CN117286099A - 用于诱导抗原特异性t细胞的方法及其在癌症或病毒感染治疗中的用途 - Google Patents
用于诱导抗原特异性t细胞的方法及其在癌症或病毒感染治疗中的用途 Download PDFInfo
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Abstract
本公开涉及一种用于诱导抗原特异性T细胞的方法及其在癌症或病毒感染治疗中的用途。本方法包括用抗原性肽、IL‑2、IL‑15,及IFN‑α培养PBMC。
Description
技术领域
本公开涉及一种用于诱导抗原特异性T细胞的方法及其在癌症或病毒感染治疗中的用途。
背景技术
癌症通常以手术、放射线疗法或化学疗法治疗。癌症手术会对身体造成物理性损伤(诸如伤口),使得在手术后难以恢复。尽管放射线疗法不会造成伤口,但放射线可能残留体内或损害非癌性细胞,造成持久性损害。化学疗法亦可通过杀死癌细胞并影响其他正常细胞而对身体造成伤害。
免疫监测在对抗癌症时扮演关键角色,并代表了一种非常吸引人的治疗方式,尤其是考虑到常规手术、放射及化学疗法在癌症管理中的诸多缺点。
在癌症免疫疗法中,免疫系统被动地或主动地用于特异性地靶向及杀死癌细胞。免疫疗法提供靶特异性,其可消除脱靶毒性,同时仍能引发有效的抗癌反应。在被动免疫疗法中,通过靶向肿瘤细胞或其微环境,可增强内源性抗肿瘤免疫反应并抑制肿瘤细胞生长。在主动免疫疗法中,免疫细胞被刺激而对抗癌症。
主动免疫疗法高度依赖抗原特异性免疫细胞的有效刺激,诸如杀伤T细胞及产生抗体的B细胞。如何诱导T细胞以产生抗原特异性为目前关注的重要课题。本公开提供一种用于诱导抗原特异性T细胞的方法及其在癌症或病毒感染治疗中的用途。
发明内容
鉴于本领域的迫切需要,本文提供在癌症治疗中安全有效的诱导抗原特异性T细胞的方法。此外,抗原特异性T细胞易于制造,其成本低廉且产量高。
在一个具体实施方案中,本公开提供一种用于诱导抗原特异性T细胞的方法,其包含:
将外周血单核细胞(PBMC)培养在包含抗原性肽、IL-2、IL-15及IFN-α的第一培养基中以获得第一细胞群;
将第一细胞群培养在包含IL-2、IL-15及IFN-α的第二培养基中以获得第二细胞群;以及
将第二细胞群培养在包含IL-2及IL-15的第三培养基中以获得抗原特异性T细胞。
在一个具体实施方案中,抗原性肽来自癌症或病毒,其浓度为1μg/mL至1mg/mL,优选为5μg/mL至500μg/mL,优选为10μg/mL至100μg/mL或更优选为10μg/mL。
在一个具体实施方案中,癌症包含鼻咽癌、膀胱癌、胆道癌、骨癌、脑肿瘤、乳腺癌、子宫颈癌、结直肠癌、结肠癌、食道癌、表皮癌、胃癌、胃肠道间质瘤(GIST)、神经胶质瘤、淋巴系的造血肿瘤、肝癌、非霍奇金氏淋巴瘤(non-Hodgkin’s lymphoma)、卡波西氏肉瘤(Kaposi’s sarcoma)、白血病、肺癌、淋巴瘤、肠癌、黑色素瘤、骨髓白血病、胰腺癌、前列腺癌、视网膜母细胞瘤、卵巢癌、肾细胞癌、脾癌、鳞状细胞癌、甲状腺癌,或甲状腺滤泡癌。
在一个具体实施方案中,癌症为爱泼斯坦-巴尔(Epstein-Barr)病毒(EBV)相关癌症。
在一个具体实施方案中,癌症为EBV阳性胃癌、EBV阳性子宫颈癌、EBV阳性伯吉特氏(Burkitt’s)淋巴瘤、EBV阳性T细胞淋巴瘤、EBV阳性乳腺癌、EBV阳性平滑肌肉瘤、EBV阳性平滑肌瘤、EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌,或EBV阳性移植后淋巴增生性疾病(PTLD)。
在一个具体实施方案中,抗原性肽包含BMLF-1、EBV肽池或CMV肽池。
在一个具体实施方案中,第一培养基、第二培养基及第三培养基是分别培养1至3天。
在一个具体实施方案中,IL-2的浓度为1至500ng/mL,优选为5至200ng/mL、10至100ng/mL、20至80ng/mL,更优选为40ng/mL。
在一个具体实施方案中,IL-15的浓度为1至100ng/mL,优选为2至50ng/mL、2.5至10ng/mL,更优选为2.5ng/mL。
在一个具体实施方案中,IFN-α的浓度为1至100ng/mL,优选为2至50ng/mL、3至25ng/mL、4至10ng/mL,更优选为5ng/mL。
在一个具体实施方案中,第一培养基、第二培养基及第三培养基进一步分别包含AIM-V培养基。
在一个具体实施方案中,第一培养基、第二培养基及第三培养基进一步包含血小板裂解液,优选为人类血小板裂解液(HPL),浓度为1至10重量%(以培养基为基准),优选为2至8重量%(以培养基为基准)、3至6重量%(以培养基为基准),更优选为4重量%(以培养基为基准)。
本公开的一些具体实施方案中提供一种组合物的用途,该组合物包含治疗有效量的抗原特异性T细胞和医药上可接受的载体,以在有需求的受试者中预防或治疗癌症或病毒感染。
本公开的又一个具体实施方案提供一种组合物的用途,该组合物包含用于制造药剂的治疗有效量的抗原特异性T细胞和医药上可接受的载体,以在有需求的受试者中预防或治疗癌症或病毒感染。
本公开进一步提供一种用于在有需求的受试者中预防或治疗癌症或病毒感染的组合物,该组合物包含治疗有效量的抗原特异性T细胞和医药上可接受的载体。
附图说明
图1说明了T细胞生长细胞因子促进的BMLF1诱导的EBV特异性CD8 T细胞活化及扩增。
图2至4说明了在第0天及第3天添加IFN-α可促进4-1BB+CD8T细胞扩增。
图5和6说明了在第0天及第3天添加IFN-α可促进4-1BB+BMLF1特异性CD8 T细胞扩增。
图7说明了九天培养期显现出最好的4-1BB+BMLF1特异性CD8T细胞扩增。
图8说明了在第0天及第3天添加IFN-α展现出最好的4-1BB+BMLF1特异性CD8 T细胞的T依赖性细胞毒性。
图9说明了不同的IL-2与IL-15协同以促进BMLF1诱导的EBV特异性CD8 T细胞反应。
图10说明了IFN-α诱导的EBV特异性T细胞(EBaT8)的表型分析。
图11说明了EBaT8的功能分析。通过PanToxilux试剂盒(OncoImmunin,Inc.)进行EBaT8的细胞毒性评估。
图12和13说明了PepTivator EBV肽池能够在IFN-α存在下诱导EBV特异性T细胞(EBaT8)扩增。
图14说明了由PepTivator EBV肽池及IFN-α生成的EBV特异性T细胞(EBaT8)呈现出T依赖性细胞毒性。
图15和16说明了PepTivator CMV肽池能够在IFN-α存在下诱导CMV特异性T细胞扩增(在第6天收获的细胞)。
图17说明了由PepTivator CMV肽池及IFN-α生成的CMV特异性T细胞呈现出T依赖性细胞毒性(在第6天收获的细胞)。
具体实施方式
本公开的前述及其他实施方式现将更详细地描述关于本文所述的其他具体实施方案。应当理解的是,本发明可以不同的形式实施,且不应被解释为局限于本文阐述的具体实施方案。而是提供这些具体实施方案使本公开彻底且完整,并将向本发明所属领域普通技术人员充分传达本发明的范围。
本文在本发明的描述中使用的术语仅出于描述某些具体实施方案的目的,而非旨在限制本发明。如在本发明的说明书及所附权利要求中所使用的,单数形式的“一”、“一个”和“该”旨在包括复数形式,除非上下文另有明确指明。
如本文所用,术语“包含(动词)”、“包含(动名词)”、“包括(动词)”、“包括(动名词)”、“具有(动词)”、“具有(动名词)”、“含有(动词)”、“含有(动名词)”、“特征在于”或其任何其他变型,旨在非排他性的涵盖,除非明确指明的任何情况。举例而言,包含一系列元素的组合物、混合物、程序或方法不一定仅局限于那些元素,而是可包括未明确列出的或此类组合物、混合物、程序或方法所固有的其他元素。
连接短语“由…组成”不包含任何未指定的元素、步骤或成分。若用于权利要求中,则这将使权利要求局限于所列举的材料,除了通常与的相关的杂质以外。当在权利要求的主体中出现短语“由…组成”,而非紧接在序言之后时,其仅局限该句中列出的元素;整体上,其他要素并不排除在该权利要求的范围内。
当申请人已经以例如“包含”之类的开放式术语定义发明或其部分,应该容易理解(除非另有说明)否则该说明应解释为亦使用术语“由…组成”来描述该发明。
本文中的所有数字可被理解为由“约”所修饰。如本文所用,术语“约”用于指明数值包括例如,测量装置的误差的固有变化,用于确定该数值的方法或研究对象的间存在的变化。通常,根据具体情况,该术语目的在于涵盖大约或少于1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%,15%,16%,17%,18%,19%,或20%的变异度。
除非明确指明仅指替代方案或替代方案是互斥的,否则权利要求中的术语“或”的使用是指“和/或”,尽管本公开支持仅提及替代方案以及“和/或”的定义。
如本文所用,“受试者”是指动物,包括,例如,被诊断患有或怀疑患有或正在发展癌症的哺乳动物受试者。在一个具体实施方案中,术语“受试者”可指患有癌症的脊椎动物或被认为需要进行癌症治疗的脊椎动物。受试者包括温血动物,例如哺乳动物,例如灵长类动物,且更优选为人类。非人类的灵长类动物亦为受试者。术语受试者包括家养动物,例如猫、狗、猿等,家畜(例如,牛、马、猪、绵羊、山羊等),以及实验动物(例如,小鼠、兔、大鼠、沙鼠、天竺鼠等)。因此,本文涵盖了兽医用途及药物制剂。
本文中,“施用(动名词)”或“施用(名词)”是指对受试者提供本申请的抗原特异性T细胞或药物组合物。举例而非限制,施用可通过肠胃外、皮下、肌内、静脉内、关节内、支气管内、腹腔内、囊内、软骨内、腔内、体腔内(intracelial)、小脑内、脑室内、结肠内(intracolic)、子宫颈管内、胃内、肝内、心肌内、骨内(intraosteal)、骨盆内、心包内、腹膜内、胸膜内、前列腺内、肺内、直肠内、肾内、视网膜内、脊髓内、滑膜内、胸腔内、子宫内、膀胱内、推注、阴道、直肠、颊、舌下、鼻内,以及透皮进行。举例而言,可通过静脉内(intravenous,i.v.)注射、皮下(sub-cutaneous,s.c.)注射、皮内(intradermal,i.d.)注射、腹膜内(intraperitoneal,i.p.)注射,或肌内(intramuscular,i.m.)注射进行注射。可采用一种或多种此类途径。肠胃外施用可例如通过推注或随时间逐渐灌注。替代地或同时地,可通过口服途径施用。
术语“治疗(动名词)”或“治疗(名词)”的使用在本文中是指对一受试者施用抗原特异性T细胞或药物组合物,其目的为治愈、减轻、缓解、补救、预防,或改善疾病、该疾病的症状、继发于该疾病的疾病状态,或对该疾病的易感性。当在权利要求和/或说明书中使用时,术语“抑制”、“减少”或“预防”或这些术语的任何变化包括任何可测量的降低或完全抑制以达到期望的结果。
可由本申请的药物组合物治疗的“癌症”包括按部位分类的那些,包括口腔及咽(嘴唇、舌头、唾液腺、口底、牙龈及其他口、鼻咽、扁桃体、口咽、下咽,其他口腔/咽)的癌症;消化系统(食道;胃;小肠;结肠及直肠;肛门、肛管,及直肠肛门;肝;肝内胆管;胆囊;其他胆道;胰腺;腹膜后;腹膜、网膜,以及肠系膜;其他消化器官)的癌症;呼吸系统(鼻腔、中耳,以及鼻窦;喉;肺及支气管;胸膜;气管、纵隔,以及其他呼吸道)的癌症;间皮瘤的癌症;骨头及关节;以及软组织,包括心脏;皮肤癌,包括黑色素瘤及其他非上皮性皮肤癌;卡波西氏肉瘤(Kaposi’s sarcoma)以及乳癌;女性生殖系统(子宫颈;子宫体;子宫、卵巢;阴道;外阴;以及其他女性生殖器)的癌症;男性生殖系统(前列腺、睾丸、阴茎,以及其他男性生殖器)的癌症;泌尿系统(膀胱;肾及肾盂;输尿管;以及其他尿道)的癌症;眼及眼框癌;脑及神经系统(大脑;以及其他神经系统)的癌症;内分泌系统(甲状腺以及其他内分泌,包含胸腺)的癌症;淋巴瘤(霍奇金氏病以及非霍奇金氏淋巴瘤)、多发性骨髓瘤,以及白血病(淋巴细胞性白血病;骨髓性白血病;单核细胞性白血病;以及其他白血病)。
可作为根据本申请的治疗组合物的合适目标,按组织学类型分类的其他癌症包括但不局限于,赘瘤,恶性;未明确分类(not otherwise specified,NOS)的癌;未分化的癌,NOS;巨细胞及梭形细胞癌;小细胞癌,NOS;乳头状癌,NOS;鳞状细胞癌,NOS;淋巴上皮癌;基底细胞癌,NOS;毛母质(Pilomatrix)癌;移行细胞癌,NOS;乳头状移行细胞癌;腺癌,NOS;胃泌素瘤,恶性;胆管癌;肝细胞癌,NOS;合并肝细胞癌及胆管癌;小梁腺癌;腺样囊状癌;腺瘤性息肉中的腺癌;腺癌,家族性结肠息肉;实体癌,NOS;类癌,恶性;细支气管肺泡腺癌;乳头状腺癌,NOS;嫌色(Chromophobe)癌;嗜酸癌;嗜氧腺癌;嗜碱性球癌;透明细胞腺癌,NOS;颗粒细胞癌;滤泡性腺癌,NOS;乳头状及滤泡性腺癌;非包膜性硬化性癌;肾上腺皮质癌;子宫内膜癌;皮肤附器癌;顶分泌(Apocrine)腺癌;皮脂腺癌;耳垢(Ceruminous)腺癌;黏液性表皮性脑膜癌;囊腺癌,NOS;乳头状囊腺癌,NOS;乳头状浆液性囊腺癌;黏液性囊腺癌,NOS;黏液腺癌;戒环细胞癌;浸润性导管癌;髓状癌,小叶癌;炎性癌;佩吉特氏病,乳腺;腺泡细胞癌;腺鳞癌;具有鳞状转移瘤的腺癌(Adenocarcinoma w/Squamous metaplasia);胸腺瘤,恶性;卵巢间质肿瘤,恶性;鞘细胞瘤(thecoma),恶性;颗粒细胞瘤,恶性;NOS;雄胚瘤,恶性;赛特利氏(Sertoli)细胞癌;睾丸间质细胞(Leydig cell)瘤,恶性;脂质细胞瘤,恶性;副神经节瘤,恶性;乳腺旁神经节瘤,恶性;嗜铬细胞瘤;血管肉瘤(Glomangiosarcoma);恶性黑色素瘤,NOS;非黑素瘤;浅表扩散黑色素瘤;巨大色素痣中的马里格黑色素瘤;上皮样细胞黑色素瘤;蓝色母斑,恶性;肉瘤,NOS;纤维肉瘤,NOS;纤维组织细胞瘤,恶性;黏液肉瘤;脂肪肉瘤,NOS;平滑肌肉瘤,NOS;横纹肌肉瘤,NOS;胚胎横纹肌肉瘤;肺泡横纹肌肉瘤;基质肉瘤,NOS;混合性肿瘤,恶性,NOS;苗勒氏混合瘤(Mullerian mixed tumor);肾母细胞瘤;肝母细胞瘤;癌肉瘤,NOS;间皮肉瘤,恶性;布伦纳瘤(Brenner tumor),恶性;叶状瘤,恶性;滑膜肉瘤,NOS;间皮瘤,恶性;精原细胞瘤;胚胎癌,NOS;畸胎瘤,恶性,NOS;甲状腺肿样卵巢瘤,恶性;绒毛膜癌;中肾上皮瘤,恶性;血管肉瘤;血管内皮瘤,恶性;卡波西氏肉瘤;血管外皮细胞瘤,恶性;淋巴管肉瘤;骨肉瘤,NOS;近皮质骨肉瘤;软骨肉瘤,NOS;软骨母细胞瘤,恶性;间质软骨肉瘤;骨巨细胞瘤;尤因氏肉瘤(Ewing’s sarcoma);齿源性肿瘤,恶性;釉质母细胞骨肉瘤;成釉细胞瘤,恶性;釉质母细胞纤维肉瘤;松果体瘤,恶性;脊索瘤;胶质瘤,恶性;室管膜瘤,NOS;星状细胞瘤,NOS;原生质星形细胞瘤;纤维性星形细胞瘤;星形母细胞瘤;胶质母细胞瘤,NOS;寡树突神经胶细胞瘤,NOS;寡树突神经胶质母细胞瘤;原始神经外胚层瘤;小脑肉瘤,NOS;神经节神经母细胞瘤;神经母细胞瘤,NOS;视网膜母细胞瘤,NOS;嗅觉神经源性肿瘤;脑膜瘤,恶性;神经纤维肉瘤;神经性瘤,恶性;颗粒细胞瘤,恶性;恶性淋巴瘤,NOS;霍奇金氏病,NOS;霍奇金氏类肉芽肿,NOS;小淋巴细胞性恶性淋巴瘤;弥漫性大细胞恶性淋巴瘤;滤泡型恶性淋巴瘤,NOS;蕈状肉芽肿;其他指定的非霍奇金氏淋巴瘤;恶性组织细胞增殖症;多发性骨髓瘤;肥大细胞肉瘤;免疫增殖性小肠疾病;白血病,NOS;淋巴白血病,NOS;浆细胞白血病;红血球性白血病;淋巴肉瘤细胞白血病;骨髓性白血病,NOS;嗜碱性球白血病;嗜酸性球白血病;单核细胞白血病,NOS;肥大细胞白血病;巨核细胞白血病;髓样肉瘤;以及毛细胞白血病。
如本文所用,“有效量”是指足以减少癌症的症状及表征的该抗原特异性T细胞或药物组合物的剂量,症状及表征包括但不局限于,体重减轻、疼痛或肿瘤块,其为在临床上可触知的团块或通过各种影像方法在放射学上可检测到的。
在某些具体实施例中,期望限制、减小或改善肿瘤或癌症病变的大小。施用途径自然地将随待靶向的病变或部位的位置及性质而变,且包括例如,局部的、肠胃外、静脉内、肌肉内,和/或全身性施用及制剂。针对目标区域,特别考虑直接注射或注射至脉管系统或器官或组织的血管及源自器官或组织的血管。局部、区域,或全身性施用亦可能是适当的。
在本文公开的内容中,使用FACS/流式细胞仪分析的细胞表面抗原的表现量或表面密度定义于表1中。表1中对各种表现量的解释为定义细胞表面抗原表现量的一个实例。应当注意的是,流式细胞术信号量强度随以下因素而变:流式细胞术、软件,以及所使用的不同抗体批次。
表1
符号 | 解释 |
- | 流式细胞术信号量强度小于或等于100(亦即,1) |
+(Dim) | 流式细胞术信号量强度介于100与101之间 |
+ | 流式细胞术信号量强度介于101与102之间 |
+(hi) | 流式细胞术信号量强度大于或等于102 |
除非另有定义,否则本文所用的所有技术及科学术语具有与本发明所属技术领域普通技术人员通常理解的相同含义。本文引用的所有出版物、专利申请、专利,以及其他参考数据通过全文引入作为参考,以用于与其中存在该参考文献的句子和/或段落有关的教导。
第I型干扰素习知可调节T细胞功能。举例而言:
1.按序信号传导:与TCR接合相关的伴随或略微延迟的第I型IFN信号传导可促进存活、效应细胞分化。
2.失序信号传导:将T细胞预先暴露于与TCR接合相关的第I型IFN可诱导STAT1依赖性抗增生及促细胞凋亡程序。
本公开提供一种用于诱导抗原特异性T细胞的方法,其包括:将外周血单核细胞(PBMC)培养在包含抗原性肽、IL-2、IL-15,及IFN-α的第一培养基中以获得第一细胞群;将第一细胞群培养在包含IL-2、IL-15及IFN-α的第二培养基中以获得第二细胞群;以及将第二细胞群培养在包含IL-2及IL-15的第三培养基中以获得抗原特异性T细胞。
抗原性肽的浓度可为1μg/mL至1mg/mL,优选为5μg/mL至500μg/mL,优选为10μg/mL至100μg/mL,或更优选为10μg/mL。
抗原性肽可来自癌症或病毒。癌症可包含鼻咽癌、膀胱癌、胆道癌、骨癌、脑肿瘤、乳腺癌、子宫颈癌、结直肠癌、结肠癌、食道癌、表皮癌、胃癌、胃肠道间质瘤(GIST)、神经胶质瘤、淋巴系的造血肿瘤、肝癌、非霍奇金氏淋巴瘤(non-Hodgkin’s lymphoma)、卡波西氏肉瘤(Kaposi’s sarcoma)、白血病、肺癌、淋巴瘤、肠癌、黑色素瘤、骨髓白血病、胰腺癌、前列腺癌、视网膜母细胞瘤、卵巢癌、肾细胞癌、脾癌、鳞状细胞癌、甲状腺癌,或甲状腺滤泡癌。
癌症可为爱泼斯坦-巴尔(Epstein-Barr)病毒(EBV)相关癌症。癌症可为EBV阳性胃癌、EBV阳性子宫颈癌、EBV阳性伯吉特氏(Burkitt’s)淋巴瘤、EBV阳性T细胞淋巴瘤、EBV阳性乳腺癌、EBV阳性平滑肌肉瘤、EBV阳性平滑肌瘤、EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌,或EBV阳性移植后淋巴增生性疾病(PTLD)。
抗原性肽可包含BMLF-1或EBV肽池。肽亦可来自巨细胞病毒(Cytomegalovirus,CMV)肽池或其他肿瘤相关抗原。
第一培养基、第二培养基及第三培养基可分别培养1至3天。另一方面,IL-2的浓度可为1至500ng/mL,优选为5至200ng/mL、10至100ng/mL、20至80ng/mL,更优选为40ng/mL。此外,IL-15的浓度可为1至100ng/mL,优选为2至50ng/mL、2.5至10ng/mL,更优选为2.5ng/mL。此外,IFN-α的浓度为1至100ng/mL,优选为2至50ng/mL、3至25ng/mL、4至10ng/mL,更优选为5ng/mL。
第一培养基、第二培养基及第三培养基可进一步包含AIM-V培养基。第一培养基、第二培养基及第三培养基可进一步包含血小板裂解液,优选为人类血小板裂解液(HPL),浓度为1至10重量%(以培养基为基准),优选为2至8重量%(以培养基为基准)、3至6重量%(以培养基为基准),更优选为4重量%(以培养基为基准)。
一种组合物,其包含治疗有效量的可由上述方法制造的抗原特异性T细胞,且组合物可与医药上可接受的载体组合。该组合物可用于在有需求的受试者中预防或治疗癌症或病毒感染。此外,组合物可用于制造药剂,以在有需求的受试者中预防或治疗癌症或病毒感染。
下列实施例将显示诱导抗原T细胞的不同策略,且最终找到其优选的程序。
实施例
实施例1
请参见图1。图1说明了T细胞生长细胞因子可促进BMLF1诱导的EBV特异性CD8 T细胞活化及扩增。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基及4% HPL中9天。在每一个特定的组中,反应PMBC分别培养于hIL-2(40ng/ml)与hIL-15(2.5ng/ml)、BMLF1肽(10μg/mL),以及BMLF1肽(10μg/mL)加上hIL-2(40ng/ml)及hIL-15(2.5ng/ml)中。在第9天,收获总细胞,并以抗4-1BB-PE、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour 750,及CD3-Krome Orange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。
如图1所示,IL-2及IL-15可诱导PMBC的分化。肽(BMLF1)的实例不诱导PMBC的分化。IL-2及IL-15与BMLF1的组合显示出PMBC的显著分化。由仅BMLF-1诱导的实例无法有效促进CD8 T细胞的复制,是因TCR阈值太高。然而,由IL-2/IL-15诱导的实例具有降低TCR阈值的功能,使得T细胞复制。因此,在IL-2/IL-15的协助下,其有助于BMLF肽诱导的CD8 T细胞的复制及4-1BB的表达。因此,IL-2及IL-15可具有单独诱导T细胞分化的功能,但与BMLF1的抗原性肽结合时将表现的更好。
实施例2
请参见图2至4。图2至4说明了在第0天及第3天添加IFN-α可促进4-1BB+CD8 T细胞扩增。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基与HPL(4%)、hIL-2(40ng/ml)及hIL-15(2.5ng/ml)中9天。在每一特定的组中,反应PMBC在第0天、第3天、第0天加上第3天或第0天加上第3天、及第6天以5ng/ml IFN-α处理。在第9天,收获总细胞,并以抗4-1BB-PE、CD8-APC-Alexa Flour700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour 750,及CD3-Krome Orange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。利用Dunnett氏多重比较检验进行数据统计分析。*,P<0.05。
如图2至4所示,此实例为针对IFN-α的不同添加时间的实验,以确认IFN-α作用时间对CD8 T细胞及4-1BB+CD8 T细胞比例的影响。在第0/3天诱导IFN-α(亦即在第0天及第3天添加IFN-α),相较于仅在第0天或第3天添加IFN-α,可诱导更多的CD8 T细胞及4-1BB+CD8T细胞(以百分比计)。然而,相较于在第0/3/6天添加IFN-α则没有差异。进一步计算每一组与不含IFN-α组的间的增加百分比差异,亦可看出,IFN-α的诱导时间必须至少在第0/3天。因此,针对下列实验,IFN-α的添加时间设定为第0/3天。
此外,如图4所示,处理IFN-α明显诱导CD8 T细胞的分化。然而,在第3天施用的治疗的增加百分比明显低于包括在第0天进行处理的组,其意味着处理IFN-α在T细胞分化的早期阶段表现的更好。
实施例3
请参见图5及6,图5及6说明了在第0天及第3天添加IFN-α可促进4-1BB+BMLF1特异性CD8 T细胞扩增。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基与HPL(4%)、hIL-2(40ng/ml)及hIL-15(2.5ng/ml)中9天。在特定的组中,反应PMBC在第0及3天以5ng/ml IFN-α处理。在第9天,收获总细胞,并以抗4-1BB-PE、BMLF1-五聚体-APC、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour750,及CD3-Krome Orange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。利用Dunnett氏多重比较检验进行数据统计分析。*,P<0.05。
如图5及6所示,IL-2及IL-15与BMLF1的组合可诱导更多PBMC分化为CD8 T细胞,且IL-2及IL-15与BMLF1和IFN-α的组合在第0/3天明显更好。
实施例4
请参见图7。图7说明了九天培养期显现出最好的4-1BB+BMLF1特异性CD8 T细胞扩增。反应PBMC培养于AIM-V培养基与HPL(4%)、BMLF1肽(10μg/mL)、hIL-2(40ng/ml)、hIL-15(2.5ng/ml)及IFN-α(5ng/ml)。在第9天及第12天,收获总细胞,并以抗4-1BB-PE、BMLF1-五聚体-APC、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour750,及CD3-Krome Orange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。
实施例4为延时实验。表明了在第0/3天添加IFN-α可在第9天诱导大部分的4-1BB+T细胞。因此,针对下列实验,将其培养时间设定在第9天。
实施例5
请参见图8,图8说明了在第0天及第3天添加IFN-α后4-1BB+BMLF1特异性CD8 T细胞展现出最好的T依赖性细胞毒性。通过PanToxilux试剂盒(OncoImmunin,Inc.)进行EBaT8的细胞毒性评估。将自体BMLF1加载的PMBC作为靶细胞,并在最佳浓度下以TFL4染色50分钟。将TFL-4标记的靶细胞和反应细胞与凋亡蛋白酶受体在37℃下共同培养30分钟。收获细胞,并经由流式细胞术分析TFL-4+受体+的信号。
如图8所示,在第0天及第3天添加了IFN-α的组呈现出更高的凋亡蛋白酶,其意味着在第0/3天诱导的分化细胞的细胞毒性高于其他两组。
实施例6
请参见图9,图9说明了不同的IL-2与IL-15协同以促进BMLF1诱导的EBV特异性CD8T细胞反应。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基与HPL(4%)、hIL-2(40ng/ml)及IFN-α(5ng/ml)中9天。在每一特定的组中,反应PMBC以2.5ng/ml或10ng/ml hIL-15处理九天。在第9天,收获总细胞,并以抗4-1BB-PE、BMLF1-五聚体-APC、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour750,及CD3-Krome Orange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。利用Dunnett氏多重比较检验进行数据统计分析。*,P<0.05。
如图9所示,IL-2以40ng/ml添加至3个组中,且IL-15分别以2.5ng/ml及10ng/ml添加至2个组中。结果显示,IL-2+IL-15+BMLF-1及第0/3天的IFN-α为最佳培养条件。图10显示了通过流式细胞术的表型分析及去颗粒分子(degranulation molecule)释放的结果。
请参见图11,图11说明了EBaT8的功能分析。通过PanToxilux试剂盒(OncoImmunin,Inc.)进行EBaT8的细胞毒性评估。自体BMLF1加载的PMBC作为靶细胞,并在最佳浓度下以TFL4染色50分钟。将TFL-4标记的靶细胞和EBaT8或旁观者细胞因子活化的T细胞与凋亡蛋白酶受体在37℃下共同培养30分钟。收获细胞,并经由流式细胞术分析TFL-4+受体+的信号。
如图11所示,在第0/3天以IL-2+IL-15+BMLF-1培养并添加IFN-α的细胞(EbaT8)具有杀死BMLF加载的靶细胞的概率。
工作实施例2
请参见图12至13的结果。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基与HPL(4%)、PepTivator EBV(0.5mg/ml)、hIL-2(40ng/ml)、hIL-15(2.5ng/ml)及IFN-α(5ng/ml)中。在第9天,收获总细胞,并以抗4-1BB-PE、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour750,及CD3-KromeOrange的mAb染色。经由Navios流式细胞仪进行样本采集,并经由Kaluza软件进行数据分析。进一步参见图14的结果,通过PanToxilux试剂盒(OncoImmunin,Inc.)进行EBaT8的细胞毒性评估。将自体PepTivator EBV加载的PMBC作为靶细胞,并在最佳浓度下以TFL4染色50分钟。将TFL-4标记的靶细胞和EBaT8与凋亡蛋白酶受体在37℃下共同培养30分钟。收获细胞,并经由流式细胞术分析TFL-4+受体+的信号。
如图12至14所示,PepTivator EBV肽池能够在IFN-α存在下诱导EBV特异性T细胞(EBaT8)扩增,且由PepTivator EBV肽池及IFN-α生成的EBaT8呈现出T依赖性细胞毒性。结果表明,4-1BB+CD8 T细胞可由EBV肽池诱导,该肽池包含不同的序列,并可产生T细胞依赖性杀伤力。因此,可提高其临床试验的可行性。
工作实施例3
请参见图15及16的结果。反应PMBC以Cell-Trace Violet标记,随后培养于AIM-V培养基与HPL(4%)、PepTivator CMV(0.5mg/ml)、hIL-2(40ng/ml)、hIL-15(2.5ng/ml)及IFN-α(5ng/ml)中。在第6天,收获总细胞,并以抗4-1BB-PE、CD8-APC-Alexa Flour 700、CD56-APC-Cy7、CD14-APC-Alexa Flour 750、CD19APC-Alexa Flour750,及CD3-KromeOrange的mAb染色。经由Navios流式细胞仪采集样本,并经由Kaluza软件进行数据分析。进一步参见图17的结果,通过PanToxilux试剂盒(OncoImmunin,Inc.)进行EBaT8的细胞毒性评估。将自体PepTivator EBV加载的PMBC作为靶细胞,并在最佳浓度下以TFL4染色50分钟。将TFL-4标记的靶细胞和EBaT8与凋亡蛋白酶受体在37℃下共同培养30分钟。收获细胞,并经由流式细胞术分析TFL-4+受体+的信号。
如图15至17所示,在第6天,PepTivator CMV肽池能够在IFN-α存在下诱导CMV特异性T细胞扩增,且由PepTivator CMV肽池及IFN-α生成的CMV特异性T细胞呈现出T依赖性细胞毒性。结果表明,4-1BB+CD8 T细胞亦可由肽池-CMV与IL-2+IL-15并在第0/3天添加IFN-α诱导,并可产生T细胞依赖性杀伤力,其意味着CMV、HPV或其他肿瘤相关抗原亦可用于以本文所述方法诱导抗原特异性T细胞扩增。
以上描述仅为示例性而非限制性。在不脱离本发明的精神及范畴下所作的任何等效修改或变化皆应包括在权利要求所界定的范畴内。
Claims (15)
1.一种用于诱导抗原特异性T细胞的方法,其包括:
将外周血单核细胞(PBMC)培养在包含抗原性肽、IL-2、IL-15,及IFN-α的第一培养基中以获得第一细胞群;
将第一细胞群培养在包含IL-2、IL-15及IFN-α的第二培养基中以获得第二细胞群;以及
将第二细胞群培养在包含IL-2及IL-15的第三培养基中以获得抗原特异性T细胞。
2.根据权利要求1所述的方法,其中所述抗原性肽来自癌症或病毒,其浓度为1μg/mL至1mg/mL。
3.根据权利要求2所述的方法,其中所述癌症包括鼻咽癌、膀胱癌、胆道癌、骨癌、脑肿瘤、乳腺癌、子宫颈癌、结直肠癌、结肠癌、食道癌、表皮癌、胃癌、胃肠道间质瘤(GIST)、神经胶质瘤、淋巴系的造血肿瘤、肝癌、非霍奇金氏淋巴瘤(non-Hodgkin’s lymphoma)、卡波西氏肉瘤(Kaposi’s sarcoma)、白血病、肺癌、淋巴瘤、肠癌、黑色素瘤、骨髓白血病、胰腺癌、前列腺癌、视网膜母细胞瘤、卵巢癌、肾细胞癌、脾癌、鳞状细胞癌、甲状腺癌,或甲状腺滤泡癌。
4.根据权利要求2所述的方法,其中所述癌症为爱泼斯坦-巴尔(Epstein-Barr)病毒(EBV)相关癌症。
5.根据权利要求4所述的方法,其中所述癌症为EBV阳性胃癌、EBV阳性子宫颈癌、EBV阳性伯吉特氏(Burkitt’s)淋巴瘤、EBV阳性T细胞淋巴瘤、EBV阳性乳腺癌、EBV阳性平滑肌肉瘤、EBV阳性平滑肌瘤、EBV阳性霍奇金淋巴瘤、EBV阳性鼻咽癌,或EBV阳性移植后淋巴增生性疾病(PTLD)。
6.根据权利要求4所述的方法,其中所述抗原性肽包含BMLF-1、EBV肽池或CMV肽池。
7.根据权利要求1至6中任一项所述的方法,其中所述第一培养基、所述第二培养基及所述第三培养基是分别培养1至3天。
8.根据权利要求1至6中任一项所述的方法,其中IL-2的浓度为1至500ng/mL。
9.根据权利要求1至6中任一项所述的方法,其中IL-15的浓度为1至100ng/mL。
10.根据权利要求1至6中任一项所述的方法,其中IFN-α的浓度为1至100ng/mL。
11.根据权利要求1至6中任一项所述的方法,其中所述第一培养基、所述第二培养基及所述第三培养基进一步分别包含AIM-V培养基。
12.根据权利要求1至6中任一项所述的方法,其中所述第一培养基、所述第二培养基及所述第三培养基进一步包含血小板裂解液,优选为人类血小板裂解液(HPL),浓度为1至10重量%,以培养基为基准。
13.一种组合物的用途,其用于在有需求的受试者中预防或治疗癌症或病毒感染,所述组合物包含治疗有效量的根据权利要求1至12中任一项所述的方法所制造的抗原特异性T细胞和医药上可接受的载体。
14.一种组合物的用途,其用于制造在有需求的受试者中预防或治疗癌症或病毒感染的药剂,所述组合物包含治疗有效量的根据权利要求1至12中任一项所述的方法所制造的抗原特异性T细胞和医药上可接受的载体。
15.一种用于在有需求的受试者中预防或治疗癌症或病毒感染的组合物,所述组合物包含治疗有效量的根据权利要求1至12中任一项所述的方法所制造的抗原特异性T细胞和医药上可接受的载体。
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