Expression and secretion of a recombinant ricin immunotoxin from murine myeloma cells

Expression plasmids carrying a humanized N901 immuno-globulinheavy chain gene (hN901HC) fused to a gene encoding the nativeB chain of ricin toxin (RTB), hN90 W CRTB, or a sugar bindingmutant of RTB, hN901HC-RTBgly, were constructed. In each case,the fused gene constructions were co-expressed in murine myelomacells (Sp2/0) with the gene for humanized N901 immunoglobulinlight chain to produce the secreted recombinant products hN901-RTBand hN901-RTBgly, respectively. When purified by affinity chromatography,both the hN901-RTB and hN901-RTBgly products were found to havean apparent molecular mass of M_r = 210 000 and to be composedof two hN901 antibody heavy chains each fused to a fulllengthcopy of RTB and two hN901 antibody light chains. In each ofthe recombinant fusions the hN901 antibody moiety retained thefull binding affinity and specificity for its cognate antigen,CD56. Moreover, when mixtures of hN901-RTB and native ricinA chain were incubated in the presence of the antigen-positivetarget cell line SW-2, antigen-specific potentiation of ricinA chain cytotoxicity was observed. It has been demonstratedpreviously that lectin activity of the B chain is essentialfor A chain cytotoxicity, and we conclude that the fused wild-typeB chain was properly folded and maintained lectin activity.These data demonstrate the feasibility of using recombinantricin B chain in an immunotoxin and of using mammalian cellculture for its expression. The use of recombinant hN901-RTBfusion protein to evaluate the contribution of the lectin activityof ricin B chain in the penetration of cell membranes by ricinA chain is proposed.     

表达人源化抗血管内皮生长因子单克隆抗体的重组CHO细胞传代稳定性评价

目的评价表达人源化抗血管内皮生长因子(vascular endothelial growth factor,VEGF)单克隆抗体(简称抗VEGF单抗)的重组CHO细胞(K11细胞)传代稳定性。方法细胞培养瓶中进行K11细胞连续传代,于培养1、4和7个月时测定K11细胞的倍增时间、单细胞抗体表达量和基因拷贝数。在传代培养过程中,分别于传代培养2、3、4、5和7个月时将K11细胞接种至全自动生物反应器中进行发酵培养,测定发酵培养产物的单抗表达量,测序单抗轻、重链基因;采用三步层析(亲和、阴/阳离子交换层析)纯化人源化抗VEGF单抗,分析人源化抗VEGF单抗的纯度、电荷异质性、一级结构和生物学活性。结果 K11细胞在细胞培养瓶中连续传代培养1、4和7个月时,对数生长期K11细胞的倍增时间分别为25、23和34 h,单细胞抗体表达量分别为15. 6、15. 0和11. 5 pg/(个·d),单细胞基因拷贝数分别为200、219和204 copies/个。生物反应器发酵培养5批单抗,纯化后单抗表达量为1. 18~2. 06 g/L,K11细胞单抗轻、重链基因测序结果均与理论序列一致,人源化抗VEGF单抗SEC-HPLC单体纯度为96. 23%~98. 21%,电荷异质性和一级结构相似,生物学活性为(0. 859~0. 901)×10^4U/mg。结论 K11细胞在工业化生产规模的培养周期内保持稳定,可满足人源化抗VEGF单抗的商品化的生产需要。     

抗氨基末端脂多糖结合蛋白基因工程抗体库的构建及体外表达筛选

目的构建人源化的单链可变区抗体库,运用体外表达技术翻译并筛选特异性的抗NHLBP抗体。方法分离健康人外周血单核细胞,抽提总RNA设计引物,扩增抗体轻重链的可变区,通过一段柔性片段将其连接成单链抗体库;应用体外表达技术翻译抗体库,并筛选出与NHLBP高度结合的单链抗体片段。结果扩增的单链抗体可变区片段分别为340bp和325bp,经连接后约为750bp。经过体外表达得到了约27000的抗体片段,经过5轮筛选进化,得到了可与NHLBP高度结合的抗体。结论已成功构建了人源化的单链抗体库;通过体外表达技术筛选到可与NHLBP高度结合的抗体。     

仙台病毒Tianjin株噬菌体Fab抗体库的构建及初步筛选

目的构建仙台病毒Tianjin株噬菌体Fab抗体库,并进行初步筛选。方法用仙台病毒Tianjin株免疫小鼠,制备脾细胞悬液,提取小鼠脾细胞总RNA,RT-PCR扩增鼠抗体轻链(κ链)和重链Fd基因,将轻链基因和噬菌粒p3MH经SacⅠ和XbaⅠ双酶切后纯化回收,经T4 DNA连接酶连接,电转化E.coli XL1-Blue,构建轻链库;将重链Fd段基因和轻链库p3MH-κ经SpeⅠ和XhoⅠ双酶切后,纯化回收并连接,电转化E.coli XL1-Blue,获得噬菌体抗体库,计算重组率和抗体库滴度。以仙台病毒Tianjin株重组蛋白HN2为抗原进行初步筛选,制备噬菌体抗体,并进行测序。结果构建的免疫噬菌体抗体库库容为1.18×107,重组率为90%,制备的噬菌体抗体滴度为1011pfu/ml;经初步筛选,噬菌体富集了约63.6倍,获得2株与重组蛋白HN2有结合活性的阳性克隆,经NCBI BLAST进行同源性分析,显示为鼠源性抗体,经IMGT分析,显示轻链基因与Vk9亚群基因的同源性为94.87%,重链基因与VH7亚群基因的同源性为97.85%。结论成功构建了仙台病毒Tianjin株噬菌体Fab抗体库,为该病毒株的诊断、疾病治疗及致病机制等研究奠定了基础。     

从半合成噬菌体抗体库筛选抗狂犬病毒人单链抗体

目的　应用纯化的狂犬病毒抗原从半合成噬菌体抗体库中筛选针对狂犬病毒的人单链抗体(ScFv)。方法　用固相化的狂犬病毒抗原对半合成抗体库进行 3轮“吸附 洗脱 扩增”的筛选 ,从第 3轮洗脱下来的克隆中获得一株有可溶性表达且特异性结合狂犬病毒抗原的ScFv ,并进行基因序列测定。结果　所获氨基酸序列经blast数据库搜索 ,与一种抗狂犬病毒免疫球蛋白的氨基酸序列同源性最高 ( 82 % )。经检索kabat数据库 ,发现其轻、重链可变区分别属于VkⅠ型、VHⅢ型。结论　从噬菌体抗体库可以方便快捷地分离到针对狂犬病毒的单链抗体 ,对狂犬病毒的预防具有重要意义     

用同系抗独特型单克隆抗体亲和层析法提纯小鼠单克隆抗体

1C_6McAb_2是抗7A_4McAb_1独特型的同系小鼠单克隆抗体。应用硫酸铵盐析,1C_6McAb_2—Sepharose 4B柱亲和层析法提纯了小鼠腹水7A_4McAb_1。获得的样品经二巯基乙醇还原,在SDS—PAGE上为两条带,分别是IgG的重链和轻链,免疫双扩散检查只与兔抗鼠IgG_1血清有沉淀反应,免疫电泳检查与兔抗鼠全血清反应只形成一条沉淀弧,纯化的McAb的PHA效价达125000/mg。本法提纯的McAb具有产量高,纯度好,特异性强等优点。     

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.     

牛坏死杆菌43K OMP单克隆抗体的制备及鉴定

目的制备牛坏死杆菌43K OMP单克隆抗体,并鉴定其生物学特性。方法经IPTG诱导表达重组43K OMP,纯化后免疫BALB/c小鼠,将小鼠脾细胞与SP2/0细胞融合,筛选分泌抗43K OMP特异性抗体的杂交瘤细胞,制备腹水,对抗43K OMP单克隆抗体进行效价、亚类、免疫原性及特异性鉴定。结果获得3株稳定表达抗43K OMP单克隆抗体的杂交瘤细胞株,取其中效价最高的1株命名为O43,其重链为IgG1,轻链为κ链,上清和腹水效价分别为1∶25 600和1∶105;该单克隆抗体可与重组43K OMP发生特异性反应,且与转染后瞬时表达43K OMP的BHK-21细胞特异性结合,发生荧光反应。结论成功表达并纯化了43K OMP,制备了牛坏死杆菌43K OMP单克隆抗体,为研究坏死杆菌的感染机制及发病机理奠定了基础。     

抗人CD25人鼠嵌合抗体在CHO细胞中的稳定表达及初步鉴定

目的在CHO细胞中稳定表达抗人CD25人鼠嵌合抗体,并对抗体活性进行初步鉴定。方法采用脂质体法将嵌合抗体真核表达质粒pOptiVEC-H和pcDNA3.3-L共转染CHO-DHFR-细胞,通过去除HT和在培养基中加入500μg/ml的Geneticine进行阳性克隆的筛选,有限稀释法对阳性克隆进行亚克隆,通过在培养基中加入浓度逐步增加的MTX提高克隆的抗体表达量。采用流式细胞术(FCM)检测嵌合抗体的抗原结合活性及人抗体重链Fc段、轻链κ链;提取细胞基因组进行PCR鉴定;抗体经超滤浓缩后,采用蛋白G亲和纯化法进行纯化,并进行Westernblot分析;体外连续培养细胞株2个月及冻存、复苏后,采用ELISA法检测抗体分泌的稳定性。结果获得稳定分泌嵌合抗体的细胞株1C1,ELISA检测抗体表达量为103ng/ml;PCR结果表明,表达的质粒已整合入细胞基因组中;FCM及Westernblot结果显示,嵌合抗体含有人抗体重链Fc段及轻链κ链,且保留了鼠抗体V区的抗原结合特异性;经蛋白G亲和纯化后,获得抗体220μg,纯度>97%;体外连续培养细胞株2个月及冻存、复苏后,抗体分泌保持稳定。结论获得了稳定表达人CD25人鼠嵌合抗体的CHO细胞株,其具有鼠源抗体的抗原结合特异性及人抗体的恒定区。     

从大容量噬菌体抗体库获取人源性抗角蛋白抗体ScFv及Diabody的构建

目的从大容量噬菌体抗体库筛选人源性抗角蛋白抗体,并构建双体抗体(Diabody)。方法以固相化的角蛋白对构建的大容量噬菌体抗体库进行筛选,经3～4轮筛选后,挑取克隆,ELISA法鉴定其特异性,并对部分抗角蛋白阳性抗体克隆基因进行DNA序列分析。选取活性好的克隆基因进行改造,构建Diabody表达载体。结果在抗体库的筛选过程中可见明显的富集现象,获得了29株可与角蛋白特异性结合的人源单链抗体,选取4个克隆基因进行序列分析,结果表明,4个克隆的轻链基因均属于λ轻链第1亚群,1号和8号克隆的重链基因属于人IgG第2亚群,6号和29号克隆分别属于第1和第3亚群。从表达抗角蛋白抗体的集落中挑选一个进行基因改造,构建的Diabody表达载体所表达的Diabody活性较高。结论利用噬菌体抗体技术获得了人源性抗角蛋白抗体,并改造成应用前景较好的Diabody,为开发银屑病治疗性抗体奠定了基础。     

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

新德里金属-β-内酰胺酶的原核表达及其多克隆抗体的制备

目的原核表达新德里金属-β-内酰胺酶(New Delhi metallo-β-lactamase-1,NDM-1),并制备其多克隆抗体。方法以临床分离的产NDM-1的臭鼻克雷伯杆菌为模板,PCR扩增NDM-1基因,克隆入pET-28a载体,构建重组原核表达质粒pET-28a-NDM-1,转化E.coli BL21(DE3)pLyss,IPTG诱导表达重组蛋白。表达的重组蛋白经硫酸铵沉淀、离子交换层析、Ni亲和层析及分子筛层析纯化后,免疫日本大耳白兔,制备NDM-1多克隆抗体。抗体经硫酸铵沉淀和SPA-Sepharose亲和层析纯化后,Western blot鉴定其特异性。结果重组表达质粒pET-28a-NDM-1经PCR及测序鉴定构建正确;表达的重组蛋白相对分子质量约为28 000,诱导表达的重组菌破菌上清存在较强的β-内酰胺酶活性,表明NDM-1蛋白为可溶性形式表达;最终纯化获得的NDM-1蛋白纯度高于95%;制备的NDM-1多克隆抗体能与诱导表达的重组菌胞外蛋白特异性结合。结论成功原核表达了NDM-1,并制备了其多克隆抗体,为NDM-1的快速检测提供了新的思路。     

二硫键稳定的抗HIV-1 gp41单链抗体的构建及原核表达

目的构建二硫键稳定的抗HIV-1 gp41单链抗体(dsFv)基因原核表达载体,并进行表达和鉴定。方法采用PCR定点突变的方法,构建含二硫键稳定的抗HIV-1 gp41单链抗体突变基因质粒pUC57-d41,BamHⅠ和HindⅢ双酶切后,定向插入pET-28a(+),转化大肠杆菌BL21(DE3),IPTG诱导表达,用SDS-PAGE、Western blot鉴定表达产物。对目的蛋白进行纯化和复性,并进行抗原结合活性及相对稳定性检测。结果重组载体pET-d41经酶切鉴定,证实构建正确。表达产物相对分子质量约为28000,与理论预期值完全相符。目的蛋白最高表达量可占菌体总蛋白的45.48%。经Ni-NTA亲和层析法纯化并复性后,蛋白纯度达95%以上,抗HIV-1 gp41 dsFv具有抗原结合活性,稳定性优于scFv。结论已成功构建了二硫键稳定的抗HIV-1 gp41单链抗体(dsFv)基因原核表达载体,并获得表达,为进一步研究其生物学功能奠定了基础。     

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a ‘wound healing’ assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.     

采用基因拼接方法构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库

目的　构建人源特异性抗乙型肝炎病毒表面抗原Fab噬菌体抗体库。方法　从抗乙型肝炎病毒表面抗体高滴度 (1:10 2 4 )的人全血中分离外周血单个核细胞 (PBMC) ,经RT PCR分别扩增出轻链可变区和重链可变区 ,再以噬菌体质粒为模板分别扩增出轻链恒定区 (Cκ)和重链恒定区 (CH1) ,将轻链可变区和轻链恒定区 (Cκ)及重链可变区和重链恒定区 (CH1)进行第 1次基因拼接 ,分别形成κ轻链和Fd重链 ,再以κ轻链和Fd重链作为模板进行第 2次基因拼接 ,形成完整的Fab基因 ,与pComb3H SS噬菌体质粒连接后 ,电穿孔转化大肠杆菌XL1 Blue。结果　通过多次电穿孔转化 ,获得总容量为 4× 10 5库容的噬菌体抗体库。结论　构建的Fab抗体库可用于筛选特异性抗乙型肝炎病毒表面抗原Fab抗体分子     

鼠源性抗EHEC O157:H7 Stx2噬菌体Fab抗体库的构建及筛选

目的构建鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,并从中筛选特异性的抗体。方法用EHEC O157∶H7 Stx2类毒素免疫BALB/c小鼠,取脾分离淋巴细胞,提取总RNA,RT-PCR分别扩增抗体轻、重链(к和Fd)基因,经双酶切依次克隆入噬粒载体pComb3X中,电转化大肠杆菌XL1-Blue,以辅助噬菌体M13K07进行超感染,构建抗EHEC O157∶H7 Stx2的Fab噬菌体抗体库。以纯化的Stx2为抗原进行筛选,获得抗EHECO157∶H7Stx2的特异性Fab抗体,Western blot法检测噬菌体抗体与毒素抗原的结合活性,并对所得阳性克隆进行基因序列分析。结果构建了一个库容为1.56×107的Fab抗体库,筛选出3株特异性较强的阳性克隆,其中2个可与Stx2A1亚单位抗原反应,1个可与Stx2B亚单位抗原反应。基因序列分析显示,轻、重链可变区氨基酸序列与GenBank中已注册的鼠免疫球蛋白可变区氨基酸序列同源性分别为98.5%和99.6%。结论已成功构建了鼠源性抗EHEC O157∶H7 Stx2噬菌体Fab抗体库,为进一步制备抗EHECO157∶H7Stx2的治疗性人源化抗体奠定了基础。     

抗CD25人鼠嵌合抗体基因真核表达质粒的构建与瞬时表达

目的构建抗CD25人鼠嵌合抗体基因真核表达质粒,并在293T细胞中进行瞬时表达。方法采用RT-PCR技术,设计针对信号肽的简并引物,钓取抗CD25杂交瘤细胞的重、轻链可变区基因,以质粒PAG4622为模板,钓取人抗体IgG1重、轻链恒定区基因,分别将重、轻链可变区基因与相应的恒定区基因进行拼接,将完整的重、轻链嵌合基因分别与真核表达载体pOptiVEC和pcDNA3.3连接,构建抗CD25人鼠嵌合抗体基因真核表达质粒,将其通过脂质体法共转染至293T细胞中进行表达。通过RT-PCR法检测嵌合抗体基因的转录水平,ELISA法检测嵌合抗体的表达量,流式细胞术(FCM)分析嵌合抗体的结合活性。结果抗CD25人鼠嵌合抗体基因真核表达质粒构建正确;RT-PCR显示嵌合抗体基因在293T细胞中成功转录;转染后48、72、96和120h,细胞培养上清中嵌合抗体的含量分别为7.47、11.72、8.02和18.28ng/ml;FCM检测其能特异性与IL-2Rα链结合。结论已成功构建了抗CD25人鼠嵌合抗体基因的真核表达质粒,并能在293T细胞中瞬时表达。     

人源化叶酸受体α抗体质控方法的建立

目的建立人源化叶酸受体α抗体的质控方法。方法应用基于表面等离子体共振(Surface plasmon resonance,SPR)技术的BIAcore3000系统测定人源化叶酸受体α单抗与重组人叶酸受体α的亲和力,从而反映该抗体的功能;采用ELISA法测定其抗原结合力;SDS-PAGE和SEC-HPLC测定其纯度;紫外分光光度法测定其蛋白含量;毛细管等电聚焦电泳法测定等电点;其他各项指标检测按《中国药典》三部(2005版)要求进行。结果人源化叶酸受体α单抗成品的平均相对百分效价为105%,RSD为6.0%。单抗成品及参考品与叶酸受体的结合力均存在量效关系,且符合四参数方程式:Y=(A-D)/[1+(X/C)B]+D,其曲线相关系数在0.98以上,成品经3次测定,相对抗原结合力平均值为97%,RSD为25%。还原SDS-PAGE分析显示,单抗成品的IgG重链和轻链百分比为98.2%;非还原SDS-PAGE分析显示,完整IgG百分比为96.7%;SEC-HPLC分析显示,单抗成品单体为99.1%,聚合体为0.9%。其他各项指标均符合《中国药典》三部(2005版)要求。结论已初步建立了人源化叶酸受体α抗体的质控方法,为该制品的质量控制奠定了基础。     

Humanization of 60.3, an anti-CD18 antibody; importance of the L2 loop

Monoclonal antibody 60.3 binds to the CD18 component of theß2 integrin family of adhesion molecules. 60.3 haspotential clinical application in blocking the neutrophilmediatedorgan damage which occurs following a myocardial infarct orhemorrhagic shock. Analysis of the nucleotide and deduced aminoacid sequences of murine 60.3 shows that the light chain containsno amino acid substitutions relative to the closest germlinesequence, while the heavy chain is heavily substituted. We reporthere the humanization of 60.3. The humanized antibody bindsto CD18-bearing cells with {small tilde}4-fold less affinitythan the murine or chimeric antibody. We have shown that modificationof amino acid L50 in the L2 loop of the humanized antibody resultsin loss of binding, while modification of a structural determinant(H71) for the H2 loop has no effect.     

抗ICAM-1单克隆抗体的制备及其活性

目的制备具有生物活性的抗人细胞间黏附分子-1(ICAM-1)单克隆抗体。方法以人ICAM-1为抗原免疫BALB/c小鼠,采用杂交瘤技术并经HAT选择培养和克隆化,筛选出稳定分泌抗人ICAM-1 McAb的杂交瘤细胞株,并对McAb进行纯化。用ELISA间接法测定效价并鉴定其亚类,Western blot鉴定其抗原特异性,细胞黏附试验检测其中和活性。结果筛选出1株可稳定分泌抗人ICAM-1抗体的杂交瘤细胞株3F2,杂交瘤染色体众数为98~104。纯化后的单抗蛋白浓度为1.253 mg/ml,纯度达83.6%,效价可达2.56×105。其分泌的抗体亚型为IgG1,腹水效价达5.12×105,可与ICAM-1特异性结合,可抑制ICAM-1与淋巴瘤细胞间的黏附,具有明显的中和ICAM-1的活性。结论已成功制备出抗ICAM-1的McAb,为其进一步的研究和应用奠定了基础。