双特异性单链抗体

双特异性单链抗体是新型基因工程抗体 ,在疾病诊断、治疗上有巨大临床应用潜力。目前已有较多各种双特异性单链抗体成功构建的报道。本文对其概念、制作方法及应用前景进行了综述     

PLGA纳米颗粒偶联的双特异性抗体制备及功能研究

目的制备一种能提高T细胞对癌细胞杀伤能力的双特异性抗体。方法利用生物可降解材料PLGA(聚乳酸-羟基乙酸共聚物)制备成纳米颗粒,然后利用化学方法在纳米颗粒表面偶联能靶向结合T细胞的CD3抗体和癌细胞标志物MUC1的抗体,利用T细胞对癌细胞的杀伤实验来检测其功能。结果成功制备出直径200 nm的PLGA纳米颗粒并偶联上CD3和MUC1抗体,功能检测得出制备的双特异性抗体能显著提高T细胞对癌细胞的杀伤率。结论制备出一种能显著提高T细胞对癌细胞杀伤率的双特异性抗体。     

自然产生的双特异性抗体——山羊抗血清中HBsAg双功能抗体的观察

在用纯ＨＢｓＡｇ免疫山羊而产生的抗血清中，发现存在一种既能同ＨＢｓＡｇ反应又能同时同正常人血清中某一成分起反应的微量的特殊抗体，验证试验的结果，证实了这一特殊抗体是一种双功能抗体，同这种功能抗体起反主尖的正常人血清成分可能是α１－抗胰蛋白酶。     

双特异性抗体对LAK细胞增殖和细胞毒作用影响的体…

将抗ＣＤ１３与抗ＨＢｓ的单克隆抗体经化学偶联得到的双特异性抗体，观察该双特异性抗体对ＬＡＫ细胞增殖和增强细胞毒性的作用。结果显示双特异性抗体显著提高ＬＡＫ细胞与２．２．１５细胞结合率；促进淋巴细胞增殖．＾１２５Ｉ－ＵｄＲ释放试验检测发现双特异性抗体增强ＬＡＫ细胞对２．２．１５细胞的细胞毒性且与抗体浓度呈正相关 。     

化学交联的抗TNF和抗CALLA双功能抗体的制备及其导向功能研究

本文探索了用抗 CALLA 和抗 TNF 的双功能抗体导向 TNF 杀伤 CALLA 阳性白血病细胞的可行性.用化学交联剂 SPDP 制备了抗 TNF 和抗 CALLA 抗体的交联物,经鉴定为双功能抗体;与 TNF 联合使用,在 CALLA 阳性白血病细胞系 nalm—6上进行体外杀伤实验。MTT 比色法测定杀伤效果。我们发现经双功能抗体导向后,nalm—6细胞对 TNF 的敏感性明显增加。     

抗人喉癌/抗VEGF双功能抗体制备及应用研究

目的：研制抗人喉癌／抗血管内皮因子（VEGF）双功能克隆抗体，用于喉癌抗血管生成治疗。方法：采用二次杂交瘤技术制备抗人喉癌／抗VEGF双功能抗体。经酶联免疫吸附试验法和SP法检测喉癌及癌前病患者血清及癌组织中VEGF的含量表达。结果：获得6株分泌抗人喉癌／抗VEGF双功能抗体的杂交瘤，经免疫组化证实与喉癌细胞特异性结合率为93％，而与血管内皮细胞结合率为89％。血清中VEGF含量表达，喉癌组与癌前病组及正常对照组相比差异均显著。IgG亚型鉴定为IgG2aBSAb抗体效价为1：25 600倍（ELISA法）。结论：二次杂交瘤法制备的双功能抗体具有均匀性、可控性、效价高、稳定性好，可用于喉癌抗血管生成治疗，动态检测可作为判断喉癌预后的客观指标。     

双功能抗体药物研究进展

抗体类药物近20年来蓬勃发展,目前全球上市的抗体药物已经有40多个品种,其治疗领域也从传统的癌症、自身免疫性疾病逐步扩展到抗感染和代谢性疾病等。2013年全球10大畅销药物中有6个是抗体类药物,包括3个自身免疫病治疗性药物和3个抗肿瘤抗体。单克隆抗体发展的同时也开启了对新结构、新功能抗体药物的探索,以期进一步优化抗体药物功能活性。抗体糖基化改造(afucosylation)、抗体-药物偶联(antibody drug conjugate,ADC)、双功能抗体(bispecific antibodies,BsAb)等都是当前抗体药物研发的热点领域。单克隆抗体能够特异性结合     

化学偶联双特异性抗体的制备及鉴定

用化学偶联的方法,制备了抗鼠IgM-抗破伤风类毒素双特异性抗体复合物,以此作为研究B细胞抗原递呈作用的工具及模拟抗原特异性B细胞的高效抗原递呈作用。经ELISA鉴定以及通过ELISA竞争抑制试验和中和抑制试验证实,此抗体复合物确有识别两种不同抗原的双特异性。     

基因工程双功能抗体的分子设计及表达

基因工程双功能抗体在临床肿瘤、病毒病等治疗和诊断方面具有广阔的应用前景,是抗体研究的热点之一。要获得有活性的双功能产物,关键在于分子设计及表达系统的选择。本文着重论述近年来基因工程双功能抗体在分子设计及表达研究方面的新进展。     

IgG型抗CD55×抗β-Gal基因工程双特异性抗体的构建

目的　探讨用生物学方法治疗病毒感染性疾病及肿瘤的新途径。方法　以有重要补体活化调节功能的膜补体调节蛋白CD5 5为靶点 ,以 β GaL为模拟病毒或肿瘤抗原 ,制备“IgG”型抗CD5 5×抗 β Gal基因工程双特异性抗体 ,并对该重组抗体真核表达后的结合活性进行初步的验证。结果　克隆的CD5 5抗体可变区片段为新的小鼠抗体可变区片段 ,经HEK 2 93细胞表达后的重组抗体显示了良好的CD5 5及Fc结合活性。结论　本研究为病毒性疾病或肿瘤的免疫学治疗提供了新的途径及实验依据     

A recombinant bispecific single-chain antibody induces targeted,supra-agonistic CD28-stimulation and tumor cell killing

Endowing tumor cells with costimulatory signals for T cell activation has emerged as a promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor-associated antigens, to achieve targeted T cell costimulation in vivo. Here we report the production and purification of rM28, a recombinant bispecific single-chain antibody directed to a melanoma-associated proteoglycan and to the costimulatory CD28 molecule on human T cells. We found that a dimer of the recombinant molecule, bound to tumor target cells, induced pronounced T cell activation in peripheral blood mononuclear cell preparations without additional TCR/CD3 stimulation being required. The lytic activity generated after 3 days of stimulation effectively prevented tumor cell growth. However, it was unspecific and predominantly mediated by non T cells. Our findings demonstrate that presentation of a CD28 antibody within a suitable recombinant, bispecific format may result in a "targeted supra-agonistic stimulation" of the CD28 molecule, which leads to effective tumor cell killing after induction of unspecifically lytic cells.     

双特异性抗体对LAK细胞增殖和细胞毒作用影响的体外研究

将抗ＣＤ３与抗ＨＢｓ的单克隆抗体经化学偶联得到双特异性抗体，观察该双特异性抗体对ＬＡＫ细胞增殖和增强细胞毒性的作用。结果显示双特异性抗体显著提高ＬＡＫ细胞与２．２．１５细胞结合率；促进淋巴细胞增殖。１２５Ｉ－ＵｄＲ释放试验检测发现双特异性抗体增强ＬＡＫ细胞对２．２．１５细胞的细胞毒性且与抗体浓度呈正相关。进一步对抗体的特异性研究表明，加入双特异性抗体后ＬＡＫ细胞对２．２．１５细胞毒性作用显著高于对照组。     

抗皂角毒、表皮生长因子受体双功能抗体的制备与体外效应的研究

目的：研究制备抗皂角毒和表皮生长因子受体双功能抗体,用以导向皂角毒杀伤肿瘤细胞。方法：在制备ＥＱ７５、ＣＹ１２．１４单克隆抗体细胞株的基础上,通过杂交杂交瘤技术制备双功能抗体细胞株。结果：双功能抗体（ＥＱ７５×ＣＹ１２．１４）与单用皂角毒杀伤肿瘤细胞（Ａ４３１）的ＩＣ５０分别为１０－９．６、１０－８．４ｍｏｌ／Ｌ。结论：双功能抗体（ＥＱ７５×ＣＹ１２．１４）杀伤肿瘤细胞（Ａ４３１）,以ＩＣ５０值计算,有效率为单用皂角毒的１５倍左右。     

抗人膀胱癌/抗VEGF双功能基因抗体制备及体外效应研究

目的：研究制备抗人膀胱癌和抗血管内皮生长因子的双功能抗体，用以导向抑制肿瘤新生血管的形成。方法：在制备抗人膀胱癌和抗血管内皮细胞生长因子单克隆抗体的基础上制备双功能抗体。结果：双功能抗体的IC50为10^-9.5，抗血管内皮生长因子抗体的IC50为10^-8.9，抗人膀胱移行细胞癌单克隆抗体的IC50为10^-8.3。结论：双功能抗体的有效率明显高于单克隆抗体。     

抗人卵巢癌×抗人CD3双特异单链抗体介导的效应细胞在体外对卵巢癌细胞的杀伤作用

目的　研究双特异单链抗体 (scBsAb)介导的Jurkat细胞 (CD3+ )及人外周血单个核细胞(PBMC)在体外对人卵巢癌细胞株SKOV3细胞的杀伤活性 ,从而探讨其用于卵巢癌治疗的可能性。方法以抗人卵巢癌×抗人CD3scBsAb激活效应细胞 ,以SKOV3为靶细胞 ,用MTT法测定不同实验条件下的杀伤活性。结果　 (1)以重组白细胞介素 2 (rIL 2 )、抗CD3单克隆抗体、抗CD2 8重链单域抗体 (VH)预刺激Jurkat及PBMC细胞比单纯用scBsAb激活效应细胞杀伤活性高 ;(2 )以Jurkat细胞或PBMC细胞作为杀伤细胞可得到相似的杀伤活性 ,最高杀伤率均在 75 %左右 ;(3)杀伤活性与scBsAb的浓度、作用时间及效靶比有关。对于Jurkat细胞 ,在抗体终浓度为 2 1μg ml,反应时间为 4 8h ,效靶比为 10∶1时达到最大杀伤率 74 .8% ;对于PBMC细胞 ,在抗体浓度为 2 0 μg ml,反应时间为 72h ,效靶比为 1∶1时达到最大杀伤率73.1%。 (4 )多因子联合应用能有效提高杀伤活性。结论　scBsAb在体外能够有效介导效应细胞杀伤肿瘤细胞 ,有明显的抗癌作用 ,具有潜在的应用前景。     

抗人膀胱癌/抗VEGF双功能基因抗体导入治疗荷瘤鼠膀胱癌的研究

目的：探讨抗人膀胱癌，抗VEGF双功能基因抗体对膀胱癌生长的抑制作用。方法：应用双功能抗体作用荷瘤鼠模型，观察抑瘤作用；应用免疫组化技术检测体内生长肿瘤中微血管密度。结果：双功能抗体对体内生长的肿瘤有明显的抑制作用，经双功能抗体治疗的瘤体中微血管密度明显低于对照组。结论：双功能抗体可以抑制肿瘤血管生成，有望为膀胱癌的治疗提供新的方法。     

Development and clinical application of bispecific antibody in the treatment of colorectal cancer

ABSTRACT

Introduction

Treatment of colorectal cancer as one of the most commonly diagnosed and a frequent cause of cancer-related deaths is of great challenges in health-related issues.     

Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. HBV envelope contains three surface antigens, called pre-S1, pre-S2 and S. For the prophylaxis of HBV infection, only an anti-S monoclonal antibody was tested for the protective efficacy against HBV infection, but it was shown to be incomplete. In addition, some immune escape mutants carrying mutations on the S antigen were reported. Therefore, a multivalent bispecific antibody rather than a single monoclonal antibody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites for each of the S and pre-S2 antigens. Each of the antigen-binding sites was composed of a single-chain Fv (ScFv). The tetravalent antibody was generated by constructing a single gene encoding a single-chain protein. This protein consisted of an anti-S ScFv whose carboxyl end was tethered, through a 45 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human γ1 constant region. The single-chain protein was expressed in Chinese hamster ovary cells and secreted in culture supernatant as a homodimeric molecule. The tetravalent bispecific antibody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater than that of either parental antibody. The tetravalent bispecific antibody is a potentially useful reagent for the prevention and treatment of HBV infection.     

One-step mixing with humanized anti-mPEG bispecific antibody enhances tumor accumulation and therapeutic efficacy of mPEGylated nanoparticles

Methoxy PEGylated nanoparticles (mPEG-NPs) are increasingly used for cancer imaging and therapy. Here we describe a general and simple approach to confer tumor tropism to any mPEG-NP. We demonstrate this approach with humanized bispecific antibodies (BsAbs) that can bind to both mPEG molecules on mPEG-NPs and to EGFR or HER2 molecules overexpressed on the surface of cancer cells. Simple mixing of BsAbs with mPEG-NPs can mediate preferential binding of diverse mPEG-NPs to cancer cells that overexpress EGFR or HER2 under physiological conditions and significantly increase cancer cell killing by liposomal doxorubicin to EGFR⁺ and HER2⁺ cancer cells. BsAbs modification also enhanced accumulation of fluorescence-labeled NPs and significantly increased the anticancer activity of drug-loaded NPs to antigen-positive human tumors in a mouse model. Anti-mPEG BsAbs offer a simple one-step method to confer tumor specificity to mPEG-NPs for enhanced tumor accumulation and improved therapeutic efficacy.