Recombinant full-size human antibody to Ebola virus

A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids, pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 × 10⁷ ± 1.5 × 10⁷ M^?1. Like the parent single-chain antibody 4D1, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.     

The production of mini antibodies against human granulocyte colony-stimulating factor using murine scFv combinatorial library

To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice. The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes. The construction of the library was based on the principle of stochastic combining of the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly4Ser)3 sequence. The scFv library was constructed using the E. coli TG1 strain and the phagemid vector pHEN1. The repertoire of the library exceeded 5 x 10(7) independent recombinant clones. The clones producing antibodies to the granulocyte colony-stimulating human factor were isolated. The affinity constants of the resulting scFv were in the range of 2 x 10(4) to 1.8 x 10(7) M-1.     

Construction of combinatorial immune library of single chain human antibodies to orthopoxviruses and selection from this library antibodies to recombinant protein prA30L of variola virus

A combinatorial immune library of human single-chain antibody fragments (scFv) was constructed on the base of genes encoding variable domains of heavy and light chains of immunoglobulins cloned from the lymphocytes of four vaccinia virus (VACV) vaccinated donors. The size of the library was 3 x 10(7) independent clones. After the library was enriched with the clones producing scFv against recombinant analogue of variola virus surface protein prA30L, a panel of unique antibodies specific to both prA30L and VACV was selected from the library. A plaque reduction neutralization test was performed for all selected antibodies and two antibodies were shown to be able to neutralize plaque formation of VACV in Vero E6 cells monolayer. Binding specificities of these antibodies were confirmed using ELISA and Western blot analysis. To determine the amino acid sequences of neutralizing antibodies their genes were sequenced.     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

The Production of Miniantibodies Against Human Granulocyte Colony-Stimulating Factor Using the Murine scFv Combinatory Library

To develop a phage display of single-chain antibodies (scFv), fractions of total cell DNA and RNA were obtained from splenocytes of naive mice. The DNA fragments encoding variable regions of light and heavy immunoglobulin chains were amplified and isolated using primers specific to the conservative regions of these genes. The construction of the library was based on the principle of stochastic combining the DNA fragments encoding the light and heavy antibody chains with the DNA linker, whose structure corresponded to the (Gly₄Ser)₃ sequence. The scFv library was constructed using the E. coli TG1 strain and the phagemid vector pHEN1. The repertoire of the library exceeded 5 × 10⁷ independent recombinant clones. The clones producing antibodies to human granulocyte colony-stimulating factor were isolated. The affinity constants of the resulting scFv were in the range of 2 × 10⁴ to 1.8 × 10⁷ M^–1.     

Construction of a combinatorial immune library of human single-chain antibodies to orthopoxviruses and selection of antibodies to recombinant prA30L of the variola virus

A combinatorial immune library of human single-chain antibodies (scAbs) was constructed using the genes coding for the variable domains of the heavy and light chains of human immunoglobulins. The genes were cloned from lymphocytes of four subjects vaccinated with the vaccinia virus (VACV). The library included 3 · 10⁷ independent clones. After enrichment with clones producing scAbs against a recombinant analog of the variola virus envelope protein prA30L, the library was used to select a panel of scAbs binding both prA30L and VACV. All scAbs selected were tested for virus-neutralizing activity, and two scAbs proved to suppress VACV plaque formation in monolayers of Vero E6 cells. The specificity of antigen binding was verified by ELISA and Western blotting. The amino acid sequences of the virus-neutralizing scAbs were determined by sequencing their genes.     

Construction and characterization of combinatorial naive phage library of human scFvs

A combinatorial phage display library of human single-chain antibody fragments (scFv) was constructed on the basis of variable domains of heavy (Vh) and light (VI) genes cloned from the lymphocytes of six healthy donors. The size of the library was 2? 10(8) independent clones. Single-chain antibodies against recombinant human TNF?, vaccinia virus and virus-like particles formed by core protein of hepatitis B virus were selected from the library. Unique scFv sequences were identified using the HaeIII fingerprinting. The specificity of the selected clones was proved by the Western-blot analysis.     

Construction and characterization of immune combinatorial cDNA library of mouse variable immunoglobuline genes

A cDNA combinatorial antibody library of mouse variable immunoglobulin fragments has been constructed from mice immunized with rhIFN-beta1b. For this purpose, cDNAS of immunoglobulin variable heavy (V(H)) and variable light (V(L)) chains genes amplified from splenocytes were joined with linker DNA to form ScFv's (single-chain Fv-antibodies). The obtained ScFv-DNA pool was cloned into a phagemid vector and used for Esherichia coli transformation. Using the phage display technique, bacterial clones producing single-chain antibodies specific to rhIFN-beta1b were selected. The following characteristics of the combinatorial library were determined in this work: abundance, functional size, and the initial ScFv-DNA diversity in the library constructed. High specificity of interaction between phage displayed ScFv's and rhIFN-beta1b has been demonstrated.     

Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.     

New monoclonal antibodies to the Ebola virus glycoprotein: Identification and analysis of the amino acid sequence of the variable domains

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.     

抗H5N1病毒嵌合IgA抗体基因的构建及其在CHO细胞中的表达

为了表达具有中和活性的抗禽流感H5N1病毒人-鼠嵌合IgA抗体,采用RT-PCR法克隆具有中和活性的抗禽流感H5N1-HA鼠源单克隆抗体的轻重链可变区基因及相应的信号肽编码序列,分别与人免疫球蛋白IgA2重链恒定区、Kappa恒定区基因拼接,构建表达质粒pEF-IGHA9和pEF-IGK9,共转染二氢叶酸还原酶缺陷型CHO(CHO-dhfr-)细胞,用ELISA检测培养上清中嵌合IgA抗体的表达,对纯化的嵌合抗体进行SDS-PAGE、Western blotting印迹分析。结果成功地在CHO细胞中表达了抗禽流感H5N1病毒人-鼠嵌合IgA抗体,为制备抗H5N1重组分泌型IgA预防性抗体制剂奠定了良好的基础。     

Antarctic teleost immunoglobulins: more extreme, more interesting

We have investigated the immunoglobulin molecule and the genes encoding it in teleosts living in the Antarctic seas at the constant temperature of −1.86 °C. The majority of Antarctic teleosts belong to the suborder Notothenioidei (Perciformes), which includes only a few non-Antarctic species. Twenty-one Antarctic and two non-Antarctic Notothenioid species were included in our studies. We sequenced immunoglobulin light chains in two species and μ heavy chains, partially or totally, in twenty species. In the case of heavy chain, genomic DNA and the cDNA encoding the secreted and the membrane form were analyzed. From one species, Trematomus bernacchii, a spleen cDNA library was constructed to evaluate the diversity of VH gene segments. T. bernacchii IgM, purified from the serum and bile, was characterized. Homology Modelling and Molecular Dynamics were used to determine the molecular structure of T. bernacchii and Chionodraco hamatus immunoglobulin domains. This paper sums up the previous results and broadens them with the addition of unpublished data.     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

By-passing immunization. Human antibodies from V-gene libraries displayed on phage.

We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

Association with beta 2-microglobulin controls the expression of transfected human class I genes

The genes encoding HLA-B27K and HLA-B27W were transfected into murine recipient cells. A monoclonal antibody HC-10, directed against free B-locus heavy chain, was the only reagent capable of efficiently detecting the HLA-B27 heavy chains in detergent lysates. These heavy chains were devoid of sialic acid. Trace amounts of HLA-B27 could be isolated with the anti-HLA-A,-B antibody W6/32, which reacts with the heavy chain beta 2-microglobulin complex. In marked contrast, HLA-A2 and -B7 genes, when transfected, yielded easily detectable amounts of antigen precipitable with W6/32, which carried the usual complement of sialic acids. Because the alpha 3 domains of HLA-B27 and HLA-B7 and the more COOH-terminal portions are identical in amino acid sequence, structural elements in the polymorphic alpha 1 and alpha 2 domains must control association of heavy chain with beta 2-microglobulin. Introduction of a human beta 2-microglobulin gene into L cells transfected with the HLA-B27 gene rescued the expression of HLA-B27 at the cell surface, as evidenced by reactivity with W6/32, surface staining, and the presence of sialic acid on the heavy chain.     

Yeast expression of a catalytic antibody with chorismate mutase activity

The catalytic antibody 1F7 promotes the rearrangement of chorismate into prephenate. We cloned and sequenced the genes encoding this catalyst to determine the origin of the observed rates and specificity. The antibody cDNAs were modified and inserted into inducible expression vectors. Simultaneous intracellular expression of the light and truncated heavy chains in strains of the yeast Saccharomyces cerevisiae lacking natural chorismate mutase resulted in the production of properly folded and assembled Fab antibody. Assembly of the light and heavy immunoglobulin chains is roughly 60-70% efficient in our in vivo system, lagging behind light chain synthesis throughout log and stationary phase. Nevertheless, high intracellular levels of functional Fab antibody (0.1% of total cellular protein) were obtained with an ultra-high copy number plasmid. As yeast-derived 1F7(Fab) catalyzes the chorismate mutase reaction with the same specific activity as antibody isolated from the hybridoma, our expression system now makes possible the application of classical and "reverse" genetics to the study and improvement of this first-generation abzyme.     

Light chain determines the binding property of human anti-dsDNA IgG autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies.     

Analysis of the expressed heavy chain variable-region genes of Macaca fascicularis and isolation of monoclonal antibodies specific for the Ebola virus' soluble glycoprotein

The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human V_H repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the V_H3 (41%), V_H4 (39%), and V_H1 (14%) subgroups used more frequently than the V_H5 (3.9%) or V_H7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human V_H genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans.