CN114213539B - 可结合cd4的纳米抗体4nb357及其应用 - Google Patents
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Abstract
本发明涉及一种可结合CD4的纳米抗体4NB357,包括3个互补决定区CDR1‑3,序列如SEQ ID NO:1‑3所示。本发明通过制备CD4蛋白、利用噬菌体库展示纳米单抗的平台技术等,筛选到特异性结合CD4的纳米抗体VHH,鉴定了其CDR序列,并构建了人源化抗体;并利用评估抗体在治疗HIV感染的疗效。本发明利用CD4结合的纳米抗体为HIV感染的临床治疗以及CD4淋巴瘤的临床治疗提供潜在的纳米抗体新药,双靶向抗体新药,基因治疗新药以及CAR细胞治疗等。
Description
技术领域
本发明涉及生物医药领域。更特别地,涉及一种可结合CD4的纳米抗体,还涉及所述纳米抗体在制备HIV感染或CD4阳性的T细胞肿瘤的治疗药物和诊断剂中的应用。
背景技术
CD4是T细胞表面上表达的重要受体分子,但同时也是病毒例如HIV等攻击T细胞的靶点。报道,HIV主要通过包膜表面的糖蛋白gp120与宿主细胞表面的特定受体(例如CD4)接触而影响病毒进入细胞的过程。抑制gp120和CD4的结合,可以有效地抑制HIV-1对宿主细胞的感染。另外,由于T细胞肿瘤也表达CD4。因此,可考虑以CD4为靶点,利用抗CD4抗体来治疗HIV感染或CD4阳性的T细胞肿瘤。
1993年,一种来源于骆驼科的新型天然抗体被发现。该抗体天然缺失轻链而只由重链组成,其重链包含两个恒定区(CH2和CH3)、一个铰链区和一个重链可变区(Variableheavy chain domain,VHH,即抗原结合位点),该重链可变区的相对分子质量约为13 KDa,仅为常规抗体的1/10,且分子高度和直径均在纳米级别,是目前可获得的最小的功能性抗体片段,因此又被称为纳米单抗(Nanobody,Nb)。因纳米单抗稳定性高(90℃条件下仍不会降解)、亲和力高、与人源抗体同源性超过80%、毒性和免疫原性均较低等特点,最近纳米单抗被广泛用于免疫诊断试剂盒研发、影像学研发以及针对肿瘤、炎症、传染病和神经系统疾病等领域的抗体药物研发。
我们期望通过新的技术手段,筛选出获得能够结合CD4的抗体,为预防和治疗CD4相关疾病例如HIV感染或CD4阳性T细胞肿瘤等提供潜在新药。
发明内容
本发明通过用抗原免疫骆驼,获取骆驼源纳米单抗及其VHH,用于治疗HIV感染的病人。基于这些研究,本发明提供了一种可结合CD4的纳米抗体,其特征在于,包括3个互补决定区CDR1-3,序列如SEQ ID NO:1-3所示。
在一个具体实施方案中,所述纳米抗体还包括4个框架区FR1-4,所述FR1-4与所述CDR1-3按顺序交错排列。例如,可将FR1-4序列设计为如SEQ ID NO:4-7所示(羊驼源),但本发明的范围不限于此。抗体的特异性识别和结合能力主要由CDR区序列决定,FR序列影响不大,可根据物种来设计,这是本领域公知的。可设计人源、鼠源或骆驼源的FR区序列来连接上述CDR,从而得到一个可结合CD4的纳米抗体。
在一个具体实施方案中,所述纳米抗体为骆驼源的VHH或人源化的VHH。
本发明还提供了上述纳米抗体在制备CD4检测剂中的应用。
本发明还提供了上述纳米抗体在制备HIV或CD4阳性的T细胞肿瘤的治疗药物中的应用。
本发明还提供了上述纳米抗体在制备针对HIV或CD4阳性的T细胞肿瘤的CAR-T治疗剂中的应用。
本发明还提供了编码上述纳米抗体的核酸。
本发明还提供了上述核酸在制备HIV感染或CD4阳性的T细胞肿瘤的治疗药物中的应用。
本发明针对HIV进行纳米抗体药物开发,通过制备CD4蛋白、免疫双峰骆驼、利用噬菌体库展示纳米单抗的平台技术等,筛选到特异性结合CD4的纳米抗体VHH,鉴定了其CDR序列,并构建了人源化的VHH-huFc1;同时利用假病毒中和实验评估4NB在治疗HIV感染的疗效。本发明利用CD4结合的纳米抗体为HIV感染的临床治疗以及CD4淋巴瘤的临床治疗提供潜在的纳米抗体新药,双靶向抗体新药,基因治疗新药以及CAR细胞治疗等。
附图说明
图1为CD4第3和4次免疫羊驼一周后的抗血清效价检测曲线;
图2为不同稀释度的第4次免疫骆驼一周后的抗血清抑制HIV假病毒体外感染ghost细胞的曲线,以免疫前的血清为对照;
图3为CD4-VHH噬菌体抗体文库为模板扩增的PCR产物的电泳图;
图4为CD4-VHH噬菌体抗体文库的淘选鉴定,其中,A为噬菌体文库针对CD4蛋白淘选后ELISA检测统计图;B为从第二轮(2nd)和第三轮(3rd)淘选后的噬菌体抗体文库各挑选40个和46个克隆进行噬菌体ELISA检测统计图;
图5为抗体4NB357中和HIV假病毒感染实验统计图,JRFL为HIV假病毒,MuLv为无关对照病毒,X轴为抗体浓度,Y轴为病毒的相对抑制率。
具体实施方式
1. 免疫原的制备
我们依据CD4蛋白序列和基因序列信息,分析并设计了可有效诱导骆驼产生针对CD4蛋白的特异性抗体的多肽CD4,在C端连接His-tag(CD4-his)或兔Fc(CD4-rFc)用于后续纯化及检测。
2. 羊驼免疫与抗血清的获得
用250 μg CD4-rFc蛋白与250 μl弗氏完全佐剂的乳化混合物对羊驼进行初免,在第14天、28天、42天用CD4-rFc蛋白与250 μl弗氏不完全佐剂加强免疫3次,第2次和第3次免疫1周后,采血检测抗血清滴度;第4次免疫1周后,采血200 ml用于噬菌体抗体库的构建。
抗血清效价通过ELISA检测,用浓度为0.5 μg/ml的CD4-his蛋白包被检测板,每孔加入梯度稀释的抗血清或者纯化的抗体100 μl(对照为免疫前骆驼血清),37℃孵育1.5 h,洗涤2次,每孔加入1:10000稀释的辣根过氧化物酶标记的Goat anti-Llamma IgG (H+L)二抗,37℃孵育1 h,洗涤4-6次后,加100 μl TMB底物,37℃孵育10 min,50 μl 0.2 M的H2SO4中止反应,测定OD 450nm。ELISA检测血清效价规定为在OD450是空白对照的2.1倍以上并且大于0.2的最高稀释倍数。
结果如图1所示,3免和4免的抗血清效价分别为3.28×106和9.84×106。由此可见,该抗原可诱导骆驼产生特异性针对CD4蛋白的高滴度抗血清。
为了进一步验证该高滴度的骆驼抗血清是否能有效阻止HIV病毒感染,进行病毒感染的中和实验。将不同稀释浓度的抗血清和免疫前血清分别与HIV病毒共同孵育60 min,随后转移到ghost细胞,48 h后通过Glo Max化学发光酶标仪(Promega)检测病毒载量并计算中和效果。中和实验结果显示,CD4诱导的抗血清抑制90% HIV感染的ID90为540倍稀释度以上(图2)。综上所述,CD4诱导了高滴度抗血清,同时该抗血清具有高效抑制HIV假病毒感染的能力。
3. VHH噬菌体库构建及淘选
收集200 ml免疫后骆驼的外周血,利用淋巴细胞分离液(GE Ficoll-Paque Plus)分离获得骆驼的PBMC,根据TRIzol操作手册,提取RNA,并利用oligo(dT)反转为cDNA,通过引物扩增,以及分子克隆等技术,将骆驼的VHH基因克隆至phagemid质粒,转化TG1细菌,得到VHH噬菌体库。
为了进一步鉴定CD4-VHH噬菌体库是否构建成功,通过PCR扩增免疫CD4骆驼的VHH目的基因,可以看出目的条带为500 bp,大小符合预期(图3),说明该CD4-VHH噬菌体抗体文库里含有VHH基因。挑选50个克隆进行测序,测序结果显示,所测序列没有完全一致的重复序列;比对结果显示,差异序列大多在CDR结合区。经检测,该构建了一个CD4-VHH噬菌体抗体文库的库容为1.6×109,阳性率为100%,序列多样性(Diversity)为100%,有效插入率(Inframe rate)大于95%。
在M13KO7辅助噬菌体的帮助下,用VHH-phagemid转化的细菌,进行噬菌体抗体库的复苏,并用PEG/NaCl进行沉淀。将包被有50 μg/ml的CD4-His蛋白进行三次富集噬菌体抗体库。将富集的噬菌体,洗脱、转化、涂板、挑取单克隆进行噬菌体与CD4蛋白ELISA的结合鉴定,将结合读值>1.0的克隆进行测序,并克隆至表达载体phv3,转染293tt细胞表达生产纳米单抗。
淘选后的文库与CD4蛋白进行结合检测。噬菌体ELISA结果显示,没有富集前的CD4-VHH噬菌体文库与CD4蛋白的结合读值为1.71,经过一轮、二轮、三轮富集后的噬菌体文库读值分别为2.5、3.1、3.1(图4A)。为了进一步验证富集后的文库中结合CD4-VHH蛋白的阳性噬菌体率,从第2轮和第3轮富集后的文库里各挑选了40、46个克隆进行单个噬菌体ELISA检测。结果显示,第2轮文库里,57.5%的单个噬菌体克隆为阳性,第3轮文库里82.6%的噬菌体克隆克隆为阳性,而且结合的平均读值在3.0左右(图4B),通过CD4蛋白淘选成功的富集了高结合力的CD4-VHH噬菌体文库。其中,筛选出抗体4NB357,CDR1-3的序列如SEQ ID NO:1-3所示,FR1-4的序列如SEQID NO:4-7所示。
4. VHH-huFc真核表达
通过分子克隆技术,将文库筛选的阳性克隆VHH基因融合人的Fc基因并插入在pCDNA3.4真核表达载体,构建形成Nb-huFc-pCDNA3.4表达质粒。将构建好的Nb-huFc-pCDNA3.4,转染293tt细胞,表达生产Nb-huFc(4NB)。收集细胞上清进行ELISA测定。
对抗体4NB357与CD4蛋白的亲和力测定试验。应用Fortebio生物分子相互作用平台检测亲和力。将抗体4NB357固化至Anti-human IgG Fc Capture Biosensors(AHC)探头上,固化时间400s,再结合抗原CD4-his蛋白,结合时间180s,解离时间180s,观察抗体-抗原的结合解离情况,由仪器拟合曲线,导出数据。亲和力测定结果如表1所示,大部分的抗体的亲和力能达到10-8或10-9(Nano mole级)。由此可见,我们获得了具有高亲和力的VHH-huFc1(C9NB)抗体,可用于检测CD4。
表1 4NB357的亲和力数据
Clone ID | Ka(1/MS) | Kd(1/S) | KD(M) | Response(nm) |
4NB357 | 9.05E+04 | <1.0E-07 | <1.0E-12 | 0.35 |
5. 4NB357阻断HIV感染ghost细胞
将抗体4NB357稀释至1 μg/ml作为起始浓度,3倍比梯度稀释,共设8个梯度,分别与JRFL病毒一起,于5% CO237℃下共孵育1个小时,添加1.0 x 104个ghost细胞,5% CO237℃温箱培养48小时后,移除细胞上清,加入100 μl/孔的Glolysis buffer(提前恢复至室温),轻晃培养板使细胞充分裂解,接着转移50 μl/孔细胞裂解物于96孔白色荧光酶标板中(Costar),再加入50 μl/孔 Brite-Glo luciferase底物(提前恢复至室温),涡旋震荡混匀,立即放入Glo Max化学发光酶标仪(Promega)进行检测。中和滴度(IC50)表述为50%抑制率时的抗体浓度。
结果如图5所示,抗体4NB357的IC50为14.7046 ng/ml。
由以上实验结果可知,抗体4NB357及其人源化形式均可对特异性地识别、结合CD4,阻断CD4的配体与CD4的结合,从而抑制与CD4通路相关的疾病,例如HIV感染、CD4阳性的T细胞肿瘤等。因为抗体4NB357能够识别细胞表面的CD4分子,因此其序列也可以应用于CAR(Chimeric Antigen Receptor,抗原嵌合受体,由VHH序列融合第三代或者第四代CD28-4-1BB-CD3zeta分子序列构成)细胞治疗肿瘤或HIV感染。
另外因为抗体4NB357能识别细胞表面的CD4分子,因此也可以通过偶联药物用于ADC(Antibody-drug conjugate,抗体偶联药物)治疗或者偶联同位素用于依赖抗体的分子影像诊断等。也可将编码抗体4NB357的核酸搭载在AAV系统中,用于治疗作用。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 源道隆(苏州)医学科技有限公司
<120> 可结合CD4的纳米抗体4NB357及其应用
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Claims (8)
1.一种可结合CD4的纳米抗体,其特征在于,包括3个互补决定区CDR1-3,序列分别如SEQ ID NO:1-3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4个框架区FR1-4,所述FR1-4与所述CDR1-3按顺序交错排列。
3.根据权利要求2所述的纳米抗体,其特征在于,所述纳米抗体为骆驼源的VHH或人源化的VHH。
4.权利要求1-3中任一项所述的纳米抗体在制备CD4检测剂中的应用。
5.权利要求1-3中任一项所述的纳米抗体在制备HIV的治疗药物中的应用。
6.权利要求1-3中任一项所述的纳米抗体在制备针对HIV的CAR-T治疗剂中的应用。
7.一种核酸,其特征在于,编码权利要求1-3中任一项所述的纳米抗体。
8.权利要求7所述的核酸在制备HIV感染的治疗药物中的应用。
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Title |
---|
Identification of a CD4-binding site-directed antibody with ADCC activity from a chronic HIV-1B′-infected Chinese donor;Yuanyuan Hu;《Virus Research》;第302卷;第1-10页 * |
人CD4 分子及其治疗性抗体的研究进展;胡迪超;《中国生物制品学杂志》;第23卷(第8期);第896-900页 * |
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