抗人P185^erbB2的scFv—Fc融合蛋白的表达及免疫功能分析

为提高鼠源单抗用于体内治疗的效应功能及降低人抗鼠抗体反应 ,将编码抗人P185 erbB2 单抗轻、重链可变区 (VL、VH)融合构建的单链抗体 (scFv)基因片段与人IgG1的Fc区基因片段融合构建了scFv Fc融合基因 ,并将其克隆到哺乳动物细胞表达载体pCIDN中。用重组载体转染CHO细胞 ,通过G4 18筛选获得稳定高表达克隆。更换无血清培养基培养 ,经重组蛋白质A亲和层析柱纯化scFv Fc融合蛋白。融合蛋白质在还原SDS PAGE中表现为5 2kD的条带 ;与SK BR 3细胞裂解液共培育可特异性地沉淀出P185 erbB2 蛋白 ;FACS分析表明融合蛋白质识别P185 erbB2 蛋白的胞外段 ;ELISA测定融合蛋白质对细胞表面抗原P185 erbB2 的亲和常数为 7.5× 10 -10 (mol/L) -1。构建scFv Fc融合基因 ,将其克隆到表达载体 ,进一步稳定表达抗P185 erbB2 的scFv Fc融合蛋白 ,为进一步的体外、体内研究鼠源单抗治疗效果创造了条件。     

人源化抗肝癌单链抗体与牛蛙核糖核酸酶融合基因的构建及表达

利用RT-PCR的方法从牛蛙肝脏中克隆牛蛙核糖核酸酶(RC—RNase)基因并进行序列测定;将人源化抗肝癌单链抗体(scFv)基因与RC—RNase基因相连接,制备scFv—RC—RNase融合基因表达质粒,转化大肠杆菌B121(DE3),用IPTG诱导进行表达。以SDS-PAGE和免疫印迹法对表达产物进行分析鉴定。序列分析表明,扩增出的RC—RNase基因片断大小约为405bpc,经SDS—PAGE和免疫印迹分析显示,scFv—RC—RNase融合基因表达质粒在大肠杆菌中的诱导表达产物出现相对分子质量约为38000的一条新生蛋白带,与预期结果相符。融合蛋白表达量占菌体总蛋白量的18．5％,主要以包涵体形式存在。表明成功地构建了抗肝癌scFv—RC—RNase融合基因,并在大肠杆菌中获得有效表达,为进一步进行肝癌的导向治疗研究奠定了基础。     

抗人CD3单链抗体四聚体基因的构建及表达

为构建和表达抗人CD3单链抗体 (scFv) 人p5 3四聚功能域融合基因 ,选用人IgG3上游铰链区作为抗人CD3scFv和人p5 3四聚功能域之间连接的linker .利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC18载体中构建pUC18 IgG3 p5 3克隆载体 .将抗人CD3scFv克隆入pUC18 IgG3 p5 3载体中 ,构建抗人CD3scFv 人p5 3四聚功能域融合基因 .经酶切鉴定及序列测定证实后 ,将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后利用流式细胞仪进行亲和活性测定 .获得了抗人CD3scFv 人p5 3四聚功能域融合基因 ,基因全长 882bp ,可编码 2 94个氨基酸 ,与已发表的抗人CD3scFv、人IgG3上游铰链区和人p5 3四聚功能域基因cDNA序列一致 .表达产物经SDS PAGE和Western印迹实验证实为约 35kD的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合人外周血单个核细胞 (PBMC)细胞 ,亲和力高于scFv ,为进一步临床应用奠定基础     

抗MRP3单链抗体-sTRAIL融合蛋白诱导U251多形性胶质母细胞瘤凋亡

采用重组PCR技术获得抗多药耐药相关蛋白3(multidrug resistance protein 3, MRP3)的单链抗体(scFv)与人源可溶性肿瘤坏死因子相关凋亡诱导配体(soluble TNF-related apoptosis inducing ligand, sTRAIL)的融合蛋白质的基因编码序列, 利用原核表达载体pMAL-c2,构建含麦芽糖结合蛋白(maltose binding protein, MBP)标签肽的antiMRP3(scFv)-sTRAIL融合蛋白, 经亲和层析柱纯化. 获得纯化的antiMRP3(scFv)-sTRAIL融合蛋白,用MRP3阳性U251多形性胶质母细胞瘤做增殖抑制实验、细胞凋亡诱导实验,结果均显示具有明显的活性, 而MBP无明显作用. 上述结果表明,成功表达了antiMRP3(scFv)-sTRAIL融合蛋白, 该融合蛋白具有诱导U251多形性胶质母细胞瘤细胞凋亡的活性, 为开发靶向性抗肿瘤药物奠定了基础.     

抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。     

人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达

在原核系统中高效表达抗狂犬病毒单链抗体scFv41,以便进一步研究其生物学功能,预测临床应用前景。以重组质粒pCANTABscFv41为模板,PCR扩增带NcoI和NotI位点的scFv41基因,克隆入原核表达载体pET-22b( ),酶切鉴定重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。竞争ELISA检测表达蛋白的特异结合活性。酶切鉴定证实scFv41基因已插入原核表达载体pETl-22b( ),重组表达质粒pET-scFv41在大肠杆菌BL21(DE3)中获得了高效表达,表达量约占菌体蛋白总量的30％。竞争ELISA检测结果表明scFv41表达蛋白可特异抑制抗狂犬病毒IGY与狂犬病毒的特异性结合。该实验为进一步研究scFv41的生物学特性和免疫保护作用,及基因工程抗体的制备奠定了基础。     

抗人整合素αvβ3-组织因子融合蛋白基因的构建和表达

目的:利用基因工程技术构建和表达抗人整合素αvβ3单链抗体(scFv)与人可溶性组织因子(sTF)的融合蛋白scFv-sTF。方法:用重叠延伸PCR技术扩增scFv基因,同时用组装PCR方法人工合成sTF基因,然后以酶切连接方式融合2个基因,并克隆至原核表达载体pQE80L中,构建表达质粒pQE80L-scFv-sTF,以重组子转化大肠杆菌M15诱导目的基因表达。结果:SDS-PAGE分析显示工程菌可以表达相对分子质量约55×103的融合蛋白scFv-sTF,Western印迹分析证实表达的目的蛋白具有6×His标签;经低剂量IPTG诱导和较低温度培养,scFv-sTF获得了可溶性表达;纯化回收目的蛋白,ELISA试验证实,该重组抗体分子具有良好的抗原结合活性。结论:构建并表达了抗人整合素αvβ3的单链抗体融合蛋白scFv-sTF,并验证了其抗原结合活性,初步证明它可以与抗原特异性结合,为进一步研究其抗肿瘤作用打下了基础。     

抗卵巢癌单链免疫细胞因子真核表达载体的构建及表达

为将细胞因子IL 2靶向卵巢癌局部 ,提高肿瘤局部细胞因子的浓度 ,构建并表达了抗卵巢癌单链免疫细胞因子IL 2 1 83B2scFv .通过基因工程将两段基因IL 2和 1 83B2scFv开放读码框架的编码序列克隆在一起 ,在CHO细胞内人巨细胞病毒启动子的作用下表达可溶性融合蛋白 .酶联免疫吸附法检测其免疫学活性 ,并检测其促淋巴细胞增殖活性 .构建成功的抗体细胞因子融合蛋白能够在哺乳动物细胞内表达并能分泌到细胞外 ,且融合蛋白既能与卵巢癌相关抗原OC1 83B2很好地结合 ,又能刺激IL 2依赖细胞株的增殖 ,为其进一步临床应用打下实验基础     

融合基因anti-erbB2 scFv-Fc-CD28-CD3(ζ)的构建及真核表达

本研究利用SWISS-MODEL预测该融合蛋白的三级结构。利用PCR的方法分别从重组pPIC9k、重组pBullet和pSecTag2B上扩增出3段基因片段,即片段anti-erbB2 scFv(简称A)、片段Fc-CD28-CD3(ζ)(简称B)和信号肽序列(简称S)。利用SOE-PCR将3段序列连接形成融合基因片段S-A-B。经TA克隆扩增及鉴定后,将融合基因片段与逆转录病毒表达载体pLNCX相连构建重组真核表达载体,电转染人淋巴瘤T细胞株Jurkat,G418筛选后用流式细胞术检测融合蛋白稳定表达情况。经预测在anti-erbB2 scFv与Fc基因片段之间不加连接肽的融合蛋白,在三级结构上可形成更佳的功能构象。经PCR、酶切及测序鉴定均证实成功构建重组真核表达载体pLNCX/S-A-B(在A与B基因片段之间不加linker)。经流式细胞术检测,在转染的Jurkat细胞中融合蛋白表达率约为56.17%。本研究应用分子克隆的方法成功地构建了重组真核表达载体pLNCX/anti-erbB2 scFv-Fc-CD28-CD3(ζ),融合基因能够在淋巴瘤T细胞株中表达,为制备含该融合基因的原代T淋巴细胞,进行erbB2过表达肿瘤的靶向基因治疗研究奠定了实验基础。     

新融合蛋白CD19scFv/CD80抗白血病作用研究

B系急性淋巴细胞白血病(B-ALL)是一种十分常见的血液系统恶性肿瘤,尽管目前化疗效果显著,但仍有部分儿童和成人B-ALL患者疗效较差、预后不佳.研究表明,ALL细胞表面免疫共刺激分子CD80(B7.1)表达降低或不表达,导致白血病细胞不能有效地被细胞毒T细胞(CTL)识别杀伤.然而,几乎所有的B-ALL细胞均表达CD19表面抗原.因此,本研究利用前期构建的CD19单链抗体(scFv)/CD80融合基因及表达的融合蛋白,通过融合蛋白中抗CD19scFv,将CD80结合到B-ALL白血病细胞表面,靶向激活CTL细胞,起到杀伤白血病细胞的作用.在体外共培养实验中,该融合蛋白结合B-ALL细胞系Nalm-6细胞后,可诱导淋巴细胞增殖,分泌细胞因子并产生较显著的特异性细胞毒作用.以Nalm-6细胞在免疫缺陷小鼠(Mus musculus)中建立白血病动物模型,CD19scFv/CD80融合蛋白联合输注淋巴细胞可显著延长小鼠的生存时间.本研究表明,该融合蛋白可使逃避免疫监视的B-ALL细胞有效地转化成抗原提呈细胞,进而激活特异性免疫反应,具有潜在的临床应用价值.     

CD40-directed scFv-TRAIL fusion proteins induce CD40-restricted tumor cell death and activate dendritic cells

Targeted cancer therapy concepts often aim at the induction of adjuvant antitumor immunity or stimulation of tumor cell apoptosis. There is further evidence that combined application of immune stimulating and tumor apoptosis-inducing compounds elicits a synergistic antitumor effect. Here, we describe the development and characterization of bifunctional fusion proteins consisting of a single-chain variable fragment (scFv) domain derived from the CD40-specific monoclonal antibody G28-5 that is fused to the N-terminus of stabilized trimeric soluble variants of the death ligand TNF-related apoptosis-inducing ligand (TRAIL). As shown before by us and others for other cell surface antigen-targeted scFv-TRAIL fusion proteins, scFv:G28-TRAIL displayed an enhanced capacity to induce apoptosis upon CD40 binding. Studies with scFv:G28 fusion proteins of TRAIL mutants that discriminate between the two TRAIL death receptors, TRAILR1 and TRAILR2, further revealed that the CD40 binding-dependent mode of apoptosis induction of scFv:G28-TRAIL is operable with each of the two TRAIL death receptors. Binding of scFv:G28-TRAIL fusion proteins to CD40 not only result in enhanced TRAIL death receptor signaling but also in activation of the targeted CD40 molecule. In accordance with the latter, the scFv:G28-TRAIL fusion proteins triggered strong CD40-mediated maturation of dendritic cells. The CD40-targeted TRAIL fusion proteins described in this study therefore represent a novel type of bifunctional fusion proteins that couple stimulation of antigen presenting cells and apoptosis induction.     

Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system

We previously isolated phage antibodies from a phage library displaying human single chain antibodies (scFvs) by screening with a mannotriose (Man3)-bearing lipid. Of four independent scFv genes originally characterized, 5A3 gene products were purified as fusion proteins such as a scFv-human IgG1 Fc form, but stable clones secreting 1A4 and 1G4 scFv-Fc proteins had never been established. Thus, bacterial expression systems were used to purify 1A4 and 1G4 scFv gene products as soluble forms. Purification of 1A4 and 1G4 scFv proteins from inclusion bodies was also carried out together with purification of 5A3 scFv protein in order to compare their Man3-binding abilities. The present studies demonstrated that 1A4 and 1G4 scFv proteins have a higher affinity for Man3 than 5A3 scFv protein, which may determine whether scFv-Fc proteins expressed in mammalian cells are retained in the ER or secreted. Furthermore, the inhibitory effects of anti-Man3 1G4 scFv and anti-Tn antigen scFv proteins on MCF-7 cell growth were evaluated. Despite the fact that no obvious difference was detected in cell growth, microscopic observations revealed inhibition of foci formation in cells grown in the presence of the anti-carbohydrate scFv proteins. This finding provides a basis for the development of cancer therapeutics.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be ka = 4.03 x 104 M-1 s-1, kd = 5.77 x 10-4 s-1, KA = 6.98 x 107 M-1, and KD = 1.43 x 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.     

A Yeast Platform for the Production of Single-Chain Antibody-Green Fluorescent Protein Fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 μg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)₃ linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

A yeast platform for the production of single-chain antibody-green fluorescent protein fusions

Fusion proteins comprised of a binding domain and green fluorescent protein (GFP) have the potential to act as one-step binding reagents. In this study, eight single-chain antibodies (scFv) and one single-chain T-cell receptor (scTCR) were secreted as fusions to GFP using a Saccharomyces cerevisiae expression system. Fusion protein secretion levels ranged over 3 orders of magnitude, from 4 microg/liter to 4 mg/liter, and correlated well with the secretion levels of the unfused scFv/scTCR. Three fusion types with various linker lengths and fusion orientations were tested for each scFv/scTCR. Although the fusion protein secretion levels were not significantly affected by the nature of the fusion construct, the properties of the fusion protein were clearly influenced. The fluorescence yield per fusion molecule was increased by separating the scFv/scTCR and GFP with an extended (GGGGS)3 linker, and fusions with scFv/scTCR at the carboxy-terminus were more resistant to degradation. By evaluating leader sequence processing and using GFP fluorescence to track intracellular processing, it was determined that the majority of fusion protein synthesized by the yeast was not secreted and in most cases was accumulating in an immature, although active, endoplasmic-reticulum (ER)-processed form. This contrasted with unfused scFv, which accumulated in both immature ER-processed and mature post-Golgi forms. The results indicated that yeast can be used as an effective host for the secretion of scFv/scTCR-GFP fusion proteins and that as a result of intracellular secretory bottlenecks, there is considerable yeast secretory capacity remaining to be exploited.     

Expression and purification of an anti-Foot-and-mouth disease virus single chain variable antibody fragment in tobacco plants

Low-cost recombinant antibodies could provide a new strategy to control Foot-and-mouth disease virus (FMDV) outbreaks by passive immunization of susceptible animals. In this study, a single chain variable antibody fragment (scFv) recognizing FMDV coat protein VP1 was expressed in transgenic tobacco plants. To enhance the accumulation of scFv protein, the codon-usage of a murine hybridoma-derived scFv gene was adjusted to mimic highly expressed tobacco genes and fused to an elastin-like polypeptide (ELP) tag. This scFv–ELP fusion accumulated up to 0.8% of total soluble leaf protein in transgenic tobacco. To recover scFv–ELP protein from the leaf extract, a simple and scalable purification strategy was established. Purified scFv–ELP fusion was cleaved to separate the scFv portion. Finally, it was shown that the purified scFv proteins retained their capacity to bind the FMDV in the absence or presence of ELP fusion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen.

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.     

Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system

The idiotype (Id)-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins are potential vaccines for immunotherapy of B-cell lymphoma. In this study, four vaccine candidates were constructed by fusing murine GM-CSF to the amino- or carboxy-terminus of the 38C13 murine B-lymphocyte Id scFv with two different arrangements of the variable regions of the heavy chain and light chain (VL-VH and VH-VL). scFv (VH-VL) and GM-CSF/scFv fusion proteins were expressed in an Escherichia coli cell-free protein synthesis system. In order to promote disulfide bond formation during cell-free expression, cell extract was pretreated with iodoacetamide (IAM), and a sulfhydryl redox buffer composed of oxidized and reduced glutathione was added. The E. coli periplasmic disulfide isomerase, DsbC, was also added to rearrange incorrectly formed disulfide linkages. The 38C13 B-lymphocyte Id scFv was expressed with 30% of its soluble yield in active form (43 microg/ml) when tested with an anti-idiotypic mAb, S1C5, as the capture antibody in radioimmunoassay. It was found that the amino-terminal GM-CSF fusion proteins, GM-VL-VH and GM-VH-VL, showed much higher activity than the carboxy-terminal GM-CSF fusion proteins, VL-VH-GM and VH-VL-GM, in stimulating the cell proliferation of a GM-CSF-dependent cell line, NFS-60. Between the two amino-terminal GM-CSF fusion proteins, GM-VL-VH showed a higher total and soluble yield than GM-VH-VL.