Companies engaged in Phage Display:  Dyax  Pharmacia Recombinant Phage Antibody System, Stratagene SurfZAP™ 

Definitions:

Phagemid: is a plasmid vector having a bacterial origin of replication and a copy of an intergenic region of a bacteriophage. The phagemid may be based on any knonw bacteriophage, including filamentous bacteriophage and lambdoind bacteriophage. The plasmid will also generaly contain a selectable marker for antbiotic resistance. Sigments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necesary for the porduction of phage particles, the mode of replication of hte plasmid changes to rolloing cirrcle replication to generate copies of one strand of the plasmid DNA and package phage partciles. The phagemid may form infectious or non-infectious pahge partciles. This term includes phagemids which contain a phage coat protein gene or fragment thereof linked to a herologous polypeptide gnee as a gene fusion such that the terologous polypeptide is dsiplayed on the surface o fthe phage partcile. (Sidhu, US 2007/0117126)

Phage vector: means a double stranded repolicative form of a bacteriophage containing a heterologocous gene and capable of replication. The pahge vector ahs a phage origina of replicaiton allowing phage replication and phage partcile formation. The pahge is preferably a filamentous bacteriphage such as an M13 phage or a derivative or a lambdoid phage such as lambda 21. (Sidhu, US 2007/0117126)

Ligation: is the process of forming phosphodiester bonds between two nucleic acid fragments. For ligation fo the two fragments, the end of the fragments must be compatible with each other. In some cases, the ends will be direclty compatible after endonuclease digestion. However, it may be necessary first to convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation. For blunting, the end the DNA is treated in a suitable buffer with the klenow fragment of DNA polymerase 1 or T4 DNA polymerase in the presence of the four deoxyribonucleotide triphosphates. The DNA is then purified by phenol-chloroform extraction and ethanol precipitation. The DNA fragments that are to be ligated together are put in solution in about equimolar amounts. The solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 ug of DNA. If the DNA is to be ligated into a vector, the vector is first linearized by digestion and the appropriate restriction endonuclease(s). The linearized fragment is then treated with bacterial alkaline phosphatase or calf interstinal phosphatase to prevent self-ligation during the ligation step. After ligation the ector with the foreign gene now inserted is purified a and transformed into a suitalbe host cell by electroporation. The trasnformed cells are generally selected by browth on an antibiotic, commonly tetracycline (tet) ro ampicillin (amp) to which they are rendered resistant due to the presence of tet and/or amp resistance genes int he vector. After selection of the transofmred cells, these cells are grown in culture and the vector DNA (pahge or phagemid vecoctor containing a fusion gnee library) may tehn be isolated. (Sidhu, US 2007/0117126)

Transcription regulatory element: will contain one or mroe of the following components: an enhancer, a promoter, an operator sequence, a repressor gene and a transcription termination sequence. (Sidhu, US 2007/0117126)

Transformant: is a cell which has taken up and maintained DNA as evidenced by the expression of a phenotype associated with the DNA (e.g., antibiotic resistance conferred by a protein encoded by the DNA). Transformation is the process whereby a cell takes up DNA and becomes a “transformant”. (Sidhu, US 2007/0117126)

Introduction:

At the biginning of the 199s, an in vitro antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particulalry antibody based drugs. They created boinatorial antibody libraries on filamentous phage to be utilzied for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 pahge derived antibodies entered lcinial studes and 14 of them ahve been approved. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.) 

One of the most successful applications of phage display has been the isolation of monoclonal antibodies using large phage antibody libraries. In these methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them (Huse, Science, 1989). In particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library. Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen. 

The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies haivng diverse and novel immunospecificties. The technoloy uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene druing the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries. Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor and CpIII membrane anchor. The diversity of a filaemntous phage based combinatorial antibody library can be increased by shuffling of the H and L chain genes. Filamentous pahge display vectors have been utilized to produce human monocloanl antibodies immunoreactive tih HIV, hepatitis B viru. (Barbas, US 6,21,558).

Proteins can be displayed on phage by cloning the corresponding gene into phage vectors (essentially the phage genome with suitable cloning sites for pIII fusions and an antibiotic reistance gene). Phage vectors carry all the genetic information necessary for phage life. With pIII fusions in phage vectors, in the absence of proteolysis, each pIII coat protein displayed on phage is fused to the heterologous polypeptide. (Vit, Methods in Enzymology, 326, pp. 480-505). For example, a bacterial host cell is transformed or infected with a bacteriphage expression vector which includes a DNA library member joined to a first nucleotide sequence encoding a tag protein. The vector also contains a second nucleotide sequence that encodes a tag ligand peptide that specifically binds the tag protein, which second sequence is joined to a hir nucleotide sequence encoding a coat protein of the bacteriphage particle. The transformed or infected host cell is then cultivated under conditions suitable for expression and assembly of the bacteriphage particles and the association of the tag protein with the tag ligand peptide on the surface of the phage particle. Particles which encode the protein of interest are then selected form the culture by an affinity enrichment technique. Dower (US 5,427,908). 

Human antibodies can be derived from phage display libraries (Hoogenboom, J. Mol. Biol. 227: 381 1991; Marks J. Mol. Biol. 222, 581-597, 1991 and Vaughan Nature Biotech 14: 309 (1996). Synthetic phage libraries can be created which use randomized combinations of synthetic human antibody V regions. By selection on antigen fully human antibodies can be made in which the V regions are very human like in nature (US 6,794, 132, 6,680,209, 4,634,666 and Ostberg (1983). 

Winter (“Making antibody fragments using phage display libraries” Nature 352, 1991) discloses using PCR to amplify the VH and Vk genes form the spleen mRNA of mice immunized with phOxand using a PCR assembly process to link these genes together randomly for expression as single-chain Fv (svFv) fragmetns. The assembed genes were cloned in a single step into the vector fd phage for display as a fusion with teh fd gene III coat protein. The initial library was diverse and sequencing revealed the presence of most VH and Vk subgroups. The library of phages was passed down a phOx affinity column and eluted with hapten. O fthe eluted clones, 13%bound to phOx and ranged form poor to strong binding in ELISA. Sequencing of 23 of these hapten binding clones showed eight different VH genes in a vareity of paring with seven different Vk genes. They also constructed a random comibnatorial library from unimmunized mice but found no phOx binding clones after two rounds of slection. Immunizaiton therefore seemes to be necesary to creat and/or enrich for VH or Vk domains with at least osme of the features required for hapten binding. 

Phage Libraries and Commercially Available Kits:

The first large non-immunised phage display library capable of yeilding high affinity antibody fragments to a given target was described in 1996. This library was constructed from antibody V genes derived form 43 donors form B cells dervied from peripheral blood lymphocytes, tonsil and bone marrow. Antibody fragments with a Kd as low as 0.3 nM were subsequently isolated directly form this library. Over the following decade various groups have constructed similarly large libraries, using scFvs, Fab fragments of VH-VL binding domains and sources of V genes ranging form immune tissues to purefly synthetic de novo constructs. Selection of such libraries to a given antigen can give rise to several hundreds to thousands of different antibodies. (Lloyd, Protein Engineering Design & selection, 22(3), 159-168, 2009

Commercially available kits for generating phage display libraries include the Pharmacia Recombinant Phage Antibody System and the Strategene SurfZAP phage display kit. (Ramasubramanyan (US 13/829989)

A complete de novo design and construction of HuCAL (human combinatorial antibody library) has been described by Knappik and by Rothe. There, the design consists of consensus VH and VL master genes that reproduce the overall human antibody repertoire in terms of structure and amino acid diversity. (Enzelberger, J Molecular Biology, 413, 2011, 261-278). 

General Scheme for Antibody Libraries

The most commonly used phage protein for displaying peptides of interest is the minor coat protein 3, which is presented 3-5 times on the M13 parrticle. The coat protein of gene 3 consist of three domains, A C terminal constant region which anchors the protein to the phage particle, and two N terminal domains, N1 and N2, mediating infectivity. N1 binds to the TolA receptor and N2 binds to the F-pilus of E. coli. Proteins of interest are usually fused to the N-terminus of the gene 3 protein. During the assembly process, resulting fusion proteins are transproted through the inner cell membrane to the bacterail periplasms and incorporated into the phage partcile, while their respective single-straned DNA gets packaged into the phage thereby coupling phenotype and genotype. Typicaly, a selection (or panning) round can be divided into several distnct steps. 1) the gene of interest, which can be a library or a single protein, is fused to gene 3. This modified phage genome is transformed into E. coli. Upon phage production, the protein of interest is displayed on the phage surface as fusion to protein 3. For the selection, phages are incubted with the target protein, immobilized either in an ELISA well or an immune test tube. Simple washing steps remove unspecific or weak binding phages. Stringency can be increased from round to round by adding more washing steps or harsher conditons. Phages are well tolerable against heat and denaturing agents. Binders are eluted by an acidic pH shift or by a tryptic digenst. The phages are then amplified in an E coli host strain, purified and enter the next round of selection. (Willemsen, “Protein Engineering” from Molecular Biomethods Handboo,, 2nd Edition) 

The process of in vitro selection of antibodies form libaries is referred to as “panning”. In vitro selection requires the immunogilixzation of the target antigen to a solid surface, such as magnetic beads, column nmatrices or plastic surfaces with high protein binding capacity as polystyrene tubes or plates. Panning in solution, which involved the use of biotinylated antigens followed by a “pull-down” with streptavidin beads, can also be done. The vast excess of non-binding antibody phage must be removed by stringent washing. Subsequently, the bound antibody phage will be eluted and reamplified by infection of E. coli and packaging with helper phage to produce a new antiboyd phage sublibrary, which will be used for another panning round until a significant enrichment of antigen specific antibody phage is acheived. Usually 2-3 panning rounds are requried to enrich antibodies even form the largest available libraries. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

M13 is a filamentous bacteriophage that has been a workhorse in molecular biology for the past 20 eyars. M13 viral particles consist of six different capsid proteins and one copy of the viral genome, as a single-stranded circular DNA molecule. Once the M13 DNA hs been introduced into a host cell like E. coli, it is converted into double-stranded, circular DNA. The viral DNA carries a second origin of replication that is used to generate the single-stranded DNA found in the viral partciles. During viral morphogenesis, there is an ordered assembly of the single-stranded DNA and the viral proteins, and the viral particles are extruded from cells in a process, much like secretion. The M13 virus is neither lysogenic nor lytic like other bacteriphage. Once cells are infectid, the M13 virus is chronically released. (Alvarex, US 5,885, 577). 

(1) cDNA is prepared from the mRNA of rearranged Ig genes from donor B cells, and heavy and light chain gene segments are amplified by PCR. The heavy and light chain cDNA are cloned into one of a number of phagment expression vectors which, after transfection into E coli results in the accumulation of Ig fab fragments in the periplasmic space. 

(2) Upon coinfection with M13 helper phage having a gene encoding the phage coat protein, phagemid particles are generated that contain plasmid DNA and that incorporate copies of the Fab fragment as part of their coat protein. Thus, a physical association is established between each antibody molecule and the DNA that encodes that antibody’s specificity. 

(3) Libraries of such Ig displaying phagemid particles can now be incubated with immobilized antigen to adsorb phage bearing antibody specificties of interest. Absorbed phage can then be eluted with acid, allowed to reinfect E coli cultures, and further propated and enriched through subsequent rounds of “panning”. Once monospecific phage are isolated further molecular manipulations of the plasmid DNA can unlink the Fab molecule from the phage surface and result in the bacterial production of solube Fab fragments.

Casey (phage display of peptides in ligand selection for use in affinity chromatography” Methods in molecular biology, February 2008) discloses a panning procedure for a random peptide library for a peptide mimotope that includes coating an ELISA plate with antibody, incobulate YT media with a colony of K91 cells, growing until log phase, washing the coating ELISA plate with PBS and blocking, taking an aliquot of the phage library and diluting it, adding it to the plate, washing the plate, eluting boudn pahge, pool elutions, add the pooled phage to stationary K91 cutulre and incubate and repeating the steps for subsequent rounds of panning). 

Examples of bacteriophage systems

Phage libraries generated form human rearranged V gene repertoires are constructed form mRNA or RNA extracted form B cells of immunized of naive donors. Immunized libraries are constructed form lymphoid tissues of individuals who carry a particular disease, such as metastatic cacner of partciular infection, or have been immunized to particular antigen. Such libraries are characteristic of a biosed antibody repertoire toward specific targets. Additionally, those antiodies tend ot ahve much hihger affinities for the desired antigen thatn antiboides isoalted form naive libaries of comparable size, becasue the VH and VL gene framgents have udnerogne the natural in vivo affinity maturation process. Naive libaries, on the other hand, represent the germline diversity of antibody repertoire. These libraries are generated form healthy donor’s mRNA or RNA without bias toward a particular disease state, and are used to yeild mAbs against unlimited range of antigens. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.)

M13 bacteriphage display: In brief, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin (Ig) genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab Ig.

pSD3 bacteriophage: 500 ug of each conjugate in PMB was emulsified with Fruednd’s complete adjuvant and injected on the back of a mouse. The spleen was isolated on day 67. Total speen RNA was isolated with a Qiagen RNease kit. Total RNA nd primr were used for first strnd cDNA synthesis using a kit. The VL library was digested with Sfi I and ligated into pSD3 and then digested with PfM I for construction of scFv phage display library by ligation of the VH ligrary. After purification of the litgated products, the DNA was transferred tito TG1 electroporation competitent cells. (Li, Biochemical and Biophysical Research communications 268, 398-404 (2000)

The HuCAL PLATINUM phage library (Bio-rad) is a synthetic library of 45 billion fully human antibodies in Fab format. Bio-Rad offers custom antigbody generation using either human combinatorial antibody libraries (HuCAL), a compilation of human antibody genes that have been made synthetically to cvoer mroe than 95% of the structural human immune repertoire, cloned in E. coli phaemid vectors of CysDisplay, a method to select antibody genes out of the library that encode for antibodies binding specifically with high affinity to just about any given antigen.   

Potential Problems

There are several difficulties associated with the generation of antibodies using bacteriophage. Purification procedures necessarily involve solubilization of protein which may render some proteins permanently denatured with concomitant destruction of antigenic sites. Such proteins cannot be bound to a solid phase and thus cannot be used to pan for phage bearing antibodies which bind to them. An example of such a protein is the human Rh antigen. To solve this problem a method was developed wherein intact RBCs were used as the panning antigen. However, it was discovered that since phage are inherently “sticky” and RBCs express a multitude of antigens on the cell surface, a sufficient amount of phage which do not express the right antibody on the surface also adhere to the RBCs. (Siegel, US 2011/0091960A1). 

Despite the advantages of antibody phage display such as bypassing animal immunization, the ability to isolate antibodies against toxic or non-imunogenic antigens and the ability to geenrate conformation specific antibodies, the vast majority of the approved therapetuic antibodies are derived form immunized mice technologies. This is becasue the filtraiton process that imposed by the immune system enables mammalins derived antibodies to have better biophysical attributes compared to antibodies generated by phage display. Antibodies direclty discoved by pahge display or engineered at some point by phage biopanning exhibit significant devleopability risks properties compared to those derived form immunized mice. Phage display derived therapeutic antibodies have higher self-interaciton and poly-reactivity due to the higher percetnage of aliphatic residuesin their CDRs compared to non-phage derive antibodeis. Additionally, antibodies selected form pahge display libraries are not glycosylated becasue they are produced in E. coli. Therefore, using eukaryotic display platforms like eyast and mammalian display would be beneficial In addition to their ability to produce glycosylated prtoeins, eyast and mammalian antibody libraries can be constructed to display full lenght antiobdies as swell as antibody fragments such as scFvs, allowing the isolation of high affintiy antibodies with definitive bioglocical characteristis. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.)

Types of Antibodies Displayed on Phage

Monoclonal antibodies represent an important class of recombinant protein therapeutics. The first approved mAb, muromonab-CD, which blocks CD3 mediated activaiton of T cells to prevent organ rejection after transplantation, was produced by hybridoma technology. However, a significant percentage of patients who were administered this murine antibody developed anti-drug antibodies. In the late 1980, recombinant DNA technology was used to substitute murine antibody sequences with human anitbody sequences to reduce immunogenicity. First, only murine constant immunoglobulin (G) (IgG) domains were replaced by human counterparts resulting in chimeric anitibodies like rituximab, which lowered the risk of immunogicity. However, the murine variable regions were still prone to generate antiidiotypic antibodies. Thus, it was also important to substitute the murine framework regions in the variable antibody domains with the closest human framework sequences, which results in humanized antibodies like daclizumab or bevacizumab. Humanization, however, did not eliminate the possibility of an immune response because the success and degree of humanization is dependent on the invidual antibody, which often requires back mutations and can involved a tremendous amount of antibody engineering effort. Moreover, the CDRs mesuating most of the interaction with the antigen are still from non-human origin, and thus, pose some risk for ADA responses. Thus, fully human antibodies were considered to be optimal solution for therapy because they are indistinguishable from those in the human body and had the lowest risk of immunogicity. Despite the tremendous success of this technology, the immunization of transgenic mice does not always result in a successful in vivo antibody response to all types of anitgences. In vitro selection technologies like antibody phage display do not depend on the in vivo immune response, and can be used to discover antibodies to almost every type of antigen and to a broader range of epitopes, which may be suppressed by the immune system. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016). 

Tow decades after McCafferty and Winter’s seminal report in 1990, mroe than 70 pahge derived mAbs entered clinical studes, and 14 of them ahve been approved. The majroity of these antiobdies are generated by three company-owned libraries. Cambridge Antibody Technology (CAT), Dyax and MorphoSys’s human combinatorial antibody libraries (HuCAL) MorphoSys’s HuCAL has the hihgest number of mAbs (20 mAbs) wehrein 19 are under clinical devleopment and one Tremfya is approved. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.)

Human mAbs: 

–anti-GM-CSF: The proinflammatory cytokine GM-CSF is produced by a variety of cells includeing activated T and B cells and plays a critical role in autoimmune diseases. MOR103 is a human mAb specific for GM0CSF and developed with phage display selection from the HuCal Gold library, followed by affinity maturation with tri-nucleotide cassette mutagenesis of CDRL3 and CDR-H2, affinity driven slection and cross-cloning of the best candidates. MOR103 inhibit human GM-CSF with an IC50 in the low picomolar range. 

–-anti-Il-23: Guselkumab (TREMFYA): is a human IgG1-lambda mAb that neutralized Il23 functions. IL-23 is a pleiotropic, herodimeric cytokines, consisting of a p19 and a p40 subunits, which are priamrly secreted by APCs. IL-23 induces TH-17 proliferation and the subsequent release of IL-17, which triggers inflammatory and autoimmune disorders such as psoriasis.

Humanized mAbs:

–anti-VEGF:

Bavacizumab is a humanized antibody dervied form teh murine antibody A.4.6.1 which binds VEGF. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

Antibody Formats used in Phage Display:

The discovery of smaller recombinant antibody formats such as variable domain (Fv; variable regions of the VH or VL, single-chain varaible domain (scFv), diabodies (bivalent scFvs), heavy-domain camelid and shark antibody fragmetns (VHHs, nanobodies) and fragment antigen binding (fab) has helped to advance antibody pahge display technology, These small er fragments are mroe amendable to expression in bacteria compared to full antibodies, which require assembly of four polypeptide chains and extnsive disulfide bond formation. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.)

Fab:

–anti-EGFR:

Necitumumab was developed from epidermal carcinoma cells which were used to screen for antibodies that blocked EGFR activation. Necitumumab binds to the receptor and blocks the binding of several relevant ligands and inhibits the proliferation of different cancer cell lines. Necitumumab was developed from the “de Haard” Dyax Fab phage display library. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

ScFV: 

Creating a combinatorial scFv library on the surface of M13 filamentous pahge has been acheived through combining populations of VH and VL domains, which are joined by a flexible, protease resistance glycine-serin linker (Fly4Ser)3, into a single DNA sequence. These antibody sequences are then introduced and cloned as a gene fusion with the bacteriophage pIII gene under the control of a wek promoter in a phagemid vector, a plasmid that carries an antiboiotic registance gene, bacterial and phage origins of replication. (Hashem “Phage display dervied monoclonal antiobdies: from bench to bedside” Frontiers in Immunogloy, 2020, volumne 11.)

–Anti-TNF (Adalimumab): Two scFv libraries were constructed, one library with VH combined with a human variable L chain repertoire and one library with VL and a H chains repertoire. Both libraries were used for panning on TNF. From the selected hybrid scFvs, human VH and VL were combined in a third library and reselected on TNF. Subsequently, CDR mutagenesis was performed, resulting in D2E7. Adalimumab was firsed approved by the FDA in 2002 for treatment of mderate to severe forms of RA. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

–Anti BLyS (belimumab): BLyS, a new member of the TNF ligand family, was suggested to be involved in monocyte-drivn B cell activation. Abut 1,200 antibodies from a scFV phage dispaly library were selected that were able to inhibit BLyS activay. Affinity maturation lead to the development of an antibody named belimumab. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

–Anti Anthrax:

Raxibacumab neutralized protective antigen (PA) which makes up lethal toxin (LT) of B. antracis. It seleccted using a naive human scFv pahge display library licnsed from CAT. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

–Anti Rabies:

Before Louis Pasteur developed a rabies vaccination, the disease was always fatal. The current post-exposure therapy is based on vaccination, as performed by Pasteur in 1885, and polyclonal anti-rabies immunoglobulines. Crucell selected a panel fo recombinant antibodies against the rabies glycoprotein from an immune scFv library. (Frenzel, MAbs, 2016, 8(7): 11771194, 2016

Particular Types of Antibody Phage Libraries:

IgE Libraries:

Steinberger “Construction of a combinatorial IgE library form an allergice patient”, 271(18) 1096710972, 1996) discloses construction of an IgE combinatorial library form a grass pollen allergic patient. cDNAs coding for IgE H chain fragments and for L chains were reverse-transcribed and PCR amplified from RNA of peripheral blood lympocytes and randomly combined in plasmid pComb3H to yield a combinatorial library. IgE Fabs with specificity for Phl p5, a major timothy grass pollen allergen, were isolated by panning. Sequence analysis showed that the 4 of the Fabs used the same H chain fragments which had combined with different kappa light chains. 

 Particular Types of Molecules Isolated from Phage Libraries:

Isolation of IgA with peptide libraries:

Ito “Human IgA-binding peptides selected form random peptide libraries” J Biological Chemistry, 287(51) 2012) discloses using disulfide construcained random peptide libaries with the T6 phage display system to isolate peptides specific to human IgA. 

Specific Phage Display Variations

RNA Display:

In RNA display, expressed proteins or peptides are linked covalently or by tight non-covalent interaction to their encoding mRNA to form RNA/protein fusion molecules. The protein or peptide component of the an RNA/protein fusion can be selected for binding to a desired target and the identity of the protein or peptide determined by sequencing of the attached encoding mRNA component. To form RNA/Protein fusions, one or mroe in vitro antiboy DNA expression libraries are transcribed to generate mRNA. Any in vitro antibody expression library is suitable (e.g., VH, VL or scFv libraries). After RNA transcription, the DNA library templates are removed by for example digesiton with DNase I. Hsieh (US 2010/0105569)

Hsieh (US 2010/0105569) discloses a method of screening an scFv antibody RNA display library which includes the steps of providing a puromycin crosslinked scFv mRNA molecule which comprises an mRNA encoding a 5 scFv and a 3′ spacer sequence which is crosslinked to puromycin at a 3′ end and a Psoralen C6 at the 5′ end, in vitro translating the puromycin-crosslinked scFv mRNA in the presence of a label under conditions that a labelled puromycin-crosslinked scFv mRNA/protein molecule is formed, purifying the molecule, subjecting the purified labeled puromycin-crosslinked scFv mRNA/protein molecule to antigen selection with at least one antigen (the library of RNA/protein fusions is screened for in vitro binding to a desired target. In general the target is bound to a solid support such as agarose beads. In one embodimetn, the target is first modified for example biotinylated and then bound via the modificaiton of a substrat such as streptavidin agarose. After binding of RNA/protein fusions the solid is washed to remove one or more itmes to remove unbound RNA/protein fusions and the RNA is then amplified. Additionally, the NA/protein fusions may be eluted form the solid support prior to amplifcaiton of the nucleic acid component. The RNA/protein fusions are eluted suing alkaline conditions for example a pH of about 8-10. In another emboidmetn, the fusions are eluted using acidc conditions, for example, using a pH of about 3-6) and recoveirng the purified labeled molecules using affinity based magnetic beads. 

Methods of isolating Antibody specific for cell antigens using Phage display: 

In one method, antibody expressing phage are incubated with a mixture of antigen expressing cells and cells which do not express antigen. The antibody expressing phage bind to the antigen expressing cells. Then, fluorescently labeled antibody is added specifically to the antigen expressing cells which can then be removed from the mixture having antibody expressing phage bound thereto using FACS (De Kruif (1995, Proc. Natl. Acad. Sci. USA, 92:3938-3942).

Siegl (US 6255455) teaches a method of isolating DNA encoding a protein which binds to a platelet by 1) mixing DNA from two different phage display libraries that comprise DNA encoding a heavy and light chain isolated form panning a population of phage against the antigen, 2) adding magnetically labeled which have the antigen on their surface, 3) incubating the phage display library with the magnetically labeled cells in the prescense of an excess of unlabeled cells which do not express the antigen, 4) isolating at least one virus vector from the mixture and obtaining its DNA encoding the H and L chains, 5) and religating the DNA for the H and L chains so as to form a hybrid virus vector encoding the L and H chains not

Screening of phage library of single-chain antibodies with multilamellar liposomes coupled to an antigen:

In biopanning procedures, plastic microplates, tubes and microbeads are widely used for adsorption of target molecuels and phage screening. However, plastic adsorbents may lead to inefficient outputs in that denaturation of target molecuels on plastic surface may cause incorrect regognition of the molecuels by phages, blocking procedures which are essential to reduce nonspecific adsorption of and and some phages may be specifically adsorbed on blocking agents. Kumada discloses  that protein-coupled lipsomes, which have been used for drug delivery, can be used as a biopanning method of phage-displayed libraries. (Kumada “Application of protein-coupled liposomes to effective affinity screening from phage library” J. Chromatography A 1080 (2005) 22-28)

Kumada (US 14/774,398, published as US 2018/0327803) discloses the following phage disply method for screening a single-chain antibody that binds to an antigen which is couple to multilamellar liposomes. 

(1) obtaining library of phages presenting single-chain antibodies:

A specific antigen (e.g., human serum-derived IgG polyclonal antiboy) is administered to an animal, the total RNA is obtained from the spleen, and a cDNA library is constructed by RT-PCR. 

Subsequently, specific primers are used to amplify the gene of the VH and VL by PCR.

Next, the PCR products are subejcted to specific restriction enzymes so they can be inserted into a phagemid vector. which is also subjected to treatment by specific restriction enzymes. The recombinant phagemid vector into which the VH and VL is introduced into host cells such as E. coli.

The transfected host cells are infected with a helper phage, followed by culturing, to obtain a library of phages presenting the single-chain antibodies in the culture supernatant. Examples of helper phages that can be used include VCSM13. 

The human serum-derived IgG polyclonal antibody-immobilized MLVs are added to an eppendorf tub followed by incubation overnight with the phage library. The resultant is centrifuged and sueprnatant removed. E coli cells which are cultured in advance are added, followed by culturing, centrigutation  to allow phages to be produced in the supernatant.. The rounds are repeated 3 times. 

(2) Selecting Phage presenting single-chain antibodies which binds to antigen coupled to multilamellar liposomes:

This step includes a step of allowing the antigen to bind to the single-chain antibodies expressed on the surface of phages in the phage library, a step of removing phages presenting single-chain antibodies which did not bind to the antigen coupled to multilamellar liposomes by washing, and a step of dissociating/eluting the phage presenting the single-chain antibody bound to the antigen coupled to multilamellar liposmes.

The antigen coupled to the multilamellar liposomes is not particularliy limited but antibodies are preferred. Kumada exmplifies exemplifies using a serium-derived IgG polyclonal antibody immobilized to MLVs. 

(3) Amplification step: The phage selected can then be amplified: First host cells are infected with the phage and then with helper phage such as VCSM13 which allows secretion of the phage presenting single-chain antibody into the culture sueprnatant. 

(4) repetition step: the above selection step may be repeated using a library of phages selected in the above described seletion step or a library of the phages amplified in the above described amplifcaiton step. By repeating one “round” it is possible to further select a phage having a higher binding capacity for the antigen. 

 encoded by the 1st and 2nd libraries and 5) incubating this hybrid with magnetically labeled cells in the presence of an excess of non-labeled cells, isolating magnetically labled cells and isolating the DNA encoding the protein from the vector.

Application of Phage Display for Production of Other Molecules:

Modified C2 domain of Protein G:

Baily, (J of Immunological Methods, 415 (2014) 24-30) discloses the following method for created of a modified C2 domain of Protein G having better affinity for the Fab portion of antibodies:

(1) Library creation: To generate the phage library, first the crystal structure of the Protein G-Fab complex was examined to identify 15 residues in Protein G that were within 5 anstromgs of the complex interfact. These residues were randomized using a soft randomizaiton strategy where at each position within the randomized codon, the wild type nucleotide as present at 70% and the remaining three nucleotides were present at 10%. Randomized primers were phosphorylated and used to anneal to prepared ssDNA a gene encoding the modifed C2 domain of Protein G, to replace stop codons within each of the randomized regions of the single stranded template. Double stranded DNA was synthesized and the phage library wes generated through electroporation of cells which were then infected with M13K07 helper phage. Phage partciles were isolated through PEG precipitation.

(2) Selection strategy: The first round of selection was performed with 1 mL of phage library resuspended in TBST. This was incubated with streptavidin coated magnetic beads with a cleavable disulfide linker. Phage particles were allowed to incubate before several washing steps to remove unbound virions. Phage particles were eluted form the beads to relaese the Fab form the resin and the resulting supernatant was used to infect log phase. M13K07 helper phage was added. Rounds two and three were performed with phage particles isolated form the overnight amplification of the previous round. Stable variants with high affinity to the Fab with a heat denaturation step followed by protein ELISA were idntified. The highest binding variant had a 100 fold higher affinity for the Fab compared to the wild type Protein G.