孵育所用培养基为AIM-V、DMEM或RPMI-1640中任一种或多种,优选为AIM-V培养基,更优选为不含任何血清的AIM-V培养基;共同孵育的时间为8-72小时,优选为24-48小时,更优选为24小时;共同孵育的温度为室温~37℃;促成熟因子选自TNF-α、IL-1β、IL-6、PGE2、IFN-γ、poly(I:C)、R848或ATP中一种或多种,优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为TNF-α、IL-1β、IL-6和PGE2,TNF-α的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-1β的工作浓度为5-50ng/mL,优选为10-30ng/mL;IL-6的工作浓度为800-1500U/mL,优选为800-1200U/mL;PGE2的工作浓度为0.5-3μg/mL,优选为0.5-1.5μg/mL;或者优选为IFN-γ、poly(I:C)和R848,或者优选为IFN-γ、poly(I:C)、R848和ATP,IFN-γ的工作浓度为10-1000IU/mL,优选为100-300IU/mL,更优选为100IU/mL;poly(I:C)的工作浓度为1-200μg/mL,优选为20-40μg/mL,更优选为30μg/mL;R848的工作浓度为0.1-50μg/mL,优选为1-10μg/mL,更优选为5μg/mL;ATP的工作浓度为0.1-10mM,优选地为0.1-5mM,更优选为1mM。
10.一种激活的免疫细胞,由权利要求4或5所述的细胞与未激活的免疫效应细胞接触而得。
11.根据权利要求10所述的免疫细胞,其特征在于,所述接触为共同孵育,共同孵育的时长为2-48小时,优选为24-48小时,更优选为24小时;共同孵育的培养基为AIM-V、DMEM或RPMI1640,优选为AIM-V培养基,更优选为含有2%v/vFBS AIM-V培养基;权利要求4或5所述的细胞与免疫效应细胞的数量比为1︰10-50,优选为1︰10-30,更优选为1︰10。
12.根据权利要求11所述的免疫细胞,其特征在于,所述AIM-V培养基中还包含IL-2,IL-2的工作浓度为10-100U/mL,优选为100U/mL。
13.权利要求1-3任一项所述的多肽组合物、和/或权利要求4或5所述的细胞、和/或权利要求10-12任一项所述的免疫细胞用于制备检测、和/或预防、和/或治疗肿瘤的药物、或试剂、或试剂盒,该药物、或试剂、或试剂盒包括但不限于生物制品。
14.一种检测、和/或预防、和/或治疗肿瘤的药物、或试剂、或试剂盒,其特征在于,含有权利要求1-3任一项所述的多肽组合物、和/或权利要求4或5所述的细胞、和/或权利要求10-12任一项所述的免疫细胞。
15.根据权利要求14所述的药物、或试剂、或试剂盒,其特征在于,还含有佐剂、和/或免疫调节剂、和/或载体、和/或赋形剂。
16.根据权利要求14所述的药物,其特征在于,所述肿瘤包括但不限于肺癌、非小细胞肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、间皮瘤、大肠癌、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体瘤和骨肉瘤、骨癌、胰腺癌、肾细胞癌、皮肤癌、前列腺癌、皮肤或眼内恶性黑色素瘤、子宫癌、肛区癌、睾丸癌、输卵管癌、子宫内膜癌、阴道癌、阴户癌、何杰金病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统癌、胆管癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、尿路上皮癌、阴茎癌、慢性或急性白血病、儿童实体瘤、淋巴细胞性淋巴瘤、肾或输尿管癌、肾盂癌、中枢神经系统肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、卡波西肉瘤、霍奇金淋巴瘤、表皮状癌、鳞状细胞癌、T细胞淋巴瘤或环境诱发的癌症。
17.一种引发体内免疫应答的方法,向体内输入有效剂量的权利要求1-3任一项所述的多肽组合物、和/或权利要求4或5所述的细胞、和/或权利要求10-12任一项所述的免疫细胞。
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