Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V_κ and V_H genes with newly designed primer sets. Initially, we amplified V_κ and V_H genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V_κ and V_H genes from the framework region 1 to the joining region. The V_κ and V_H genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.     

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.     

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.     

人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.     

Rapid cloning of any rearranged mouse immunoglobulin variable genes

Immunoglobulins (Ig) have been the focus of extensive study for several decades and have become an important research area for immunologists and molecular biologist. The use of polymerase chain reaction (PCR) technology has accelerated the cloning, sequencing, and characterization of genes of the immune system. However, cloning and sequencing the Ig variable (V) genes using the PCR technology has been a challenging task, primarily due to the very diverse nature of Ig V region genes. We have developed a simple, rapid, and reproducible PCR-based technique to clone any rearranged mouse Ig heavy or light chain genes. A close examination of all Ig heavy and light chain V gene families has resulted in the design of 5 and 3 universal primers from regions that are highly conserved across all heavy or light chain V gene families, and the joining or constant regions, respectively. We present our strategy for designing universal primers for Ig V gene families. These primers were able to rapidly amplify the rearranged Ig V genes, belonging to diverse Ig V gene families from very different cell lines, i.e., J558, MOPC-21, 36–60, and a chicken ovalbumin specific B-cell hybridoma. In addition, the present study provides the complete alignment of nucleotide sequences of all heavy and light chain variable gene families. This powerful method of cloning Ig V genes, therefore, allows rapid and precise analysis of B-cell hybridomas, B-cell repertoire, and B-cell ontogeny.The nucloetide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number U32111     

Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> ISP5230

Consensus amino acid sequences of FADH₂-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a β–α–β fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [α-³²P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes. Journal of Industrial Microbiology & Biotechnology (2002) 29, 1–5 doi:10.1038/sj.jim.7000263 Received 25 June 2001/ Accepted in revised form 02 April 2002     

The V<Subscript>H</Subscript> gene repertoire of splenic B cells and somatic hypermutation in systemic lupus erythematosus

In systemic lupus erythematosus (SLE) it has been hypothesized that self-reactive B cells arise from virgin B cells that express low-affinity, nonpathogenic germline V genes that are cross-reactive for self and microbial antigens, which convert to high-affinity autoantibodies via somatic hypermutation. The aim of the present study was to determine whether the V_H family repertoire and pattern of somatic hypermutation in germinal centre (GC) B cells deviates from normal in SLE. Rearranged immunoglobulin V_H genes were cloned and sequenced from GCs of a SLE patient's spleen. From these data the GC V gene repertoire and the pattern of somatic mutation during the proliferation of B-cell clones were determined. The results highlighted a bias in V_H5 gene family usage, previously unreported in SLE, and under-representation of the V_H1 family, which is expressed in 20–30% of IgM⁺ B cells of healthy adults and confirmed a defect in negative selection. This is the first study of the splenic GC response in human SLE.     

Synthetic antibody libraries focused towards peptide ligands

Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V_H) germ line gene 3-23, which was fused to a variant of the human variable light chain (V_L) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V_H) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V_H only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V_H had been subjected to diversification.     

Contribution of VH Replacement Products in Mouse Antibody Repertoire

V_H replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged V_H gene and the 23-bp RSS from an upstream unrearranged V_H gene. Due to the location of the cRSS, V_H replacement leaves a short stretch of nucleotides from the previously rearranged V_H gene at the newly formed V-D junction, which can be used as a marker to identify V_H replacement products. To determine the contribution of V_H replacement products to mouse antibody repertoire, we developed a Java-based V_H Replacement Footprint Analyzer (V_HRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify V_H replacement products. The overall frequency of V_H replacement products in these IgH genes is 5.29% based on the identification of pentameric V_H replacement footprints at their V-D junctions. The identified V_H replacement products are distributed similarly in IgH genes using most families of V_H genes, although different families of V_H genes are used differentially. The frequencies of V_H replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified V_H replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of V_H replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

Human V_H single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human V_H domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (V_HH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human V_H single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human V_H domains and the selection of binders against a diverse set of antigens. Unlike “camelized” human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the V_H/V_L interface. Structure determination in complex with hen egg white lysozyme revealed an extended V_H binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human V_H domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.     

Generation and characterization of an immune combinatorial cDNA library of variable genes of mouse immunoglobulins

A combinatorial cDNA library of mouse variable immunoglobulin genes from mice immunized with recombinant human interferon β1b (rhIFN-β1b) has been constructed. For this purpose, cDNA of variable genes of heavy (V _H ) and light (V _L ) immunoglobulin chains amplified from splenocytes were joined by linker DNA to form single-chain antibodies (single-chain Fv-antibodies, or ScFv’s). The ScFv-DNA pool thus obtained was cloned into a phagemid vector and used to transform Escherichia coli. Bacterial clones that produce single-chain antibodies that are specific to rhIFN-β1b ScFv’s were selected using the technique of phage display. Such characteristics of generated library as abundance, functional size, and initial diversity of the ScFv-DNA sequences were established in the study. A high degree of specificity of interaction of selected phage displayed ScFv’s with rhIFN-β1b has been demonstrated. Original Ukrainian Text ? M.V. Pavlova, P.V. Gilchuk, Ia. O. Pokholenko, I.S. Nikolaiev, V.A. Kordium, 2008, published in Tsitologiya i Genetika, 2008, Vol. 42, No. 2, pp. 10–15.     

Locus-specific vector/primer systems for rapid cloning of allelic variants

We have developed a rapid cDNA cloning procedure which uses a single-stranded (ss) vector/primer in which the primer sequence is locus-specific. Vector/primers were constructed by substituting a specific oligodeoxynucleotide primer sequence in place of the polylinker in M13mp19. The ss vector/primer is linearized and used to prime cDNA synthesis. Recircularized DNA is then used directly to transform competent bacterial hosts. As no intermediate column purifications or extractions are necessary, the entire procedure is performed in a single tube, contributing to the overall simplicity of the protocol. The primary use for this kind of vector/primer system will be for cloning and sequencing multiple allelic variants of polymorphic loci which contain a conserved 3' sequence. The two vector/primers we report here are specific for HLA-DQ beta genes and for human Ig variable regions associated with IgM antibodies.     

Conserved amino acid networks involved in antibody variable domain interactions

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody V_H and V_L function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which V_H and V_L domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (?‐values). We provide rosters of the key conserved amino acid pairs in antibody V_H and V_L domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in V_H and V_L are at or adjacent to the V_H–V_L interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the V_H domain, but not the V_L domain, residue pairs at the variable‐constant domain interface (V_H–C_H1 interface) are also strongly conserved. The same network of conserved V_H positions involved in interactions with both the V_L and C_H1 domains is found in camelid V_HH domains, which have evolved to lack interactions with V_L and C_H1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Systematic Engineering of Optimized Autonomous Heavy-Chain Variable Domains

Autonomous heavy-chain variable (V_H) domains are the smallest functional antibody fragments, and they possess unique features, including small size and convex paratopes, which provide enhanced targeting of concave epitopes that are difficult to access with larger conventional antibodies. However, human V_H domains have evolved to fold and function with a light chain partner, and alone, they typically suffer from low stability and high aggregation propensity. Development of autonomous human V_H domains, in which aggregation propensity is reduced without compromising antigen recognition, has proven challenging. Here, we used an autonomous human V_H domain as a scaffold to construct phage-displayed synthetic libraries in which aspartate was systematically incorporated at different paratope positions. In selections, the library yielded many anti-EphA1 receptor V_H domains, which were characterized in detail. Structural analyses of a parental anti-EphA1 V_H domain and an improved variant provided insights into the effects of aspartate and other substitutions on preventing aggregation while retaining function. Our naïve libraries and in vitro selection procedures offer a systematic approach to generating highly functional autonomous human V_H domains that resist aggregation and could be used for basic research and biomedical applications.     

Anti-diabetic effects of vanadium(III, IV, V)–chlorodipicolinate complexes in streptozotocin-induced diabetic rats

Vanadium(III, IV, V)–chlorodipicolinate (dipic-Cl) complexes, including H[V^III(dipic-Cl)₂] · 5H₂O (V₃dipic-Cl), V^IVO(dipic-Cl)(H₂O)₂ (V₄dipic-Cl) and K[V^VO₂(dipic-Cl)] (V₅dipic-Cl), were prepared with the indicated oxidation states. Our aim was to evaluate the anti-diabetic effects of V₃dipic-Cl, V₄dipic-Cl and V₅dipic-Cl in streptozotocin-induced diabetic rats. Vanadium complexes were orally administered to diabetic rats at concentrations of 0.1–0.3 mg/ml in the drinking water. We found that vanadium–chlorodipicolinate (V–dipic-Cl) complexes at the concentration of 0.1 mg/ml did not exhibit blood glucose-lowering effects when administered to diabetic rats for 20 days. However, the levels of fasting blood glucose in diabetic rats were decreased after treatment with 0.3 mg/ml of V₄dipic-Cl and V₅dipic-Cl complexes for the following 20 days. Although administration of both V₄dipic-Cl and V₅dipic-Cl significantly lowered diabetic hyperglycemia, the vanadium intake from administration of V₄dipic-Cl is nearly 1.5-fold greater compared to that of V₅dipic-Cl. Treatment with the H₂dipic-Cl ligand and all three V–dipic-Cl complexes significantly lowered serum cholesterol, while administration of the V₅dipic-Cl complex lowered serum cholesterol significantly more than administration of the ligand alone. Treatment with ligand alone did not have an effect on serum triglyceride, while administration of the V₄dipic-Cl and V₅dipic-Cl significantly lowered the elevated serum triglyceride associated with diabetes. Oral administration of the ligand and all V–dipic-Cl complexes did significantly lower diabetes elevated serum alkaline phosphatase. Treatment with H₂dipic-Cl ligand and V₄dipic-Cl and V₅dipicCl significantly lowered diabetes elevated aspartate amino transferase. These results indicate that the health of the treated animals did not seem to be further compromised compared to that of diabetic animals. In addition, oral administration of H₂dipic-Cl, V₃dipic-Cl, V₄dipic-Cl and V₅dipic-Cl did not alter diabetic serum creatinine and blood urea nitrogen levels, suggesting no significant side effects of vanadium treatment on renal functions at the dose of 0.3 mg/ml in diabetic rats. The results presented here suggest that the anti-diabetic effects of treatment with V–dipic-Cl complexes were likely associated in part with the oxidation state of vanadium.     

Rapid construction and characterization of synthetic antibody libraries without DNA amplification

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V_H) and light (V_κ) libraries. Four high quality, chemically synthesized polynucleotides (90–140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10⁹ transformants could be synthesized within 1 day. Fusion to β‐lactamase and selection on ampicillin resulted in 3.7 × 10⁸ V_H and 6.9 × 10⁸ V_κ clones highly enriched for full‐length, in‐frame genes. High‐throughput 454 DNA sequencing of >250,000 V_H and V_κ genes from the pre‐ and post‐selection libraries revealed that, in addition to the expected reduction in reading‐frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V_H/V_κ‐β‐lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions. Biotechnol. Bioeng. 2010; 106: 347–357. © 2010 Wiley Periodicals, Inc.     

Isolation and characterization of 12 microsatellite loci from <Emphasis Type="Italic">Suzukia shikikunensis</Emphasis> (Lamiaceae), a genus endemic to Taiwan and Ryukyus

Suzukia shikikunensis Kudo is an endemic plant in Taiwan and suffers habitat destruction caused by human overexploitation. In this study, we developed 12 microsatellite primer pairs for genetic study. These markers were screened for 24 samples collected from wild populations distributed in Taiwan, and for a sister species S. luchuensis collected from Yonaguni and Lutao Islands. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.65–0.922 and 0–0.625, respectively. All loci were significantly deviated from Hardy–Weinberg equilibrium due to the heterozygote deficiency. These primers amplifying microsatellites in the two species may provide a useful tool for population genetics to establish conservation strategy.