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.     

从scFv噬菌体库分离特异性的人源化抗D-dimer抗体

目的 从scFv(单链Fv)噬菌体抗体库分离出对D-dimer有特异性的人源化单克隆scFv.方法 对Tomlinson scFv噬菌体文库进行3轮淘洗,富集特异性的抗D-dimer抗体并进行ELISA 验证.通过酶联免疫检测和双脱氧终止法基因测序,获取特异性的人源化单克隆抗体.结果 3轮淘洗选择出38个抗D-dimer噬菌体抗体,酶联免疫和基因测序分析后,20个不同的全长单克隆抗D-dimer scFv噬菌体抗体被筛选出来,3轮选择后阳性克隆获取率为100%;分泌性抗体ELISA结果显示单克隆anti-D-dimer噬菌体顺利表达了抗体蛋白;5个A450值较高的单克隆中,3个显示了对D-dimer的高特异性和亲和力.结论 抗体噬菌体展示技术是分离获取人源化特异性anti-D-dimer抗体的高效快速方法.

Abstract：

Objective To isolate specific humanized anti-D-dimer scFv(single chain Fv) antibody from scFv phage libraries. Methods Isolate anti-D-dimer positive clones from Tomlinson I + J phage libraries by three rounds of panuing, then sequence monoclonal genes by bideoxy-mediated chain termination and express soluble scFv antibody; Pick out anti-D-dimer antibodies with high specificity and affinity by ELISA.Results After three rounds of selection from human scFv phage libraries Tomlinson I and J, 38 monclonal specific anti-D-dimer scFv fragments were selected. By polyclonal and monoclonal phage ELISA and gene sequencing, 20 different full-length monoclonal scFv phages were identified, the result of soluble scFv ELISA showed that 20 full-length monoclonal scFv were expressed smoothly. According to the result of soluble scFv ELISA, in 5 scFv antibodies with high value of A450 selected, 3 scFv antibody fragments showed high specific and affinity. Conclusion Antibody phage display was an effective, rapid method to isolate anti-D-dimer antibodies with high specificity and affinity.     

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.     

人噬菌体抗体库中异常重组子的分析

目的 阐明在噬菌体抗体库技术中，选择适当限制性内切酶酶切位点的重要性，并介绍人抗体可变区胚系基因的限制酶谱。方法 从正常人外周血提取淋巴细胞总RNA，用RT－PCR扩增IgM和IgG1的Fd片段及κ链基因，重组型载体p3MH中构建噬菌体抗体库。以限制性内切酶消化及电泳分析所获重组克隆；用PCGENE软件分析人抗体可变区胚系基因的限制性内切酶谱。结果 在构建抗体库的过程中，发现高频率的异常重组子克隆。经序列分析证实，在人VH基因片段中，存在用于克隆轻链的Sac I 位点。对人全部功能性可变区胚系基因进行限制性内切酶谱分析，发现人VH等Ⅳ家族的11个成员均含有SacI位点，其它限制酶切位点在抗体可变区胚系基因中具有不同的出现率。结论 构建抗体库时，用于重组可变区基因的酶切位点，对库的构建具有重要的影响，因此，应对表达载体所用的限制性内切酶进行精心选择。现在比较广泛使用的pCOMB系统载体不利于良好性能抗体库的构建。     

A rapid method for generating and characterizing anti-variable region monoclonal antibodies

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.     

人源Fab噬菌体抗体库的构建与抗精氨酸加压素抗体的筛选

目的:构建人源天然Fab噬菌体抗体库,筛选抗精氨酸加压素特异性抗体并进行初步鉴定。方法:从18位健康成人的外周血淋巴细胞,提取总RNA。利用RT-PCR扩增人Fab抗体基因片段,将其克隆至噬菌粒载体pComb3XSS内,构建人源天然Fab噬菌体抗体库。以固相化的精氨酸加压素为靶抗原对抗体库进行五轮筛选后,随机挑取50个单克隆进行phage-ELISA检测,阳性克隆行DNA测序分析。结果:成功构建库容为2.4×108的噬菌体抗体库,从中筛选到6株阳性克隆能够与精氨酸加压素特异性结合,其中阳性值最高的C4克隆行DNA测序分析证实其重链属IgG亚类,轻链为λ链。结论:构建大容量人源天然Fab抗体库,从中获得了特异性抗精氨酸加压素人源Fab抗体片段,有望为临床上精氨酸加压素的快速检测技术的建立奠定基础。     

Recombinant antibodies to small analytes and prospects for deriving them from synthetic combinatorial libraries

During the past 4 years, several laboratories have developed new methods of cloning antibody combining site genes from hybridomas or B‐lymphocytes, and functionally expressing them in bacteria, yeast, mammalian cells or plants. At least three research groups have also constructed ‘antibody display’ libraries with vastly diverse sequence permutations of combining site genes in bacteriophage. These semi‐synthetic combinatorial libraries present an antibody repertoire orders of magnitude greater than that accessed by conventional hybridoma technology. They may yield antibodies not obtainable through conventional immunization. We have cloned the immunoglobulin genes from a hybridoma specific for the phenylurea herbicide, diuron, into a phage display vector. The cloned antibody fragments (Fabs) were as sensitive as the parent monoclonal antibody for detecting free diuron in competition enzyme immunoassays. We also derived diuron hapten‐specific clones from synthetic combinatorial phage libraries, but only a few weakly recognized free diuron. The relative merits of deriving Fabs from synthetic phage display libraries, and the steps in engineering new specificities and other properties into recombinant antibodies, are discussed.     

Quantum leap of monoclonal antibody (mAb) discovery and development in the COVID-19 era

In recent years the global market for monoclonal antibodies (mAbs) became a multi-billion-dollar business. This success is mainly driven by treatments in the oncology and autoimmune space. Instead, development of effective mAbs against infectious diseases has been lagging behind. For years the high production cost and limited efficacy have blocked broader application of mAbs in the infectious disease space, which instead has been dominated for almost a century by effective and cheap antibiotics and vaccines. Only very few mAbs against RSV, anthrax, Clostridium difficile or rabies have reached the market. This is about to change. The development of urgently needed and highly effective mAbs as preventive and therapeutic treatments against a variety of pathogens is gaining traction. Vast advances in mAb isolation, engineering and production have entirely shifted the cost-efficacy balance. MAbs against devastating diseases like Ebola, HIV and other complex pathogens are now within reach. This trend is further accelerated by ongoing or imminent health crises like COVID-19 and antimicrobial resistance (AMR), where antibodies could be the last resort. In this review we will retrace the history of antibodies from the times of serum therapy to modern mAbs and lay out how the current run for effective treatments against COVID-19 will lead to a quantum leap in scientific, technological and health care system innovation around mAb treatments for infectious diseases.     

Novel monoclonal antibodies recognizing different subsets of lymphocytes from the common marmoset (Callithrix jacchus)

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.     

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.     

Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries

We introduce a procedure for the rapid generation of fully human antibodies derived from "Fab-on-phage" display libraries. The technology is based on the compatibility of display vectors and IgG expression constructs, and allows reformatting of individual Fab clones to IgG, as well as reformatting of antibody repertoires. Examples of batch reformatting of an uncharacterized Fab repertoire and of a pool of Fabs, previously analyzed at the phage level, are presented. The average transient expression levels of the IgG constructs in HEK293T cells are above 10 microg/ml, allowing the use of conditioned media in functional assays without antibody purification. Furthermore, we describe a high-throughput purification method yielding IgG amounts sufficient for initial antibody characterization. Our technology allows the generation and production of antigen-specific complete human antibodies as fast or even faster than raising monoclonal antibodies by conventional hybridoma techniques.     

A human cDNA expression library in yeast enriched for open reading frames

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.     

Antibody-dependent cell-mediated cytotoxicity is induced by a single-chain Fv-protein III fusion in the presence of a rabbit anti-protein III polyclonal antibody

The use of phage-displayed antibody libraries has enabled the isolation of several thousand cancer-specific monoclonal antibodies. To further select for clones among these antibodies which have therapeutic potential for cancer, several types of in vitro anti-tumor assay, such as an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, are required. The cytotoxic activities of effector cells are triggered by the binding of the Fc portion of IgG to its receptor, necessitating the conversion of a candidate clone with a single-chain variable fragment (scFv) form into a human IgG form. In the laboratory however, this conversion process is expensive and involves laborious steps such as the cloning of mammalian cells that contain an IgG expression vector, the subsequent production of protein, and affinity purification. In our current study, we show that an original fusion of scFv and protein III, a coat protein of the M13 bacteriophage, can induce ADCC activity towards its target cells in the presence of a rabbit anti-protein III polyclonal antibody. Our modified assay method thus enables the more rapid selection of potentially therapeutic clones from phage-displayed antibody libraries.     

CɛmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B lymphocytes

Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of C?mX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore virus-like particles formed by hepatitis B virus core antigen (HBcAg) that harbors the entire C?mX peptide or its fragments as immunogens for inducing anti-C?mX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant C?mX-containing human IgE.Fc in ELISA and Western blot analyses, and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice were able to cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG could mediate ADCC in human mIgE-expressing A20 murine B-cell lymphoma. These studies suggest that HBcAg-C?mX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.     

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS

Background: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). Objectives: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. Study design and results: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. Conclusions: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.     

Multiple-antigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens

Antibody phage display is a powerful tool for the generation of monoclonal antibodies against virtually any given antigen. Chickens are phylogenetically more distant from humans compared to other laboratory animals, such as mice and rats. Therefore, the use of chickens is especially beneficial when generating recombinant antibodies against human autoantigens, which are often highly conserved among mammals. Another advantage of using chickens in antibody phage display is that the preparation of single chain variable fragment (scFv) antibody libraries is faster and easier compared to preparing such libraries from other species, as only two primer sets are needed for amplification of the chicken variable heavy chain (V(H)) and variable light chain (V(L)) genes. In the present study we explored the possibility to immunize chickens with antigen cocktails for the generation of recombinant antibody fragments directed to a range of human autoantigens. Two pairs of chickens were immunized with two cocktails of seven recombinant autoantigenic proteins, libraries were prepared and panned on the individual proteins. The polyclonal chicken sera reacted strongly with most of the antigens used for immunization. By creating and screening single-chain variable fragment antibody phage display libraries, recombinant monoclonal antibody fragments were isolated successfully against the autoantigens annexin XI, centromere protein B, heat shock protein B3, DNA topoisomerase I, histidyl tRNA synthetase, Ro52, Ro60, Rpp30 and U1A. In conclusion, the immunization of only four chickens with two distinct pools of a total of 14 autoantigenic proteins allowed the isolation of scFvs against nine of these antigens.     

A rapid and efficient method for generating anti-variable region monoclonal antibodies using type-1 interferons as immune modulators

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.     

人源性噬菌体抗体库的构建及HBsAg人单抗的筛选

用聚合酶链伸反应从人外周血淋巴细胞克隆出Ｆｄ段和ｋ链基因，重组到表达载体ｐＣＯＭＢ３中，构建了人源性噬菌体抗体，将乙肝病毒表面抗原固相化对噬菌体抗体库进行了三轮“吸附－洗脱－扩增”的筛选，使特异性噬菌体抗体得到了富集，筛选到了抗ＨＢｓＡｇ的人单抗。     

Microbial platform technology for recombinant antibody fragment production: A review

Recombinant antibody fragments are being used for the last few years as an important therapeutic protein to cure various critical and life threatening human diseases. Several expression platforms now days employed for the production of these recombinant fragments, out of which bacterial system has emerged a promising host for higher expression. Since, a small antibody fragment unlike full antibody does not require human-like post-translational modification therefore it is potentially expressed in prokaryotic production system. Recently, small antibody fragments such as scFvs (single-chain variable fragments) and Fabs (antibody fragments) which does not require glycosylation are successfully produced in bacteria and have commercially launched for therapeutic use as these fragments shows better tissue penetration and less immunogenic to human body compared to full-size antibody. Recently developed Wacker’s ESETEC secretion technology is an efficient technology for the expression and secretion of the antibody fragment (Fab) exceeded up to 4.0?g/L while scFv up to 3.5?g/L into the fermentation broth. The Pfenex system and pOP prokaryotic expression vector are another platform used for the considerably good amount of antibody fragment production successfully. In this review, we summarize the recent progress on various expression platforms and cloning approaches for the production of different forms of antibody fragments in E. coli.     

Isolation of human Fab antibodies specific for the low-affinity IgE receptor (CD23) by selecting a hierarchical antibody library system against B lymphoblastic IM-9 cells

The development of human antibodies specific for certain B cell markers is required to generate therapeutic antibody leads with improved therapeutic indices against B-cell lymphomas. To meet this demand, we selected a primary human antibody library, HuDVFab-8L, against human B lymphoblastic IM-9 cells via a ‘Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)’ cell panning approach. Six Fab clones that specifically bound to IM-9 cells were successfully isolated. Among these clones, two clones (IM-L6-E and IM-L8-G), were found to be specific for CD23 (Fc?RII). Affinity maturation of these Fab clones was then performed in a hierarchical manner by constructing secondary antibody libraries through combining heavy (H) chains of two Fabs with the human kappa L chain sublibrary HuNL-D3 followed by biopanning against the CD23 antigen. Clone IM-L6-5, one of the affinity maturated Fab derivatives from IM-L6-E, has a binding affinity of k_D ≈ 30 nM to soluble CD23. In addition, IM-L6-5 Fab is able to bind to an inducible form of CD23 expressed on U937 cells upon IL-4 stimulation, and inhibits binding of human IgE to CD23. Since the Fab IM-L6-5 is derived from a fully human naïve origin, we believe that IM-L6-5 can be utilized for the development of a therapeutic mAb which may have an improved therapeutic index over lumiliximab, a primatized anti-CD23 mAb, for the treatment of CLL or allergic diseases.