Selection Of Antibody Libraries Research Articles

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Phage Display Selection of Antibody Libraries: Panning Procedures.

The mining of naive, immune, and synthetic antibody repertoires by phage display has been widely applied to the de novo generation and in vitro evolution of monoclonal antibodies from multiple species. Once built, phage display antibody libraries can be selected by a variety of different strategies tailored toward the desired antigen binding properties. Here, we describe the selection of antibody libraries generated in a phage display vector of the pComb3 phagemid family. The approach includes panning procedures for immobilized antigens, biotinylated antigens in solution, and cell surface antigens. Although the typical format of these antibody libraries is human Fab or chimeric nonhuman/human Fab, the basic selection strategies provided in this protocol are compatible with a variety of formats.

Phage Display Selection of Antibody Libraries: Screening of Selected Binders.

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.

The pComb3 Phagemid Family of Phage Display Vectors.

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

In vivo Phage Display: A promising selection strategy for the improvement of antibody targeting and drug delivery properties.

The discovery of hybridoma technology, described by Kohler and Milstein in 1975, and the resulting ability to generate monoclonal antibodies (mAbs) initiated a new era in antibody research and clinical development. However, limitations of the hybridoma technology as a routine antibody generation method in conjunction with high immunogenicity responses have led to the development of alternative approaches for the streamlined identification of most effective antibodies. Within this context, display selection technologies such as phage display, ribosome display, yeast display, bacterial display, and mammalian cell surface display have been widely promoted over the past three decades as ideal alternatives to traditional hybridoma methods. The display of antibodies on phages is probably the most widespread and powerful of these methods and, since its invention in late 1980s, significant technological advancements in the design, construction, and selection of antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. With evolving novel disease targets and the emerging of a new generation of therapeutic antibodies, such as bispecific antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cell therapies, it is clear that phage display is expected to continue to play a central role in antibody development. Nevertheless, for non-standard and more demanding cases aiming to generate best-in-class therapeutic antibodies against challenging targets and unmet medical needs, in vivo phage display selections by which phage libraries are directly injected into animals or humans for isolating and identifying the phages bound to specific tissues offer an advantage over conventional in vitro phage display screening procedures. Thus, in the present review, we will first summarize a general overview of the antibody therapeutic market, the different types of antibody fragments, and novel engineered variants that have already been explored. Then, we will discuss the state-of-the-art of in vivo phage display methodologies as a promising emerging selection strategy for improvement antibody targeting and drug delivery properties.

Discovery of internalizing antibodies to basal breast cancer cells.

We present a strategy to discover recombinant monoclonal antibodies (mAbs) to specific cancers and demonstrate this approach using basal subtype breast cancers. A phage antibody library was depleted of antibodies to common cell surface molecules by incubation with luminal breast cancer cell lines, and then selected on a single basal-like breast cancer cell line (MDA-MB-231) for binding associated receptor-mediated endocytosis. Additional profiling against two luminal and four basal-like cell lines revealed 61 unique basal-specific mAbs from a pool of 1440 phage antibodies. The unique mAbs were further screened on nine basal and seven luminal cell lines to identify those with the greatest affinity, specificity, and internalizing capability for basal-like breast cancer cells. Among the internalizing basal-specific mAbs were those recognizing four transmembrane receptors (EphA2, CD44, CD73 and EGFR), identified by immunoprecipitation-mass spectrometry and yeast-displayed antigen screening. Basal-like breast cancer expression of these four receptors was confirmed using a bioinformatic approach, and expression microarray data on 683 intrinsically subtyped primary breast tumors. This overall approach, which sequentially employs phage display antibody library selection, antigen identification and bioinformatic confirmation of antigen expression by cancer subtypes, offers efficient production of high-affinity mAbs with diagnostic and therapeutic utility against specific cancer subtypes.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Domain structure of growth signalobodies critically affects the outcome of antibody library selection.

Wide applications of antibodies have demanded rapid and easy methods for isolating high-affinity antibodies. We recently developed an antibody screening system in mammalian cells using a growth signalobody, which is a single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a target oligomeric antigen. However, we have never investigated how the domain structure of signalobodies affects the outcome of library screening. In this study, we screened naïve scFv library-inserted signalobodies having distinct extracellular and transmembrane (TM) domains. Although the previously constructed signalobody with the extracellular D1/D2 domains of erythropoietin receptor had recovered the clones with high affinity against a target antigen and with low background cell growth, its D1/D2-deficient variant which was tested in this study recovered the clones with low affinity against a target antigen and with considerable background cell growth. In addition, mutagenesis in the TM domain lowered the level of the background cell growth. These results suggest that the D1/D2 domains increase a threshold to activate signalobodies, thereby selecting clones with high affinity against a target antigen and that the TM domain could be engineered to minimize background growth signalling.

A novel platform for antibody library selection in mammalian cells based on a growth signalobody

While many antibody-screening methods in vitro have been developed, these methods need repeated cycles of panning or sorting procedures to isolate antigen-specific antibodies. Here we developed a new antibody selection system based on antigen-dependent growth of mammalian cells. In this system, a growth signalobody library, which is a naïve single-chain Fv (scFv) library/cytokine receptor chimera that can transduce a growth signal in response to a specific antigen, is expressed in murine interleukin-3-dependent Ba/F3 cells. Simple culture of the cells in an antigen-containing medium results in growing cells with a high-affinity scFv gene, leading to selection of the scFv specific to the target antigen without panning/sorting procedures. To demonstrate this system, we used the SD1D2g signalobody having the signaling domain of gp130 and fluorescein-conjugated BSA as a target antigen, and investigated whether a fluorescein-specific scFv could be selected from a naïve scFv library. As a result, we successfully obtained fluorescein-binding scFv clones, and the scFv clone with the highest affinity was most abundantly selected, having the same sequence as the clone, which had been obtained through phage display. These results demonstrate the utility of our system as an affinity-based scFv selection method based on growth advantage of mammalian cells.

An efficient strategy for cell-based antibody library selection using an integrated vector system

BackgroundCell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.ResultsA mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.ConclusionsThis study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

An improved yeast transformation method for the generation of very large human antibody libraries

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Antibody affinity maturation in vitro using unconjugated peptide antigen

Selection of antibody library in vitro is almost always performed on a certain solid-phase with immobilized antigen. However, for the selection of small molecule binders, conjugation of the antigen to a carrier molecule is indispensable, which often leads to the selection of unwanted binders such as conjugate-binders or those with insufficient specificity. Here we describe a rapid and efficient way to improve the affinity of an anti-small molecule antibody without antigen derivatization. The method is based on the open-sandwich (OS) principle, which utilizes the antigen-dependent stabilization of antibody variable domain Fv. We used an anti-osteocalcin C-terminal peptide Fv that showed a good response but with moderate sensitivity in OS ELISA as a model. By selecting PCR-randomized V(H)-displaying phages for superior binders to the immobilized V(L) fragment in the presence of limited amount of antigen peptide, V(H) mutants that show superior detection sensitivity in OS ELISA were obtained, and were characterized to retain improved antigen-binding affinity. Furthermore, saturation mutagenesis of a mutant resulted in further improvement in sensitivity. This 'OS-selection' will be the first to select anti-small molecule antibodies without using conjugated antigens, and especially useful in the affinity maturation of antibodies whose Fv has limited stability in the absence of antigen.

Identification of MCAM/CD146 as the Target Antigen of a Human Monoclonal Antibody that Recognizes Both Epithelioid and Sarcomatoid Types of Mesothelioma

The prognosis for patients diagnosed with mesothelioma is generally poor, and currently available treatments are usually ineffective. Therapies that specifically target tumor cells hold much promise for the treatment of cancers that are resistant to current approaches. We have previously selected phage antibody display libraries on mesothelioma cell lines to identify a panel of internalizing human single chain (scFv) antibodies that target mesothelioma-associated, clinically represented cell surface antigens and further exploited the internalizing function of these scFvs to specifically deliver lethal doses of liposome-encapsulated small molecule drugs to both epithelioid and sarcomatous subtypes of mesothelioma cells. Here, we report the identification of MCAM/MUC18/CD146 as the surface antigen bound by one of the mesothelioma-targeting scFvs using a novel cloning strategy based on yeast surface human proteome display. Immunohistochemical analysis of mesothelioma tissue microarrays confirmed that MCAM is widely expressed by both epithelioid and sarcomatous types of mesothelioma tumor cells in situ but not by normal mesothelial cells. In addition, quantum dot-labeled anti-MCAM scFv targets primary meosthelioma cells in tumor fragment spheroids cultured ex vivo. As the first step in evaluating the therapeutic potential of MCAM-targeting antibodies, we performed single-photon emission computed tomography studies using the anti-MCAM scFv and found that it recognizes mesothelioma organotypic xenografts in vivo. The combination of phage antibody library selection on tumor cells and rapid target antigen identification by screening the yeast surface-displayed human proteome could be a powerful method for mapping the targetable tumor cell surface epitope space.

Antibody library selection by the β-lactamase protein fragment complementation assay

Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of beta-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.

Selection of functional human antibodies from retroviral display libraries

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 103-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.

Phage display and colony filter screening for high-throughput selection of antibody libraries.

During the last 12 years, antibody combinatorial libraries have provided a new approach for the construction and production of reagents and drugs based on the human monoclonal antibodies. Studies employing antibodies or antibody mimics have become an important part of the explosive growth of proteomics. This places tremendous emphasis on the new approaches for faster library screening, improved methods of selection and evaluation of novel applications. The phage display system, together with its variants of ribosome and bacterial display, is the most extensively used method for the rapid screening of large antibody libraries. However, in the last two years the need to improve selection methods together with a complex patent situation regarding the phage display system, has also directed research towards the possibility of performing antibody selection by colony filter screening. Here, we summarise the results obtained by these different methods of selection comparing their efficacy and advantages.