Isolation of human single chain antibodies (scFv) against human TNF-alpha from human peripheral blood lymphocyte-SCID mice

By developing an appropriate immunization protocol for SCID (hu-PBL-SCID) mice engrafted with human peripheral blood lymphocytes in combination with scFv phage display library, we were able to establish an efficient strategy to obtain human scFv clones against a human self-antigen, TNF-alpha. The mice pretreated with gamma-radiation (3Gy) and anti-asialo GM1 antibody were immunized with a mixture of human TNF-alpha-keyhole limpet hemocyanin and Freund's adjuvant. Human antibody maturation was suggested to be induced in the mice with the immunization protocol. The scFv clones obtained from the mice were shown to exhibit binding affinities in the range of 10(7)-10(8) M(-1). Together with our previously published work on the isolation of respiratory syncytial virus neutralizing scFvs, the results of this study have implicated that this combined approach is one of the effective alternatives for the cloning of human monoclonal antibodies specific for a wide range of antigens of interest.     

从大容量人源噬菌体抗体库中筛选抗黑素瘤特异性抗体

目的:用黑素瘤(MM)细胞系筛选大容量人源噬菌体抗体库,获得能与MM细胞特异性结合的人源性单链抗体(scFv)。方法:用完整的MM细胞对大容量人源噬菌体抗体库进行4轮“吸附-洗脱-扩增”筛选,并通过细胞ELISA法对随机挑选的克隆进行抗原结合活性测定,得到的阳性克隆进一步做DNA序列测定和免疫组化染色鉴定。结果:从80个随机挑选的克隆中,筛得1株抗MM细胞的抗体,该抗体对人的鳞癌细胞、角质形成细胞、黑素细胞、角蛋白、胰蛋白酶、转铁蛋白及小鼠IgG等细胞或抗原的结合反应均呈阴性。免疫组化染色显示,该抗体与MM细胞系Libr的染色呈阳性,对黑素细胞和痣细胞的染色呈阴性。DNA测序结果表明,该抗体VH属于人IgGVH5亚群,VL属于人Vκ亚型。结论:通过筛选噬菌体抗体库获得到1株抗MM细胞的特异性抗体,为MM的靶向治疗研究奠定了基础。     

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.     

A novel variable antibody fragment dimerized by the dHLX peptide with enhanced affinity against amantadine compared to its corresponding scFv antibody

Amantadine (AMA) is an illegally used antiviral drug in the poultry industry, it is necessary to establish a fast, accurate and time-saving detection method for poultry food. The antibody-based immunoassay can achieve fast and accurate requirements. We developed a recombinant antibody-based specificity immunoassay for AMA. In the recombinant antibody, the heavy chain variable region (VH) is connected covalently with the light chain variable region (VL) by the artificial linker. Here, two recombinant antibodies’ single-chain variable fragment (scFv) and scFv-dHLX were constructed and functionally expressed in the periplasm of Escherichia coli. The helix-turn-helix peptide was utilized to dimerize VH and VL similar to the IgG counterpart. The ScFv-dHLX protein showed a higher binding ability and affinity resulting in improvement of in vitro affinity activity over its corresponding scFv. Our results not only indicated scFv-dHLX as an alternative for scFv in analytical application, but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced affinity.     

从噬菌体抗体库筛选人源性抗白细胞介素8抗体

目的: 从噬菌体抗体库中筛选人源性抗IL- 8单链抗体(scFV)。方法: 采用原核表达载体pRSET- IL- 8在大肠杆菌BL21(DE3)中表达IL- 8 His融合蛋白, 并通过亲和层析纯化。以该融合蛋白为固相抗原, 对噬菌体抗体库进行 3轮淘筛, 并对所获阳性克隆进行抗原结合活性测定和DNA序列测定。结果: 获得 2株特异性抗IL- 8噬菌体抗体。对其DNA序列测定结果表明, 其VH基因均属于人IgGVH3亚群, Vλ基因分别属于人VλDPL5和VλDPL2亚群。结论: 利用噬菌体抗体库技术可不经免疫制备人源性抗IL- 8抗体, 为银屑病及相关疾病的临床应用提供了条件。     

Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses.

BACKGROUND: Recombinant antibodies expressed in plants ('plantibodies'), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis. OBJECTIVES: In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants. STUDY DESIGN: An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants. RESULTS: High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway. CONCLUSION: This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a 'plantibody'-mediated resistance to tospoviruses.     

抗人SARS病毒单链抗体库的构建

目的:利用细胞内重组的方法,构建大库容量的人源性抗SARS病毒单链抗体(scFv)库,为筛选SARS病毒抗原的人源性抗体奠定基础。方法:取6例SARS康复患者的80mL外周血,分离淋巴细胞后提取总RNA。分离纯化mRNA并反转录成cDNA后,扩增抗体重链、轻链可变区基因片段。然后经重叠延伸PCR装配成scFv基因并克隆入噬粒载体pDAN5中,电转化大肠杆菌TG1构建初级抗体库。制备初级库噬菌体后,感染大肠杆菌BS1365进行细胞内重组制备次级抗体库。结果:初级库库容量为3×109,在大肠杆菌BS1365中重组后得到3×1011的次级抗体库。结论:细胞内重组方法的应用可使大库容量抗体库的构建变得简单易行。构建的抗SARS病毒单链抗体次库不仅接近人类天然抗体库的水平,而且多样性好,为筛选抗SARS病毒抗原的抗体提供了保证。     

全人源抗肝癌噬菌体单链抗体的筛选与鉴定

目的: 构建人源抗肝癌噬菌体单链抗体库, 从中筛选抗肝癌单链抗体(scFv), 并对其特异性进行鉴定.方法: 采用体外致敏法和EBV转化技术联合噬菌体展示技术构建噬菌体抗体库.对初级抗体库进行亲和富集及ELISA筛选, 获得的阳性克隆进行免疫组化鉴定并测序.结果: scFv基因与载体连接后得到库容量为1.0×108的初级噬菌体抗体库.对抗体库进行3轮正负淘选和富集后, 随机挑取2 798个克隆进行ELISA, 发现3个克隆对HepG2呈强阳性反应, 而与QSG-7701等人正常细胞系呈弱阳性或不反应.对克隆SA3进行免疫细胞化学鉴定, 结果与ELISA一致.免疫组织化学鉴定表明SA3与肝癌组织和肝组织阳性率的差别有统计学意义.结论: 构建了库容量达1×108的全人源抗肝癌噬菌体抗体库.通过淘选富集、 ELISA和免疫组织化学鉴定获得特异性较强的噬菌体克隆, 为肝癌的临床诊断及导向治疗奠定了基础.     

A scFv antibody fragment as a therapeutic candidate to neutralize a broad diversity of human IFN-alpha subtypes

Despite their significant role in maintaining the normal physiology, cytokines may cause pathological conditions when they are overproduced. In this way, the increased expression of human interferon alpha (hIFN-) is associated with acute viral infections, inflammatory disorders and several autoimmune illnesses, where the cytokine may be a factor in either initiating or maintaining the disease.

Currently, there are several mAbs marketed for a variety of indications and many more in clinical trials, in which IFN- represents a potential target for antibody-based therapy.

A panel of 11 murine mAbs was prepared using recombinant hIFN-_2b as immunogen, all of which bound to the native form of the cytokine with affinity constants ranging from 1.7 × 10⁷ M^− 1 to 1.4 × 10¹⁰ M^− 1. An epitope mapping protocol demonstrated four spatially distinct areas of the protein recognized by the mAbs. Taking into account the characterization of the antibodies and their ability to inhibit the IFN- biological activity, four mAbs were selected to produce scFv fragments. One of these fragments (CA5E6) was able to neutralize a wide spectrum of subtypes of the IFN- family, including the recombinant cytokines hIFN-_2a and hIFN-_2b and a heterogeneous collection of IFN- produced by activated leukocytes and Namalwa cells.

With the aim of improving the affinity of the selected fragment, a standard error-prone PCR method was carried out. By using this strategy, it was possible to generate a new fragment (EP18) with increased affinity and ability to neutralize a broad diversity of IFN- subtypes. Consequently, the scFv EP18 represents a potential therapeutic agent for those immune and inflammatory diseases which are associated with an increased IFN- expression.     

Recent de novo origin of human protein-coding genes

The origin of new genes is extremely important to evolutionary innovation. Most new genes arise from existing genes through duplication or recombination. The origin of new genes from noncoding DNA is extremely rare, and very few eukaryotic examples are known. We present evidence for the de novo origin of at least three human protein-coding genes since the divergence with chimp. Each of these genes has no protein-coding homologs in any other genome, but is supported by evidence from expression and, importantly, proteomics data. The absence of these genes in chimp and macaque cannot be explained by sequencing gaps or annotation error. High-quality sequence data indicate that these loci are noncoding DNA in other primates. Furthermore, chimp, gorilla, gibbon, and macaque share the same disabling sequence difference, supporting the inference that the ancestral sequence was noncoding over the alternative possibility of parallel gene inactivation in multiple primate lineages. The genes are not well characterized, but interestingly, one of them was first identified as an up-regulated gene in chronic lymphocytic leukemia. This is the first evidence for entirely novel human-specific protein-coding genes originating from ancestrally noncoding sequences. We estimate that 0.075% of human genes may have originated through this mechanism leading to a total expectation of 18 such cases in a genome of 24,000 protein-coding genes.New genes are a rich substrate for evolution to act upon. New genes frequently arise through duplication of existing genes, or through fusion, fission, or exon shuffling between genes (Long et al. 2003). Origination of genes from noncoding DNA is extremely rare: A few eukaryotic examples are known in yeast and Drosophila (Levine et al. 2006; Begun et al. 2007; Cai et al. 2008; Zhou et al. 2008) and a very recent paper reported initial evidence for this process in a primate ancestor (Toll-Riera et al. 2009). No cases have been previously reported in human.Analysis of the differential presence and absence of genes in different genomes is hampered by incomplete genome sequence and annotation artifacts (Clamp et al. 2007). We undertook a rigorous and systematic analysis of the human genome to identify protein-coding genes with no counterpart in the chimp and macaque genomes. Essential to this analysis is an extremely strict and conservative set of criteria to exclude artifacts due to annotation errors or sequencing gaps. The central pillar of this analysis is a synteny framework to examine candidate novel genes. The synteny approach allowed us to pinpoint the expected location of the gene in other primate genomes and meticulously examine that region for evidence of protein-coding capacity. After careful exclusion of all cases where there might be an ortholog in another genome or where the annotated human gene is unreliable, we identified three novel human protein-coding genes that have originated from noncoding DNA since the divergence with chimp.     

A neutralizing recombinant human antibody Fab fragment against Puumala hantavirus

A combinatorial human antibody Fab pComb3H library, generated from splenic lymphocytes of a Puumala hantavirus (PUUV) immune individual, was selected against PUUV using the phage display technique. Panning was carried out with antigens immobilized by MAbs directed to the two PUUV envelope glycoproteins G1 and G2. Thirteen Fabs, with reactivity directed to PUUV and specifically the G2 protein, as assessed by immunofluorescence and ELISA respectively, were isolated in crude preparations. By a focus reduction neutralization test (FRNT), four of the 13 crude Fab preparations exhibited type-specific neutralization of PUUV (strain Sotkamo) with 44-54% reduction in the number of foci. After affinity purification, the four Fab clones exhibited 50% focus reduction of PUUV at concentrations below 2 microg/ml. Sequencing of the heavy and light chain complementarity determining regions (CDR) 1-3 showed that the four selected clones were identical within the antibody binding regions. In inhibition tests with the PUUV G2-specific MAbs, 4G2 and 1C9, a new epitope important for neutralization, designated as G2-a3, was defined. This epitope, overlapping partially the neutralizing epitope recognized by the human MAb 1C9, seems to be unique for the PUUV serotype since none of the Fab clones neutralized any of the other hantaviruses tested.     

Direct isolation of recombinant human antibodies against group B Neisseria meningitidis from scFv expression libraries

The successful generation of human antibodies from large nai;ve antibody libraries requires iterative selection steps. Here, we describe a new and fast method for the isolation of high affinity antibodies directly from human single chain Fv antibody (scFv) expression libraries. Escherichia coli scFv expression libraries were made from peripheral blood lymphocytes from four individuals vaccinated with group B Neisseria meningitidis outer membrane vesicle (OMV) vaccine. Forty thousand clones were directly screened for antibodies binding N. meningitidis strain 44/76 (B:15:P1.7,16). Of 430 specific clones detected, 225 candidates were isolated and re-screened against the N. meningitidis strains NZ-98/254 (B:4:P1.7b,4) giving 4% cross-reactive clones. Antibodies were further characterized by DNA sequencing, ELISA and surface plasmon resonance (SPR) analysis, showing broad V-gene diversity and nanomolar scFv affinities. Antibodies derived by this method may assist in the discovery and development of new vaccine antigens as well as therapeutic antibody agents for the treatment of meningococcal diseases.     

Expression of a recombinant human anti-MUC1 scFv fragment in protease-deficient Escherichia coli mutants

The proteolytic activity of Escherichia coli periplasmic proteases can affect the expression efficiency of many heterologous proteins such as antibody fragments that are transported to the host periplasm for folding. We investigated whether four E. coli strains that were deficient in the periplasmic proteases tsp, protease III, degP and ompT, in different combinations, affect the expression levels of an anti-MUC1 scFv fragment. The ompT protease appeared to be involved in partial degradation of the scFv since degradation products were observed in all ompT unmutated strains in Western blotting, whereas such products were absent in the ompT mutated strains. The HM120 strain that contained most mutations, expressed the scFv protein efficiently but the level of functional antibody activity was low. This was probably due to an accumulation of incorrectly folded antibody molecules in the periplasm as it was characterised by low enzyme immunoassay reactions in contrast to the intense staining of the tag in Western blots. Improved understanding of the periplasmic protease involvement in the process of the antibody expression in bacteria may allow us to design host E. coli strains that are more efficient in producing functional antibodies.     

Production and characterization of a single chain variable fragment (scFv) against the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a mycotoxin produced by certain fungi that infest cereal grains. A hybridoma cell line producing a monoclonal antibody (Mab) was used as the starting point in the development of a recombinant single chain variable fragment antibody (scFv) recognising DON. The scFv and Mab were characterised using two immunoassay formats: competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and biolayer interferometry (BLI). Using CD-ELISA the IC₅₀s for DON were 36.1 and 13.8 ng/ml for assays based on the scFv and Mab, respectively. The cross-reactivity to DON analogs was very similar for the scFv and the Mab. The real-time binding of the antibodies to an immobilised DON-protein conjugate was also monitored. In competitive BLI assays the IC₅₀s using the scFv and Mab were 68.3 and 15.8 ng/ml, respectively. The results suggest that sensitivity of assays, but not selectivity, was affected by removal of the constant regions of the Mab.     

Single-chain variable fragment (scFv) antibodies against rotavirus NSP4 enterotoxin generated by phage display

The rotavirus non-structural NSP4 protein causes membrane destabilization as well as an increase in intracellular calcium levels in eukaryotic cells and induces diarrhea in young mice, acting as a viral enterotoxin. In this study the phage display technique was used to generate a panel of single-chain variable fragment (scFv) antibodies specific for the NSP4 protein of the human rotavirus strain Wa from a human semi-synthetic scFv library. After several rounds of panning and selection on NSP4 adsorbed to polystyrene tubes, individual scFv were isolated and characterised by fingerprinting and by sequencing the VH and VL genes. The isolated scFv antibodies specifically recognize NSP4 in enzyme immunoassay and in Western blot. Four truncated forms of the NSP4 protein were constructed which allowed us to map the binding region of the selected scFv antibodies to the C-terminal portion of NSP4. The isolated scFv antibodies constitute valuable tools to analyse the mechanisms of NSP4 functions.     

Expression,production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10⁻⁸ M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10⁻⁷ M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.     

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.