一、Bispecific antibodies introduction

Bispecific antibodies usually do not occur in nature but are constructed by recombinant DNA or cell-fusion technologies. Most are designed to recruit cytotoxic effector cells of the immune system effectively against pathogenic target cells. This complex task explains why, after more than 15 years of extensive research, many different formats of bispecific antibodies have been developed but only a few have advanced to clinical trials. Here, we give a brief history of bispecific antibodies and review very recent progress towards formats that are beginning to solve the major issues of earlier formats. These improved bispecific antibodies are expected to show clinical efficacy in patients with cancer and other diseases, in a way that monoclonal antibodies have shown in recent years.

Until now, the hybridoma technology invented by Ko¨hler and Milstein to generate monoclonal antibodies has nourished the hope for therapeutic breakthroughs in diseases with high medical needs not served sufficiently by conventional therapies. The hallmark of monoclonal antibodies is their specific binding to a particular antigen, which enables them to find their target precisely in vivo while ignoring antigen-negative sites. Bound to a target, therapeutic antibodies can deliver a toxic payload, act as agonists or antagonists of receptors, or as neutralizers of ligands. Antibodies might even bind many targets that are not recognizable by small-molecule drugs.

Monoclonal antibodies of the IgG type contain two identical antigen-binding arms and a constant fragment, (Fc)g. The Fc part enables the antibody to function as an adaptor protein, linking antibody-bound cells to immune cells bearing Fcg receptors. Because there are different Fcg receptors and other proteins binding to Fc portions of antibodies, such as complement, monoclonal antibodies can mediate multiple effects ranging from the recruitment of immune effector functions to mere increase of serum half-life by retention of IgG on non-signaling Fc receptors. It was observed recently that human antibodies of the IgG4 type can exchange their halves with each other, potentially creating antibodies with dual specificity [2]. However, the biological relevance of this observation remains obscure.

For treatment of malignant diseases, monoclonal antibodies typically need to be modified to enhance efficacy and to use them in humans. One important modification is the reduction of immunogenicity of rodent monoclonal antibodies by chimerization, humanization throughgrafting of complementarity determining regions (CDRs) or using various technologies for recovery of fully human antibodies, such as phage display libraries or transgenic mice expressing human antibody repertoires. Reduced immunogenicity of antibodies can prolong their half life and, in the absence of a neutralizing immune response, enable prolonged treatment. Another important modifi-cation is arming the humanized antibody with additional cytotoxic mechanisms, be it radioisotopes, bacterial toxins,

inflammatory cytokines, chemotherapeutics or prodrugs. There is a growing number of approved cancer therapeutics that are efficacious either as chimerized antibody or humanized IgG1,or as conjugate with chemotherapeutics or a radioisotope. In spite of this progress, the efficacy of monoclonal antibodies for cancer treatment is still limited, leaving great potential for further improvements. One class of antibody derivatives with the promise of enhanced potency for cancer treatment are bispecific antibodies.

二、双特异抗体简介

双特异抗体（Bispecific antibody）是含有两个不同配体结合位点的免疫球蛋白分子。自然状态下不存在双特异性抗体，只能通过特殊方法进行制备。以往双特异抗体的制备方法有化学交联法，杂合F（ab'）2 分子法和鼠杂交瘤法等。化学交联法生产双特异抗体的异源性，批与批之间的不稳定性，以及抗体特异性易受某些修饰或不当连接而改变的特性，使得该法生产的双特异抗体不适于体内使用。以巯基交联蛋白酶消化片断F（ab'）生产的双特异杂交分子，成分虽较均一，但费时费力，且产量很低。杂交瘤法生产的双特异抗体，来源可靠，但由轻链、重链随机配对产生的多种可能抗体形式，使得双特异抗体生产、纯化变得非常困难。

随着基因工程抗体技术研究的深入，尤其是单链抗体的出现，为基因工程双特异单链抗体的研制奠定了基础。单链抗体（Single2Chain Fv，scFv）是利用DNA 重组技术将抗体重链可变区（VH）和轻链可变区（VL）基因通过一短肽链（linker）连接后融合表达出来的抗体片断。近年来，将噬菌体展示技术（phage display）应用于svFv 筛选，可直接从杂交瘤和外周血淋巴细胞提取mRNA，构建单链抗体库，从而更易获得高特异性、高亲合力scFv。scFv 有与天然抗体相同的抗原结合特征，同时缺乏Fc段，具有分子量小，穿透力强，体内循环半衰期短及免疫原性低等特点，且易与效应分子相连构建多种新功能抗体分子，是构建免疫毒素或双特异抗体的理想元件。因而，近年来scFv 已成为抗体研究领域内的热点。根据不同研究、应用目的，采用基因工程、蛋白质工程方法，将两条不同来源的svFv 组合成具有两种不同抗原结合特征的新型抗体即为双特异单链抗体（bispecific single2chain Fvs ，bisFvs）。bisFvs 分子是仅相当于F（ab）大小，由于其具有独特的与两个抗原位点结合的能力，因此无论是作为导向药物载体，效应细胞识别、连接，还是作为免疫阻断抗体，免疫诊断试剂等等，都具有更为广阔的应用前景。目前，人们已在许多领域，尤其是肿瘤的诊断、治疗等方面对bisFvs 的应用进行了偿试。

三、双特异单连抗体的制作方法

制备bisFvs 的核心是将两条scFv 以一定方式连接起来，并使其各自保留与特异性抗原结合的能力。长期以来，随着抗体工程技术的发展，人们对基因工程bisFvs 的制作方法进行了多种探讨，逐步摸索出了一些成功的制作途径。按bisFvs 分子连接方式的不同，可将这些途径归为3 类

1、非共价健二聚体

这一方法最早由Huston 以及Holliger 等创建，制备出名为“双体”（diabody）的双特异抗体，随后在一些研究中得到进一步探讨和应用。其方法是用一短的氨基酸linker （3 - 15 氨基酸）将一抗体的重链（VHA）与另一抗体的轻链（VLB ）连接起来，构成杂合scFv ，同样再以VLA 和VHB 构建杂合scFv ，两条杂合scFv 在同一表达系统，同时分别表达，由于短的linker 的限制，同一条肽链内的两个V 区之间不能匹配，只能与另一条杂合scFv 中相应同源V 区相匹配，重新聚合成具有两个抗原结合位点的二聚体。通过分泌性原核表达体系，可直接获得有功能的bisFvs 分子。经计算机摸拟分析，两scFv 呈两个位点相背的空间结构。另外，研究表明，减少linker 长度至3 个氨基酸以下，还可获得三聚体或四聚体多特异性抗体。该设计方法已成功应用于肿瘤特异性抗原及效应细胞相互作用等多项研究中。然而，也有人认为，该设计中片段之间为非共价连接，其稳定性较差；短的linker 将限制其柔韧性，并进而对两细胞间连接造成负面影响。在折叠过程中，非匹配的VH、VL 片段之间的相互作用也可能对双特异抗体的形成产生不利影响，且体系中会有一些单体及不同聚合体成份的污染。

2、共价连接双特异单链抗体

该方法在首先获得有功能的scFv的基础上，根据特定研究目的，将两种具有不同抗原结合特征的scFv ，用一段多肽linker直接连接起来，在原核或真核表达体系进行表达，经必要的复性或纯化过程，就可获得bisFvs。该方法的关键是要选择有一定的柔韧性，不影响两端scFv复性、结合特征的适当linker。目前用于该设计方法的linker 有：25 氨基酸残基的205c linker，以2C11CH1片段为主的23 氨基酸残基linker，24氨基酸残基的CBH1 linker，114氨基酸残基的ETA Ⅱ区段linker，（Gly4Ser）3及Ser2 （Gly4Ser）3linker等，均获得了有功能的双特异抗体分子。Coloma等将一种scFv直接融合表达于另一种scFvC 端或铰链区之后也获得了有功能的bis2Fv。Helfrich 等还构建了专门用于表达bisFvs的载体，可直接将两个scFv片段克隆于该载体的两个克隆位点，两位点之间是一固定的25 氨基酸残基linker，经克隆表达，就可生产出各种bisFvs ，使得这一过程得以程式化。由于本设计中，两scFv之间以共价键相连，因而相对于diabody ，其稳定性会有所提高，更易于纯化和大量生产。较长片段linder的应用也使两抗体间有较大的自由度。

3、应用亮氨酸拉链、螺旋－转角－螺旋等蛋白质结构域将两单链抗体连接起来

Kruif等将小鼠IgGC3上段铰链区和Fos或Jun亮氨酸拉链区融合于scFv蛋白，建立了依赖亮氨酸拉链的二聚化设计方案，可以将从噬菌体抗体筛选出的scFv直接克隆入该系统，获得二聚化biscFvs。Kostelny、Pack等也都以亮氨酸拉链结构域为基础成功获得了bisFvs。Kalinke、Pack等将螺旋－转角－螺旋结构融合于两条单链抗体C端经大肠杆菌表达系统，就可聚合表达出bisFvs。Dubel等将核心2链亲合素与scFv片段融合表达，该嵌合蛋白可形成四聚体，其C端插入的半胱氨酸，使其具有形成共价双功能分子的能力。