CN102321173A - 人源化巨噬细胞抑制因子1单克隆抗体及其应用 - Google Patents
人源化巨噬细胞抑制因子1单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种人源化抗巨噬细胞抑制因子1单克隆抗体及其应用,所述抗体包含:重链可变区,该重链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ ID NO:1、2和3所示的氨基酸序列;和轻链可变区,该轻链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ IDNO:4、5和6所示的氨基酸序列。本发明还涉及编码所述抗体的多核苷酸,含有所述抗体的药物组合物以及所述抗体在肿瘤治疗中的应用。本发明提供的单克隆抗体免疫原性低、亲和力高、特异性强,为***提供一个新的可能。
Description
技术领域
本发明属于生物医药工程技术领域,涉及一种人源化抗巨噬细胞抑制因子1(MIC-1)单抗隆抗体及其应用。
背景技术
巨噬细胞抑制因子1(Macrophage inhibitory cytokine-1,MIC-1)是人转化生长因子β(Transforming growth factor-β,TGF-β)超家族中的一个重要分支成员,也被称为生长分化因子-15(growth differentiation factor-15,GDF-15)等,因其能够显著抑制活化巨噬细胞产生TNF-α而得名[Bootcov,1997]。
人类基因组中MIC-1基因定位于染色体19p13.1,包括2个外显子(309bp和891 bp)和一个内含子(1820 bp)[Fairlie et al,1999]。MIC-1基因编码相对分子质量25×103的MIC-1蛋白,由308个氨基酸多肽组成,包括29个氨基酸信号肽、167个氨基酸前肽和112个氨基酸成熟区。两条多肽链经二硫键连接成前体蛋白后由furin样蛋白酶进行剪切,最终形成由二硫键连接的同源二聚体成熟蛋白,分子量25KD[Fairlie LN et al,1999]。成熟MIC-1蛋白分泌出细胞后进入血液循环发挥细胞因子的内分泌作用[Bauskin et al,2005]。MIC-1主要以前体形式存在于细胞内,在不同的生理及病理条件下水解后释放到血液中。
MIC-1作为一种细胞因子作用于细胞表面TGFβ受体,广泛参与细胞凋亡、分化、增殖和机体炎症反应的信号传递,并在抑制肿瘤细胞增长、促进凋亡、维持胎盘功能和胚胎发育、调节和保护中枢神经等方面具有重要作用[Lee DH et al,2003]。正常生理状态下,MIC-1高表达于胎盘组织,而在***、结肠、肾、脑、肝、胰腺等组织中呈低表达。病理状态如肿瘤、急性损伤和炎症,MIC-1表达可显著提高[Bauskin et al,2006]。
MIC-1主要是通过p53、PI3K/AKT/GSK3β信号通路、死亡受体和PLAU/uPAR***发挥抑制肿瘤的作用。在乳腺癌细胞中,MIC-1作为p53的靶基因过表达时导致肿瘤细胞细胞凋亡和细胞停止于G1期[Bauskin ARet al,2006];在研究结肠癌细胞系HCT-116时发现MIC-1作为PI3K/AKT/GSK3β通路的靶目标,能够促进肿瘤细胞的凋亡[Yamaguchi Ket al,2004];Jang等人研究则证实MIC-1能够明显诱导DR-4和DE-5的表达从而促使肿瘤细胞凋亡增加和细胞活力下降,但MIC-1作用的受体和传导通路还不是非常清楚[Jang TJ et al,2004];在结肠癌细胞中,MIC-1通过抑制PLAU/uPAR***的活性,从而促使肿瘤细胞凋亡[Yang H et al,2009]。
MIC-1同时也具有促进肿瘤细胞的增殖、侵袭和转移作用,Sung等人研究发现通过V600EB-Raf信号通路可促进MIC-1表达及分泌,从而促进肿瘤血管的生成使肿瘤增殖[Sung Jin Huh et al,2010];MIC-1还可通过促进ErBb2受体的超激活并伴随EGFR、ErBb2等酪氨酸磷酸化从而激活ERK1/2和Akt,促进肿瘤细胞的侵袭能力(Kim et al 2008);Lee等人发现胃癌中MIC-1过度表达能显著增加尿激酶纤维蛋白溶酶原激活剂uPA和uPAR的活性,通过胞外信号转导激酶1/2相关途径来上调uPAu/PAR活性***,诱导肿瘤细胞的侵袭性并促进胃癌细胞的恶性程度[Lee DH,2003]。
MIC-1与肿瘤相关的厌食症和体重下降之间也存在一定关系,MIC-1能够与下丘脑的TGF-β受体II相互作用,促进ERK-1/2和类***样神经肽并抑制神经肽Y的功能,从而达到对食欲的控制作用,使患者发生厌食症和体重下降并最终降低患者的生存时间[Johnen et al,2007]。F.E.Wiklund等对876名男性和324对双胞胎的血清MIC-1水平和死亡率之间进行了中位观测时间分别为5.3年和9.1年的回归性流行病学研究,结果表明MIC-1能够预示包括肿瘤在内的多种原因导致的死亡(p=0.0001),在排除年龄、BMI、吸烟史的干扰后,MIC-1高血清水平组与正常水平组相比有较高的肿瘤死亡风险(OR=2.74,95%CI 1.70-4.22)。
上述的研究结果表明,MIC-1参与机体多种生理及病理过程,并在其中发挥各种各样的作用。我们设想如果通过抗体阻断MIC-1的作用途径,有可能在肿瘤的治疗中发挥作用。在胰腺癌为例,使用自主研发的鼠抗人MIC-1抗体腹腔注射治疗胰腺癌荷瘤裸鼠(PANC-1和SW1990),与对照相比实验组肿瘤显著缩小,切片免疫组化分析显示CD31和MMP2水平降低,微血管数量明显减少。提示MIC-1抗体可通过阻断MIC-1与受体的结合发挥了其抗肿瘤作用。MIC-1单抗通过抑制MIC-1的功能可望给肿瘤患者提供新的治疗选择。
鼠单抗在人体内诱导内源性抗抗体可分为两类,抗独特性抗体和抗种型抗体。前者通过中和抗原结合部位而消除单抗结合能力;后者除了加速单抗的清除外,还有可能会引起过敏性休克。据报道,多数鼠源性单抗药物在肠癌等患者中的HAMA发生率高达100%,HAMA对注入的单抗药物起中和作用,从而抵消其疗效。解决这些问题,要从抗体质量、抗体理化性质、用药途径等多个环节进行改善,目前避免或减少HAMA反应的主要途径是使鼠源性单抗人源化或研制完全人源化抗体。但完全人源化抗体在研制时往往遇到抗体亲和力下降、活力不高、稳定性差或产量低等瓶颈问题。
综上所述,MIC-1是一个非常重要的细胞因子,本领域有必要进一步研究和开发抗MIC-1抗体药物,特别是免疫原性低、亲和力高、特异性强的人源化单克隆抗体,为肿瘤的治疗提供新的途径。
发明内容
为解决上述问题,本发明提供了一种新的MIC-1单克隆抗体。本发明还提供了所述新的MIC-1单克隆抗体的人源化抗体。
本发明利用基因工程技术对自主研制的鼠源抗体进行改造,在维持其抗原特异性的基础上,使其主要氨基酸组成来自人类抗体,从而降低其免疫原性,成为理想的临床药物。
因此,本发明一方面提供一种MIC-1单克隆抗体,其包含:
重链可变区,该重链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ ID NO:1、2和3所示的氨基酸序列;和
轻链可变区,该轻链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ ID NO:4、5和6所示的氨基酸序列。
在本发明的一个实施方案中,本发明提供上述MIC-1单克隆抗体的人源化形式,其中,重链可变区的框架区氨基酸序列与人胚系基因位点VH4-59编码的框架区相同;轻链可变区的框架区氨基酸序列与人抗体胚系基因位点VL 08编码的框架区相同。
本发明又一方面提供一种药物组合物,其包含本发明的MIC-1单克隆抗体和药用载体。
本发明还一方面提供一种多核苷酸,其编码本发明的MIC-1单克隆抗体。
本发明还一方面提供一种表达载体,其包含本发明的多核苷酸。
本发明还一方面提供一种宿主细胞,其包含本发明的表达载体,所述宿主细胞可以是真核宿主细胞或原核宿主细胞。
在一个实施方案中,本发明还提供本发明的MIC-1单克隆抗体在制备***的药物中的应用。
根据本领域的知识和本发明提供的实验数据,本领域技术人员可知本发明的MIC-1单克隆抗体可治疗的肿瘤包括胰腺癌、食管癌、胃癌、肠癌、肺癌、乳腺癌、卵巢癌和肝癌。优选的,所述肿瘤为胰腺癌,食管癌、胃癌、肠癌和肝癌。
在一个实施方案中,本发明提供的MIC-1单克隆抗体为IgG2型抗体,并且与MIC-1的亲和力为1×10-9M-1×10-8M。
本发明通过对自主研制的鼠源抗MIC-1抗体进行人源化改造,研制出具有亲和力高、免疫原性低的新型人源化抗MIC-1单克隆抗体,本发明的抗体能够阻断MIC-1与细胞表面受体的结合,从而抑制肿瘤的转移和血管生成,为***提供了一种可能。
本发明的其他方面由于本文的公开内容,对本领域的技术人员而言是显而易见的。本说明书和序列表的中的序列如不一致,以说明书中记载的序列为准。
附图说明
从下面结合附图的详细描述中,本发明的上述特征和优点将更明显,其中:
图1鼠抗MIC-1单克隆抗体对裸鼠移植人PANC-1体内抑瘤作用。
图2鼠抗MIC-1单克隆抗体对裸鼠移植人SW1990体内抑瘤作用。
图3.PANC-1荷瘤小鼠MIC-1抗体给药后的肿瘤体积变化(天)。
图4.SW1990荷瘤小鼠MIC-1抗体给药后的肿瘤体积变化(天)。
具体实施方式
实施例1杂交瘤细胞株分泌的单克隆抗体的制备
本发明用毕赤酵母表达的人MIC-1蛋白(通过将MIC-1编码区克隆至pPIC9K质粒中,经GS115表达;融合蛋白通过硫酸铵沉淀上清和SOURCE-30Q阴离子层析纯化。pPIC9K质粒和GS115菌株购自Invitrogen公司)。常规方法免疫健康BALB/C雌鼠(体重14~16g)。首次免疫采用MIC-1与等量弗氏完全佐剂乳化后腹腔注射,剂量为100μg/只。14天后抗原与弗氏不完全佐剂乳化腹腔注射加强免疫,此后每隔2周加强免疫1次。免疫后10天开始小鼠尾静脉采血,采用间接ELISA方法检测血清效价,直至血清效价达到1∶106以上。融合前3天血清效价最高的小鼠不加佐剂尾静脉注射100μg抗原加强免疫。采集强化免疫3天后的小鼠血液,分离血清作为阳性对照。无菌取该免疫鼠脾细胞,按10∶1比例将免疫后脾细胞与SP2/0细胞在50%PEG4000(购自Sigma公司)作用下融合,用HAT培养液悬浮再分种于已铺好滋养细胞的96孔细胞培养板中,放入37℃、5%CO2培养箱中。采用间接ELISA法检测筛选特异性抗体,选取效价较高的阳性孔进行有限稀释法克隆,扩大培养并建株冻存。采用秋水仙素阻抑法对杂交瘤细胞染色体进行分析。
单抗生物学特性鉴定①Dot ELISA鉴定单抗特异性:培养甲醇酵母GS115,离心收集菌体,沉淀用PBS悬浮,超声波裂解后离心取上清。剪取一定大小的硝酸纤维素膜(NC),用去离子水浸透,晾干,取菌液5μL点于NC膜上,37℃干燥30分钟,用含100mL/L小牛血清的PBST封闭,4℃过夜;PBST洗涤3次,5分钟/次;再浸入工作浓度的mAb溶液中,37℃孵育2h,PBST洗涤3次;转入工作浓度的羊抗鼠HRP-Ig(G+M)酶标抗体溶液中,37℃孵育1h,PBST洗涤3次;以DAB显色液显色。同时设阳性抗血清作为对照。②Western blot分析:选用12%分离胶对MIC-1和甲醇酵母总蛋白同时进行SDS-PAGE分析,再将蛋白从SDS聚丙烯酰胺凝胶转移至NC膜上,用含50g/L脱脂奶的PBS封闭,室温轻摇过夜;充分洗涤后,加入稀释后的腹水mAb作用1h;充分洗涤后,加入工作浓度的HR标记的羊抗鼠IgG抗体,作用1h;充分洗涤后,用DAB显色液显色,蒸馏水终止反应。
用单抗亚类鉴定试剂盒鉴定单抗亚类。以不同抗原浓度进行抗体倍比稀释的间接ELISA检测,绘制反应曲线,以曲线上最大A450值一半时的抗体浓度计算单克隆抗体的亲和常数,确定抗体与抗原的结合能力(2×10-9M)。抗体亲和常数(Ka)按如下公式计算[6]。Ka=(n-1)/2×(n[Ab]-[Ab]t)。[Ab]:表示抗原浓度为[Ag]时A=1/2Amax对应的抗体浓度;[Ab]t:表示抗原浓度为[Ag]t时A=1/2Amax对应的抗体浓度;[Ag]/[Ag]t:表示抗原浓度;n:为抗原[Ag]与[Ag]t间的稀释倍数。
腹水单抗的制备及分析纯化用0.5mL/只剂量的无菌弗氏不完全佐剂腹腔注射致敏8周龄的雌性BALB/C小鼠,1周后注射2×106个杂交瘤细胞。视小鼠的腹部膨大情况于10~14天后收集腹水,间接ELISA方法测定腹水单抗效价,BCA试剂盒法测定蛋白浓度。腹水的纯化采用硫酸铵沉淀后蛋白G亲和层析纯化,对上述方法纯化的腹水进行SDS-PAGE泳,薄层扫描分析凝胶,根据IgG区带占全部区带面积的百分比,计算单抗的纯度和回收率。
实施例2抗MIC-1人源化抗体的制备
鼠抗体在人体内会产生免疫排斥反应,因此只能用于急症的治疗,一旦人体内产生针对鼠抗体的抗抗体,作为药物的鼠抗体即失效。为了克服这一缺点,本发明对鼠抗体进行了人源化,制备了人-鼠嵌合抗体,该抗体的可变区来自鼠抗体杂交瘤细胞株分泌的单克隆抗体,不变区序列来自人的IgG2。这种抗体保持了鼠单抗的抗原结合特异性,同时降低了在人体内诱导的免疫排斥反应。制备嵌合抗体的步骤如下:
从上述杂交瘤细胞株中分离纯化mRNA,使用oligo-dT获得cDNA,通过PCR方法分别扩增抗体轻链和重链可变区,
轻链引物为
5’-GAYATTG TG MTSACMCARWCTMCA-3’(SEQ ID NO:7)和
5‘CTCCAGATGTTAACTGCTCAC3’(SEQ ID NO:8);
重链引物为
5’-ATGSARGTNMAGCTGSAGSAGTC-3’(SEQ ID NO:9)和
5’-GGTCAAGG TCACTGGCTCAGG-3’(SEQ ID NO:10)。
其中R=G或A,Y=T或C,M=A或C,S=G或C,W=T或A,N=G或A或T或C。将PCR产物分别克隆到T载体中测序;将鼠源抗体的重链可变区CDR移植到4-59框架上,产生人源化的重链可变区。重链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ IDNO:2和SEQ ID NO:3所示;重链可变区氨基酸序列如SEQ ID NO:11所示。
SEQ ID NO:1:Asp Gly Met Tyr Thr Leu Ala Lys Ser Tyr Lys Gly
SEQ ID NO:2:Tyr Ser His Ser Gln Pro Trp Lys Tyr Asn Gly Leu Asp Gly
SEQ ID NO:3:Met Trp Gly Asp Ile Trp Asn Lys Gly Gln Ser Asp Ala Tyr
SEQ ID NO:11:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr LeuSer Leu Thr Cys Thr Val Ser Asp Gly Met Tyr Thr Leu Ala Lys Ser Tyr Lys Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ser His Ser Gln Pro Trp Lys Tyr Asn Gly Len Asp Gly Arg Val Thr Ile Ser Val Asp Thr SerLys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyrTyr Cys Ala Arg Met Trp Gly Asp Ile Trp Asn Lys Gly Gln Ser Asp Ala Tyr TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser。
将鼠源抗体的轻链可变区CDR移植到VL08框架上,产生人源化的轻链可变区。轻链可变区的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示。轻链可变区氨基酸序列如SEQ ID NO:12所示。
SEQ ID NO:4:Ser Gln Tyr Thr Ser Ala Lys Lys Gly
SEQ ID NO:5:Gly His Ser Trp Lys Asn Gly Leu Asp Tyr
SEQ ID NO:6:Tyr Ile Gly Pro Trp Gly Gln Gln Leu Ala The
SEQ ID NO:12:
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg ValThr Ile Thr Cys Ser Gln Tyr Thr Ser Ala Lys Lys Gly Trp Tyr Gln Gln Lys ProGly Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gly His Ser Trp Lys Asn Gly Leu Asp Tyr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrPhe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Tyr Ile Gly Pro Trp Gly Gln Gln Leu Ala The Phe Gly Gly Gly Thr Lys Val Asp Ile Lys
用常规方法将人源化抗体重链可变区连接到人IgG2恒定区(其编码序列如SEQ ID NO:13所示)形成全长基因,人源化轻链可变区连接到人K链恒定区(其编码序列如SEQ ID NO:14所示)形成全长基因。构建可编码嵌合抗体的融合基因,将该嵌合抗体轻链的编码基因***到有选择性标记(GPT)和基因表达调控区的表达载体PCI-GPT(以Promega公司pCI质粒为模板,将鸟嘌呤磷酸核糖转移酶的编码基因***PCI的多克隆位点获得PCI-GPT表达质粒)中,得到该嵌合抗体轻链表达载体pCI-gpt-L;将将该嵌合抗体重链的编码基因***到有选择性标记(DHFR)和基因表达调控区的表达载体PCI-DHFR(以Promega公司pCI质粒为模板,将二氢叶酸还原酶的编码基因***PCI的多克隆位点获得PCI-DHFR表达质粒)中,得到该嵌合抗体重链表达载体pCI-gpt-H;用电转染的方法将上述两个载体一同导入哺乳动物细胞NS/0中。用霉酚酸酯在含有黄嘌呤的培养基中筛选转染细胞,获得稳定转染的细胞株。按照实施例1中的方法进行分泌抗体的鉴定,结果显示嵌合抗体保留了杂交瘤细胞株分泌单克隆抗体的特异性和亲和力。
SEQ ID NO:13
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCC CCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO:14
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
实施例3、抗MIC-1抗体在胰腺癌小鼠模型中的应用
取经体外细胞培养已在裸鼠体内皮下传代生长良好的人胰腺癌PANC-1和SW1990(购自ATCC,编号分别为CRL-1469和CRL-2172)肿瘤结节,超净台中无菌操作制成瘤细胞液,接种于裸鼠腋窝皮下,7x106细胞/鼠。将动物分别随机分为4组,每组6只,称体重标号,PANC-1和SW1990共8组。实验分别为模型对照组、阳性药键择(Gem 50mg/kg)组、鼠抗hMIC-1单克隆抗体高剂量(10mg/kg)组、鼠抗hMIC-1单克隆抗体低剂量(2mg/kg)组。实验组在接种肿瘤后肿瘤体积生长约80~100mm3第10日、13日尾静脉注射鼠抗hMIC-1单克隆抗体液各1次,以后每周相同时间尾静脉注射鼠抗hMIC-1单克隆抗体液各1次,实验全程共注射鼠抗hMIC-1单克隆抗体液8次。阳性药键择(Gem 50mg/kg)组在接种肿瘤后第10日腹腔注射1次,以后每周相同时间腹腔注射1次,实验全程共注射4次。模型对照组相同时间腹腔注射0.9%无菌Nacl注射液10ml/kg,实验全程共注射8次。各组给药时间从接种肿瘤后第10天开始。于实验开始后每4日用卡尺测一次皮下肿瘤体积,日常观察荷瘤鼠表现,末次给药结束后,于肿瘤接种后第37日处死动物。
实验结果表明,鼠抗hMIC-1单克隆抗体对人胰腺癌PANC-1具有明显抑瘤活性(10mg/kg,Biw,iv×8),肿瘤抑制率67.65%,抑瘤效应与剂量间具有量效关系,抑瘤效果优于键择腹腔给药组(Gem 50mg/kg,qw,ip×4)(P<0.01)(表1、图1、图3)。实验第六周结束后对人胰腺癌PANC-1实验,鼠抗人hMIC-1单克隆抗体组(10mg/kg,Biw,iv×8)动物肿瘤体积平均为559mm3,而荷瘤模型对照组动物肿瘤体积平均为1685mm3,键择腹腔给药组(Gem 50mg/kg,qw,ip×4)动物肿瘤体积平均为765mm3,鼠抗hMIC-1单克隆抗体高剂量组实验结束时肿瘤体积与荷瘤模型对照组比较差异极显著P<0.01。对人胰腺癌SW1990实验,鼠抗hMIC-1单克隆抗体组(10mg/kg,Biw,iv×8)动物肿瘤体积平均为1045mm3,而荷瘤模型对照组动物肿瘤体积平均为1737mm3,键择腹腔给药组(Gem 50mg/kg,qw,ip×4)动物肿瘤体积平均为881mm3,鼠抗hMIC-1单克隆抗体高剂量组实验结束时肿瘤体积与荷瘤模型对照组比较差异显著P<0.05(图2、图4)。
表1:鼠抗hMIC-1单克隆抗体对裸鼠移植人胰腺癌抑瘤作用观察
ΔN.S.:生理盐水
*Gem或MIC-1 vs对照
进行移植瘤组织病理学检查,光镜下可见荷瘤模型对照组癌组织,肿瘤细胞结构清楚,细胞完整,癌细胞圆形或椭圆形,核大,核仁明显,细胞大小不等,癌细胞被纤维组织分隔成大小不等的癌巢,其中可见腺管、腺腔样结构。鼠抗hMIC-1单克隆抗体组可见肿瘤细胞被破坏,光镜下可见有大量淋巴细胞浸润,肿瘤细胞明显坏死,细胞溶解。表明鼠抗hMIC-1单克隆抗体对肿瘤细胞具有明显抑制作用。
从实验结束后石蜡切片CD34免疫组化染色结果表明,鼠抗hMIC-1单克隆抗体组(10mg/kg,Biw,iv×8)移植瘤肿瘤组织内可见形态不规则、且无明显管腔形成的新生血管,血管分布以肿瘤组织边缘处多见。血管明显少于荷瘤模型对照组,两者有显著性差异,阴性对照组移植瘤中微血管密度显著高于鼠抗hMIC-1单克隆抗体组(P<0.01)。
Claims (8)
1.一种MIC-1单克隆抗体,其包含:
重链可变区,该重链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ ID NO:1、2和3所示的氨基酸序列;和
轻链可变区,该轻链可变区的CDR1、CDR2和CDR3氨基酸序列分别为SEQ ID NO:4、5和6所示的氨基酸序列。
2.根据权利要求1所述的MIC-1单克隆抗体,其中,重链可变区的框架区氨基酸序列与人胚系基因位点VH4-59编码的框架区相同;轻链可变区的框架区氨基酸序列与人抗体胚系基因位点VL 08编码的框架区相同。
3.一种药物组合物,其包含权利要求1或2所述的MIC-1单克隆抗体和药用载体。
4.一种多核苷酸,其编码根据权利要求1或2所述的MIC-1单克隆抗体。
5.一种表达载体,其包含权利要求4所述的多核苷酸。
6.一种宿主细胞,其包含权利要求5所述的表达载体。
7.根据权利要求1或2所述的MIC-1单克隆抗体在制备***的药物中的应用。
8.根据权利要求7所述的应用,其中所述的肿瘤包括胰腺癌、食管癌、胃癌、肠癌、肺癌、乳腺癌、卵巢癌和肝癌,优选的,所述肿瘤为胰腺癌,食管癌、胃癌、肠癌和肝癌。
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CN116444667B (zh) * | 2023-06-13 | 2023-09-01 | 上海驯鹿生物技术有限公司 | 一种靶向gdf15的全人源抗体及其应用 |
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