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、雙特異抗體簡(jiǎn)介

雙特異抗體（Bispecific antibody）是含有兩個(gè)不同配體結(jié)合位點(diǎn)的免疫球蛋白分子。自然狀態(tài)下不存在雙特異性抗體，只能通過特殊方法進(jìn)行制備。以往雙特異抗體的制備方法有化學(xué)交聯(lián)法，雜合F（ab'）2 分子法和鼠雜交瘤法等。化學(xué)交聯(lián)法生產(chǎn)雙特異抗體的異源性，批與批之間的不穩(wěn)定性，以及抗體特異性易受某些修飾或不當(dāng)連接而改變的特性，使得該法生產(chǎn)的雙特異抗體不適于體內(nèi)使用。以巰基交聯(lián)蛋白酶消化片斷F（ab'）生產(chǎn)的雙特異雜交分子，成分雖較均一，但費(fèi)時(shí)費(fèi)力，且產(chǎn)量很低。雜交瘤法生產(chǎn)的雙特異抗體，來源可靠，但由輕鏈、重鏈隨機(jī)配對(duì)產(chǎn)生的多種可能抗體形式，使得雙特異抗體生產(chǎn)、純化變得非常困難。

隨著基因工程抗體技術(shù)研究的深入，尤其是單鏈抗體的出現(xiàn)，為基因工程雙特異單鏈抗體的研制奠定了基礎(chǔ)。單鏈抗體（Single2Chain Fv，scFv）是利用DNA 重組技術(shù)將抗體重鏈可變區(qū)（VH）和輕鏈可變區(qū)（VL）基因通過一短肽鏈（linker）連接后融合表達(dá)出來的抗體片斷。近年來，將噬菌體展示技術(shù)（phage display）應(yīng)用于svFv 篩選，可直接從雜交瘤和外周血淋巴細(xì)胞提取mRNA，構(gòu)建單鏈抗體庫(kù)，從而更易獲得高特異性、高親合力scFv。scFv 有與天然抗體相同的抗原結(jié)合特征，同時(shí)缺乏Fc段，具有分子量小，穿透力強(qiáng)，體內(nèi)循環(huán)半衰期短及免疫原性低等特點(diǎn)，且易與效應(yīng)分子相連構(gòu)建多種新功能抗體分子，是構(gòu)建免疫毒素或雙特異抗體的理想元件。因而，近年來scFv 已成為抗體研究領(lǐng)域內(nèi)的熱點(diǎn)。根據(jù)不同研究、應(yīng)用目的，采用基因工程、蛋白質(zhì)工程方法，將兩條不同來源的svFv 組合成具有兩種不同抗原結(jié)合特征的新型抗體即為雙特異單鏈抗體（bispecific single2chain Fvs ，bisFvs）。bisFvs 分子是僅相當(dāng)于F（ab）大小，由于其具有*的與兩個(gè)抗原位點(diǎn)結(jié)合的能力，因此無論是作為導(dǎo)向藥物載體，效應(yīng)細(xì)胞識(shí)別、連接，還是作為免疫阻斷抗體，免疫診斷試劑等等，都具有更為廣闊的應(yīng)用前景。目前，人們已在許多領(lǐng)域，尤其是腫瘤的診斷、治療等方面對(duì)bisFvs 的應(yīng)用進(jìn)行了償試。

三、雙特異單連抗體的制作方法

制備bisFvs 的核心是將兩條scFv 以一定方式連接起來，并使其各自保留與特異性抗原結(jié)合的能力。長(zhǎng)期以來，隨著抗體工程技術(shù)的發(fā)展，人們對(duì)基因工程bisFvs 的制作方法進(jìn)行了多種探討，逐步摸索出了一些成功的制作途徑。按bisFvs 分子連接方式的不同，可將這些途徑歸為3 類

1、非共價(jià)健二聚體

這一方法zui早由Huston 以及Holliger 等創(chuàng)建，制備出名為“雙體”（diabody）的雙特異抗體，隨后在一些研究中得到進(jìn)一步探討和應(yīng)用。其方法是用一短的氨基酸linker （3 - 15 氨基酸）將一抗體的重鏈（VHA）與另一抗體的輕鏈（VLB ）連接起來，構(gòu)成雜合scFv ，同樣再以VLA 和VHB 構(gòu)建雜合scFv ，兩條雜合scFv 在同一表達(dá)系統(tǒng)，同時(shí)分別表達(dá)，由于短的linker 的限制，同一條肽鏈內(nèi)的兩個(gè)V 區(qū)之間不能匹配，只能與另一條雜合scFv 中相應(yīng)同源V 區(qū)相匹配，重新聚合成具有兩個(gè)抗原結(jié)合位點(diǎn)的二聚體。通過分泌性原核表達(dá)體系，可直接獲得有功能的bisFvs 分子。經(jīng)計(jì)算機(jī)摸擬分析，兩scFv 呈兩個(gè)位點(diǎn)相背的空間結(jié)構(gòu)。另外，研究表明，減少linker 長(zhǎng)度至3 個(gè)氨基酸以下，還可獲得三聚體或四聚體多特異性抗體。該設(shè)計(jì)方法已成功應(yīng)用于腫瘤特異性抗原及效應(yīng)細(xì)胞相互作用等多項(xiàng)研究中。然而，也有人認(rèn)為，該設(shè)計(jì)中片段之間為非共價(jià)連接，其穩(wěn)定性較差；短的linker 將限制其柔韌性，并進(jìn)而對(duì)兩細(xì)胞間連接造成負(fù)面影響。在折疊過程中，非匹配的VH、VL 片段之間的相互作用也可能對(duì)雙特異抗體的形成產(chǎn)生不利影響，且體系中會(huì)有一些單體及不同聚合體成份的污染。

2、共價(jià)連接雙特異單鏈抗體

該方法在首先獲得有功能的scFv的基礎(chǔ)上，根據(jù)特定研究目的，將兩種具有不同抗原結(jié)合特征的scFv ，用一段多肽linker直接連接起來，在原核或真核表達(dá)體系進(jìn)行表達(dá)，經(jīng)必要的復(fù)性或純化過程，就可獲得bisFvs。該方法的關(guān)鍵是要選擇有一定的柔韌性，不影響兩端scFv復(fù)性、結(jié)合特征的適當(dāng)linker。目前用于該設(shè)計(jì)方法的linker 有：25 氨基酸殘基的205c linker，以2C11CH1片段為主的23 氨基酸殘基linker，24氨基酸殘基的CBH1 linker，114氨基酸殘基的ETA Ⅱ區(qū)段linker，（Gly4Ser）3及Ser2 （Gly4Ser）3linker等，均獲得了有功能的雙特異抗體分子。Coloma等將一種scFv直接融合表達(dá)于另一種scFvC 端或鉸鏈區(qū)之后也獲得了有功能的bis2Fv。Helfrich 等還構(gòu)建了專門用于表達(dá)bisFvs的載體，可直接將兩個(gè)scFv片段克隆于該載體的兩個(gè)克隆位點(diǎn)，兩位點(diǎn)之間是一固定的25 氨基酸殘基linker，經(jīng)克隆表達(dá)，就可生產(chǎn)出各種bisFvs ，使得這一過程得以程式化。由于本設(shè)計(jì)中，兩scFv之間以共價(jià)鍵相連，因而相對(duì)于diabody ，其穩(wěn)定性會(huì)有所提高，更易于純化和大量生產(chǎn)。較長(zhǎng)片段linder的應(yīng)用也使兩抗體間有較大的自由度。

3、應(yīng)用亮氨酸拉鏈、螺旋－轉(zhuǎn)角－螺旋等蛋白質(zhì)結(jié)構(gòu)域?qū)蓡捂溈贵w連接起來

Kruif等將小鼠IgGC3上段鉸鏈區(qū)和Fos或Jun亮氨酸拉鏈區(qū)融合于scFv蛋白，建立了依賴亮氨酸拉鏈的二聚化設(shè)計(jì)方案，可以將從噬菌體抗體篩選出的scFv直接克隆入該系統(tǒng)，獲得二聚化biscFvs。Kosny、Pack等也都以亮氨酸拉鏈結(jié)構(gòu)域?yàn)榛A(chǔ)成功獲得了bisFvs。Kalinke、Pack等將螺旋－轉(zhuǎn)角－螺旋結(jié)構(gòu)融合于兩條單鏈抗體C端經(jīng)大腸桿菌表達(dá)系統(tǒng)，就可聚合表達(dá)出bisFvs。Dubel等將核心2鏈親合素與scFv片段融合表達(dá)，該嵌合蛋白可形成四聚體，其C端插入的半胱氨酸，使其具有形成共價(jià)雙功能分子的能力。