Transgenic Animals: 

A major obstacle in the clinical use of monoclonal antibodies in humans is that such antibodies are usually mouse antibodies and thus recognized as foreign, inducing an anti-isotype response. Thus efforts are underway to try and produce human monoclonal antibodies.

Transgenic mice: One approach is to incorporate genes encoding human antibody within mice. Scid-human mice, for example, contain human B and T cells and following immunization of such mice, activated human B cells can be isolated and used to produce human monoclonal antibodies. It has also been described that the monozygous deletion of the antibody heavy chain joining rejgion (JH) gene in chimeric and germ-line mutant mice results in complete inhibiition of endogenous antibody production. Transfer of the human germ line immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies upon anitgen challenge (Jakbovits, Proc. Natl. Acad. Sci, USA, 90: 2551 (1993).

Rajpal (US 13/857,867) discloses generating human monoclonal antibodies to human IL-6 using human Ig transgenic mouse strains which express human antibodies that are indistinguishable from antibodies isolated form humans. In brief, the transgenic mice strains were immunized with human IL-6splenocytes and/or lymph node lymphocytes isolated from mice producing anti-IL-6 antibodies were fused to mouse myeloma cells, cells were cultured and then supernatants from individual wells were screen for the present of human gamma, kappa antibodies and then screen for human anti-IL-6 monoclonal IgG antibodies, followed by cloning and sequencing of the anti-IL-6 antibodies. Amino acid substitution variants of the anti-IL-6 antibody 9C8 in the heavy chain variable domain were made at various positions including 68 (N68T) either singly or in combination both in the context of an IgG1 or an IgG2 format. Amino acid substitution variants of the anti-IL-6 antibody 9C8 in the light chain variable domain were made at 92 (K92N) both as an IgG1 or an IgG2 format. 

Commerical transgenic mice systems include HuMAB Mouse® (Medares, Inc., ) KM Mouse® (Medarex, Inc.) and XenoMouse®(Amgen) (Ramasubramanyan (US 13/829989) 

Transgenic chickens: Zhu show that a mAb produced in the tubular gland cells of the chicken oviduct is secreted ito egg white to yield eggs containing miligram quantities of the antibody. (Nature Biotechnology, 23(9), 2005. 

Antibody production in birds:

When compared with other immunogloical hosts, such as mice, rats and rabbits, chicken have various advantages. First,chickens can geenrate antibodies agaisnt conserved mammlain molecules because molecules that are less immunogenic in mammals might be immunogenic in chickens due to their genetic distance. Second, chicken antibodies agasint human antigens may cross-react with mosue homologues. In fact, a lot of chicken mAbs agaisnt PrP reacts with human, mouse, sheep and govine PrP. This is an important property for developmetn of therpaetuic antibodeis becasue they must be evaluated in mouse models before clinical application, and thus they are required to recognie both the human antigens and mouse homologues. Theird, chicken mAbs are easily generated by hybridoma technology. Lastly, chicken antiobdies can be easily humanized and the resulting antibodies retain both high specificity and affinity for the antigen when compared with parental antibodies. (Nishibori, “Humanization of checken monoclonal antibody using a phage-display system” (2006) Molecular immunology. 43; 634-642. 

Avian Influenze A virus M2 protein immunization:

Sinkells, Virology Journal 2013, 10: 206 (2013) discloses giving chicken a primer-boost vacination with recombinant M2 protein construct and determining specific antibody responses in serum before vaccination and after vaccination. 

Chicken Egg Yolk Immunoglobulin:

–Chiken immunized with Rotavirus:

Sarker (J Pediatric Gastoenterology and Nutrition, 32: 19-25, 2001). disclsoes that hyperimmune egg yolk given to children with known rotairus diarrhea was significantly reduced. 

Mammalain Expression Systems: 

Bansal disclsoes cloning of heavy and light chain variable domains into BioAtla’s proprietary mammalian expression system. 

Chinese haster ovary (CHO) cell based systems remain by far the most common mammalian cell line in use for mAb production. (Gary Wash “Biopharmaceutical benchmarks 2018” Nature Biotechnology, 36(12), 2018)

Adaptor molecule Act 1 (Act1) deficient B cells: 

Li (US20070028316) discloses methods of producing a hybridoma which expresses a monoclonal antibody that is specifically directed to an antigen by introudcing the antigen into a transgenic non-human mammal whose genome includes a disruption of the Act1 gene. The antibodies which specifically bind to the antigen are produced in the mammal and B cells are isolated and a B celss which expresses an antibody that specifically recognizes the antigen is selected. The selected B cell is fused with an immortal cell, thereby producing a hybrodoma that expressed a monoclonal that is specifically directed to the antigen. The invention is based on the discovery that a genetic deficiency in the adapter molecule, Act1, for CD40 and BAFF results in a dramatic increase in peripheral B cells, which culminates in lymphadenopathy and splenomegaly, hypergammaglobulinemia, hyper humoral immune respones and production of autoantibodies. 

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. 

Epstein-Barr Virus (EBV) Transformation:

B cell immortalization by Epstein-Barr vius (EBV) is an established method for antibody production. EBV infects B cells via their CD21 receptor and subsequently transforms them into continually dividing, lymphoblastoid cell lines that produce antibodies representing the humoral immune response in vivo. The major advantage of B cell immortalization when compared to other antibody producing techniques is the generation of fully human antibodies that truly reflect both the specificity and diversity of the human imune response. Fraussen (J. of Autoimmunity 35 (2010) 130-134)

EBV in combination with CpG oligonucleotides

Esslinger (WO2008/001372) discloses isolation of memory B cells from human peripheral blood lymphocytes by a two step selection prcedure. The B cell marker CD22 was used for positive selection of B cells using the MACS technology. PBL were labeled using MACS conjugated anti human CD22 mAbs, phycoerythrin conjugated mAbs aanti human IgD and APC conjugated antibodies anti human IgM, IgA, CD3, CD8, CD56. Pan B cells were isolated by positive selecting CD22 positive cell using a midi MACS device followed by selection of phycoerythrin and APC negative cells using a MoFlo cell soter. CD22 positive IgM-, IgD-, IgA- B cells where then incubated with EBV containing supernatant obtained from B95-8 cells in the presence of CpG2006.

Fraussen (J. of Autoimmunity 35 (2010) 130-134) discloses a B cell immortalization method using simultaneous B cell stimulation by CpG2006 and B cell infection by EBV, followed by an additional CpG2006 and Il-2 stimulus. 

Funaro (WO2007/068758) discloses a method for immortalizing a population of cells that secrete antibodies which includes selecting the antibody producing cells by using B cell markers such as CD22, stimulating them with a stimulating agent such as an activator of TLRs expressed on B cells (activators include CpG2006 and IL-2), eliminating the stimulating agent from the cell culture, selecting the population of stimulated cells that express antibodies of specific isotypes, exposing the population of selected and stimulated cells to an immortalizing agent, elimaiting the immortalizing agent from the cell culture.

Hallybone (WO2004/076677) discloses a method for producing a clone of an immortalised human B memory lymphocyte using the steps of transforming human B memory lymphocytes using EBV in the presence of a polyclonal B cell activator such as CpG2006.

Simmons (PLoS Med 2007, 4(5): e178) discloses using epstein-Barr virus to immotalize memory B cells from aduls who had recovered from infections with highy pathogenic avian influenza (HPAI) H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cells secreting neutrlizing antibodies were cloned and mAbs purified.

Traggiai (Nature Medicine, 10(8) 2004, pp. 871-) discloses an improved method for Epstein-Barr virus transformation of human B cells which was used to analyze memory B cells from a patient who recovered from severe acute respiratory syndrome coronavirus (SARS0CoV) infection. According to the method, peripheral blood was collected from a patient who had recovered from SARC. Memory B cells were isolated by binding to CD22 microbeads followed by depletion of cells carrying IgM, IgD and IgA by cell sorting. Memory B cells were seeded in plates with medium containing CpG 2006 in the presence of EBV. After 2 weeks, the culture supernatants were screened for specific antibodies.

EBV Stimulation but not Immortilization:

Hayday (US 14/368,749) discloses a method of producing human antibody by providing a B cell culture from peripheral blood mononuclear cells (PBMC) which are subjected to a polyclonal B cell activator such as EBV, under conditions which are sufficient for stimulating/activating proliferation and immunoglobulin secretion but wihout relying on the immortalizing properties of EBV.  In contrast to EBV based methods where the B lymphocytes are immortalized, the cells are only simulated/activated with EBV by incubating the cells with EBV for a limited time and then either diluting the EBV out such as by seeding in a different medium with a second polyclonal B cell activator such as CpG2006. 

Enhancing telomerase activity in B lymphocytes:

Esslinger (US12/991422 and WO2010/003529) discloses lentivector-mediated gene transfer of human Telomerase-Reverse-Transcriptase (hTert), the catalytic protein subunit of human telomerase, into blood dervied membory B cells to immortalize or prolong the life span of the human memory B cells.

Kohler and Milstein (1975) were the first to demonstrate that somatic cells could be fused with murine myelomas and that monoclonal antibodies with unique specificities could be produced. A vast array of monoclonal antibodies have now been produced. In the generation of most monoclonal antibodies, mice are immunized against a specific antigen, and their cells are fused with the mouse myeloma cell. The procedure is as follows: (1) spleen cells from immunized mice are fused, using polyehtylene glycol, with myeloma cells which are rendered drug sensitive by a mutation in a growth essential gene HGPRT (hypoxanthin-guanine-phosphoribosyltransferase). (2) the cell mixture is then cultured in medium containing the selective drug. The immune cells, although not sensitive to HGPRT, survive for only about one week in culture. The myeloma cells are drug senstivie and die within a week. The only cells that survive are those hybrid myeloma cells that obtained a normal HGPRT gene from the immune cells. These hybridomas can grow continuosly in vitro and some secrete antibody. (3) Using appropriate screening technology, clones of cells that secrete antibody of interest can be identified and expanded in vitro or in vivo to obtain large quantities of monoclonal antibody that can subsequently be purified to homogeneity.For more information see (Nelson, J Clin Pathol: Mol Pathol 2000, 53: 111-117) and for an 

example protocol of this method see (US Patent Application No 12/596747; Okumura (US2006/246550).  See also Greenfield “Geernating Moncolonal Antibodies, Chapter 7, 2014 Cold Spring Harbor Laboratory Press)

A more detailed procedure is the following: 1st, B-cell hybridomas are formed by fusing primed B cells and myeloma cells. This procedure involves 1) priming a mouse with a given antigen. 2) fusing spleen cells from that primed mouse with mouse myeloma cells using plyethylene glycol. The myeloma cells have the immortal growth properties of a cancer cells but do not secret their own antibody gene product. The myeloma cells also lack the ability to produce an enzyme known as HGPRT which is necessary in the synthesis of nucleotides by the salvage pathway. 3) Growth of the cells on a HAT medium which contains aminopterin which blocks the synthesis of nucleotides by the de novo pathway. When the de novo pathway is blocked, cells try to synthesize nucleotides by the salvage pathway. Fused hybridomas will be able to do this since they will contain the full complement of necessary enzymes (spleen cells which are HGPRT+ ) but myeloma cells which have not fused will not be able to grow. Unfused spleen cells do not need to be selected at all because they are terminal cells.

2nd, the resulting clones are screened for those clones which secrete the antibody with the desired specificity. The supernatant of each hybridoma culture contains its secreted antibody and can be assayed for a particular antigen specificity as by ELISA (antigen that reacts with the desired antibody is bound to microtiter wells).Once the hybridoma secreting a monoclonal antiboyd of the desired specificity has been identified, it is growth in tissue culture flasks or can be grown in the periotneal cavity of histocompatible mice.

Agilent Technologies.     Element Biosciences.   Illumina. MGI Tech. Oxford Nanopore Technologies  PacBioPacBio. Qiagen. Roche. Singular Genomics Thermo Fisher Scientific

Ultima Genomics  Twist Biosciences

single cell sequencing:

10x Genomics (conoply biosciences) (single cell RNA sequencing, kits for B and T cell receptor profiling  ArgenTAG  Fluent Biosciences

Enzymatic sequencing:

Ansa Biotechnologies    Moligo Technologies   NunaBio

Introduction:

The most popular second-generation sequencing platofrms are the 454 sequencing system (Roche), the SOLiD system (Life Technology) and the HiSeq and Genome Analyzer platforms (Illumina). Recently, to overcome the limitations of second generation sequencing due to reverse transcription and PCR amplificaiton, third generaiton sequencing platforms ahve been developed based on direct single molecule sequencing. Another benefit of third generaiton platforms is the decrease in indirect data; measurements are directly linked to the nucleotide sequence rather than being converted into quantitative data for base calling fro captured images. Third generation sequencing offers the following advnatages over second generation systems: higher throughput, higher fold coverage in minutes, higher consensus accuracy, longer read lengths and the need for smaller amounts of starting material. Monsuro, “Next generation sequencing: new tools in immunology and hematology” Blood Res 2013, 48: 242-9).

There has been a rapid proliferation in the number of next-generation sequencing (NGS) platforms, including Illumina, the Applied Biosystems SOLID System, 454 Life Sciences (Roche), Helicos HeliScope, Complete Genomics, Pacific Biosciences PacBio and Life Technologies torrent. Template preparation consists of building and amplifying a library of nucleic acid (genomic DNA or cDNA). Sequencing libraries are constructed by shearing the DNA sample into fragments of about 500 p or less and ligating adapter sequences (synthetic oligonucleotides of a known sequence) onto the ends of the DNA fragments. Once constructed, libraries are clonally amplified in preparation for sequencing. Depending on the platform, the amplification method can vary. For instance, the Life Technologies Ion Torrent PGM platform utilises emulsion PCR on the One Touch system to amplify single library fragments onto microbeads, wheras the Illumina MiSeq instrument utilises bridge amplificaiton to form template clusters on a flow cell. (Stambrook, “Next-generation sequencing technologies: breaking the sound barrier of human genetics” Mutagenesis, 2014, 29(5), 303-310). 

Next-geenration sequencing (NGS), also called massive parallel sequencing, was developed in the last decade and allows simultaneous sequencing of millions of DNA fragments, without previous sequence knowledge. This advanced techology ahs been a true revolution copared with the traditional sequencing methods, in which one or a few relativey short fragments of DNA, previously amplified by PCR, could be sequenced per tube. With NGS, the today promise is that a coplete genome can be sequenced in a few days for less than 1k per gehome. (Kamps, “Next-generation sequencing in oncology: genetic diagnosis, risk prediction and cancer classification” International J. of Molecular Sciences, 2017, 18, 308)

Next generation sequencing (NGS) refers to a procedure similar to capillary electrophoresis based sequencing in which DNA polymerase catalyzes the incorporation of fluorescently labeled desoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during sequential cycles of DNA synthesis. During each cycle, at the point of incorporation, the nucleotides are identified by fluorophore excitation. Instead of sequencing a single DNA fragment, the process extends across millions of fragments in a massively parallel manner. (Maher Albitar, Neogenomics, US Patent No: 10,253,370). In practice, this technology increases sequencing thorugh-put by attaching millions of DNA fragments to a solid surface or support, and simultaneously sequencing all fragments in parallel. Current methods generally involve randomly breaking the sample into fragments and building fragment libraries. The fragment libraries are then prepared for sequencing by ligating specific adaptor oligonucleotides to both ends of each fragment, and subsequently using these as sequencing templates. The typical output of NGS is a list of billions of short sequences (25 to 400 bp), called reads, associated with quality scores. The molecular reliability of NGS data depend on 3 criteria: depth fo coverage, heterogeneity, and accuracy of sequencing. Depth of coverage indiates the number of times that a given nucleotide is sequenced (for example a 5x indiates that each nucleotide of the target region was sequenced, on average, 5 times). Heterogeneity is a measure of uneven sequencing depth of coverage along the lenght of the expressed region. Finally, accuracy of sequencing is indicated by the quality of the base calls or quality scores (i.e., the quality scores assigned to each base call in automated sequencer trace, known as phred scores). Monsuro, “Next generation sequencing: new tools in immunology and hematology” Blood Res 2013, 48: 242-9).

The analysis of the billions of short sequence reads generated by NGS platforms requires powerful computational tools. Such tools must be able to align reads to a reference transcriptome or genome sequence to identify and quantify expressed gene isoforms (transcriptome profiling), and to perfom differential expression analysis between specimens (expression quantification). As a fule, the estimation of expression levels in RNA-seq analysis is perfomred in 2 steps: (1) sequence alignments to a reference genome and (2) quantificaiton of gene isoform expression levels. Since the entire process reqires several comptuer programs to be used (whose parameters must be tuned according to the goal of the study), researches tend to prepare their pipeline of programs to analyze RNA-seq sampels in an automated and simple manner. Monsuro, “Next generation sequencing: new tools in immunology and hematology” Blood Res 2013, 48: 242-9).

RNA can be isolated from antibody-secreting cells and libraries prepared by RT-PCR which can then be sequenced using the Illumina MiSeq platform. The robustness of the antibody repertoire data can then be assessed based on clonal identificaiton defined by amino acid sequence of either full-lenght VDJ region or the CDR3. (Ereif, “Quantitative assessment of the robustness of next-generation sequencing of anitobdy variable gene repoertoires from immunized mice” (2014) BMC immunology. 15(40). 

High-throughput DNA sequencing can be used to analyze the VL and VH gene repertories derived form the mRNA transcripts of fully differentiated mature B cells, antibody-secreting BMPCs, from immunized mice. After bioinfomratic analysis, several abundant VL and VH gene sequences can be identified with the repertoire of each immunized mouse. VL and VH genes can be parted acording to their relative frequencings within the repertoire. Antibody gene can then be synthesized by oligonucleotide and PCR assembly by automated liquid-handling robots. Recombinant antibodies are then expressed in bacterail and mammalian systems as single-chain variable fragmetns (scFv) and full-lenght IgG, respetively. (Reddy, “Monoclonal antibodies isolated without screwewning by analyzing the variable-gene repertoire of plasma cells” (2010) Nature Biotechnology, 28(9); 965-969. 

Multiomics uses NGS techniques to collect unbiased data from different biologcial levels “omes” in one experiment –1. proteomics, 2-transcriptomics 3-epigenetics and 4 -genomics. Doing this allows researcers to examine the same problem from multiple angles. Multiomics has been instrumental in udnerstanding why different patients with the sae type of cancer have variable respones to the same treatment. Single omics may not adequately explain these different resposne rates given taht cancers contain heterogeneous populations of cells which change and evolve in response to treatment. (Multiomics: An Overview of Useful Methods and Applications” Illumina)

NGS Methods:

The NGS workflow includes the basic steps of (1) the sequencing library is prepared by random fragementaion of the DNA or cDNA sample, followed by 5′ and 3′ adapter ligation. Alternatively, “fagmentation” combines the fragmentation and ligation reactions into a single step to increase the efficiency of the library preparation step. Adapter-ligated fragments are then PCR amplified and gel purified. (2) for cluster generation, the library is loaded into a flow cell where fragmetns are captured on a law of surface-bound oligos complementary to the libary adaptorers. Each fragment is then amplified into distinct, clonal clusters through bridge amplificaiton When cluster generation is completed, the templates are ready for sequencing; (3) seqeuncing reagents including fluorescently labeled nucleotides, are added and the first base is incorporated. The flow cell is imaged and the emission from each cluster is recorded. The emission wave-lenghts and intesities are used to identify the bases. (4) newly identified sequence reads are aligned to a reference genome. After alignment, differences between the referenced genome and the newy sequenced reads can be idnetified. (Maher Albitar, Neogenomics, US Patent No: 10,253,370).

Different approaches can be used according to the needs and the questions to be addressed. The initial input material can be genomic DNA 9DNA-seq), messenger or non-coding RNA (RNA-seq) or any nucleic/ribonucleic material obtained after specific procedrues. The implemtntaion of NGS technology can be visualised as four major blocks: (Kamps, “Next-generation sequencing in oncology: genetic diagnosis, risk prediction and cancer classification” International J. of Molecular Sciences, 2017, 18, 308)

(1) Library perpation or sample processing: the material is first fragmented mechanically or enzymatically to yield fragments whose size is compatible with the sequencer (small fragmetns of 200-300 nucleotides for short read sequencing, longer for teh long read sequencing). This material can be enriched to analyse a limited number of genetic regions (e.g., disease gene panels or microbes or all coding exons of the human genome from about 21,000 genes; Whole-exome sequenencing, WES). The complete genomic DNA can also be sequenced (Whole-Genome Sequencing, WGS) and it does not reqire any enrichment step. The regions that are intended to be analysed are defined reigon of inteterest (ROIs). An amplification step through PCR with 4-12 cycles is performed in most cases. During this step, poper linekrs and barcodes are attached to the DNA fragments adn are necessary for subsequent analyses by the sequencer. DNA barcodes, which are unique nucleotide tags (6-8 nt) allow pooling sampels togetehr oin one single flowcell for teh sequencing reaciton.

(2) Sequencing: 

For most clinical applications, the use of gene-panels to sequence only a discrete number of genes of interest has been the method of choice, because of its cost efficiency, and because at the same time it achieves high coverage of ROIs and offers implicity in the raw and subsequent data analyses. When the number of enes sequenced is restricted to the few already analysed in previous diagnostic tests using traditional methods, this is normally called targeted re-sequencing. Different protocals are available to design and capture panels of genes and other ROIs. In most cases, companies providing the library prepration kits offer online use friendly tools to design the hydridisation probes or the PCR oligos to enrich the desiged ROIs. Envirhcmetn can be obtained via solid phase hydridisation, in soclution hybridisation (ost fequently sued) or PCR based enrichment and is followed by amplificaiotn via ultiplex PCR, folling circule amplificaiton (HaloPlex) or amplicon based microdroplet PCR (RainDance technology). The latter presetns the advantage of simultaneously amplifying a large number of targeted regions into separate micro drops, thus keeping each amplification separate form teh others and limiting the distrurbance due to primer pair interactions. A cheap and flexible method to capture small regions of the genome for NGS analyses is the Molecualr Inversion Probe. 

(3) Initial quality and raw data analyses: 

(4) Variant calling and data interpretation. This step is dependent on the specific applciaiton. 

Sequencing Before and After Immunziation:

Ankeny (US Patent Application 16/060,304, published as US 2020/0407426) discloses that prior to immunization, the non-immune repertories was sampled from venous blood from hens. Hens were hyperimmunized (booster) and 13 days following the primary hyperimmunization, the post-state repertoire was sampled form venous blood. PMBCs were isolated from anticoagulated bood by density gradient centrifugation, isolated PMBCs were then extracted using the mir-Vana kit form Thermo Fisher, following the PBMC enrichment, cell pellets were lised and mRNAs isolated and reverse transcribed, the variable regions of the H and L chains amplified, sequenced and cloned. Knowing the repertoire prior to hyperimmunization can allow for calculation of clonal frequency changes that can be explained by B cell clonal expansion during the adaptive immune response. A variety of sequencing methods can be used. In one embodiment, Ion Torrent sequencing libaries are prepared and sequenced. Other methods include Illuminia, 454 pyrosequencing as well as 3rd generation systems such as single-molecule real-time sequencing. Following amplication of VL and VH genes, purified VL and VH amplicon libraries were prepared using the Ion Torrent’s ion Plus Library Preparation Kit. Amplicon libraries recieved unique sequencing barcodes. Abundant CDRL3 sequences were matched to the candidate VH sequences by relative rank orders from immunized repertories. As a resutl of clustering by similarity, cominant CD3R3 sequences for each reperotire were revealed. CDR3 lenghts of the two immune states’ most abundant sequences can be significanlty different. 

Swinkels (Virology Journal 2013, 10: 206) disclsoes determing the M2 specific antibody response (from influenza virus) in the serum befroe vaccination, 3 weeks after vaccination and two weeks after booster from chickens. 

Ma (“characteristics peripheral blood IgG and IgM heavy chain complementarity determinign region 3 repertoire before anda fter immunation with recombinant HBV vaccine” published January 23, 2017). discloses  high-througput sequences of BCR heavy chain CDR3 repertories in 3 healthy volunteeers before and after the thrid immunizaiton with recombinant HBV vaccine. The Roche 454 Genome Sequncer FLX system was used to perform a comparative analysis of IgM and IgG H chain CDR3 repertories. In the producedure, PBMCs  were isolated from heparin treated periopheral blood using denstiy gradeint centrifugation. Total RNA was extracted from PBMCS, then reverse transcribed into cDNA and PCR was performed to amplify human BCRH hain CDR3 repoertories. 

Affinity Purification – Mass Spectometry – Next-generation DNA sequencing

Cheung “A proteomics approach for the identificaiton and cloning of monoclonal antibodies from serum” 2012, Nature Biotechnology, 30(5): 447-452) discloses a proteomics approach that identifies antigen-specific antibody sequences directly from circualting polyclonal antibodies in the serum of an immunized animals. The approach includes affinity purification of antibodies with high specific activity and then analyzing digested antibody fractions by MS. High-confidence petpide spectral matches of antibody variable regions are obtaine by searcing a reference database created by next-generation DNA sequencing of the B-cell immunoglobulin repertoire of the immunized animals. Finally, H and L chain sequences are paired and expressed as recomibnant monoclonal antibodies. 

Sequencing Directly from PCR Products:

Large numbers of templates for DNA sequencing can be produced via PCR directly from plaques, colonies or genomic DNA. Sequencing direclty from PCR products has many advantages over subcloning such as removing the need for template prepration. It is also highly amenable to automation. However, a problem is the subsequent purificaiton of the amplified products prior to DNA sequencing. (Hawkins”Solid-phase reversible immobilizaiton for the isolation of PCR products” Nucleic Acids Research, 1995, 23(22) discloses a method for producing aulity DNA sequencing template form PCR products termed SPRI “solid-phase reversible immobilization”. The SPRI employs a carboxyl coated magnetic partcile which can reversibly bind DNA in the presence of polyethylene glycol (PEG) and salt. 

mRNA Sequencing:

Messenger RNA accounts for about 2% of the whole-transcriptome and is composed of poly-A-tailed RNA that codes for proteins. mRNA sequecing (mRNA-Seq) provides an unbiased and complete view of the coding transcriptome. Compared to Ttoal RNA-Seq, mRNA-Seq allows researchers to focus on the coding transcriptome which also means less but targeted data. Illumina reccomends the Illumina Stranded mRNA Prep which is a simple, scalable rapid library preprations solution for anlyzing teh coding transcriptome with as little as 25ng of RNA input. Sequencing can be done with either the NextSeq 1000/NextSeq 2000 or NovaSeq 6000. Data analysis can be perofrmed using the DRAGEN RNA Pipeline or Differential Expression apps to obtain differentail expression results at the gene and transcript levels. (Multiomics: An Overview of Useful Methods and Applications” Illumina)

Whole-Genome Sequecing (WGS): 

WGS analyzes the whole gehome of a population of cells or of tissue samples. Using WGS, researchers can uncover genetic events that contribute to disease beyond protein coding variants. WES allows researchers to analyze the portion of the genome responsible for coding proteins (the exome). While the exome represents less than 2% of the entire genome, it accounts for 85% of diases related variants. using WES, researchers can study which protein coding variants contribute to disease or dysfunciton. After DNA extraction, libraries ofr WES can be prpared using the Illumina DNA prep with enrichment. Illunina reccomends the NextSeq 200 or NovaSeq ^000 sequencers for WES. Illumina recommends using the DRAGEN platofrm either on BaseSpace Sequence Hub or on a DRAGEN server to obtain data from WES. In BaseSpace Sequence Hub, you can monitor runs in real time while securely streaming data directly from the intruments into the ecosystem. (Multiomics: An Overview of Useful Methods and Applications” Illumina)

Whole-genome sequencing of bacterial pathogens:

Peacock “Rapid single-colony whole-genome sequencing of bacterail pathogens” J. Antimicrob Chemother 2014, 69, 1275-1281) discloses that rapid benchtop sequencers can provide multiple pieces of clinically relevant information in a single process. The starting material for bacterail WGS is typically purified DNA extracted form liquid culture. But sequencing can also be done directly form a single bacterail colony on primary isolation plates. 

Proteomic Methods:

CITE-Seq uses oligonucleotide-labeled antibodies to measure proteins and RNA in teh same experiment. CITE-Seq is a high throughput multiomic tool that allows researchers to study protein expression and the intricacies of the cellular transcriptome both at the single cell level and for spatial anlysis. (Multiomics: An Overview of Useful Methods and Applications” Illumina)

Epigenomics Methods:

ATAC-Seq is an epigenomic discovery tool for mapping chromatin accessibility across the genome. This approach analyzes DNA accessbility using the Tn5 transposase. The Tn5 transposase inserts sequencing adapters into open chromatin regions. Researchers can then use sequencing to locate regions of increased chromatin accessbility. ATAC-Seq allows researchers to study how these regions impact gene expression and can be used to study both single cells and cell populations. (Multiomics: An Overview of Useful Methods and Applications” Illumina)

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Definitions:

VHH refers to an antibody single variable domain (VH or VL) polypeptide that specifically binds antigen. While the antigen binding unit of a naturally occuring antibody in humans and most other mammals is generally known to be comprised of a pair of V regions (VL/VH), camelid species express a large proportion of fully functional, highly specific antibodies that are devoid of light chain sequences. (see Hamers-Casterman, Nature, 363, 1993). 

VHHs were discovered in 1993 at the University of Brussels. Only a fraction of the immune repertoire of the animals of the camelid family comprise the heavy chain antibodies. The other fraction are normal classical antibodies. The heavy chains of these so called VHH bind their antigen by one single domains, the variable domain of the heavy immunoglobuilin chain, referred to as VHH. With a MW of about 12 kDa, the VHH domain is the smallest known intact binding fragment dervied from a funcitonal immunoglobuilin.

antibody single domain or single domain antibody refers to antibody fragments that include either a VH or VL domain of antibody, that is, a single monomeric variable antibody domain. Unlike whole antibodies, single domains do not exhibit complement system triggered cytotoxicity, as they lack an Fc region. Antibody single domains often have peptide chains of about 110 amino acids and MW in the range of 12-15 kDa. They are much smaller than intact antibodies which generally are about 150-160 kDa, being composed of two heavy chains and two light chains. Antibody single domains are also smaller than Fab fragments (about 50 kDa; one light chain and half a heavy chain) and single-chain variable fragments (about 25 kDa; a light chain variable domain and a heavy chain variable domain). The small size of antibody single domains allows better solubility and better permeability in tissues, as well as greater heat-resistance and stability towards detergents and urea. Small size often leaves also to a short serum half-life, as smaller molecuels are eliminated renally more readily. Nonetheless, small size facilitates binding to hidden epitopes that may not be accessible to intact antibodies or larger antibody fragments. (Castanheira, WO 2013/043070). 

A dAb is either the variable domain of an antibody heavy cahin (VH domain) or teh variable domain of an antibody light chain (VL domain) each dAb therefore contains three of the six natturally occurring complementary determining regions (CDRs) from an antibody. Although it might seem surprising that three CDR regions are sufficient to confer antigen-binding specificity and high affinity, Darwinian evolution has itself arrived at that very solution in camelids, which product antibodies including only a heavy chain. The antigen ginding site of these antibodies consists of a single unpaired varable domain (referred to as VHH). Holt, “Domain antibodies: proteins for therapy” TRENDS Biotech. 21(11), 2003).

Types of Domain Antibodies: VHH/Domain antibodies (dAb) (also known as “domain antibody” or “single domain antiboy” or “single variable domain” or “immunogloublin single variable domain or dAb or nanobody®)

Single domain (SdAb): One class of antibodies are single domain antibodies (or “nanobodies”). Unlike standard human antibodies which are exclusively expressed as paired species containing both heavy and light chains, single domain antibodies contain either only the H chain variable region or only the L chain variable region, while preserving the antigne-binding properties of conventional antibodies. They are able to demonstrate high specificity and affinity similar to standard IgG antibodies but are much smaller in size. which is useful in a number of appliations such as biosensor, diagnostic and therapeutic applications. Although single domain antibodies exist as monomers, they can dimerize to form dimers. For example, light chain dimers known as Bence-Jones proteins have previously been described. (Christei (US 16/497,788, published as US 20200033363). See also particular types of antibodies purified in outline 

The single antigen domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies which need to interact with another variable domain (.e.g. through a VH/VL interaction to form a functional antigen binding domain. Domain antibodies will often comprise at least one intradomain disulfide bridge between cysteine 22 and cysteine 92. Sometimes, nanobodies comprises a disulfide bond between CDR3 and Cys45 in the framework region FR2. There may also be a disulfide bond between CDR2 and CDR3 for example between cystein 45 and CDR3 or between cysteine 50 and CDR3. (Schotte US13/266503 and US2012/0157664)

Camedlid VHH antibodies:

In 1993 Hamers-Casterman discovered a novel class of IgG antibodies in Camelidae (camels, dromedaries and LLamas). These antibodies are devoid of light chains and thus called “heavy-chain” IgGs or HCab. They have a MW of about 95 kDa instead of the about 160kDa for conventional IgG antibodies. Their binding domains consist only of the heavy-hain variable domains. Since the first constant domain (H1) is absent (spliced out during mRNA processing due to loss of a splice consensus signal, the variable domain (VHH) is immediately followed by the hinge region, the CH2 and CH3 domains. Although HCAbs are devoid of light chains, they have an authentic antigen binding repertoire.(Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15).

Due to structural differences compared to VHs of normal heterotetrameric IgGs, VH domains of the heavy chain dimer IgGs are called variable domain of the heavy-chain of heavy chain antibody (VHH). (Honda, US 2005/0037421).

The crystal and solution structures of several dAbs have been solved and show that isolated human VH and camelid VHH dAbs adopt a beta-sheet structure similar to a VH or VL immunoglobuilin fold in a conventional antibody. By contrast, some of the CDR structures seen in camel VHH dAbs are different fom those in naturally paired VH domains. A comparison of the structures of camelid CDRH1 and CDRH2 with the set of loops found in human and mouse antibodies has revealed significant differences in their main chain conformations. In addition, the CDRH3 region of naturally occurring camelid VHH antibodies are, on average, longer than the CDRH3 region s of naturally paired VH domains. Camelid VHH sequences also contain amino acid substitutiosn in their framework regions taht are not observed in paired VH domains and which might have evolved to accommodate the absence of the VL and constant CH1 domains. Holt, “Domain antibodies: proteins for therapy” TRENDS Biotech. 21(11), 2003).

Diversity of Single Domain Antibodies

Single domain antibodies interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH-VL combinatorial diversity, these heavy chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions (Muyldermans “Single domain camel antibodies: current status” Reviews in Mol. Biotech., 74 (2001) 277-302).

Analysis of the differences in amino acid sequence between the VHs of these camel heavy chain only antibodies and the VH domains form conventional human antibodies has been used to design an altered human VH domain. This “camelized VH” is a small and efficient recognition unit (“Riechmann “Single domain antibodies: comparison of camel VH and camelised human VH domains” J. Immunol. Methods, 231 (1999), 25-38).

The VH germline segments are highly diveeast of the single domain was at least fivefold higher than the scFv, and the ability of the single domain intraody to inhibit huntington aggregation, which has been implicated in the pathogenesis of Huntington’s disease was confiremd in a cell free in vitro assay as well as in a mammalian cell culture model of HD. ytoplasmic expression levels in y of this variable L chain doamin and it was found to retain full binding activity to huntingtin. Crse, leading to a broad structural repertoire of the antigen-binding loops. Seven VHH subfamilies are known, of which five have been confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrated that the rearranged VHHs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and high incidence of nucleotide inertions or deletions. These diversificaiton processes are driven by hypermutation and recombination hotspots embedded in the VHH germline genes at the regions affecting the structure of the antigen-binding loops. (Nguyen, EMBO Journal, 19(5), 2000, pp. 921-930)

The diversity of antibody repertoire of camelids is determeined by the CDR1-3 in the VH or VHH regions. The CDR3 in the camel VHH region is characterized by its relatively long lenght aeveraging 16 amino acids. For example, compared to the CDR3 of mouse VH having an average of 9 amino acids, the CDR3 of camel IGG is very long. (Honda US 2005/0037421).

The simpler structure of camelid VHH does not decrease their affinity or specificty due to the presence of an extremely variable CDR (Victor de Lrenzo Prieto, US2008/0280346).

Advantages of Single Domain Antibodies:

Advantages of VL:

Colby (J. Mol. Biol. 2004, 342, 901-912) discloses using yeast surface display to affinity mature an scFv pool against huntington form a non-immune human antibody library. Colby then analyzed the location of the binding site of the mutant with the highest affinity and discovered that the paratope was mapped exclusively to the light chain domain of the scFv. A single domain antibody was then constructed consisting soley of this variable light chain domain and was found to retain full binding activity to huntingtin. A potential advantage of using single-domain intrabodies rather than scFv for treatment of HD via gene therapy arises form the redued mass of the smaller intrabody. A hallmark of HD is the formation of intranuclear inclusions, so the ability of an intrabody to diffuse into the nuecleus an d and prevetn aggregation may be critical. While scFvs, with a MM of 25-30 kDa are close to the MM cutt-off of nuclear pores, single domain intrabodies are only half that size and should more readily diffuse into the nuclear subcompartment. 

Advantages of VHHs

Since the VH region of a heavy chain dimer IgG does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is mutated to hydrophilic amino acids residues. As a result, VHH have exellent solubility. Furthermore, VHHs derived form acmels and LLmas have very high thermostability compared to mouse heterotetrameric antibodies. The use of VHH derived form these species can provide, for example, molecles that maintain their antigen binding ability even at 90C. (Honda US 2005/0037421).

Single Domain Antibodies as Multimers (e.g., Dimers)

(Castanheira, WO 2013/043070) discloses more than one single dimain of an immunoglobulin as a dimer, trimer, tetramer, etc such as an anti-TNF alpha polypeptide that includes two antibody single domains in the form of a dimer. Dimer formats include, e.g., VH-VH dimers, VL-VL dimers, and VH-VL dimers. VL single dimains that showed particularly high affintiy bidning to human TNF alpha were sequenced. Two VL domains showing high affintiy binding were selected for combination as dimers. As a spacer between the two VL domains, either a 8 amino acid GlySer linker, 13 amino acid GlySer linker or 18 amino acid GlySer linker was used. Dimer combinations were cloned and expressed and purified. 

VL-VL Dimers:

It has been shwon that free immunoglobulin light chains assocaite as dimers, thus burying the hydrophobic surface that are normally involved in the interactions with the heavy chain. (Nymalm, J Structural Biology 138 (2002) 171-186). An unusual mode of domain-domain association is the VL-VL interaction, found in most cases of Bence-Jones disease and many myelomas lacking the H chain. This closely mimics the interace geometry of VL/VH, although its ontact area is 18% less. (Burrone, J. Mol. Biol. (2003), 333, 355-365). 

Christ (US 16/497,788, published as US 20200033363) discloses immunoglobulin homodimers that include identical light chain variable region (VL) monomers able to bind to an antigen. The single domain immunoglobulin homodimer is produce using V and J segments of the human antibody light chain family which are amplified by PCR and cloned into the phage display vector pHEN1. V segment diversity was introduced at CDR1 and CDR2 positions while J segment diversity was introduced by PCR using degenerate TRIM oligonucleotides. Combinatorial diversity was generated through recombination of the V and J segments using splice overlap extension PCR followed by cloning into display vector and transformation into E coli. This resulted in a library of phage cosntructs with each bacteriophage displaying multiple copies of the receptor on the tip of its filamentous surface. These multiple copies of each receptor were found to be able to dimerize, for example, upon binding an antigen. The phage library was for example panned against hen egg-white lysozyme (HEL) which had been biotinylated and the HEL antigen was captured using nuetravidin coated wells. For rounds 2 and 4 of the selection, amgnetic streptavidin beads were used as an alternative means of capture to prevent the selection of binder against the capture reagents. After four rounds of selection, binders were identified by soluble fragment ELISA. This selection step allowed the identification of antigen specific phage clones. Regions encoding single-domain immunoglobulin homodimer from selected clones were amplified by PCR and subsequently cloned into the periplasmic expression vector which were transformed into E. coli. The single domain homidmers were purified using Protein L affintiy resin. Next, a dimierization screening step using size exclusion chromatography coupled with multi-angle laser lgiht scattering (SECMALLS) was used to measure the MW of the immunoglboullin-antigen complex. By measuring the MW of the immunoglobulin-antigen complex, immunoglobulin alone and antigen alone, a stoichiometry of immunoglobulin-antigen complext was found to be 2:1. The measured MW were consistent with two Ig domains bound to a single HEL molecule in the immunoglobulin-antigen complex. 

VH-VH Dimers:

Functional VH dimers selected from phage display libraries can be secreted efficiently from both bacteria and mammalian cells in different formats. (Burrone, J. Mol. Biol. (2003), 333, 355-365).

The novel iimmunoglobulin isotype novel antigen recetpor (IgNAR) found in cartilagenous fish is composed of a heavy cahin homodimer that does not assocaite with light chains. The variable regions of igNAR function as independent domains similar to those found in the heavy chain immunoglobulins of Camelids. (Dooley, Molecular Immunogloby 40, 2003, 25-33). 

Isolation/Purification (See also purification of antibodies)

The variable fragments of heavy chains of immunoglobulins devoid of light chains can be prepared starting from immunoglobulins obtainable by purification from serum of camelids according to the process for the purification as described in WO94/04678. 

IgG homeostasis: (see right hand panel)

IgG participates in defending against viral antigens, whereas IgM is predominantly active in antibacterial and antitoxin immune responses (US2013/0052208). 

Antibodies belong to a family of globular proteins called immunoglobulins. IgG is the most common. Eight percent of all the antibodies present in the blood are IgG. IgG is a relatively large molecule (about 150 kDa). It has four subclasses, IgG1, IgG2, IgG3 and IgG4. (see below). Chakrabarti, “separation of monoclonal antibodies by analytical size exclusion chromatography” Antibody engineeering, Cahpt 7, 2018).

Which Isotype to choose?

The human IgG compartment consists of four distinct subclasses, designated IgG1 (6.98 mg/ml in serum) , IgG2 (3.80 mg/ml) , IgG3(0.51 mg/ml) and IgG4 (0.56 mg/ml). The principal biological activities of IgGs are related to their effector functions, including activation of complement and binding Fcgamma receptors to mediate antibody-dependent cellular cytotoxicity. Although their heavy chains are about 95% similar in sequence homology, IgG subclasses express unique effector activities. In general, protein antigens characteristically elicit IgG1 and IgG3 responses, and these isotypes are capable of activating all Fc receptors and the C1 component of complement. Induction of IgG4 subclass appears to be characteristic of chronic anitgen stimulation, such as observed in autoimmune disease. IgG4 exhibits restricted Fc receptor activation and does not activate C1q. The IgG2 sublcass often predominantes in response to carbohydrate antigens and also exhibits restricted Fc receptor and C1q activation. (Taylor, Gynecologic Oncology 116, (2010) 213-221). 

Different IgG isotypes, such as IgG1, 2, 3, and 4, demonstrate unique recognition and activation profiles when interacting with favrious FcyRs. IgG1 has proved to be most effective in complement dependent (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). This corresponds with the fucntion of this highest cirulating serum IgG subclass for binding exogenous pathogens and for effective destrcution and clearance of antigens via activation of various effector mechanisms. IgG3 has been shown to be as effective as IgG1 in complement activation and cell mediated toxicity, whereas IgG2 and IgG4 isotypes are relatively inactive in eliciting effector functions. (Drug Discov. Today, 2006 Jan; 11(1-2):81-8).

A recent paper comparing all four human isotypes of an anti-GXM antibody in a Crytococcus neoformans infeciton model is evidence that there are no general rules for istype slection. Contrary to expectations that an active istype might have superior bactericidal function, IgG2 or IgG4 were optimal, whereas IgG1 was determiental. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Specific Isotypes

Structurally, the human IgG subclasses are remarkably similar with an overall >95% sequence identity. This implies that relatively few amino acid differences unerlie functional variation between for example IgG1 and IgG. (Rispens “Mechanisms of Immunoglobulin G4 Fab-arm Exchange” J Am Chem Soc 2011, 10302-10311). 

IgG1: The most abundant isotype of human serum antiboides is IgG1. IgG1 is a glycoprotein whose N-linked carbohydrate is located in the CH2 domain of its H chain. Mainy studies show that thsi carbohydrate is critical in maintaning the structure and funciton of IgG1. Dorai (Hybridoma, 10(2), 211-217 (1991)) show that aglycosylated IgG1 failed to exhibit any ADCC activity but a significant level of CDC activity was retained. Accordingly, some, but not all, effector functions of a human IgG1 Ab are affected by aglycosylation.  

IgG1 has been the most popular choice for antibody isotype choice and has been approved for over 13 chimeric, humanized or fully human antibodies compared with only two instances for IgG4 and one fo IgG2. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

IgG2: The IgG2 sublcass often predominates in response to carbohydrate antigens and also exhibits restricted Fc receptor and C1 q activation. For example, in acute lymphocytic leukemia (ALL), elevated levels of 9-O-acetylated sialic acid specific IgG2 were induced, which were unable to trigger activaiton of Fcy receptors, the complement cascade and cell mediated cytotoxicity, although its glycotope binding ability was unaffected. The subclass switching of anti-9-OAcSa to IgG2 in ALl was linked with alterations in the tumor cell’s glycosylation profile. (Taylor, Gynecologic Oncology 116, (2010) 213-221).

Human IgG2 antibodies have been shown to consist of three major disulfide structural isoforms IgG2-A, -B and -A/B. This structural heterogeneity is due to different light chain to heavy chain connectivity in each isoform. It has been shown that the individual isoforms can have unique and different structural and functional properites, including differences in potency or other quality attributes including Fcy receptor binding, viscosity, stability, and particle formation. (Dillon, US14/363735)

The natural IgG2 response is typically directed twoard carbohydrate antgiens, which are arrayed in repetitive patterns on bacterial surfaces. Dimers of these often low affinity IgG2s can enhance protection and provide an evolutionary advantage. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Panitumumab is a human IgG2k mAb that binds specifically to EGFR with high affinity, and not to the other ErbB family members. Treatment with panitumumab has been associated with low immunogenicity and low risk of hypersensitivity reacitons. The fully human IgG2 mAb, panitumumab is devoic of natural killer (NK) cell mediated classical ADCC. The IgG2 isotype was chosen to minimiz potential toxicity to EGFR expressing normal tissues form ADCC and CDC. A novel anti-EGFR mAb, denoted as Pan-P, has been developed with the goal of enhancing ADCC and target selective activity and using probody technology where an epitope blocking peptide was tethered to Pan via a tumor specific protease selective linker. Pan-P showed almost 40 fold weaker affinity compared with Pan, but funcitonal activity was restored to a similar extent as Pan when Pan-P was selectively activated by urakinase-type plasminogen activator (uPA)  (Yang, “Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency” mAbs (2015). 

IgG3: Antibodies desigend for slective eradication of cancerl ells typically require an active isotype that permits complement activaiton and effector mediated cell killing by antibody dependent cell mediated cytotoxicity. Although IgG1 and IgG3 both meet these criteria, IgG3 has not been used for therpaeutic antibody development probably because of a shomewhat shorter half life susceptibility of the longer hinger region to proteolysis and extensive allotypic polymorphism. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

IgG4:

IgG4s possess several properties that make them attractive therapeutic candidates. For example, IgG4s have long serum half-lives and low Fc function and/or effector function. However, IgG4 antibodies also have unusual properties which are undesirable in vivo: they are unstable, dynamic molecules which engage in Fab arm exchange. An administered therapeutic IgG4 antiboy may exchange with endogenous IgG4 antibodies with undesired specificites. (Brower, US 14/668820, published as US 2016/0108127). 

–Structure of IgG4:

For the CH1 domain, the IgG4 has a Cys at position 151, which is used for linking to the light chain. In contrast, IgG1 has a Ser at this position, lining to the light chain via Cys220 in the hinge, which is one of the three extra amino acids in the IgG1 hinge compared to IgG4. At positions 137-138 both IgG2 and IgG4 have Glu-Ser, whreas IgG1 has Gly-Gly. IgG4 also has Lys at position 196, which is Gln in IgG1 as wella s in IgG2. The IgG4 hinge 9as well as the IgG2 hinge) is three amino acids shorter than the hinge of IgG1. Similar to IgG1, IgG4 has two cysteins that are available for the covalent interaciton between the H chains. The CH2 domain of IgG4 has its most marked ifferences with IgG1 in a surface exposed patch of the C temrinal part of the doamin which in 3D structure is clsoe to the hinge. Starting at position 337, the IgG4 sequences reades GLPSS, that of IgG1: ALPAP. The IgG4 patch is clearly much less hydrophobic than the IgG1 patch. (Shuurman, Immunology, 2002, 105, 9-19). 

–Fab Arm Exchange (IgG4 shuffling):

IgG4 antibodies are known to undergo a process called Fab arm exchange, also known as IgG4 shuffling in which the heavy chains separate and randomly re-assocaite to produce a mixed population of IgG4 molecules with randomized heavy-chain and light-chain pairs. 

It has been demonstrated that a Serine to Proline mutation at position 228 using the EU index in the hinge region of human IgG4 results in the reduction of IgG4 “half-antibody” molecules and reduced heterogeneity/shuffling of IgG4 molecules. 

Relative to the Cys-Pro-Pro-Cys motif in IgG1, the IgG4 sequence appararently increases hinge flexibility, allowing justaposition of the two intrachain cysteines and bringing intra and interchain disulfide bonds into equilibrium. When the interchain bonds of IgG4 break and the intrachain bonds are created, new interchain bonds can be formed. The relative concentrations of different IgG4 molecuels (and their derived half molecules) determine whether these new interchain disulfide bonds are formed between two identical or two different IgG4 half molecules, leading to bivalent moelcules with mono or bispecificity, respectively. To address this problem, antibody engineers introduced a serine to proline substitution that made the middle hinge region of IgG4 more like that found in IgA which gave a more homeogenous monospecific product but also extended serum half-life and improved tissue distribution. (Salfeld “isotype selection in antibody engineering” Nautre Biotechnology, 25,(12), 2007).

Comapred to IgG1, two structural features of IgG4 are important for the exchagne reaction. First, the CH3 domain of IgG4 is necessary for exchange. The (noncovalent) interheavy chain interactions in the CH3 domains are most likely significantly weaker, thereby allowing dissociation and inevitable step in the exchange process. Second, a serine instead of proline at position 228 in the core hinge further facilitates exhcange, probably because the hinge is more easily reduced, since the exchange reaction can be observed at higher concentrations of GSH also if this feature is absent. Introducing boht features in IgG1 results in a mutant antibody that is able to exchange half-molecules similar to IgG4. (Rispens “Mechanisms of Immunoglobulin G4 Fab-arm Exchange” J Am Chem Soc 2011, 10302-10311).

companies:  Secretory IgA 

IgA synthesis exceeds the combined total of all the other immunoglobulin classes. 

Where IgA is found, Generally

IgA may be monomeric (i.e., a single molecule), dimeric (composed of two molecules) or trimeric (composed of three molecules). IgA monomers are joined together as dimers at the constant regions of their heavy chains by a J chain. IgA is secreted as one of two subclasses, IgA1 and IgA2. IgA1 predominates in the cirulcating blood wherein most of it occurs as a monomer. Most IgA on mucosal surfaces, such as the surfaces of the trachea, bronchi, and broncioles in the lungs, occurs as dimers or timers joined by J chains. IgA dimers and trimers have an increased ability to bind to and agglutinate target molecules which are more readily phagocytosed. IgA dimers and timers, because of the presence of their J chains, have the ability to attach to secretory component (see below). WO 02/076502. 

IgA Isotypes: 

There are two isotypes of IgA, IgA1 and IgA2.  The main difference between IgA1 and IgA2 subclasses is a 13 amino acid deletion in the IgA2 hinge region. This segment in IgA1 contains several serine and threonine amino acid residues that are O-glycosylated.

On mucosal surfaces (e.g., gut, respiratory tract, genital tract) both IgA1 and IgA2 are present, synthesized by local B cells. In the blood, IgA1 predominates, produced by B cells in the bone marrow, lymph nodes and spleen. IgA is the predominant class in external secretions such as breast milk, saliva, tears and mucus of the bronchial, enitourinary and digestive tracts.

IgA1: is heavily glycosylated and consists of 8% carbohydrate. The five O-linked glycosylation sites at the hinge region between the CH1 and CH2 domains of the alpha chain on IgA1 are distinctive and unusual features that are not found in most of the serum proteins, including IgA2. The O-glycosylation oat the hinge region of IgA1 has been reproted as significant with respect to hepatic clearance of IgA1 by asiloglycoprotein receptor (ASGPR). (Leung, Kindey International, 59, 2001, pp. 277-285) 

gA2: Three allotypes of IgA2(1) have been described; IgA2m and IgA2m(2).

IgA2 is only present in small amounts in human serum and makes up about 16% of the total IgA (Leibl, J. Chromatography B, 678 (1996) 173-180)

Secretory Component (SC):

In nature: 

Human secretory component (SC) is assocaited with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived form teh polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. (Prinsloo, Protein Expr Purif 2006, 47(1) 179-85, 2005).

The term “secretory component” refers to a protein that specifically binds to J-chain-containing immunoglobulin, and is related to or derivable form identical to an extracellular portion of the polymeric immunoglobuliln receptor (pIgR). SC is the extracellular portion of the polymeric immunoglobulin receptor (pIgR), which usually gets assocaited during secretion with dimeric or polymeric IgA or pentameric IgM comprising a J chain. J chain containing IgA/IgM binds to the polymeric immunoglobulin receptor at the basolateral surface of epithelia cells and is taken up into the cell by transcytosis. This receptor complex then transits through the cellular compartments before being transported to the luminal surface of the epithelial cells. The transcytosed IgA/IgM-pIgR complex is then released through proteolysis, and the part of the polymeric immunoglobulin receptor (pIgR), referred to as the natural secretory component, stays associated with the J chain containing IgA/IgM, releasing secretoy IgA/IgM. (Corthesy, WO 2013/132052). 

Secretory component (Sc) exists in three molecuelar forms: as a membrane protein expressed on the outer surface of epithelial cells, it acts as a receptor for polymeric immunoglobulins. SC forms part of secretory IgA and IgM molecuesl in mucosal fluids where it is also found as a free glycoprotein. SC interacts with a specific binding site on the Fc region of SIgA and IgM, stabilises their quaternary structures and increases their resistance to proteolytic digestion (DOE, “The intestinal immune system” Gut, 1989, 30, 1679-1685).

The first line of defense against pathogens consists of mucosal secretions, with secretory IgA being one of the main effectors. Secretoy IgA is transported by the polymeric immunoglobulin receptor (pIgR) from usbmucosal sites into the lumen. The pIgR is expressed by lepithelial cells in a variety of mucosal tissues and in rodents is further implicated in transport into bile through the liver. The receptor can bind dimeric IgA, polymeric IgA and IgM but not monomeric IgA or IgG. The complex is internalized at the basolateral surface of the epithelium and transcytosed to the apical plasma membrane, where the extracellulur portion of the receptor called secretory component (Sc) is released by proteolytic cleavage and remains bound to IgA within a complex termed secretory IgA (sIgA). (J. Biological Chemistry, 274(44), 1999). 

Polymeric IgA binds to the polymeric immunoglobuilin receptor on the basolateral surface of epithelial cells and is taken up by the cells by transcytosis. The receptor-IgA complex then passes through the cell before being secreted on the luminal side, still attached to the receptor. Proteolysis of the receptor occurs and the dimeric IgA along with its secretory components which is wrapped around the the IgA are free to difuse throughout the lumen. Proteolytic components protect IgA from digestion by gastric acids and enzymes of the digestive system. 

recobminant production:

Human secretory component can be produced by recombinant techinques. 

Secretory IgA

Where sIgA is found in vivo

Mucosal surfaces comprising the GI, respiratory and urogenital mucosae represent a large port of entry for most of the pathogens and thus have to be efficiently protected. The goal is achieved by a combination of constitutive, non specific substances (e.g., mucus, lyzozyme, lactoferrin and defensins and specific immune mechanisms mediated by cellular and antibody resposnes. In vivo, the chief antibody at mucosal surfaes is secretory IgA (SIgA), a complex structure of IgA produced by activated B cells in the mucosal epithelium and of the secretory component (SC), a polypeptide of 85 kDa corresponding to the extracellular part of the poly immunoglobulin receptor (pIgR) expressed by epithelial cells (RFavre, J. Chromatography B, 786, 2003, 143-151).

Structure of sIgA and How Produced:

Unlike IgA in the serum and crebrospinal fluid which largely comprisees 7S monomers, secretory IgA is an 11S dimer made up of two IgA monomers joined by a covalently linked peptide named J chain. Both IgA and IgM plasma cells synthesise J chain which greatly enhances binding of an additional polypeptide, secretory component (SC), thereby completing the asembly of secretory IgA and IgM molecuels. One molecuel of J chain joins two IgA monoemrs whereas three or more J chain molecuels are involved in forming the IgM pentamers within the plasma cell before secretion. (DOE, “The intestinal immune system” Gut, 1989, 30, 1679-1685). 

In vivo, sIgA is produced by two different cell types, the plasma cell and the epithelial cell. Plasma cells synthesize and assemble alpha H and L chains with J chains into polymeric IgA. The pIgA secreted by the plasma cell binds to a polymeric Ig receptor (pIgR) expressed on the basolateral surface of the mucosal epithelium. The IgA-pIgR complex is transcytosed to the apical surface. During transit, a disulfide bond is formed betweent he IgA and the pIgR. At the apical surface, the IgA molecuel is released by proteolytic cleavage of the receptor. This cleavage results in a fragment, aobut 70k MW, bieng retained on the IgA moelcule. This fragment is the SC fragment, which is attached by disulfide bonds to the IgA moelcule. The IgA-SC complex is thereby rleased into external secretions. (Morrison (US 6,300,104).

Production of sIgA in vitro

Kelly (US 2015/0017181) discloses that plasma dervied immunoglobulin mixed with secretory component leads to the formation fo secretory-like IgA and/or secretory like IgM, i.e., J chain containing IgA dimers, tetramers or other polymer forms or J chain containing IgM pentamers complexed with SC even without purificaiton from plasma prior to mixing witht he SC. However, it is preferred to enrich the composition for such J chain containingIgA and/or L chain-containing IgM prior to mixing with the SC. 

Morrison (US 6,300,104; see also WO98/57993) discloses a method of producing secretory Ig molecules, such as secretory IgA, for treating infection, by transfecting a cell producing an IgG with a polynucleotide encoding an SC.

Regulation of IgA Production 

Polymeric Ig receptor (pIgR) and the asialoglycoprotein binding receptor (ASGR) expressed by the liver are important in the regulation of serum IgA levels. IgA2 has been showed to be cleared more rapidly than IgA1 from the circulation (Rifai, J. Exp. Med. 191(12), 2000, 2171-2181).

Where and How IgA is found:

IgA comprises dimers of IgA monomers, lined by the so-called J-chain and having a secretory component, is important with defense against certain bacteria like gonorrhea and viruses like polio. Secretory IgA can survive in harsh environments such as the disgestive and respiratory tracts which make it a preferred immunoglobulin for application in products for treating and/or preventing infection and/or inflammation of mucosal surfaces, such as the gastro intestinal mucosa, mucosa of the respiratory tract and also of the skin. In contrast to erum, in which IgG predominates, the predominant type of antibody in milk is IgA. IgA in milk is also structurally different from that in the serum. In serum, IgA is structurally similar to IgG although a small proportion consists of a dimeric immunoglobulin (i.e., 2 IgA monomers coupled together, by a polypeptide called the J-chain). Milk IgA is predominatly secretory (IgA, a dimeric molecule with two additional peptide chains termed J-chain and secretory component). 

IgA only represent about 3-4% of teh total protein of normal human serum. In plasma, IgA monomers exist in an equilibrium with non-covalently associated IgA dimers; however, J chain containing IgA dimers are also present. Total dimeric IgA constitues aobut 10-25% of total IgA. In the secretions of the mucous membranes and glands, J-chain containing IgA dimers with an additional secretory component are predominant. (Menyawi US 14/377,535)

For a nice list of monoclonal antibodies currently on the market see (Shukla, “Recent advances in large-scale production of monoclonal antibodies and related proteins” Trends in Biotechnology, 28(5) pp. 253-261)

IL-1:

Anakinra targets the effects of IL-1 by competing with IL-1 for the Il-1 receptor. 

–IL-1alpha: Hsieh (US  8,383,778) discloses anti-IL-1alpha antibodies for treating diseases. 

 IL-6:

Rajpal (US 13/857,867) discloses generating human monoclonal antibodies to human IL-6 using human Ig transgenic mouse strains which express human antibodies that are indistinguishable from antibodies isolated form humans (see also monoclonal antibody production on the right).

Tocilizumab targets the IL-6 receptor. 

TNF-alpha:

–Infliximab is a chimeric anti TNF-alpha mAb.

 The EMA has approved Resmisma (or Inflectra), a biosimilar or originator infliximab (Remicade).

–adalimumab 

In the early 19902, Cambridge Antibody Technology Limited (CAT) and Knoll AG (“knoll”) (CAT changed its name to MedImmune Limited in 2007) begain a collaboration to develop therapeutic human antibodies and, in 1995, entered intoa Development and License Agreement. The collaboration led to the antibody adalimumab, the active ingredient of Humira. Uner the 1995 agreement, knoll recivd a license from CAT to certain patents including US 6,248,516 and US 7,306,907 and agreed to pay royalties on sales of certain antibodies reulting form the collaboration, including Humira. Abbvie (a spin off of Abbott Laboratories) is Knoll’s  successor in interest under the 1995 Agreemnt. 

Adalimumab binds to TNF-alpha and inhibits its interaction with cell surface receptors. Adalimumab refers to a FDA approved fully humanized IgG1 TNF alpha inhibitor monoclonal antibody (tradename Humira). Each IgG antibody molecule comprises two kappa light chains and two human IgG1 heavy chains, with a total MW of 148 kDa. Each light chain consists of 214 amino acids and each heavy chain consists of 451 amino acid residues. Adalimumab was derived form murine monoclonal antibody MAK195 using guided selection phage display and is produced in CHO cells. Physico-chemical studies reveal that adalimumab is present in three major fomrs, corresponding to molecules carrying two, one or no C terminal lysine. (Nti-Gyabaah, US 14/355014).

Adalimumab was approved by the FDA in 2002 and the European Agenecy for teh Evaluation of Medical (EMEA) products in 2003 for the treatment of rheumatoid arthritis. It was subsequently approved for the treatment of other TNF mediated chronic inflammatory diseases including psoriatic arthritis, chronic plaque psoriasis, ankylosing spondylitis, Crohn’s disease and polyarticular juvenile idiopathic arthritis. It can be used alone or in combination with methotrexate (MTX) or other nonbiological disease modifying anti-rheumatic drugs (DMARDs. For the treatment of RA, adlaimumab is typically administered by subcutaneous infection at 40 mg every one or two weeks. It is marketed in prefilled syringes and as an autoinjeciton device called HUMIRA Pen, which is typically used by patients for self administration. The prefilled syringes and autoinjector comprise 40 mg of adalimumab in 0.8 ml of a buffered solution of mannitol, citric aci monohydrate, sodium citrate, disodium phosphate dehydrate, sodium chloride and poysorbate 80.  (Nti-Gyabaah, US 14/355014)

— etarnercept is a TNF alpha receptor fusion protein. 

Antibodies to Complement Pathway Components

Preclinical studies using animal models and human derived samples demonstrate that inhibition of complement ameliorates many inflammatory and autoimmune disease manifestations. The AP pathway has been shown to play a particular important role (for review of AP pathway in various diseases see Holers “The spectrum of complement altnerative pathway-mediated diseases, Immunological Revies, 2008).  

Factor B: 

Factor B is a 90 kDa single chain glycoprotein composed of 5 protein domains. The amino terminal region (Ba) consists predominantly of 3 short consensus repeats (SCRs) domains found in complement regulatory proteins. The carboxy-terminal region (Bb) consists of a type A domain found in von Willebrand factor and complement receptors, followed by a trypsin like serine protease domain. Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722. 

Monoclonal antibodies to human factor B which prevent factor B binding to C3b are known (Clardy, “Complement Activation by whole endotoxin is blocked by a monoclonal antibody to Factor B. Infect. and Immun. 1994, 62, 4549-4555).

Bansal (US7,959,919B2, US2005/0107319 and US7,959,919) discloses a method of inhibiting complement activation in the treatment of vsirous disorders by using factor B inhibitors such as antibodies to human factor B. 

–Factor Ba: 

Hourcade (J. Biological Chemistry, 270(34), 1995, pp. 19716-19722) disclose using site directed mutagenesis of recombinant factor B and monoclonal antibody epitope mapping to identify 3 sites on factor Ba; site 1 which is a stretch of 19 contiguos amino acids in short consensus repeat 1 that forms the epitope of a monoclonal antibody that effectively blocks factor B function, site 2, composed of a contiguous amino acids in short connsensus repeat 2 and site 3, consisting of 7 contiguous amino acids in short consensus repeat 3 were also defined by mutations that reduced factor B hemolytic activity ot 3% or less. Further analyses indicated that sites 2 and 3 contribute to factor B-C3b interactions. 

Bansal (US12501165) discloses a method of inhibiting factor Ba dependent complement activation in bood of a subject by administering anti-factor Ba antibodies which specifically bind to factor Ba protein sequences involved in Factor B binding to C3b. 

The protein Ba (cleaved from Factor B) is a large protein. Any of a multiude of antiboides can be produced against various protein motifs of, and locations on, the protein (Bansal, US13/646286). 

Hourcade (J. Biological Chemistry, 270(34), pp. 19716-19722, 1995) discloses an anti-Ba mAb that blocks factor B hemolytic activity and appears to interfere with the normal binding of factor B to C3b. 

Tanaka (Immunology, 73, 1991, pp.383-387) discloses a murine monoclonal antibody directed against the Ba domain of factor B that enhanced binding of intact factor B with cell bound C3b and the haemolytic activity of factor B. 

Ueda (J. Immunology) 138(4), 1143-1149, 1987) discloses two antibodies that react with distinct epitopes on Ba and inhibited the hemolytic activity of B. The two anti-Ba Mabs also inhibited binding of B to C3b, suggesting that the binding site for C3b on intact B is located on the Ba portion of the molecule. 

–Factor Bb: 

Bansal (US12/675220, published as US2010/0239573) teaches a method of inhibiting AP by administering antibodies that specifically binds to an epitope of the Bb segment of factor B. 

Ueda (J. Immunology) 138(4), 1143-1149, 1987) discloses two antibodies which reacted with the same or adjacent peptides on the Bb fragment of B and inhibited the hemolytic activity of B. 

Factor D antibodies: The following patents disclose inhibition of complement activation using factor D antibodies; Fung (US 81,124,090, US8,236,317B2; see also US2002/0081293). Ann (US 8,193,329B2).

The following applications disclose anti-factor D antibodies for the treatment of complement associated eye conditions such as chroidal neovascularization (CNV) and age-related macular degeration (AMD) (Fung US application # 12/092346; Hass US 8268310)

Tanhehco (Transplantation Proceedings, 31, 2168-2171 (1999) discloses an anti factor D antibody, MAb 166-32 which inhibits the AP.

For anti-factor D antibodies and cardiopulmonary Bypass see Undar (Ann Thorac Surf 2002, 74, 355-62). 

C3b: is a large protein and thus multiple antibodies can be produced against various segments of this protein. Tthere exists multiple sites where an antibody might bind and inhibit the protein’s activity in any variety of ways. Depending on how and where an antibody binds to C3b, the effect of that antibody could range from inconsequential to complete inhibition (Bansal, US13/646286). 

Bansal (US2010/0111946) discloses the use of anti-C3b antibody to inhibit C3 dependent complement activation. The anti-C3b antibody specifically binds to C3b protein sequences involved in C3b binding to properdin or Factor B or C3b cleavage from C3. 

DiLillo (Mol Immunol., 2006, 43(7): 1010-9) discloses selective inhibition of the AP by an anti-C3b/iC3b monoclonal antibody and by a chjimeric, deimmunized form of the antibody. 

Taylor (WO2006/012621) discloses the use of an antibody directed against C3b as a means of inhibiting the AP. 

C5: 

Eculizumab and pexelizumab® (Soliris®; Alexion Pharmaceuticals) are FDA approved anti-C5 antibodies.  

Antibodies which bind to complement C5 and prevent C5 form being cleaved are disclosed in US Patent No: 6,355,245 and include both a whole or full lenght antibody (eculizumab) and a single chain antibody (pexelizumab). A similar antibody against mouse C5 is called BB5.1 (see Rother, WO2005/110481).

Eculizumab is an approved antibody directed against complment compoonent C5 for the disease paroxysmal nocturnal hemoglobinuria.

Rother (WO2010/054403) discloses inhibitors of C5 such as a C5 antibody and methods for using the compositions to treat complement assocaited disorders. 

Evans (US6,355,245) beaches using anti-C5 antibodies to treat glomerulonephritis.

Zhou (US2014/0056888) teaches highly concentration aqueous solutions of an anti-C5 antibody which can be advantageously administered to a patient in a small volume. 

Methods for identifying the epitope to which a particular antibody binds are known in the art. For example, the binding epitope of an anti-C5 antibody can be identified by measuring the binding of the antibody to several overlapping epptide fragments of a C5 protein. Each of the different overlapping pepties is then bound to a unique address on a solid support (e.g., separate wells of a multi-well assay plate). Next, the anti-C5 antibody is interrogated by contacting it to each of the peptides. Unbound anti-C5 antibody is removed by washing. Next, a detectably-labeled secondary antibody that binds to the anti-C5 antibody, if present in a well of the plate is contacted to each of the wells and unbound secondary antibody is removed by washing. The presenceof the detectable signal produced by the detetably-labeled secondary antibody in a well is an indicaiton that hte anti-C5 antibody binds to the particular peptide fragment assocaited with the well. A particular epitope to whcih the antibody binds can also be identified usign BIAcore chromatographic techniques. (Rother, (PCT/US2009/063929)

–C5a: Synthetic peptides derived from C5a are known antagonists of C5a. However, the antagonistic activity is highly dependent upon the structure of the peptides. For example, whereas a 14 amino acid residue peptide from the C5a C-terminal region can function as an antagonist of C5a, 5-8 amino acid residue peptide analogues of the same region have been reported to be agonists rather than antagonists (Kaneko, Immunology, 1995, 86, 149-154).

Wang (US7,279,158) dicloses use of compounds such as antibodies that block complement component C5 to treat established joint inflammation (arthritis).

–C5a receptor (CD88): Several antagonists for C5a receptor (CD88) have been described, including peptides, a non-peptidic compound, C5a mutants and anti-C5R-antibodies. All of these compounds are potent CD88 antagonists in vitro; however, only the C5a mutants C5aRAM and jun/fos-A8, the cyclic peptide AcPhe[AcF-(pdChaWR), and a nonpeptidc antagonist have been proven useful in vivo (Otto, J. Biological Chemistry, 279, 1, 2004, 142-151).

–C5b antibodies: are known in the art (Evans, US6,355,245) and commercially available anti-C5b antiboides are available. 

Properdin antibodies: (see also Properdin under “complement” for structure of properdin)

Brunden and Bansal disclose anti-human properdin MoAbs which blocked activation of the AP in vitor and in ex vivo models of CPB (Molecualr Immunology, 36, No. 4-5). 

Gupta-Bansal discloses an anti-properdin antibody which caused nearly complete inhibition of the C3a and C5b-9 formation in a cardiopulmonary bypass model (Molecular Immunology 37 (2000) 191-201). 

Gupta-Bansal discloses a number of patents claiming anti-properdin antibodies: US6,333,034, issued 12/25/2001 (inhibit AP); US 2003/0198636 (US 6,333,034, issued 12/25/2001) (US2002/0015701) (US7,423,128, issued 9/9/2008) (US2006/0093599 (binds epitope within a thrombospondin type 1 repeat of propderin)); US13/849092 (anti-properdin antibody useful for inhibiting AP that is specific to amino acid sequence at N-terminal end of properdin). 

Holers (US2006/0292141) teaches antagonists to complement proteins such as properdin. Holers teaches that properdin has the sequence

Song (US12/663,690, US 2010/0263061; see also PCT/US08/07270 and WO/2008/154018) also disclose anti-properdin mAbs which selectively inhibit AP and have no effect on the AP amplifcation loop of the CP.

Bansal discloses (US 12/920,997, US 2011/008340, now US 8,435, 512; see also PCT/US2008/068530; see also US 13/849092 (also teaching anti properdin antibody that competitively inhibits binding of an antibody to a proeprdin sequence)) discloses an antibody that specifically binds to an epitope of the N terminus end of properdin that blocks the alternative pathway activaiton without affecitng the classical pathway activation. See also US8,664,362 and 14/195458 disclosing anti-properdin antibody that can bind to the N-terminal domain of properdin to inhibit the bidning of properdin to C3b. 

Properdin is a large protein with many potential sites where antibodies can bind. Not all, or even most, of these antibodies will necessarily have any therapeutic value (Bansal, US13/646286). 

Antibodies to Complement Regulatory Proteins

CD59: CD59 inhibits MAC formation by specifically binding to complement proteins C8alpha and C9. Giddings (Nature Structural & Molecular Biology, 11(12), 2004). Qin (US 13/391124) discloses antibodies that bind to CD59, inhibiting binding of CD59 to C8 and/or C9. The antibodies bind the same epitope of CD59 as domain 4 of intermedilysin (ILYd4). The antibodies were identified by taking erythrocytes from humans or hCD59RBC trasngenic mice that express only human CD59 and incubating them with a candidate antibody, then incubation with ILYd4 or mouse anti-hCD59, followed by FITC conjugate anti-His antibody or FITC-conjugated secondary antibody. Cells incubated with antibodies that bound to the same epitope as ILY4 did not stain with either ILYd4 plus FITC anti-HIS or FITC secondary antibody. 

Qin (US13391124) disclose antibodies that bind to CD59 and inhibit binding of CD59 to complement proteins C8 and C9. The antibodies bind to the same epitope as ILYYd4 and thus inhibit binding of ILYd4 to CD59. Intermedilysin (ILY) is a cytoxin secreted by Streptococcus intermedius which lyses only human cells due to its receptor specificity for human CD59 via its domain 4 (ILYd4). ILYd4 binds to AA42-58 in hCD59, which also participates in the binding to C8 and C9. A truncated ILYd4 has been shown to abrogate hCD59 function to facilitate CDC effect on cancer and HIV. Hu (“Domain 4 of ILY sensitizes antibody therapy on cancer and HIV through abrogating human CD59 function”) showed ILYd4 sensitized a Rituximab-resistance B lymphoma cells to Rituximab treatment without toxicity effects and sensitized HIV to complement mediated virolysis activated to HIV patient’s antibody. The alpha helical region, specifically, residues 42-58 of huCD59 contains the ILY-binding site. This same region is also recognized and bound by complement proteins C8alpha and C9. Thus the domain 4 of ILY and complement proteins C8alpha and C9 recognize the same or overlapping regions of huCD59. Giddings (Nature Structural & Molecular Biology, 11(12), 2004) disclsoses an antibody (10G10) that completely blocked ILY binding and cytolytic activity to huCD59. 

Sims (US2003/0166565) teaches compounds such as an antibody which specifically bind to amino acids 42-58 of huCD59 and thereby block CD59 binding to C9 (¶s17, 41; claims 28-29).