CN117069847A - 靶向bcma抗体及其相关产品和医药用途 - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001116—Receptors for cytokines
- A61K39/001117—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR] or CD30
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明属于生物技术领域、生物医药领域,具体涉及靶向BCMA抗体及其相关产品和医药用途。具体地,所述抗体包含可变轻链和可变重链,所述可变轻链中包含轻链CDR1、轻链CDR2、轻链CDR3,所述轻链CDR1序列如SEQNO.1所示,所述轻链CDR2的氨基酸序列如SEQ ID NO.2所示,所述轻链CDR3如SEQ ID NO.3或SEQ ID NO.4所示。通过本发明所提供的人源化抗体制备CAR‑NK细胞,所获得的CAR‑NK细胞的阳性率稳定性和杀伤能力高。
Description
技术领域
本发明属于生物技术领域、生物医药领域,具体涉及靶向BCMA抗体及其相关产品和医药用途。
背景技术
BCMA(B Cell Maturation Antigen,即CD269)全称为B细胞成熟抗原,是肿瘤坏死因子受体(TNF)超家族的成员,可结合B细胞激活因子和增殖诱导配体,优先在成熟B淋巴细胞中表达,多发性骨髓瘤(Multiple Myeloma,MM)癌症细胞上BCMA的表达水平显著高于健康的浆细胞。越来越多的证据表明,BCMA是用于治疗多发性骨髓瘤的合适的靶抗原之一。
由于伦理的限制,抗体的筛选不能用人体来做筛选。因此,目前抗体的筛选都是基于试验动物获得,比如:小鼠,兔子,羊驼等等。小鼠的筛选成本最低,因此应用最为广泛,所以获得的抗体大多数都是鼠源的。近年来单抗类药物,以及后续CAR-T、CAR-NK细胞免疫治疗手段的涌现,以试验动物为基础的抗体筛选应用范围进一步扩大。随着单抗、CAR-T、CAR-NK细胞产品的广泛应用,研究人员发现直接应用鼠源等动物来源的抗体及嵌合抗原受体(CAR),进入人体后,被人体的免疫***视作异种、异源蛋白,会产生明显的抗原抗体反应,人体内会出现大量的抗体,进而影响到鼠源抗体在人体内的治疗效果。解决这个问题的一个有效途径就是利用生物信息学的数据及知识,对筛选出来的鼠源抗体的核酸序列进行密码子的优化,使其更符合在人体内的表达习惯。同时再利用生物信息学的算法,在不改变CDR区域的前提下,对抗体的框架区进行人源优化,最终使鼠源抗体的序列尽可能转变成接近人体内天然的抗体序列。进行上述优化以后的抗体就称为人源化抗体,在人体内不会出现明显的抗原抗体反应,保证了抗体药物、CAR-T、CAR-NK最大的治疗效果。
发明内容
本发明首先通过筛选试验获得了鼠源的靶向BCMA抗原的鼠源抗体序列,对鼠源抗体序列进行优化后获得了数个人源化的靶向BCMA抗原的scFv抗体序列。再构建包含该抗体的嵌合抗原受体,并将该嵌合抗原受体转导进入人的NK细胞,成功制备了CAR-NK细胞,该CAR-NK细胞在体内外实验中都取得了很好的治疗效果。通过人源化抗体所获得的CAR-NK细胞的阳性率稳定性和杀伤能力均高于鼠源抗体。
具体地,本发明提供了以下技术方案:
第一方面,本发明提供了一种靶向BCMA的抗体或其抗原结合片段,所述抗体包含可变轻链和可变重链,
所述可变轻链中包含轻链CDR1、轻链CDR2、轻链CDR3,所述轻链CDR1序列如SEQNO.1所示,所述轻链CDR2的氨基酸序列如SEQ ID NO.2所示,所述轻链CDR3如SEQ ID NO.3或SEQ ID NO.4所示;
所述可变重链中包含重链CDR1、重链CDR2、重链CDR3;所述重链CDR1序列如SEQNO.5所示,所述重链CDR2的氨基酸序列如SEQ ID NO.6所示,所述重链CDR3如SEQ ID NO.7或SEQ ID NO.8所示。
优选地,所述抗体还包括轻链FR1、轻链FR2、轻链FR3、轻链FR4、重链FR1、重链FR2、重链FR3和重链FR4。
优选地,所述轻链FR1可以如SEQ ID NO.9-10任一所示。优选地,所述轻链FR2可以如SEQ ID NO.11-12任一所示。优选地,所述轻链FR3可以如SEQ ID NO.13-14任一所示。优选地,所述轻链FR4可以如SEQ ID NO.15-16任一所示。优选地,所述重链FR1可以如SEQ IDNO.17-18任一所示。优选地,所述重链FR2可以如SEQ ID NO.19-21任一所示。优选地,所述重链FR3可以如SEQ ID NO.22-24任一所示。优选地,所述重链FR4可以如SEQ ID NO.25-26任一所示。
优选地,所述抗体的链接方式是可变轻链-连接子-可变重链。
更优选地,所述抗体是人源化的。具体地,所述人源化抗体的表达更稳定,对靶细胞的杀伤性更强。
更优选地,所述抗体的完整序列如SEQ ID NO.28-35所示。
如本文所用,术语“抗原结合片段”是指由抗体的一部分形成的抗体片段,其包含一或多个CDR或与抗原结合但不包含完整天然抗体结构的任何其它抗体片段。抗原结合片段的实例包括但不限于双功能抗体、Fab、Fab'、F(ab')2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双功能抗体(ds双功能抗体)、单链抗体分子(scFv)、scFv二聚体(二价双功能抗体)、双特异性抗体、多特异性抗体、骆驼化单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。抗原结合片段能够结合至与亲本抗体所结合相同的抗原。
更优选地,所述抗体进一步包含重链恒定区和轻链恒定区。
另一方面,本发明提供了一种抗体复合物,所述抗体复合物是在前述抗体或其抗原结合片段的氨基端和/或羧基端以肽键连接结构域所构成的。
优选地,所述结构域包括铰链区、跨膜结构域和/或信号转导结构域。
优选地,所述信号转导结构域包含共刺激结构域和/或胞内信号传导结构域。
优选地,所述铰链区包括CD8铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。
优选地,所述铰链区是CD8铰链区,其氨基酸序列如SEQ ID NO.45所示。
优选地,所述跨膜结构域包括蛋白质的跨膜结构域,所述蛋白质包括:2B4基因表达的蛋白、T细胞受体的α、β或ζ链,CD28,CD3ε,CD45,CD4,CD5,CD8,CD9,CD16,CD22,CD33,CD37,CD64,CD80,CD86,CD123,CD134,CD137和CD154。
优选地,所述跨膜结构域是CD8跨膜结构域,其氨基酸序列如SEQ ID NO.46所示。
优选地,所述共刺激结构域包括4-1BB、2B4、CD3ζ、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)的功能性信号传导结构域。
优选地,所述共刺激结构域是4-1BB共刺激结构域,其氨基酸序列如SEQ ID NO.47所示
优选地,所述胞内信号传导结构域包括CD3-ζ、FcεRIγ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的一种蛋白或多种蛋白的任意组合的信号传导区。
优选地,所述胞内信号传导结构域的氨基酸序列如SEQ ID NO.48所示。
更优选地,所述抗体复合物即为嵌合抗原受体(CAR)。
优选地,所述结构域还包括信号肽。
优选地,本发明选用CD8信号肽进行测试。
如本文所用,嵌合抗原受体(chimeric antigen receptor,CAR)是一种人工合成的受体,能引导免疫细胞特异性地追踪识别和清除表达相关靶配体的肿瘤细胞,通常由能够识别肿瘤相关抗原的胞外结合域(一般来自单抗的抗原结合区的scFv片段)、铰链域、跨膜域和胞内信号结构域,具有高度靶向性。
另一方面,本发明提供了一种编码前述抗体的核酸分子。
优选地,所述核酸分子如SEQ ID NO.37-44所示。
另一方面,本发明提供了一种编码前述抗体复合物的核酸分子。
另一方面,本发明提供了一种载体,所述载体包含前述编码抗体的核酸分子和/或前述多肽复合物DNA分子,或者,所述载体表达前述靶向BCMA的抗体或其抗原结合片段,或,抗体复合物。
优选地,所述载体是表达载体。
优选地,所述载体是病毒表达载体。
优选地,所述病毒表达载体包括慢病毒载体、腺病毒载体、腺相关病毒表达载体或其他类型病毒载体。
另一方面,本发明提供了一种宿主细胞,所述宿主细胞包含前述抗体、前述多肽复合物、前述编码抗体的DNA分子、前述多肽复合物的DNA分子和前述载体中的一种或多种。
优选地,所述宿主细胞包括原核细胞和真核细胞。
优选地,所述原核细胞是细菌细胞,如农杆菌、大肠杆菌。
优选地,所述细菌细胞包括革兰氏阴性微生物和革兰氏阳性微生物。
优选地,所述真核细胞是真菌细胞,即酵母细胞。
优选地,所述真核细胞是动物细胞、昆虫细胞、植物细胞。
优选地,所述动物细胞包括人源细胞或非人源的细胞。
优选地,所述人源细胞是免疫细胞。
优选地,所述免疫细胞包括T细胞、B细胞、K细胞和NK细胞中的一种或多种。
优选地,所述免疫细胞是NK细胞或T细胞。
优选地,所述免疫细胞是自体或异体的。
优选地,所述免疫细胞来源于外周血(PB)、脐带血(UCB)、人类胚胎干细胞(HESC)、诱导多能干细胞(IPSC)或商品化细胞系。
另一方面,本发明提供了一种抗体偶联物,所述抗体偶联物包含前述抗体,还包含选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子或酶。
另一方面,本发明提供了一种治疗BCMA相关疾病的药物组合物,所述药物组合物中含有前述抗体或其抗原结合片段、前述抗体复合物、编码前述抗体的核酸分子、编码前述抗体复合物的核酸分子、载体、宿主细胞、抗体偶联物中的一种或多种;和/或药学上可接受的载体。
优选地,所述药物组合物中还可以包含其他活性成分,如舒尼替尼(Sunitinib,Sutent)、易瑞沙(Iressa、吉非替尼片)、它赛瓦(Tarceva,特罗凯)、赫赛汀(Herceptin,Trastuzumab)、阿瓦斯汀(avastin,Bevacizumab)、三氧化二砷、反式维甲酸、万珂(Velcade,bortezomib)、替莫胺(Temodar)、爱必妥(Erbitux,cetuximab)、大沙替尼(dasatinib Sprycel)、索拉非尼(sorafenib,Nexavar)、巾白尼单抗(Vectibix,panitumumab)、替吉奧(TS-1)、Ixempra(ixabepilone)等。
优选地,所述药物组合物中还可以包含药学上可接受的辅料。
优选地,所述药学上可接受的辅料包括稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
优选地,所述药物联用组合物的剂型包括胶囊剂、片剂、丸剂、散剂、颗粒剂、乳液、溶液、悬浮液、糖浆或酊剂。
另一方面,本发明提供了一种检测BCMA的方法,所述方法包括将前述抗体或其抗原结合片段与待检测样品接触的步骤。
优选地,所述抗体或其抗原结合片段是具有可检测标记的抗体。
优选地,所述可检测标记包括酶、荧光材料、发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。
优选地,所述方法还包括检测待测样品与抗体结合后的反应的步骤。
优选地,所述待测样品包括生理体液,所述生理体液包括血液、血清、血浆、唾液、眼睛分泌物、脑脊液、脓、渗出液、乳汁、汗液、眼泪、耳流出物、痰、淋巴液、尿液、粪便;所述待测样品还包括组织,所述组织包括肺、脾和肾。
优选地,所述待测样品中含有B细胞。
优选地,所述检测是非诊断目的的。
另一方面,本发明提供了一种治疗癌症的方法,所述方法包括向受试者施用前述抗体或其抗原结合片段、前述多肽复合物、编码前述抗体的核酸分子、编码前述抗体复合物的核酸分子、前述载体、前述宿主细胞或前述药物组合物。
优选地,所述施用方式包括肠道给药或非肠道给药,如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔黏膜、眼、肺和呼吸道、皮肤、***、直肠等。
最优选地,所述癌症是多发性骨髓瘤(MM)。
优选地,所述治疗的方法还可以与其他治疗方式例如:化学疗法、放射疗法、免疫疗法、基因疗法、外科手术等。
另一方面,本发明提供了前述抗体或其抗原结合片段、前述多肽复合物、编码前述抗体的核酸分子、编码前述抗体复合物的核酸分子、前述载体、前述宿主细胞或前述药物组合物中任意一种或多种在结合BCMA中的应用。
另一方面,本发明提供了前述抗体或其抗原结合片段、前述多肽复合物、编码前述抗体的核酸分子、编码前述抗体复合物的核酸分子、前述载体、前述宿主细胞或前述药物组合物中任意一种或多种在制备治疗癌症或表达BCMA的免疫细胞介导的免疫病症的药物中的应用。
优选地,所述癌症包括浆细胞恶性肿瘤或B细胞恶性肿瘤。
优选地,所述癌症包括白血病、淋巴瘤或骨髓瘤。
优选地,所述癌症包括BCMA高表达相关的癌症。
最优选地,所述癌症包括多发性骨髓瘤(Multiple Myeloma,MM)、霍奇金淋巴瘤、白血病、胶质母细胞瘤、非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma,NHL)、骨髓发育异常综合症(MDS)、骨髓增生综合症(MPS)、MGUS(意义未明的单克隆丙种球蛋白病,monoclonalgammopathy of undetermined significance)、孤立性浆细胞瘤(SolitaryPlasmacytoma)、Waldenstrom巨球蛋白血病(Macroglobulinemia)、原发性***性淀粉样变性(AL Amyloidosis)、浆细胞白血病(Plasma Cell Leukemia)、伯基特氏淋巴瘤(Burkett's lymphoma)
最优选地,所述癌症是多发性骨髓瘤(MM)。
优选地,所述免疫细胞是B细胞。
优选地,所述免疫病症包括类风湿性关节炎、全身性红斑狼疮(SLE)、I型糖尿病、哮喘、异位性皮炎、过敏性鼻炎、血小板减少性紫癜、多发性硬化症、牛皮癣、修格兰氏综合症、桥本氏甲状腺炎、格雷夫氏病、原发性胆汁性硬化、韦格纳氏肉芽肿病、结核病以及移植物抗宿主疾病。
附图说明
图1是鼠源抗体对BCMA的亲和力测试结果图,其中,A是H929空白,B是H929+PE,C是阴性对照,D是阳性对照,E是鼠源抗体。
图2是对各种BCMA-CAR在NK细胞中的表达情况进行检测的结果图,其中,A是阴性对照,B是鼠源抗体,C是人源化抗体V0,D是人源化抗体V1,E是人源化抗体V2,F是人源化抗体V3,G是人源化抗体V4,H是人源化抗体V6,I是人源化抗体V7,J是人源化抗体V8。
图3是对比各种BCMA-CAR的杀伤能力的检测结果图,其中,A是阴性对照,B是鼠源抗体,C是人源化抗体V0,D是人源化抗体V1,E是人源化抗体V2,F是人源化抗体V3,G是人源化抗体V4,H是人源化抗体V6,I是人源化抗体V7,J是人源化抗体V8。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例一、鼠源抗体对骨髓瘤细胞亲和力的测定
获得鼠源抗体并检测抗体对骨髓瘤细胞的亲和力,所述鼠源抗体的氨基酸序列如SEQ ID NO.27所示,核苷酸序列如SEQ ID NO.36所示。
测定方法:
1、培养H929骨髓瘤细胞,进行细胞计数,然后取5×105个细胞置于1.5ml EP管中,1000rpm,5min离心,去上清;
2、加500μl PBS重悬细胞,然后1000rpm,5min离心,去上清;
3、将空白细胞培养上清与抗体生产上清分别重悬待测细胞,置于4℃,避光反应30min,然后1000rpm,5min离心,去上清;
4、加500μl PBS重悬细胞,然后1000rpm,5min离心,去上清;
5、加100μl二抗(PE anti-human IgG Fc Anti body,原液用PBS经过1:600稀释后使用)重悬细胞,置于4℃,避光反应30min,然后1000rpm,5min离心,去上清;
6、加500μl PBS重悬细胞,然后1000rpm,5min离心,去上清;
7、加400μl PBS重悬细胞,然后上机检测。
结果证明本发明提供的鼠源BCMA抗体对H929细胞有很好的亲和力,PE荧光标记的H929细胞阳性率如表1统计,检测结果如图1所示。
表1、PE阳性率统计
分组 | APC荧光阳性率 |
H929空白 | 0.05% |
H929+PE | 0.01% |
阴性对照 | 0.05% |
阳性对照 | 98.09% |
BCMA-scFv候选 | 73.05% |
实施例二、人源化抗体构成的CAR向细胞的转导率检测
获得人源化抗体
对鼠源抗体进行人源化,具体地,获得的各个人源化抗体的轻链CDR1序列如SEQNO.1所示,轻链CDR2的氨基酸序列如SEQ ID NO.2所示,轻链CDR3如SEQ ID NO.3或SEQ IDNO.4所示;各个人源化抗体的重链CDR1序列如SEQ NO.5所示,重链CDR2的氨基酸序列如SEQID NO.6所示,重链CDR3如SEQ ID NO.7或SEQ ID NO.8所示。
在以上CDR(Complementarity-Determining Region,亦称为HypervariableRegion,超变区域)不变的情况下,还测试了不同的Framework Region(骨架区域,FR),确认了在以上CDR不变的情况下,由不同FR组成的抗体均具有功能。具体地,
轻链FR1可以如SEQ ID NO.9-10任一所示,轻链FR2可以如SEQ ID NO.11-12任一所示,轻链FR3可以如SEQ ID NO.13-14任一所示,轻链FR4可以如SEQ ID NO.15-16任一所示,重链FR1可以如SEQ ID NO.17-18任一所示,重链FR2可以如SEQ ID NO.19-21任一所示,重链FR3可以如SEQ ID NO.22-24任一所示,重链FR4可以如SEQ ID NO.25-26任一所示。
进一步制备人源化抗体V0、V1、V2、V3、V4、V6、V7、V8进行测试,具体地,人源化抗体V0、V1、V2、V3、V4、V6、V7、V8氨基酸序列依次如SEQ ID NO.28-35所示,编码核酸的序列依次如SEQ ID NO.37-44所示。
构建嵌合抗原受体
在以上鼠源抗体和人源化抗体前连接CD8信号肽(SEQ ID NO.45所示),在鼠源抗体后依次连接CD8铰链区+跨膜域(SEQ ID NO.46所示)、4-1BB共刺激结构域(SEQ ID NO.47所示)、胞内信号传导结构域(SEQ ID NO.48所示)得到BCMA-CAR-V0/V1/V2/V3/V4/V6/V7/V8进行转导。
也即本发明通过以下结构验证CAR的功能:CD8信号肽-抗体-CD8铰链区-CD8跨膜域-4 1BB共刺激结构域-胞内信号传导结构域)。
制备CAR-NK细胞
使用三代慢病毒包装***共四质粒***,包装三质粒pRSV-Rev,pMDLg/pRRE,pMD2.G,穿梭pLVX-puro,HEK293T细胞作为病毒生产细胞,混合比例pLenti-puro:pRSV-Rev:pMDLg/pRRE:pMD2.G2:1:1:1,转染试剂使用脂质体mirusTransIT-293(mir2700),脂质体和DNA比例为3:1,转然后,三天收集上清,高速离心40000g6小时。
将病毒按照感染浓度为10MOI直接加入NK细胞的培养基,同时加入polybrene 0.8μg/ml,孵育6小时候,去除含有病毒NK培养基,加入新鲜的NK培养基,继续培养,在不同时间点通过流式检测确认CAR阳性率。
结果分析
表2、BCMA-CAR表达及稳定性流式检测结果
人源化BCMA-CAR表达及稳定性流式检测结果统计如表2所示。结果显示BCMA-CAR-V0/V1/V2/V3/V4/V6/V7/V8均可以很好的转导NK细胞,各组在Day2的转导率均能达到70-90%之间。
Day4开始BCMA-CAR的表达趋于稳定,各组间表达的稳定性差异显现出来。鼠源组BCMA-CAR表达下降明显,下降至30.5%,且分群不明显。人源化各组表达的稳定性也有不同,其中BCMA-CAR-V0/V1/V2/V6四组表达稳定性较好,可见细胞分群明显,CAR-NK细胞占比分别为:19.99%,14.2%,15.45%,17.01%。在histogram图中可以看出右侧形成了明显的BCMA-CAR-NK细胞群。
Day6-Day8鼠源组BCMA-CAR表达进一步下降,下降至11.29%,CAR-NK细胞分群不明显。人源化各组中BCMA-CAR-V0/V1/V2/V4/V6五组表达稳定性好,分群明显,CAR-NK细胞占比分别为:18.35%,12.83%,14.5%,14.3%,14.47%。
与鼠源的BCMA-CAR相比,人源化的BCMA-CAR在NK细胞膜上的表达更稳定,更均匀,能够形成较为明确一群表达CAR的NK细胞群(V0/V1/V2/V4/V6)。histogram图上,BCMA-CAR-NK细胞显示为右侧小的细胞峰,该峰越明显就意味着CAR的表达越均匀,越稳定。
实施例三、细胞杀伤性实验
将各组BCMA-CAR-NK细胞扩增,培养,计数。
培养带有绿色荧光的靶细胞系:H929-GFP细胞,计数。
按效靶比1:2,将BCMA-CAR-NK细胞与H929-GFP细胞共培养。
不同的时间点(24小时,48小时)流式检测H929-GFP细胞在共培养体系中的比例。
结果分析
表3、效靶比1:2的细胞杀伤检测结果
效靶比1:2的细胞杀伤检测结果统计如表3所示。在24小时,鼠源BCMA-CAR-NK组H929-GFP细胞占比45.16%,人源化BCMA-CAR-NK各组中,H929-GFP细胞占比较鼠源组更低,其中:BCMA-CAR-V0/V4/V7/V8四组中H929-GFP细胞占比分别为:30.5%,32.17%,30.68%,30.4%。
在48小时,鼠源BCMA-CAR-NK组H929-GFP细胞占比22.78%,人源化BCMA-CAR-NK各组中,H929-GFP细胞占比较鼠源组显著降低,其中:BCMA-CAR-V0/V4/V7/V8四组中H929-GFP细胞占比分别为:3.45%,4.3%,4.17%,4.66%。在histogram图上显示为右侧小的细胞峰越下,甚至消失,就意味着杀伤能力越强。
与鼠源的BCMA-CAR-NK细胞相比,人源化的BCMA-CAR-NK细胞具有更好的杀伤能力,在48小时,已经能够将肿瘤细胞基本清除干净,不同的人源化改造在杀伤能力上也不同,但总体来看,杀伤能力均优于鼠源的BCMA-CAR-NK细胞。
Claims (10)
1.一种靶向BCMA的抗体或其抗原结合片段,所述抗体包含可变轻链和可变重链,
所述可变轻链中包含轻链CDR1、轻链CDR2、轻链CDR3,所述轻链CDR1序列如SEQ NO.1所示,所述轻链CDR2的氨基酸序列如SEQ ID NO.2所示,所述轻链CDR3如SEQ ID NO.3或SEQID NO.4所示;
所述可变重链中包含重链CDR1、重链CDR2、重链CDR3;所述重链CDR1序列如SEQ NO.5所示,所述重链CDR2的氨基酸序列如SEQ ID NO.6所示,所述重链CDR3如SEQ ID NO.7或SEQID NO.8所示;
优选地,所述抗体还包括轻链FR1、轻链FR2、轻链FR3、轻链FR4、重链FR1、重链FR2、重链FR3和重链FR4;
优选地,所述轻链FR1可以如SEQ ID NO.9-10任一所示;
优选地,所述轻链FR2可以如SEQ ID NO.11-12任一所示;
优选地,所述轻链FR3可以如SEQ ID NO.13-14任一所示;
优选地,所述轻链FR4可以如SEQ ID NO.15-16任一所示;
优选地,所述重链FR1可以如SEQ ID NO.17-18任一所示;
优选地,所述重链FR2可以如SEQ ID NO.19-21任一所示;
优选地,所述重链FR3可以如SEQ ID NO.22-24任一所示;
优选地,所述重链FR4可以如SEQ ID NO.25-26任一所示;
优选地,所述抗体的链接方式是可变轻链-连接子-可变重链;
更优选地,所述抗体是人源化的;
更优选地,所述抗体的完整序列如SEQ ID NO.28-35中一种或多种所示;
更优选地,所述抗体进一步包含重链恒定区和轻链恒定区。
2.一种抗体复合物,所述抗体复合物是在权利要求1所述抗体或其抗原结合片段的氨基端和/或羧基端连接结构域所构成的;
优选地,所述结构域包括铰链区、跨膜结构域和/或信号转导结构域;
优选地,所述信号转导结构域包含共刺激结构域和/或胞内信号传导结构域;
优选地,所述铰链区包括CD8铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合;
优选地,所述铰链区是CD8铰链区,其氨基酸序列如SEQ ID NO.45所示;
优选地,所述跨膜结构域包括蛋白质的跨膜结构域,所述蛋白质包括:2B4基因表达的蛋白、T细胞受体的α、β或ζ链,CD28,CD3ε,CD45,
CD4,CD5,CD8,CD9,CD16,CD22,CD33,CD37,CD64,CD80,
CD86,CD123,CD134,CD137和CD154;
优选地,所述跨膜结构域是CD8跨膜结构域,其氨基酸序列如SEQ ID NO.46所示;
优选地,所述共刺激结构域包括4-1BB、2B4、CD3ζ、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1、ICOS和4-1BB的功能性信号传导结构域;
优选地,所述共刺激信号结构域有一个、两个、三个或更多个;
优选地,所述共刺激结构域是4-1BB共刺激结构域,其氨基酸序列如SEQ ID NO.47所示
优选地,所述胞内信号传导结构域包括CD3-ζ、FcεRIγ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的一种蛋白或多种蛋白的任意组合的信号传导区;
优选地,所述胞内信号传导结构域的氨基酸序列如SEQ ID NO.48所示;
优选地,所述结构域还包括信号肽;
优选地,所述信号肽是CD8信号肽。
3.一种编码权利要求1所述抗体或其抗原结合片段的核酸分子;
优选地,所述核酸分子如SEQ ID NO.37-44所示。
4.一种编码权利要求2所述抗体复合物的核酸分子。
5.一种载体,所述载体包含权利要求3所述核酸分子和/或权利要求4所述核酸分子,或者,所述载体表达权利要求1所述抗体或其抗原结合片段或权利要求2所述抗体复合物;
优选地,所述载体是表达载体;
优选地,所述载体是病毒表达载体;
优选地,所述病毒表达载体包括慢病毒载体、腺病毒载体、腺相关病毒表达载体或其他类型病毒载体。
6.一种宿主细胞,所述宿主细胞中包含权利要求1所述抗体或其抗原结合片段、权利要求2所述抗体复合物、权利要求3所述核酸分子、权利要求4所述核酸分子、权利要求5所述载体;
优选地,所述宿主细胞包括原核细胞和真核细胞;
优选地,所述真核细胞包括动物细胞、昆虫细胞、植物细胞;
优选地,所述动物细胞包括人源细胞或非人源的细胞;
优选地,所述人源细胞包括免疫细胞或体细胞;
优选地,所述免疫细胞包括T细胞、B细胞、NKT细胞和NK细胞中的一种或多种;
优选地,所述免疫细胞是NK细胞;
优选地,所述NK细胞来源于外周血、脐带血、人类胚胎干细胞、诱导多能干细胞或细胞系。
7.一种抗体偶联物,所述抗体偶联物包含权利要求1所述抗体或其抗原结合片段,所述抗体偶联物还包含选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子或酶。
8.一种治疗BCMA相关疾病的药物组合物,所述药物组合物中含有权利要求1所述抗体或其抗原结合片段、权利要求2所述抗体复合物、权利要求3所述核酸分子、权利要求4所述核酸分子、权利要求5所述载体、权利要求6所述宿主细胞;和/或药学上可接受的载体;
优选地,所述药物组合物中还可以包含药学上可接受的辅料;
优选地,所述药学上可接受的辅料包括稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合;
优选地,所述药物组合物中还包含以下任意一种或多种:舒尼替尼、易瑞沙、它赛瓦、赫赛汀、阿瓦斯汀、三氧化二砷、反式维甲酸、万珂、替莫胺、爱必妥、大沙替尼、索拉非尼、巾白尼单抗、替吉奧、Ixempra。
9.一种检测BCMA的方法,所述方法包括将权利要求1所述抗体或其抗原结合片段与待检测样品接触的步骤;
优选地,所述抗体或其抗原结合片段是具有可检测标记的抗体;
优选地,所述可检测标记包括酶、荧光材料、发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子;
优选地,所述方法还包括检测待测样品与抗体结合后的反应的步骤;
优选地,所述待测样品包括生理体液,所述生理体液包括血液、血清、血浆、唾液、眼睛分泌物、脑脊液、脓、渗出液、乳汁、汗液、眼泪、耳流出物、痰、淋巴液、尿液、粪便;所述待测样品还包括组织,所述组织包括肺、脾和肾;
优选地,所述待测样品中含有B细胞;
优选地,所述检测是非诊断目的的。
10.权利要求1所述抗体或其抗原结合片段、权利要求2所述抗体复合物、权利要求3所述核酸分子、权利要求4所述核酸分子、权利要求5所述载体、权利要求6所述宿主细胞或权利要求8所述药物组合物在制备治疗疾病的药物、制备BCMA检测产品、结合BCMA中的应用,所述疾病包括癌症或表达BCMA的免疫细胞介导的免疫病症;
优选地,所述癌症包括白血病、淋巴瘤或骨髓瘤;
优选地,所述癌症包括浆细胞恶性肿瘤或B细胞恶性肿瘤;
优选地,所述癌症包括BCMA高表达的癌症;
优选地,所述癌症是多发性骨髓瘤;
最优选地,所述癌症包括多发性骨髓瘤、霍奇金淋巴瘤、白血病、胶质母细胞瘤、非霍奇金氏淋巴瘤、骨髓发育异常综合症、骨髓增生综合症、MGUS、孤立性浆细胞瘤、Waldenstrom巨球蛋白血病、原发性***性淀粉样变性、浆细胞白血病、伯基特氏淋巴瘤;
优选地,所述免疫细胞是B细胞;
优选地,所述免疫病症包括类风湿性关节炎、全身性红斑狼疮、I型糖尿病、哮喘、异位性皮炎、过敏性鼻炎、血小板减少性紫癜、多发性硬化症、牛皮癣、修格兰氏综合症、桥本氏甲状腺炎、格雷夫氏病、原发性胆汁性硬化、韦格纳氏肉芽肿病、结核病以及移植物抗宿主疾病。
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