CN117551201A - 一种靶向siglec-6的同种异体通用car-t细胞及其制备方法及用途 - Google Patents
一种靶向siglec-6的同种异体通用car-t细胞及其制备方法及用途 Download PDFInfo
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Abstract
本发明提供了一种靶向SIGLEC‑6的同种异体通用CAR‑T细胞及其制备方法及用途,涉及生物医药技术领域。本发明以人SIGLEC‑6为靶点制备了抗SIGLEC‑6纳米抗体,所述纳米抗体包括包含CDR1、CDR2和CDR3的重链可变区:所述CDR1如SEQ ID NO.1所示,CDR2如SEQ ID NO.2所示,CDR3如SEQ ID NO.3所示;或者,所述CDR1如SEQ ID NO.5所示,CDR2如SEQ ID NO.6所示,CDR3如SEQ ID NO.7所示。该纳米抗体可特异性结合SIGLEC‑6抗原,亲和力在10‑10~10‑11M之间,属于高亲和力纳米抗体。由该纳米抗体制备了同种异体通用CAR‑T细胞,该CAR‑T细胞具有良好的抗肿瘤活性,可应用于制备预防、诊断和治疗SIGLEC‑6阳性的白血病和淋巴瘤,特别是AML、CLL、MALT淋巴瘤和克隆性肥大细胞疾病等,具有良好的应用前景。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种靶向SIGLEC-6的同种异体通用CAR-T细胞及其制备方法及用途。
背景技术
白血病,是一类起源于造血干细胞的恶性克隆性疾病。根据白血病细胞的分化程度和自然病程,白血病可分为急性和慢性两大类。根据主要受累的细胞系列可将急性白血病分为急性髓系白血病(AML)和急性淋巴细胞白血病(ALL)。慢性白血病则分为慢性髓系白血病(常称为慢性粒细胞白血病,CML)、慢性淋巴细胞白血病(CLL)及少见类型白血病。AML是一种侵袭血液及骨髓的肿瘤,是白血病家族中成年人最常见的类型之一,它进展快速,极具侵袭性。正常情况下,骨髓会产生髓系造血干细胞,造血干细胞又经过分化,最终分化为各种成熟的血细胞。但如果体内基因出现突变,会导致髓系原始细胞无法正常分化为正常的血细胞(红细胞、血小板、白细胞),进而引发AML。多数病例病情急重,预后凶险,如不及时治疗常可危及生命。
CAR-T疗法,就是嵌合抗原受体T细胞免疫疗法,英文全称Chimeric AntigenReceptorT-Cell Immunotherapy。这是一种治疗肿瘤的新型精准靶向疗法,近几年通过优化改良在临床肿瘤治疗上取得很好的效果。靶向CD19的自体CAR-T细胞在复发/难治性急性B淋巴细胞白血病(B-ALL)的治疗,以及靶向BCMA在多发性骨髓瘤的治疗中均取得了革命性突破,多款CAR-T产品获批上市。
CAR-T细胞出色的临床治疗效果,激起了人们使用CAR-T细胞治疗AML的兴趣。由于CAR-T细胞靶向细胞表面分子,所以希望靶抗原在肿瘤细胞上均匀表达,并且在健康细胞和组织上具有最小的表达甚至不表达。这对AML的CAR-T治疗具有挑战性,因为已知的大多数候选抗原都在健康的HSC/P和先天免疫细胞上又表达。临床研究证实,针对髓系抗原,例如CD123、CD33的CAR-T细胞已被证明是清髓性的,并且需要异基因造血干细胞移植(alloHSCT)来重建造血。在一项使用同种异体CD123-CAR-T细胞产品的首次人体临床试验中,临床使用CD123特异性CAR-T细胞导致了显著的毒性和致命的严重不良事件,该试验已被暂停。有研究者利用CRISPR/Cas9基因编辑敲除供体HSC/P细胞中的CD33或改变其抗原表位,阻止CD33-CAR-T细胞对HSC/P的清除,然而这一策略的临床实施是极其费力、复杂和昂贵的,基因编辑后的造血干细胞会带来更大的非预期遗传毒性风险,包括恶性转化的风险。AML中其他潜在CAR靶点包括FLT3、CLL-1、CD7、Lewis-y抗原等。研究显示FLT3-CAR-T细胞在小鼠异种移植物中效果很好,而在另一项研究中因其在HSC/P上表达,CAR-T细胞治疗仍会清除造血干细胞;C型凝集素样分子-1(CLL-1)同时表达于AML母细胞、肺和胃肠道上皮细胞上;CD7仅在约30%的AML患者中表达,并在T细胞上高水平表达,需从T细胞中敲除CD7来制造抗CD7-CAR-T细胞,这使临床应用变得复杂。
唾液酸结合免疫球蛋白样凝集素(Siglecs)是一种细胞表面受体免疫球蛋白超家族,主要由白细胞表达,并与人类免疫细胞中的抑制性信号传导有关。值得注意的是,Siglec-2(CD22)和Siglec-3(CD33)是Siglec超家族的成员,并且分别作为血液系统恶性肿瘤如B细胞急性淋巴细胞白血病(B-ALL)和AML中的CAR靶抗原。令人鼓舞的是,CD22靶向的CAR-T细胞在复发/难治性(R/R)B-ALL患者中显示出完全缓解,这表明靶向具有良好表达谱的Siglecs可以诱导白血病缓解并有可能治愈患者。Siglec-6属于CD33相关的Siglec亚家族,其结构与Siglec-3(CD33)密切相关。Siglec-6由三个细胞外免疫球蛋白(Ig)结构域和两个细胞内免疫受体酪氨酸抑制基序(ITIM)基序组成。由于这些ITIM基序,Siglec-6被认为与其他CD33相关的Siglec一样,是激活途径的调节因子。
Siglec-6在约60%的AML病例中表达,但在HSCs上表达很少,造血系统中的表达仅限于记忆B细胞和嗜碱性粒细胞群。此外,Jetani等人还证明siglece-6可能是B细胞慢性淋巴母细胞瘤(B-CLL)的有用靶点。Siglec-6在原代B细胞中表达,在CLL和MALT淋巴瘤中高表达。Siglec-6在造血系统外,除胎盘和人肥大细胞外未发现表达;与其他Siglec蛋白不同,它在NK细胞、T细胞、中性粒细胞、巨噬细胞和单核细胞中均无表达。因此,Siglec-6是一个比较理想的AML CAR-T治疗靶点。
在血液系统恶性肿瘤治疗中,自体CAR-T细胞免疫疗法的抗肿瘤效果显著,极大的提高了多种血液瘤患者的生存率。自体CAR-T细胞输注患者后不会发生免疫排斥反应且在体内持续发挥作用。但诸多不利因素限制了此细胞疗法的应用,例如患者经过多线治疗后体内T细胞数量减少或T细胞功能受损质量下降,制备周期长、成本高等,患者往往错过了最佳治疗窗口。随着CRISPR/Cas9等多种基因编辑技术的快速发展,对健康供者来源T细胞的TCR、B2M和CD52等介导免疫排斥反应的基因敲除,从而最大程度降低GvHD和HvGR,制备成“现货型”同种异体通用CAR-T细胞(UCAR-T)。UCAR-T细胞疗法能够克服自体CAR-T细胞大部分缺点并可通过标准化流程进行规模化制造,从而实现CAR-T细胞治疗产品的批量化生产和产业化,降低患者治疗成本。
CAR-T细胞制备,需要筛选能够特异结合靶抗原的特异单链抗体(scFv)或纳米抗体Nanobody(Nb)。Nb被认为是目前发现的最小的(15kDa)具有结合完整抗原功能的抗体分子,与其他基因工程抗体相比,Nb具有高稳定性、高亲和力、特异性强、高可溶性、与不同蛋白分子偶联的灵活性和易改造及优良的组织穿透性等特点,受到各领域的高度关注,在疾病治疗和检测等领域具备广阔的开发和应用价值。相比scFv,Nb由于其稳定性和小分子量,近些年在CAR-T制备中显示了更多的优势和应用价值。
白血病和淋巴瘤,特别是AML、CLL、MALT淋巴瘤和克隆性肥大细胞疾病,目前缺乏安全有效的治疗方法,Siglec-6是一个有价值的CAR-T细胞治疗靶标。研究一种靶向Siglec-6的CAR-T细胞具有重要意义。
发明内容
本发明的目的在于提供一种靶向SIGLEC-6的同种异体通用CAR-T细胞及其制备方法及用途。以解决目前AML缺乏有效治疗策略的问题。
本发明是这样实现的:
第一方面,本发明提供了一种抗SIGLEC-6的纳米抗体,所述纳米抗体包括包含CDR1、CDR2和CDR3的重链可变区:
所述CDR1如SEQ ID NO.1所示,CDR2如SEQ ID NO.2所示,CDR3如SEQ ID NO.3所示;或者,所述CDR1如SEQ ID NO.5所示,CDR2如SEQ ID NO.6所示,CDR3如SEQ ID NO.7所示。
进一步地,所述纳米抗体还包括骨架区;
优选地,所述纳米抗体为单价纳米抗体、多价纳米抗体、多特异性抗体和融合型纳米抗体中的至少一种;
更优选地,所述纳米抗体为单价纳米抗体时,所述纳米抗体的氨基酸序列如SEQID NO.4或SEQ ID NO.8所示。
本发明的抗SIGLEC-6的纳米抗体只含有VHH区。
第二方面,本发明还提供了一种抗体,其包括前述的纳米抗体,或包括前述的纳米抗体的重链可变区。
第三方面,本发明还提供了一种基因片段或包含所述基因片段的重组载体,所述基因片段的核苷酸序列如SEQ ID NO.9或SEQ ID NO.10所示;所述基因片段编码前述的纳米抗体或所述基因片段编码前述的抗体;
优选地,所述重组载体为质粒或病毒;
更优选地,所述病毒为腺病毒、腺相关病毒、逆转录病毒、慢病毒或溶瘤病毒。
第四方面,本发明还提供了一种宿主细胞,其包含前述的重组载体;
优选地,所述宿主细胞选自原核宿主细胞、真核宿主细胞和噬菌体中的至少一种;
更优选地,所述原核宿主细胞为大肠杆菌、链霉菌、枯草杆菌或分枝杆菌;
和/或,所述真核宿主细胞为动物细胞、植物细胞或真菌;
进一步优选地,所述动物细胞选自哺乳动物细胞、昆虫细胞或秀丽隐杆线虫;
和/或,所述真菌选自酿酒酵母、毕赤酵母、汉森酵母、假丝酵母、乳克鲁维酵母、构巢曲霉、粟酒裂殖酵母和解脂耶罗酵母中的任一种;
更进一步优选地,所述哺乳动物细胞选自293细胞、293T细胞、293FT细胞、CHO细胞、COS细胞、小鼠L细胞、LNCaP细胞、633细胞、Vero、BHK细胞、CV1细胞、Hela细胞、MDCK细胞、Hep-2细胞和Per6细胞中的任一种。
第五方面,本发明还提供了前述的纳米抗体,或前述的抗体的制备方法,所述制备方法为培养前述的宿主细胞,以获得抗体。
第六方面,本发明还提供了一种免疫缀合物或药物组合物,其包括前述的抗SIGLEC-6的纳米抗体或前述的抗体;
优选地,所述免疫缀合物还包括治疗剂;
和/或,所述药物组合物包括药用赋形剂、载体和稀释剂中的至少一种;
更优选地,所述治疗剂包括免疫检查点相关制剂、毒素、因子、化疗药物、放射性核素、激酶抑制剂和细胞毒性剂中的至少一种。
第七方面,本发明还提供了一种嵌合抗原受体,所述嵌合抗原受体的抗原结合结构域包括前述的纳米抗体或前述的抗体;
优选地,所述嵌合抗原受体还包括信号肽、铰链区、跨膜区和信号转导结构域;
更优选地,所述信号转导结构域选自CD3ζ、4-1BB、CD27、CD30、CD40、CD54、CD83、PD-1、ICOS、淋巴细胞功能相关抗原-1、CD2、CD7、CD270、CD273、CD274、DAP10、SIGLEC-63、CD134、CD150和CD152中的至少一种;
和/或,所述跨膜区为CD28跨膜结构域、CD8跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种;
和/或,所述铰链区为IgG1FC-CH2-CH3铰链区、IgG2FC-CH2-CH3铰链区、IgG4FC-CH2-CH3铰链区、CD28铰链区和CD8α铰链区中的一种或多种;
和/或,所述信号肽为CD8α信号肽、IL-2信号肽或GM-CSF信号肽;
更优选地,所述信号转导结构域包括CD3ζ和4-1BB胞内区。
第八方面,本发明还提供了一种靶向SIGLEC-6的CAR-T细胞,其包括如前述的嵌合抗原受体;
优选地,所述CAR-T细胞选自同种异体通用型CAR-T细胞和自体CAR-T细胞中的至少一种;
更优选地,所述通用型CAR-T细胞为敲除了B2M和TRAC基因的CAR-T细胞。
第九方面,本发明还提供了前述的纳米抗体、前述的抗体、前述的基因片段或包含所述基因片段的重组载体、前述的宿主细胞、前述的嵌合抗原受体或前述的CAR-T细胞在制备预防或治疗肿瘤的产品中的用途,或在制备检测SIGLEC-6的产品中的用途,或在制备联合治疗药物中的用途;
优选地,所述预防或治疗肿瘤的产品为免疫细胞、试剂、试剂盒、药物和药物组合物中的至少一种;
和/或,所述检测SIGLEC-6的产品为试剂、试剂盒或基因芯片;
更优选地,所述治疗肿瘤的产品为靶向SIGLEC-6以治疗或辅助治疗肿瘤的药物;
进一步优选地,所述肿瘤为原发性肿瘤或继发性肿瘤;
更进一步优选地,所述肿瘤为SIGLEC-6阳性的白血病或淋巴瘤;更优选地为AML、CLL、MALT淋巴瘤或克隆性肥大细胞疾病。
与现有技术相比,本发明的有益效果为:
本发明以人SIGLEC-6为靶点,通过噬菌体展示技术制备了抗SIGLEC-6纳米抗体,且均可特异性结合SIGLEC-6抗原,亲和力在10-10~10-11M之间,均属于高亲和力纳米抗体。
由此制备了同种异体通用CAR-T细胞,经过验证,该CAR-T细胞具有良好的抗肿瘤活性,可应用于制备预防、诊断和治疗SIGLEC-6阳性的白血病和淋巴瘤,特别是AML、CLL、MALT淋巴瘤和克隆性肥大细胞疾病等,具有良好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明实施例1中利用间接ELISA分析SIGLEC-6纳米抗体与SIGLEC-6抗原的结合特异性的结果。
图2为本发明实施例3中利用间接ELISA分析SIGLEC-6纳米抗体与SIGLEC-6抗原的结合能力的结果。
图3为本发明实施例5中B2M和TRAC敲除效率检测结果。
图4为本发明实施例5中CAR结构示意图。
图5为本发明实施例6中通用CAR-T细胞对SIGLEC-6阳性细胞系U937的杀伤效率检测结果。
图6为本发明实施例7中通用CAR-T细胞对AML小鼠移植模型生存期分析结果。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
除非另外指明,否则实践本发明将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:ALaboratory Manual)》,第二版(Sambrook等人,1989);《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编,1984);《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编,1987);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《哺乳动物细胞用基因转移载体(Gene Transfer Vectors forMammalian Cells)》(J.M.Miller和M.P.Calos编,1987);《当代分子生物学方法(CurrentProtocols in Molecular Biology)》(F.M.Ausubel等人编,1987);《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编,1994);以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编,1991),所述文献中的每个文献均通过引用明确并入本文中。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
第一方面,本发明提供了一种抗SIGLEC-6的纳米抗体,所述纳米抗体包括包含CDR1、CDR2和CDR3的重链可变区:
所述CDR1如SEQ ID NO.1所示,CDR2如SEQ ID NO.2所示,CDR3如SEQ ID NO.3所示;或者,所述CDR1如SEQ ID NO.5所示,CDR2如SEQ ID NO.6所示,CDR3如SEQ ID NO.7所示。
进一步地,所述纳米抗体还包括骨架区;
优选地,所述纳米抗体为单价纳米抗体、多价纳米抗体、多特异性抗体和融合型纳米抗体中的至少一种。
名词解释:
单价纳米抗体:是用特异性的抗原从纳米抗体库中筛选得到的抗原特异性的纳米抗体,因其表面有大量的亲水残基,能保持严格的单体结构,且仅以这种单体形式就能高特异性、高亲和力地与其抗原相结合。
多价纳米抗体:多价抗体是识别同一种表位的单价抗体的聚合物,比对应的单价纳米抗体具有更高的抗原亲和力。多特异性抗体是识别不同表位的单价抗体的聚合物,能结合不同靶目标或同一靶目标的不同表位,比单价抗体具有更高的抗原识别能力。而纳米抗体结构简单,只有一个结构域,可通过短小的连接序列聚合在一起,从而转换成多价和多特异性的形式。
融合型纳米抗体:纳米抗体具有严格的单体特点且其相对分子质量很小,很容易通过基因工程技术与其他结构(如BSA、IgG-Fc等)结合形成新的融合分子,如能延长其半衰期的酶、抗菌肽或显影物质等。在新的融合分子中,纳米抗体与其靶抗原定向结合,与纳米抗体融合的部分就能发挥相应的功能。在临床,医生希望药物能在体内停留足够长的时间,然而纳米抗体的血液清除速度却很快,不利于它所携带的药物发挥作用。所以,通过基因技术将纳米抗体VHH和寿命较长的分子融合在一起,可以提高纳米抗体在血液中的存在时间即延长其半衰期,从而达到更好的治疗效果。
更优选地,所述纳米抗体为单价纳米抗体时,所述纳米抗体的氨基酸序列如SEQID NO.4或SEQ ID NO.8所示。
第二方面,本发明还提供了一种抗体,其包括前述的纳米抗体,或包括前述的纳米抗体的重链可变区(VHH)。
抗体可以为全长抗体、重链抗体、嵌合抗体、多特异性抗体(例如双特异性抗体、三特异性抗体、四特异性抗体等)、鼠源抗体、人源化抗体或抗原结合片段中的任意一种。抗原结合片段选自抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种,只要它们展示出所期望的抗原结合活性均可行。
本发明所述的“嵌合抗体”是将非人源性抗体的可变区与人抗体的恒定区或骨架区融合而成的抗体,可以减轻非人源性抗体诱发的免疫应答反应。
上述抗原结合片段也即抗体的功能性片段,通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本发明记载的内容容易理解到,上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本发明公开了完整抗体的结构基础上,本领域技术人员容易获得上述的功能性片段。
上述抗原结合片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪,比如Applied BioSystems等销售的自动肽合成仪合成获得。
第三方面,本发明还提供了一种基因片段或包含所述基因片段的重组载体,所述基因片段的核苷酸序列如SEQ ID NO.9或SEQ ID NO.10所示;所述基因片段编码前述的纳米抗体或所述基因片段编码前述的抗体。
考虑到密码子的简并性,可在其编码区,在不改变氨基酸序列的条件下,编码上述抗体的基因序列可以进行修改,获得编码相同抗体的基因;也可以根据表达抗体宿主的密码子偏爱性,人工合成改造基因,以提高抗体的表达效率。
重组载体为表达载体或克隆载体,优选为表达载体,可以指任何重组的多核苷酸构建体,该构建体可通过转化,转染或转导的方式将目的DNA片段直接或间接(如包装成病毒)导入宿主细胞内,进行目的基因表达。
其中一种类型的载体是质粒,即环状双链DNA分子,可将目的DNA片段连接至质粒环中。另一种类型的载体为病毒载体,其可将目的DNA片段连接包装至病毒基因组中(如腺病毒,腺相关病毒,逆转录病毒,慢病毒,溶瘤病毒)。这些载体进入宿主细胞后,可以进行目的基因的表达。
第四方面,本发明还提供了一种宿主细胞,其包含前述的重组载体。
优选地,所述宿主细胞选自原核宿主细胞、真核宿主细胞和噬菌体中的至少一种;
更优选地,所述原核宿主细胞为大肠杆菌、链霉菌、枯草杆菌或分枝杆菌;
和/或,所述真核宿主细胞为动物细胞、植物细胞或真菌;
进一步优选地,所述动物细胞选自哺乳动物细胞、昆虫细胞或秀丽隐杆线虫;
和/或,所述真菌选自酿酒酵母、毕赤酵母、汉森酵母、假丝酵母、乳克鲁维酵母、构巢曲霉、粟酒裂殖酵母和解脂耶罗酵母中的任一种。假丝酵母例如选自白色假丝酵母或光滑假丝酵母。
更进一步优选地,所述哺乳动物细胞选自293细胞、293T细胞、293FT细胞、CHO细胞、COS细胞、小鼠L细胞、LNCaP细胞、633细胞、Vero、BHK细胞、CV1细胞、Hela细胞、MDCK细胞、Hep-2细胞和Per6细胞中的任一种。其中的293系列细胞、Per6细胞和CHO细胞是用于生产制备抗体或重组蛋白的常用哺乳动物细胞,为本领域普通技术人员所熟知。
第五方面,本发明还提供了前述的纳米抗体,或前述的抗体的制备方法,所述制备方法为培养前述的宿主细胞,以获得抗体。具体地,本发明对宿主细胞的培养条件没有具体地限定,基于常规技术知识可获得能够使得宿主细胞表达产生所述抗体的培养条件。
第六方面,本发明还提供了一种免疫缀合物或药物组合物,其包括前述的抗SIGLEC-6的纳米抗体或前述的抗体;
优选地,所述免疫缀合物还包括治疗剂;
更优选地,所述治疗剂包括免疫检查点相关制剂、毒素、因子、化疗药物、放射性核素、激酶抑制剂和细胞毒性剂中的至少一种。
免疫检查点相关制剂包括不限于:抑制性第二信号分子的抗体、PD-L1抑制剂、PD-1/PD-L1单抗药物。抑制性第二信号分子可以是PD-1;CTLA-4;PD-1和CTLA-4。
免疫检查点抑制剂治疗的相关生物标志物包括PD-L1、MSI/bMSI、TMB/bTMB、TNB及EGFR突变、ALK融合、TP53突变、KRAS突变。
在本发明应用较佳的实施方式中,PD-1/PD-L1单抗药物选自如下组中的至少一种:纳武单抗(Nivolumab)、派姆单抗(Pembrolizumab)、皮地利珠单抗(Pidilizumab)、BMS-936559、阿特珠单抗(Atezolizumab)、AMP-224、AMP224、AUNP12、BGB108、MCLA134、MEDI0680、PDROOl、REGN2810、SHR1210、STIAllOX、STIAlllO、TSR042,BMS-936558、BGB-A317、BCD-100和JS001。
在一种可选的实施方式中,上述化疗药物选自紫杉烷类、长春花生物碱类,蒽环霉素类,表鬼臼毒素类,酪氨酸激酶抑制剂,夫拉平度、伊利替康及其代谢物SN-38、拓扑替康、替尼泊苷、依托泊苷、伊马替尼、吉非替尼、达努塞替、多柔吡星、柔红霉素、米托蒽醌、甲氨蝶呤、喜树碱和沙奎那韦中的任意一种或多种。
本发明所述的“药物组合物”表示组合在一起以实现某种特定目的的至少一种药物以及任选地可药用载体或辅料的组合。在某些实施方案中,所述药物组合物包括在时间和/或空间上分开的组合,只要其能够共同作用以实现本发明的目的。一些药物组合物是通过联合施用一些可药用成分或化合物,达到增强本发明的生物功效或减小药物副作用(例如,可以和其他抗肿瘤药物联合使用,增强抗肿瘤效果)。另一些药物组合物的目的是促进对生物体的给药,利于活性成分的吸收,增强稳定性或靶向性,延长半衰期,进而更好的发挥本发明的生物功效。
优选地,所述药物组合物包括药用赋形剂、载体和稀释剂中的至少一种;
本发明的优选技术方案中,上述载体为药学上可接受的载体,药学上可接受的载体包括但不限于聚乙烯吡咯烷酮及其衍生物、聚乙烯醇及其衍生物、甲基纤维素及其衍生物、乙基纤维素及其衍生物、羟丙基纤维素及其衍生物、淀粉及其衍生物、聚乙二醇及其衍生物、乳糖、蔗糖、甘露醇、海藻糖、山梨糖醇、糊精、微晶纤维素、丙烯酸树脂、磷酸氢钙、硬脂酸钙、硬脂酰富马酸钠、二氧化硅、二氧化钛、滑石粉、色靛中的一种或其组合。
赋形剂包括至少一种极性有机溶剂和至少一种增稠剂。
稀释剂例如选自药学上可接受的水或盐。
第七方面,本发明还提供了一种嵌合抗原受体,所述嵌合抗原受体的抗原结合结构域包括前述的纳米抗体或前述的抗体;
优选地,所述嵌合抗原受体还包括信号肽、铰链区、跨膜区和信号转导结构域;
更优选地,所述信号转导结构域选自CD3ζ、4-1BB(CD137)、CD27、CD30、CD40、CD54、CD83、PD-1、ICOS(CD278)、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、CD270、CD273、CD274、DAP10、SIGLEC-63、CD134、CD150和CD152中的至少一种;
和/或,所述跨膜区为CD28跨膜结构域、CD8跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种;
和/或,所述铰链区为IgG1FC-CH2-CH3铰链区、IgG2FC-CH2-CH3铰链区、IgG4FC-CH2-CH3铰链区、CD28铰链区和CD8α铰链区中的一种或多种;
和/或,所述信号肽为CD8α信号肽、IL-2信号肽或GM-CSF信号肽;
更优选地,所述信号转导结构域包括CD3ζ和4-1BB胞内区。
第八方面,本发明还提供了一种靶向SIGLEC-6的CAR-T细胞,其包括如前述的嵌合抗原受体;
优选地,所述CAR-T细胞选自同种异体通用型CAR-T细胞和自体CAR-T细胞中的至少一种;
更优选地,所述通用型CAR-T细胞为敲除了B2M和TRAC基因的CAR-T细胞。
第九方面,本发明还提供了前述的纳米抗体、前述的抗体、前述的基因片段或包含所述基因片段的重组载体、前述的宿主细胞、前述的嵌合抗原受体或前述的CAR-T细胞在制备预防或治疗肿瘤的产品中的用途,或在制备检测SIGLEC-6的产品中的用途,或在制备联合治疗药物中的用途;
优选地,所述预防或治疗肿瘤的产品为免疫细胞、试剂、试剂盒、药物和药物组合物中的至少一种;
和/或,所述检测SIGLEC-6的产品为试剂、试剂盒或基因芯片;
更优选地,所述治疗肿瘤的产品为靶向SIGLEC-6以治疗或辅助治疗肿瘤的药物;
进一步优选地,所述肿瘤为原发性肿瘤或继发性肿瘤;
更进一步优选地,所述肿瘤为SIGLEC-6阳性的白血病或淋巴瘤;更优选地为AML、CLL、MALT淋巴瘤或克隆性肥大细胞疾病。
本发明所述的“治疗”包括治愈、改善、降低患者的病情或病理特征,或抑制病情的恶化。
上述试剂或试剂盒中抗体或其功能性片段标记有可被检测的标记物。
可被检测的标记物是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质,通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中,可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在实际的使用过程中,本领域技术人员可以根据检测条件或实际需要选择合适的标记物,无论使用何种标记物,其均属于本发明的保护范围。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1、抗SIGLEC-6蛋白纳米抗体的筛选与制备
1、SIGLEC-6重组蛋白表达及纯化
将含有SIGLEC-6胞外段基因(该基因的基因号为NP_001236.4,第一位到1041位碱基,该基因对应UniProtKB编号O43699-1的第一位氨基酸到第347位氨基酸)的重组质粒pVax-SIGLEC-6(ECD)-mFc转染至HEK293T细胞中,表达5天后收集培养上清,利用NTA-Ni柱通过亲和层析的方式纯化获得高纯度的重组蛋白SIGLEC-6-mFc。
2、动物免疫
将1ml的SIGLEC-6-mFc重组蛋白(1mg/ml)与等体积弗氏佐剂混合后对双峰驼通过颈部及背部皮下注射,连续免疫4次,每次间隔2周,最后一次免疫后第7天采集外周血。
3、VHH噬菌体抗体文库的构建及淘选
(1)外周血淋巴细胞分离
从颈部静脉无菌采集外周抗凝血,先用等体积RPMI-1640培养基稀释。再用Ficoll-Paque Plus淋巴细胞分离液分离获得外周血淋巴细胞,得到的淋巴细胞可直接提取总mRNA或冻存于-80℃备用。
(2)VHH基因扩增
提取淋巴细胞总mRNA,反转录获得cDNA,利用巢式PCR扩增VHH基因,第一轮PCR所用引物为(CALL001;CALL002),利用琼脂糖凝胶电泳分离并回收约700bp条带;然后以回收的700bp产物为模板进行第二轮PCR扩增(VHH-FOR;VHH-REV),利用琼脂糖凝胶电泳分离并回收400bp条带。
(3)VHH噬菌体展示载体构建
将回收的400bp PCR产物和噬菌体展示载体pMECS均用Pst I和Not I双酶切,然后利用T4 DNA连接酶连接。
(4)电转
将连接产物电转至大肠杆菌TG1感受态细胞中并涂布于LB/Amp-Glu平板,37℃培养过夜后,收集菌苔,加入甘油进行保存,即为制备的噬菌体文库。
(5)噬菌体文库多样性和库容的测定
将电转化产物进行10倍稀释,然后涂布于LB/Amp-Glu平板,37℃培养12h后,计算转化子数量,最终得到库容量为3.94×109的噬菌体文库。
(6)抗SIGLEC-6蛋白特异性纳米抗体的筛选
利用噬菌体展示技术经过3轮筛选后随机挑选96个克隆扩大培养,利用单克隆ELISA鉴定能够特异性结合SIGLEC-6蛋白的纳米抗体,结果表明96个克隆中共有83个为阳性克隆;对阳性克隆测序结果比对分析,共获得5株抗SIGLEC-6的特异性纳米抗体。
(7)抗SIGLEC-6蛋白纳米抗体的特异性分析
以(6)中筛选获得的5株纳米抗体的重组上清为一抗分别与SIGLEC-6重组蛋白及其他无关抗原(BCMA-mFc、CD123-mFc、CD5-His、CD7-His、和CD276-His)共孵育,利用HRPanti-M13抗体检测,结果如图1所示,获得的2株纳米抗体(#23,#63)与SIGLEC-6蛋白能够结合,且不与其他无关抗原反应,表明上述2株纳米抗体具有良好的特异结合活性。2株纳米抗体的氨基酸序列信息如表1所示。2株纳米抗体基因的核苷酸序列信息如表2所示。
表1.纳米抗体氨基酸序列信息
表2.纳米抗体基因核苷酸序列信息
表3.VHH基因扩增所需引物序列
SEQ ID NO.13所示的核苷酸序列中R表示为A或G。
实施例2、抗SIGLEC-6蛋白特异性纳米抗体的制备
以实施例1步骤3(6)中鉴定的阳性克隆为模板扩增VHH基因并通过同源重组的方式构建至真核表达载体pcDNA3.1-MCS-hFc中,构建成功后将其转染至HEK293T细胞中,表达5天后收集上清,利用NTA-Ni柱通过亲和层析的方式纯化获得实施例1中的重组纳米抗体。
实施例3、抗SIGLEC-6蛋白纳米抗体与抗原结合检测
将实施例2中制备的2株特异性纳米抗体与包被于酶标板中的SIGLEC-6重组蛋白(200ng/孔)共孵育,利用HRP anti-human抗体进行检测,结果如图2所示,上述2株纳米抗体与SIGLEC-6蛋白均具有良好的结合活性。
实施例4、纳米抗体与SIGLEC-6蛋白的亲和力检测
将上述纳米抗体(#23,#63)利用HEK293T细胞进行表达、纯化,然后与包被于CM5芯片上的抗原SIGLEC-6-mFc利用Biacore 8k仪器检测结合亲和力。结果如表4所示,两株纳米抗体与SIGLEC-6蛋白的亲和力介于10-10~10-11M。
表4.抗SIGLEC-6纳米抗体与SIGLEC-6蛋白结合亲和力和动力学分析
实施例5、通用CAR-T细胞的制备
利用CRISPR基因编辑技术敲除健康供者T细胞中TRAC和B2M基因,获得TRAC-/B2M-T细胞;将含有本发明SIGLEC-6纳米抗体基因(SIGLEC-6纳米抗体基因的核苷酸序列如SEQID NO.9或SEQ ID NO.10所示)的CAR慢病毒感染上述TRAC-/B2M- T细胞制备获得通用CAR-T细胞。体内外杀伤实验结果表明,本发明制备的靶向SIGLEC-6同种异体通用CAR-T细胞均具有良好的抗肿瘤活性。可应用于制备预防、诊断和治疗SIGLEC-6阳性的白血病和淋巴瘤,特别是AML、CLL、MALT淋巴瘤和克隆性肥大细胞疾病中的至少一种。
具体地,UCAR-T细胞制备
(1)B2M和TRAC基因的敲除
采集健康供者外周血并分离淋巴细胞,利用CD3/CD28磁珠刺激活化增殖获得高纯度的T细胞,再将靶向B2M和TRAC的sgRNA(表5)及Cas9蛋白复合物电转至T细胞中,电转72h后流式细胞术检测结果显示B2M和TRAC基因的敲除效率分别为66.10%和67.46%(图3),并利用含有抗β2m和HLA抗体的磁珠通过反向纯化的方式获得高纯度的B2M-/TRAC-T细胞。
表5.敲除B2M和TRAC基因的sgRNA序列
(2)CAR慢病毒载体构建
以实施例1步骤3(6)中鉴定的阳性克隆为模板,扩增靶向SIGLEC-6的两条纳米抗体基因(#23和#63),并利用同源重组方式克隆至CAR慢病毒表达质粒载体(pSLCAR-BBz)中。该慢病毒质粒载体的CAR表达区主要包含以下元件:CD8α信号肽、抗原结合域、CD8α铰链区、CD28跨膜域、4-1BB胞内信号转导结构域和CD3ζ信号转导结构域(如图4所示)。
(3)慢病毒感染制备UCAR-T细胞
将本发明中制备的CAR慢病毒质粒与包装质粒psPAX2和pMD2.G共转染HEK293T细胞收集细胞上清超速离心浓缩后获得CAR慢病毒,再将其感染B2M-/TRAC-T细胞制备成UCAR-T细胞。本发明中不同抗体的靶向SIGLEC-6-UCART细胞中CAR的阳性率分别为85.3%(纳米抗体#23)和83.25%(纳米抗体#63),表明UCAR-T细胞制备成功。
实施例6、SIGLEC-6-UCART细胞体外抗肿瘤实验
通过体外共培养实验检测实施例5制备的SIGLEC-6-UCART细胞抗肿瘤效果;将购自ATCC的U937细胞(SIGLEC-6表达阳性)制备成稳定表达luciferase的细胞系U937-luciferase,并将其与UCAR-T细胞按照不同效靶比(4:1、2:1、1:1和1:2)共培养24h后,加入荧光素钾盐避光反应5min,置于酶标仪中检测荧光强度并计算杀伤效率。结果如图5所示,抗SIGLEC-6的同种异体通用CAR-T细胞UCAR-23和UCAR-63细胞对U937细胞均具有显著的杀伤活性,而阴性对照组没有明显的杀伤作用。UCAR-23、UCAR-63均为本发明制备的UCAR-T细胞,靶向CD19的scFv制备的通用CAR-T设置为阴性对照UCAR-CTRL。
实施例7、体内检测通用CAR-T细胞在B-ALL小鼠移植模型中的抗肿瘤活性
将购自ATCC的THP-1和U937细胞制备成稳定表达luciferase的细胞株,并分别按照每只2×106个细胞通过尾静脉接种至6~8周龄的NCG小鼠中,接种10天后在活体成像仪中检测肿瘤生长情况并将其随机分为3组,每组5只。分别于接种肿瘤细胞后第10天,将实施例5制备的靶向SIGLEC-6的UCAR-T细胞及其对照UCAR-T细胞(UCAR-CTRL)通过尾静脉接种。观察统计实验组和对照组每只小鼠的生存状态,观察统计时间长度为60天。利用Kaplan-Meier法绘制小鼠生存曲线并通过log-rank(Mantel-Cox)检验统计比较各组小鼠生存期的差异性。
结果如图6所示,靶向SIGLEC-6的UCAR-T(UCAR-Nb23和UCAR-Nb63)细胞处理组生存期显著长于阴性对照组(UCAR-CTRL),表明本发明中的靶向SIGLEC-6的UCAR-T细胞均有效的控制了AML细胞的增殖与生长并显著延长了小鼠生存期。
本发明以人SIGLEC-6为靶点制备了抗SIGLEC-6纳米抗体,其可特异性结合SIGLEC-6抗原,亲和力在10-10~10-11M之间,属于高亲和力纳米抗体。由纳米抗体制备了同种异体通用CAR-T细胞,该CAR-T细胞具有良好的抗肿瘤活性,可应用于制备预防、诊断和治疗SIGLEC-6阳性的白血病和淋巴瘤,特别是AML、CLL、MALT淋巴瘤和克隆性肥大细胞疾病等,具有良好的应用前景。
Claims (10)
1.一种抗SIGLEC-6的纳米抗体,其特征在于:所述纳米抗体包括包含CDR1、CDR2和CDR3的重链可变区:
所述CDR1如SEQ ID NO.1所示,CDR2如SEQ ID NO.2所示,CDR3如SEQ ID NO.3所示;或者,所述CDR1如SEQ ID NO.5所示,CDR2如SEQ ID NO.6所示,CDR3如SEQ ID NO.7所示。
2.根据权利要求1所述的纳米抗体,其特征在于:所述纳米抗体还包括骨架区;
优选地,所述纳米抗体为单价纳米抗体、多价纳米抗体、多特异性抗体和融合型纳米抗体中的至少一种;
更优选地,所述纳米抗体为单价纳米抗体时,所述纳米抗体的氨基酸序列如SEQ IDNO.4或SEQ ID NO.8所示。
3.一种抗体,其特征在于:其包括权利要求1或2所述的纳米抗体,或包括权利要求1或2所述的纳米抗体的重链可变区。
4.一种基因片段或包含所述基因片段的重组载体,其特征在于:所述基因片段的核苷酸序列如SEQ ID NO.9或SEQ ID NO.10所示;所述基因片段编码权利要求1或2所述的纳米抗体或所述基因片段编码权利要求3所述的抗体;
优选地,所述重组载体为质粒或病毒;
更优选地,所述病毒为腺病毒、腺相关病毒、逆转录病毒、慢病毒或溶瘤病毒。
5.一种宿主细胞,其特征在于:其包含权利要求4所述的重组载体;
优选地,所述宿主细胞选自原核宿主细胞、真核宿主细胞和噬菌体中的至少一种;
更优选地,所述原核宿主细胞为大肠杆菌、链霉菌、枯草杆菌或分枝杆菌;
和/或,所述真核宿主细胞为动物细胞、植物细胞或真菌;
进一步优选地,所述动物细胞选自哺乳动物细胞、昆虫细胞或秀丽隐杆线虫;
和/或,所述真菌选自酿酒酵母、毕赤酵母、汉森酵母、假丝酵母、乳克鲁维酵母、构巢曲霉、粟酒裂殖酵母和解脂耶罗酵母中的任一种;
更进一步优选地,所述哺乳动物细胞选自293细胞、293T细胞、293FT细胞、CHO细胞、COS细胞、小鼠L细胞、LNCaP细胞、633细胞、Vero、BHK细胞、CV1细胞、Hela细胞、MDCK细胞、Hep-2细胞和Per6细胞中的任一种。
6.权利要求1或2所述的纳米抗体,或权利要求3所述的抗体的制备方法,其特征在于:所述制备方法为培养权利要求5所述的宿主细胞,以获得抗体。
7.一种免疫缀合物或药物组合物,其特征在于:其包括权利要求1或2所述的抗SIGLEC-6的纳米抗体或权利要求3所述的抗体;
优选地,所述免疫缀合物还包括治疗剂;
和/或,所述药物组合物包括药用赋形剂、载体和稀释剂中的至少一种;
更优选地,所述治疗剂包括免疫检查点相关制剂、毒素、因子、化疗药物、放射性核素、激酶抑制剂和细胞毒性剂中的至少一种。
8.一种嵌合抗原受体,其特征在于:所述嵌合抗原受体的抗原结合结构域包括权利要求1或2所述的纳米抗体或权利要求3所述的抗体;
优选地,所述嵌合抗原受体还包括信号肽、铰链区、跨膜区和信号转导结构域;
更优选地,所述信号转导结构域选自CD3ζ、4-1BB、CD27、CD30、CD40、CD54、CD83、PD-1、ICOS、淋巴细胞功能相关抗原-1、CD2、CD7、CD270、CD273、CD274、DAP10、SIGLEC-63、CD134、CD150和CD152中的至少一种;
和/或,所述跨膜区为CD28跨膜结构域、CD8跨膜区、CD134跨膜区、CD137跨膜区、ICOS跨膜区和DAP10跨膜区中的一种或多种;
和/或,所述铰链区为IgG1FC-CH2-CH3铰链区、IgG2FC-CH2-CH3铰链区、IgG4FC-CH2-CH3铰链区、CD28铰链区和CD8α铰链区中的一种或多种;
和/或,所述信号肽为CD8α信号肽、IL-2信号肽或GM-CSF信号肽;
更优选地,所述信号转导结构域包括CD3ζ和4-1BB胞内区。
9.一种靶向SIGLEC-6的CAR-T细胞,其特征在于:其包括如权利要求8所述的嵌合抗原受体;
优选地,所述CAR-T细胞选自同种异体通用型CAR-T细胞和自体CAR-T细胞中的至少一种;
更优选地,所述通用型CAR-T细胞为敲除了B2M和TRAC基因的CAR-T细胞。
10.权利要求1或2所述的纳米抗体、权利要求3所述的抗体、权利要求4所述的基因片段或包含所述基因片段的重组载体、权利要求5所述的宿主细胞、权利要求8所述的嵌合抗原受体或权利要求9所述的CAR-T细胞在制备预防或治疗肿瘤的产品中的用途,或在制备检测SIGLEC-6的产品中的用途,或在制备联合治疗药物中的用途;
优选地,所述预防或治疗肿瘤的产品为免疫细胞、试剂、试剂盒、药物和药物组合物中的至少一种;
和/或,所述检测SIGLEC-6的产品为试剂、试剂盒或基因芯片;
更优选地,所述治疗肿瘤的产品为靶向SIGLEC-6以治疗或辅助治疗肿瘤的药物;
进一步优选地,所述肿瘤为原发性肿瘤或继发性肿瘤;
更进一步优选地,所述肿瘤为SIGLEC-6阳性的白血病或淋巴瘤;更优选地为AML、CLL、MALT淋巴瘤或克隆性肥大细胞疾病。
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