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1.
Engineered bispecific antibodies that recruit cytotoxic lymphocytes to kill specific tumor cells have been showing promising clinical results. Here, we describe a bispecific single-chain Fv (scFv) immunofusion or BIf to target CD123(+) leukemia, that contains an anti-CD123 scFv fused at the N-terminus of human IgG1 hinge-C(H)2-C(H)3, and an anti-CD3 scFv fused at C-terminus. When expressed from transfected CHO-S cells, CD123xCD3 BIf forms a homodimer that provides a structure of N-terminal tumor-targeting domain that closely resembles natural antibody. The CD123xCD3 dimeric structure also provides binding affinity to CD123(+) tumor cells with a Kd of 10(-10) M, one to two orders of magnitude stronger than traditional bispecific antibody constructs. The location of the anti-CD3 scFv at C-terminus of BIf reduces the binding affinity to CD3(+) T cells by two orders, which could help to prevent non-specific T-cell activation. CD123xCD3 BIf is able to achieve T-cell-mediated target cell killing activities at low pM levels with E/T ratios as low as 2. Overall, the inclusion of human IgG1 constant region in BIf construct increases target cell-binding affinity; potentially increases serum half-life by its larger size and FcRn-mediated salvage system; and includes the abilities to activate the additional antibody-mediated cellular cytotoxicities. 相似文献
2.
Furuta M; Uchikawa M; Ueda Y; Yabe T; Taima T; Tsumoto K; Kojima S; Juji T; Kumagai I 《Protein engineering, design & selection : PEDS》1998,11(3):233-241
An expression system for mono- and bivalent single-chain Fv fragments
(scFv) of a human antibody against D antigen in the Rh blood group system
was established in Escherichia coli. The cDNA encoding the Fv fragment of
the anti-D monoclonal antibody D10 was cloned using the polymerase chain
reaction and expressed in E.coli by fusing with a peptide tag link in the
C-terminus of the light chain variable region. The scFv fragment expressed
by the bacteria produced specific agglutination of human D positive red
cells in the presence of an anti- peptide tag antibody. Flow cytometric
analysis clearly indicated that the bacterially prepared scFv showed high
specificity and affinity for D antigen, which was identical with that of
the parental IgG. In order to construct bivalent D10 scFv for use in direct
cell agglutination, the scFv was fused with a dimeric protein, bacterial
alkaline phosphatase (BAP). The fusion protein produced significant
agglutination of human red blood cells with D antigen, confirming that the
bacterially expressed fusion protein is a functional bivalent antibody
fragment. Specific agglutination of D positive red cells by D10 scFv-BAP
was enhanced in the presence of anti-BAP antibody, suggesting that further
multimerization of scFv led to highly efficient cell agglutination. By
grafting BAP enzymatic activity into the scFv fragment (enzyme-linked
scFv), blood typing could conveniently be performed. These results indicate
that bacterially expressed scFv and scFv-BAP would be of practical use in
blood typing. The system reported here could also be applied to the
examination of other cell surface antigens and cell agglutination.
相似文献
3.
目的对人源抗乙型肝炎病毒表面抗原的基因工程IgG全抗体进行表达、纯化及初步鉴定。方法用含Fc片段抗-HBsAg Fab抗体基因的载体pAC-HBs-Fc,与杆状病毒线性DNA共转染昆虫细胞sf9,产生重组抗-HBsAg的全抗体。以不同浓度的HBsAg包被酶标板孔,用间接ELISA法检测培养上清中抗体的表达及特异性;用蛋白G亲和层析柱进行抗体的纯化,并对纯化的抗体进行SDS-PAGE、Western blot和竞争性ELISA分析。结果上清中表达的重组IgG抗体仅与HBsAg呈阳性反应,特异性良好,纯化后其纯度达97.1%。经SDS-PAGE和Western blot分析可见,IgG抗体轻链和重链的相对分子质量分别约为27000和55000,为人源IgG抗体。CHO表达的HBsAg和血源性HBsAg能竞争性抑制该重组IgG抗体与E.coli表达的HBsAg反应,其抑制率分别为55.9%和81.9%。结论人源抗乙型肝炎病毒表面抗原的基因工程IgG全抗体可在杆状病毒载体表达系统中成功表达。 相似文献
4.
《中国生物制品学杂志》2010,(10)
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。 相似文献
5.
Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction 总被引:1,自引:0,他引:1
Kolly R Thiel MA Herrmann T Plückthun A 《Protein engineering, design & selection : PEDS》2007,20(2):91-98
Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells. 相似文献
6.
目的构建抗人CD4嵌合抗体稳定表达细胞株,并对表达产物进行鉴定。方法采用脂质体法将抗人CD4嵌合抗体质粒pHDC4稳定转染CHO-DHFR-细胞,经选择性培养、有限稀释法克隆和MTX加压筛选抗人CD4嵌合抗体稳定表达株,经细胞工厂扩大培养。收集培养上清,经Protein A亲和层析纯化目的抗体,激光共聚焦显微镜观察抗体基因在细胞内的表达,并分别进行质谱分析、蛋白质N-末端测序和平衡解离常数(KD)的测定。结果构建的抗人CD4嵌合抗体稳定表达细胞株的抗体表达水平为4.29~10.52μg/ml;BCA测得纯化回收后的抗体表达水平为12.68 mg/L;激光共聚焦检测显示,抗CD4嵌合抗体稳定表达细胞株可稳定表达人的恒定区和鼠的可变区;质谱分析表明,其含有鼠源性和人源性成分;蛋白质N-末端氨基酸测序表明,其轻链与亲本抗体N-末端氨基酸序列完全一致;其KD为2.67×10-9M。结论成功构建了抗人CD4嵌合抗体稳定表达细胞株,其表达的抗人CD4嵌合抗体保留了亲本抗体的抗原结合特异性和亲和力。 相似文献
7.
目的在小鼠骨髓瘤细胞SP2/0中表达抗人T淋巴细胞CD4人-鼠嵌合抗体。方法真核表达质粒PAG4622+CD4VL和PAH4604+CD4VH经酶切和序列测定后,采用电穿孔转染技术将二者共转染SP2/0细胞,经组胺醇和霉酚酸联合筛选阳性克隆,通过ELISA、流式细胞术、RT-PCR和DNA测序的方法进行初步鉴定。结果真核表达质粒经酶切和测序鉴定证明构建正确。获得2株分泌抗人T淋巴细胞CD4人-鼠嵌合抗体的阳性SP2/0细胞克隆0925CASP2/0和1107CASP2/0,嵌合抗体表达量为0.5~2ng/ml。结论已成功地在SP2/0细胞中表达了抗人T淋巴细胞CD4人-鼠嵌合抗体,为该抗体在其他真核细胞中的高效表达及临床应用奠定了基础。 相似文献
8.
Dr. Dominic P. H. M. Heuts Martin J. Weissenborn Dr. Rouslan V. Olkhov Dr. Andrew M. Shaw Dr. Jennet Gummadova Dr. Colin Levy Prof. Dr. Nigel S. Scrutton 《Chembiochem : a European journal of chemical biology》2012,13(16):2384-2391
CD73 is a dimeric ecto‐5′‐nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto‐5′‐nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer‐dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease. 相似文献
9.
Dr. Carlo Baggio Dr. Elisa Barile Dr. Gianluigi Di Sorbo Prof. Dr. Thomas J. Kipps Prof. Dr. Maurizio Pellecchia 《ChemMedChem》2016,11(10):1097-1106
The cell surface receptor CD44 is a glycoprotein belonging to the hyaluronan‐binding proteins, termed hyaladherins. CD44 is expressed in a wide variety of isoforms in many cells and, in particular, is present on the surface of malignant cells where it is involved in the onset and progression of cancer. In a first attempt to identify novel CD44‐binding agents, we first characterized, with NMR spectroscopic techniques, several agents that were reported to bind to human CD44 (hCD44). To our surprise, however, none of these putative CD44‐binding agents, including a peptide that is in phase 2 clinical trials (A6 peptide) and a recently reported fragment hit, were found to interact significantly with recombinant hCD44(21–178). Nonetheless, we further report that a fragment‐screening campaign, with solution NMR spectroscopy as the detection method, identified a viable fragment hit that bound in a potentially functional pocket on the surface of CD44, opposite to the hyaluronic acid binding site. We hypothesize that this pocket could be indirectly associated with the cellular and in vivo activity of the A6 peptide, which would provide a novel framework for the possible development of therapeutically viable CD44 antagonists. 相似文献
10.
Wang Z Mathias A Stavrou S Neville DM 《Protein engineering, design & selection : PEDS》2005,18(7):337-343
Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements. 相似文献
11.
Wu Anna M.; Tan Giselle J.; Sherman Mark A.; Clarke Patrick; Olafsen Tove; Forman Stephen J.; Raubitschek Andrew A. 《Protein engineering, design & selection : PEDS》2001,14(12):1025-1033
A series of single-chain anti-CD20 antibodies was produced byfusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions,designated scFv-Fc. The initial scFv-Fc construct was assembledusing an 18 amino acid (aa) linker between the antibody light-and heavy-chain variable regions, with the Cys residue in theupper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20scFv-Fc retained specific binding to CD20-positive cells andwas active in mediating complement-dependent cytolysis. Size-exclusionHPLC analysis revealed that the purified scFv-Fc included multimericas well as monomeric components. Variant scFv-Fcs were constructedincorporating four different hinges between the scFv and Fcregions, or three different linkers in the scFv domain. Allformed multimers, with the highest level of multimerizationfound in the scFv-Fc with the shortest linker (8 aa). Eliminationof an unusual salt bridge between residues L38 and H89 in theVL-VH domain interface failed to reduce the formation of higherorder forms. Structural analysis of the scFv-Fc constructedwith 18 or 8 aa linkers by pepsin or papain cleavage suggestedthe proteins contained a form in which scFv units had cross-pairedto form a `diabody'. Thus, domain exchange or cross-pairingappears to be the basis of the observed multimerization. 相似文献
12.
Simon K. Krebs Marlitt Stech Felix Jorde Nathanaël Rakotoarinoro Franziska Ramm Sophie Marinoff Sven Bahrke Antje Danielczyk Doreen A. Wüstenhagen Stefan Kubick 《International journal of molecular sciences》2022,23(22)
Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 StarTM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC50 value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required. 相似文献
13.
Aggen DH Chervin AS Insaidoo FK Piepenbrink KH Baker BM Kranz DM 《Protein engineering, design & selection : PEDS》2011,24(4):361-372
Single-chain antibody fragments (scFv), consisting of two linked variable regions (V(H) and V(L)), are a versatile format for engineering and as potential antigen-specific therapeutics. Although the analogous format for T cell receptors (TCRs), consisting of two linked V regions (Vα and Vβ; referred to here as scTv), could provide similar opportunities, all wild-type scTv proteins examined to date are unstable. This obstacle has prevented scTv fragments from being widely used for engineering or therapeutics. To further explore whether some stable human scTv fragments could be expressed, we used a yeast system in which display of properly folded domains correlates with ability to express the folded scTv in soluble form. We discovered that, unexpectedly, scTv fragments that contained the human Vα2 region (IMGT: TRAV12 family) were displayed and properly associated with different Vβ regions. Furthermore, a single polymorphic residue (Ser(α49)) in the framework region conferred additional thermal stability. These stabilized Vα2-containing scTv fragments could be expressed at high levels in Escherichia coli, and used to stain target cells that expressed the specific pep-HLA-A2 complexes. Thus, the scTv fragments can serve as a platform for engineering TCRs with diverse specificities, and possibly for therapeutic or diagnostic applications. 相似文献
14.
15.
目的建立高表达抗人TNF-α人-鼠嵌合抗体的工程细胞株。方法将构建的抗人TNF-α人-鼠嵌合抗体基因真核表达载体转染CHO细胞,并采用DHFR/MTX基因扩增策略,经反复加压和克隆筛选,提高抗体的表达量。用ELISA、Westernblot和体外中和试验分析比较抗人TNF-α人-鼠嵌合抗体与鼠亲本单抗的特性。结果获得的表达抗人TNF-α嵌合抗体的转染细胞系2F5,在静止培养4d后工程抗体的表达量约80mg/L。所表达的工程抗体优于鼠亲本单抗F7/2,并具有良好的特异性和中和活性,其相对亲和力约为2.1×10-10mol/L。结论已成功建立了高表达的嵌合抗体工程细胞株,其抗体具有潜在的临床应用前景。 相似文献
16.
McAlister MS; Davis B; Pfuhl M; Driscoll PC 《Protein engineering, design & selection : PEDS》1998,11(10):847-853
CD5 is a type-I transmembrane glycoprotein found on thymocytes, T-cells and
a subset of B-cells. The extracellular region consists of three domains
belonging to the scavenger receptor cysteine-rich (SRCR) superfamily for
which the three-dimensional polypeptide fold is as yet unknown.
Glycosylated CD5 domain 1 (CD5d1) has been obtained by expression by
secretion from both Chinese hamster ovary (CHO) cells and Pichia pastoris.
Recombinant CD5d1 expressed in this manner was shown to be correctly folded
by binding to anti-CD5 L17F12/Leu1 monoclonal antibody. Preliminary nuclear
magnetic resonance (NMR) spectra obtained for CD5d1 (residues 1-118) had
spectral dispersion typical of a folded protein, but otherwise of such poor
quality that NMR structural studies were not feasible. The analysis of
glycoproteins by NMR is frustrated by sample heterogeneity and poor
spectral quality associated with glycan resonance overlap and the potential
for increased line-widths due to the large hydrodynamic volume. In order to
pursue NMR structural studies of CD5d1 it was necessary to optimize the
quality of NMR spectra of CD5d1. A range of constructs of varying length
and carbohydrate content were expressed in CHO cells and in P. pastoris. In
addition the P. pastoris CD5d1 proved susceptible to N-glycan cleavage with
endoglycosidase H. The protein products were characterised using size
exclusion chromatography, NMR measurement of translational self- diffusion
coefficients and two-dimensional 1H nuclear Overhauser effect spectroscopy
experiments. Removal of an eight residue O-glycosylated C- terminal
peptide, in particular, resulted in significant improvements in the quality
of the CD5d1 NMR data, while retaining native protein structure.
Two-dimensional heteronuclear NMR spectroscopy of nitrogen- 15 isotope
labelled deglycosylated CD5d1 (residues 1-110) prepared from P. pastoris
suggests that this protein product is now amenable to solution structure
determination.
相似文献
17.
Caldas Cristina; Coelho Veronica P.C.V.; Rigden Daniel J.; Neschich Goran; Moro Ana Maria; Brigido Marcelo M. 《Protein engineering, design & selection : PEDS》2000,13(5):353-360
The 6.7 murine monoclonal antibody (mAb) recognizes the humanCD18 antigen and is therefore of interest as an anti-inflammatoryagent. The 6.7 heavy variable chain (VH) was humanized usingthe closest human germline sequence as the template on to whichto graft the murine complementary determining regions (CDRs).Two versions were proposed, one in which the residue proline45 of the murine form was maintained and another in which thisframework residue was changed to the leucine found in the humansequence. These VH humanized versions were expressed in theyeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs),with the VL from the murine antibody. The scFv from the murineantibody was also expressed. The binding activities of the murineand both hemi-humanized scFvs were determined by flow cytometryanalysis. All the constructions were able to recognize humanlymphocytes harboring CD18, indicating successful humanizationwith transfer of the original binding capability. Some differencesbetween the two hemi-humanized versions were observed. The methodused was simple and straightforward, with no need for refinedstructural analyses and could be used for the humanization ofother antibodies. 相似文献
18.
A Synthetic MUC1 Anticancer Vaccine Containing Mannose Ligands for Targeting Macrophages and Dendritic Cells
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Markus Glaffig Natascha Stergiou Dr. Sebastian Hartmann Prof. Dr. Edgar Schmitt Prof. Dr. Horst Kunz 《ChemMedChem》2018,13(1):25-29
A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose‐receptor‐positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non‐mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4+ T cells in the local lymph organs. Comparison of di‐ and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose‐coupled vaccine and the non‐mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells. 相似文献
19.
目的构建人源核糖体展示单链抗体(scFV)库。方法从人外周血单个核细胞中提取总RNA,反转录为cDNA,以此为模板,设计多对具有简并性特点的引物,PCR扩增人免疫球蛋白重链可变区(VH)和轻链可变区(VL)基因,在其两端加上核糖体展示所需元件,并通过重叠PCR法将VH和VL经(Gly4Ser)3短肽连接成单链抗体,构建核糖体展示库。结果利用不同引物进行PCR时,绝大多数引物能扩增出300~400 bp的VH和VL片段;通过大引物扩增成功加上了核糖体展示所需元件,重叠PCR体外连接成约900 bp大小的scFv基因,大量扩增得到核糖体展示库。结论已成功构建了人源核糖体展示scFv库,为人源抗体药物的开发奠定了基础。 相似文献
20.
Kim GB Wang Z Liu YY Stavrou S Mathias A Goodwin KJ Thomas JM Neville DM 《Protein engineering, design & selection : PEDS》2007,20(9):425-432
T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo. 相似文献