An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.     

从人源大容量天然抗体库中筛选到抗禽流感病毒H5N1单链抗体

目的从人源大容量天然抗体库中筛选到抗禽流感病毒H5N1单链抗体。方法运用噬菌体表面呈现技术,用纯化的H5N1禽流感血凝素蛋白HA（A/Anhui/1/2005）对人源大容量天然抗体库进行富集筛选,对获得的阳性克隆进行序列分析,并将其克隆入携带人源Fc段的表达载体,实现scFv-Fc融合抗体的分泌型表达。用ELISA、IFA对所获人源单抗的功能特性进行鉴定。结果经过3轮富集筛选,共获得了82株特异性结合H5N1禽流感病毒HA的人源scFv单链抗体,分别属于5个不同的抗体序列,ELISA、IFA表明从抗体库筛选获得的5株人源单抗与禽流感病毒H5N1及其HA具有较好的特异性,而与甲1型和甲3型人流感病毒无交叉反应。结论从人源大容量天然抗体库中利用高通量的筛选策略在短时间内获得抗禽流感病毒H5N1人源单抗,对于禽流感的紧急预防和治疗具有重要意义。     

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

AIM：To screen human single chain Fv antibody(scFv)against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.METHODS:The phage antibody library was panned by HCVE2 antigen,Which was coated in microtiter plate.After five rounds of bipanning,56phage clones were identified specific toHCVE2antigen,The selected scFv clones were digested by SfiI/NotI and DNAwas sequenced.Then it was subcloned into the vector pCANTAB5Efor expression as E-tagged soluble scFv.The liver tissue sections from normal person and patients with choronic hepatitis B and chronic hepatitis C were immunostained with HCVE2 scFv antibody.RESULTS:The data of scFv-E2DNAdigestion and DNA sequencing showed that the scFv gene is composed of 750bp.ELISAand immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitisCE2 antigen has a specific binding character with hepatitis virus E2antigen and paraffin-embedded tissue,but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.CONCLUSION:We have successfully screened and identified HCVE2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitisC.     

Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10⁹ members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.     

Quantitative analysis of the effect of the mutation frequency on the affinity maturation of single chain Fv antibodies

Random mutagenesis and selection using phage or cell surface display provides an efficient method for affinity maturation of single chain Fv (scFv) antibodies, thereby improving function in various applications. To investigate the effects of mutation frequency on affinity maturation, error-prone PCR was used to generate libraries containing an average (m) of between 1.7 and 22.5 base substitutions per gene in a high affinity scFv antibody that binds to the cardiac glycoside digoxigenin. The scFv antibody libraries were displayed on Escherichia coli, and mutant populations were analyzed by flow cytometry. At low to moderate mutation frequencies with an average mutation rate of m 相似文献   

Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the α_vβ₃ integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-α_vβ₃ interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to α_vβ₃. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.     

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the V_H chains and V_κ chains of all three scFv antibodies were derived from V_H3 and Vκ1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.     

Human recombinant single-chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage-display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single-chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti-HVR1 scFv clones from two patient-derived phage-displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross-reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the VH chains and Vkappa chains of all three scFv antibodies were derived from VH3 and Vkappa1 family germline V-genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV-infected patient sera. In addition, scFv S52/20 blocked the binding of HCV-susceptible Molt-4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad-spectrum blocking scFvs.     

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.     

Thyroid peroxidase autoantibodies obtained from random single chain FV libraries contain the same heavy/light chain combinations as occur in vivo

Three combinatorial libraries were constructed from unpurified, CD19(+), and antithyroid peroxidase (anti-TPO) B cells extracted from thyroid tissue of Graves' disease patients. Fifteen of the 41 randomly derived anti-TPO single chain variable region fragments (scFvs), showed VH1-3/V lambda 1-51 or VH1-69/V lambda 1-40 heavy/light chain pairing similar to that obtained with TPO-specific scFv derived from an in-cell library. One VH1-3/V lambda 1-51 scFv, A16, showed exactly the same nucleotide sequence as in-cell scFv ICB7, demonstrating that in vivo rearrangement can be obtained from a random combinatorial library. The majority of the scFvs used a heavy chain gene derived from the VH1-3 gene segment, whereas the light chain gene segments used were more heterogeneous, with dominance of the V kappa 1-39 and V lambda 1-51 gene segments. The anti-TPO scFvs showed high affinities to TPO, with values between 0.77 and 12.3 nM, and defined seven antigenic regions on the TPO molecule. The anti-TPO fragments, particularly VH1-3/V lambda 1-51 randomly associated scFv B4, which mimic natural H/L pairing, and VH1-3/V lambda 1-40 in-cell-derived scFv ICA5, efficiently displaced the TPO binding of serum autoantibodies from 20 Graves' disease patients. Our study directly demonstrates that antibodies derived from combinatorial libraries are likely to represent in vivo pairing, leading to high affinity antibody fragments mimicking the binding of serum autoantibodies to TPO.     

Characterization of human blood group scFv antibodies derived from a V gene phage-display library

Summary. We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kp^b and HI. The scFvs were isolated from a phageantibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17 600 sites on cDE/cDE red cells with an association constant (K_a), of 5.2 × 10⁷ m ¹ and a rate constant for dissociation (k_off) of 1.9 × 10² s^-1. The anti-E scFv recognized 29 800 and 39 800 sites on cDE/cDE red cells in two experiments with K_as of 8.4 × 10⁶ and 4.4 × 10⁷ m ^-1. The k_off for this antibody was 2.7 × 10^-2s^-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affnities typical of the primary immune response can be obtained from a phage-display library.     

日本血吸虫单克隆抗独特型抗体NP30单链抗体基因的构建和表达

目的　构建日本血吸虫单克隆抗独特型抗体NP30单链抗体 (scFv)基因。　方法　通过PCR方法体外扩增并经测序验证的重链、轻链可变区 (VH、VL)基因先后重组入原核表达质粒 pTHA90相应的位点上 ,中间通过一连接肽 (Gly4 Ser) 3基因连接构建成单链抗体基因 (scFv) ,连接产物转化相应受体菌Top1 0 ,提取质粒 ,酶切鉴定重组克隆。表达产物经ELISA方法测定活性。　结果　重组克隆经酶切鉴定可见预期大小的片段 ,表明重组成功。表达产物经ELISA检测 ,OD4 92 值为 1 .0 6 ,高于阴性对照 3倍以上 ,证实具有与相应抗原结合的能力。　结论　成功地构建了scFv基因 ,且其表达产物保留了抗体的亲和性和特异性     

Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

AIM:To construct the natural immune Fab antibody phagedisplay libraries of colorectal cancer and to select antibodiesrelated with colorectal cancer.METHODS:Extract total RNA from tissue of local cancermetastasis lymph nodes of petients with colorectal cancer.RT-PCR was used to amplify the heavy chain Fd and lightchain k and the amplification products were insertedsuccessively into the vector pComb3 to construct the humanlibraries of Fab antibodies.They were then panned by phagedisplay technology.By means of Dot immunoblotting andEUSA,the libradse were identified and the Fab phageantibodies binding with antigens of colorectal cancer wereselected.RWSULTS:The amplified fragments of Fd and k gained byRT-PCR were about 650bp.Fd and k PCR products weresubsequently inserted into the vector pComb3,resulting in arecombination rate of 40% and the volume of Fab phagedisplay library reached 1.48×10~6.The libraries wereenriched about 120-fold by 3 cycles of adsorption-elution-multiplication(penning).Dot immunoblotting showed Fabexpressions on the phage libraries and ELISA showed 5clones of Fab phage antibodies which had binding activitieswith antigens of colomctal cancer.CONCLUSION:The nstuml immune Fab antibody phagedisplay libraries of colorectal cancer were constructed.Theycould he used to select the relstive antibodies of colorectalcancer.     

Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.     

Combinatorial surrobody libraries

A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a “surrobody” to distinguish it from the canonical antibody molecule.     

Libraries against libraries for combinatorial selection of replicating antigen–antibody pairs

Antibodies are among the most highly selective tight-binding ligands for proteins. Because the human genome project has deciphered the proteome, there is an opportunity to use combinatorial antibody libraries to select high-affinity antibodies to every protein encoded by the genome. However, this is a large task because the selection formats used today for combinatorial antibody libraries are geared toward generating antibodies to one antigen at a time. Here, we describe a method that accelerates the identification of antibodies to a multitude of antigens simultaneously by matching combinatorial antibody libraries against eukaryotic antigen libraries so that replication-competent cognate antigen–antibody pairs can be directly selected. Phage and yeast display systems are used because they each link genotype to phenotype and can be replicated individually. When combined with cell sorting, the two libraries can be selected against each other for recovery of cognate antigen–antibody clones in a single experiment.     

丙型肝炎病毒非结构蛋白NS5抗独特型人源单链可变区抗体的筛选与鉴定

目的：制备丙型肝炎病毒（HCV）非结构蛋白NSS（NS5）的抗独特型单链可变区抗体scFv(抗－Id scFv)，为研制HCV NS5的抗－Id scFv疫苗奠定基础。方法：采用噬菌体表面展示技术，将HCV NS5单克隆抗体固相包被于Nunc板，从噬菌体单链可变区抗体库中经过5轮“吸附－洗脱－扩增”筛选过程，随机挑选80个克隆，利用酶联免疫吸附试验（ELISA）、交叉反应和竞争抑制实验，对其进行免疫学检测，获得与HCV NS5单克隆抗体结合活性较强的抗－Id scFv阳性克隆，并对HCV NS5特异性抗－Id scFv的编码序列进行序列测定分析。结果：筛选得到的HCV NS5抗－Id scFv片段由786bp组成，具有结合HCV NS5单克隆抗体的生物学活性和特异性。结论：用噬菌体抗体库技术能够成功地获得HCV NS5的抗－Id scFv。本实验结果为开展用抗－Id scFv防治丙型肝炎的研究创造了条件。     

Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem.

The properties of naiveté and large diversity are considered to be essential starting features for combinatorial antibody libraries that eschew immunization by evolution in vitro. We have prepared large libraries with such properties by using random oligonucleotide synthesis, which has the potential to create approximately 10(20) complementarity-determining regions for antibody heavy chains. When combined with light chains and expressed on phage surfaces, high-affinity antibodies could be selected from 5.0 x 10(7) Escherichia coli transformants. Remarkably, antibodies selected only for binding displayed both general structural features known to be important in nature's own antibodies and specific consensus sequences thought to be critical for interaction with the hapten against which the library was selected. Semisynthetic and ultimately totally synthetic combinatorial libraries when coupled with mutation and selection procedures should replace immunization for generation of reagent, therapeutic, and catalytic antibodies.