See also Immunization and ways to increase immunogenicity                See also B cell activation . See also cell culture in Biotechnology

Introduction

Since the development of the hybridoma technology by Kohler and Milstein 35 years ago, several methods for teh generation of mAbs have been developed. Such methods include B-cell immortalizaiton through genetic reprogramming by means of Epstein-Barr virus or retrovirus-mediated gene transfer, cloning of V genes by single-cell PCR and approaches for in vitro discovery that involve the display and screening of recombinant antibody libraries. (Reddy, “Monoclonal antibodies isolated without screwewning by analyzing the variable-gene repertoire of plasma cells” (2010) Nature Biotechnology, 28(9); 965-969.

Specific recognition of foreign antigen in vivo by cell surface immunoglobulin (sIg) on B cells takes place in the T cell zones of the secondary lymphoid tissues. There, antigen activated B and T cells interact resulting in induced proliferation and subsequent differentiation of the B cells into antibody producing cells. The antigen specific T cell help is initiatetd through the T cell receptor (TcR). Interaction between APCs, e.g., B cells and specific T helper cells, results in activaiton of the T cells which enables them to provide stimulatory signals for B cells, transmitted either through cognate or non-cognate mechnaisms.

Human mAbs appear to be the most suitable for in vivo applications because unlike mAbs from other species, they are minimally immunogenic in humans. However, human mAbs against human antigens are difficult to obtain mainly because humans are generally tolerant to their own antigens. Several approaches to circumvent this problem have been developed. One approach is to create chimeric antibodies that contain V regions from mAbs raised in experimental animals immunized with the desired human antigen and C regions from human antibodies. A further refinement of this approach is to limit the nonhuman V region sequence in the chimeric antibody to the antigen CDR (humanization). A third approach is to select an antibody with the desired specificity from combinatorial libraries encoding the human antibody gene repertorie usually in comibnation with phage display technology. A fourth approach is to generate genetically manipulated mice, able to produce human antibodies by eliminating endogenous mouse anitbody genes and inserting human antibody gene elements. 

A completely different approach is to create a human immunoadhesins which is a chimeric protein that contains two parts: one part is derived from an IGg molecule and the other from a protein that possesses an adhesion or a binding function, hence the name “immunoadhesin”. This type of molecule has also been referred to as an Ig or an Fc fusion protein. (Ashkenazi “Immunoadhesins: An alternative to Human Monoclonal Antibodies”. 

Techniques for monoclonal antibody production:

Hybridoma technology: The production of mAbs by hybridoma technoloyg was a significant milestone for teh generation of antibodies for therapeutic use. As this technology is based on the fusion of antibody producing spleen cells from immunized mice or rats with immortal myeloma cell lines, its main obstacle is the inefficient immune response to highly toxic or conserved antigens. In addition, nearly all antibodies which are currently in clinical development are of human origin or at least humanized in some aspect to prevent immunoglnicity. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) and Hammerling, Monoclona Antiboides and T Cell Hybridomas, 563-681 (Elsevier, NY 1981).

immortalization of Bells with Epstein-Bar virus; A disadvantage of this method is the extensive subcloning and overall shotgun approach limit the number of useful antibodies that an be produced even over extensive periods of time. 

Production of B-cell hybridomas,

Humanization of antibodies from other species, This method may not provide a true evaluation of the epitope specificites that humans gennerate in vivo, limiting the sue of theis technique for applications such as epitope discovery and vaccine development or evaluation. The Fab that is produced in other species must be cloned and fused to a human Fc backbone and expressed in a human cell line. These humanizing techniques represent a significant outlay of time and resources. 

Phage display libraries or geernating antiboides recombinantly form isolated single B cells. Allthough pahge display uses fully human heavy and light chain variable genes, the H and L chains are randomly paired in vitro, and so are more likely to induce anaphylactic responses as foreign proteins or to be autoreactive if therapeutic uses are the goal. 

Transgenic animals: especially mice, have been developed which contain a human immunoglobulin gene hypertoire, sovling the problem of immunogenicity but not the need of an efficient immune response after immunization. (Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Isolation of antibody-secreting cells (ASCs); In this method, ASCs are isolated from whole blood after vaccination and sorted by flow cytometry into single cell plates. The ASC are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. (Wilson, Nat Protoc. 2009; 4(3); 372-384 (2009). 

Ribosome Display:

Ribosome display is a technology for the in vitro selection and evolution of very large protein libraries. The main feautre distinguishing this technique form other selection techniques such as phage isplay is that the entire procedure is performed in vitro, without using cells at any step. In ribosome display genotype and phenotype are linked through ribosomal complesxes, consisting of messenger RNA, ribosome and encoded protein that are used for selection. In ribosome display, a library of scFvs is transcribeda and translated in vitro. The resulting mRNA lacks a stop condon, giving rise to linked mRNA-ribosome-scFv complexes. These are directly used for selection on the immobilized target. The mRNA incorporated in bound complexes is eluted and purified. Reverse transcription-PCR can introduce mutations and yields a DNA pool enriched for binders (Hanes, “Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display” Nature Biotechnology, 16, 2000). 

Production of Monoclonal Antibodies using Mass Spectrometry

Polakiewicz (US 2012/0308555) discloses methods for creating a monoclonal antibodies that includes obtaining collecting a population of immunoglobulins either from an animal or from tissue culture sueprnatants of B cells , subjecting the antibodies to protein A or G affinity chromatography to purify them, optionally further enriching the antibody pool for an antigen of choice which can be placed on a solid surface, treating the bound antibodies with a protease which specifically cuts near the hinge region, rinsing away nonbinding proteins and eluting the antigen specific antibodies, followed by the mass spectra of the collected polyclonal antiobdies (proteins may also be digested to smaller fragments that are amenable to MS) by correlating the observed mass spectra with predicted masses. Predicted MS information  can for example be determined by using standard publicly available software algorithm tools including for example Sequest software, the Sequest 3G software or Mascot software.  Finally immunoglobulin H or L chains are slected based on parameters such as the number of unique peptides, the spectrum share and the amino acid sequence coverage.  In a separate interesting embodiment, elderly individuals known to have been exposed to the 1918 influenza virus are screened for the presence of serum antibodies that can neutralize the virus, bloo is drawn, B lymphocytes isolated, nuecliec acid obtained and the nucleic acid molecuels obtained are used to populate the genetic matrial database. Alternatively, serum and/or plasma is collected, antibodies present in the serum and/or plasma are isolated such as by protein A chromatography and th antibodies are further enriched  by passing the serum antibodies over a second column coated with 1918 virus. The boudn antibodies are next treated with a protease (e.g., papain) that specfically cuts near the inge region and the non-adherent Fc protion removed. Finally, the bound Fab or Fab2 fragments are treate with trypsin to generate peptide fragments and then analyzed by MA. Using an algorithm such as Sequest, the observed tandem MAS of the peptides are correlated with the rpedicted mass spectra from the nuecliec acid sequences extracted from teh pateints’ B lymphocytes.. Using this process, at least one peptide found within the predicted amino acid sequence of a unique immunoglobulin chain of the genetic material database may be identified. The DNA may be synthesized and then subcloned into expression vectors which are transfected into CHO cells. 

 Culturing Techniques to produce mAbs:

Conventional antibody production has involved the immunization of animals (i.e., mice) with a target antigen, such as a virus, bacterial or foreign prodtein. The immunized mice produce on the order of 104-105 antibody secreting cells (ASCs) each with the capacity to product a unique (monoclonal) antibody specific to the target antigen. The ASCs are then harvested form the immunized animals and screened in order to select which cells are producing antibodies of desired affinity and selectivity to the target antigen. Since isngle ASCs do not produce antiobdies in sufficiently large quantities for binding afffinity measurements, each ASC is clonally expanded. Primary ASCs do not grow efficiently in laboratory tissue cultures; thus clonal expansion may be acheived by fusing ASCs to murine myeloma (cancer) cells to product immotaglized, antibody secreting (hybridoma) cells. A limitation of hybridoma technology is the low efficiency of the fusion process. For example, wehreas an immune response may product on the order of 105-105 antibody secreting cells, a typical fusion will yeild less than 100 viable hybridomas. Therefore, fusions from hundreds to thousdands of animals are required to fully sample the diversity of antibodies produced in an immune response, making the hybridoma approach both time-consuming and expensive. Singhai (US 10,107,812)

An important bottleneck in the developmetn of mAb production processes is the need to gerneate cell lines that produce large quantities of antibodies. After transduction of the gene of interest, stable clone selection with the desired product quality can take several months. This is normally the longest step in the development of new protein production processes. Most production cell lines have been generated by performing limiting dilution of a transduced pool fo cells in multiwell plates, with often more than 1000 wells screened due to the low cell plating efficiency and the need to analyze many candidates. This method reuqires at least 2 weeks of culture to allow accumulation of detectable mAbs concetnraitons before a first measurement can be made. Lowest producing clones are eliminated while highest producers are advanced to the next phage of scale-up, a labroious process that is often repeated for subsequent rounds of sub-cloning to ensure the generation of cloned cell lines. Hansen (US 10,738,270)

In an effort to increase throughput and accelerate the identificaiton of high producing cells,s everal FACS based methods have been devloped. Cell soritng strategies can be coupled with single-cell deposition, hence eliminating the need for sub-cloning. Hansen (US 10,738,270)

Microfluidics:

Hansen (US 10,738,270) discloses a method for culturing single cells by introduction a populartion of cells into a microfluidic device that includes 1,600 to 20,000 microfluidic chambers wherein each chamber includes an inlet wherein single cells of the populaiton are rentained in different microfluidic chambers such that a plurality of individual clonal cell populations are retaied in the same microfluidic chamber as their respective parental cells. 

Singhai (US 10,107,812) discloses a method of assaying for a binding interation between a protein produced by an antibody producing cell and a biomolecule such as a fluorescently labeled antigen by retaining the cell in a chamber having an inlet and outlet, exposing the protein produced by the cell to a capture substrate (e.g., microsphere, polystyrene bead, antibody capture bead) to bind the protein produced by the cells and flowing a fluid that includes the biomolecule through the inlet into the chamber and out the outlet and determining binding interactions between the prtoein produced by the cell and the biomolecule. 

Recombinant Technology: 

Another approach is to engineer monoclonal antibodies using recombinant DNA technology. These vectors are then transfected into Ab lacking myeloma cells. For example, one can clone recombinant DNA containing the promoter, leader, and variable region sequences from a mouse antibody gene and the constant region exons from a human antibody gene. The antigenic specificity of this mouse-human chimera is derived from the mouse DNA whereas its isotype which is determined by the constant region is derived from the human DNA. Since the constant regions are encoded by human genes, these antibodies have fewer mouse antigenic determinants and are far less immunogenic than mouse monoclonal antibodies when administered to humans. 

In particular, mRNA encoding variable region (V region) of the antibody of interest is otbtained from the hybridoma producing the antibody of interest. Total RNA is obtained by the conventional method well-informed to those in the art such as guanidine ultracentrifugation. Then, target mRNA is obtained from the total RNA by using mRNA purificaiton kit (Pharmacia) or QuickPrep mRNA purificaiton kit (Pharmacia). From the obtained mRNA, cDNA of variable region of the antibody is synthesized by using reverse transcriptase. If necessary, RACE PCR can be used for cDNA synthesis or amplifcation. The synthesized cDNA encoding variable region is introduced into an expression vector comprising DNA encoding constant region (C region). This expression vector can contain regulatory sequence such as promoter, enhancer, replication origin, polyadenylation signal, and ribosome binding site, etc. Once host cells are transformed with this expression vector, they can produce antibody.

Isolation of antibody from antibody-secreting cells (e.g., PBMCS)

B-cell maturation terminates with the formation of plasma cells, which represent <1% of all lymphoid cells but are responsibel for the overwhelming majority of antibodies in circulation. The bone marrow constitutes the major compartment where plasma cells reside and produce antibodies for prolonged periods of time, whereas plasma cells present in secondary lymphoid organs are often sort lived. In mice, a stable and highly enriched antigen-specific BMPC population of 15 to the 5 cells (10-20%) of all BMPC) appears 6 days after secomndary immunizaiton and persists for prlonged peridos. In contrast, the increase in size of the splenic plasma cell populaiton is highly transient, peaking at day 6 an rapidly declining to <10 to the 4 cells by day 11. Notably, BMPCs are long lived and thus responsible for making the stable circulating population of antibodeis in serum, which in turn is likely to play a dominant role in pathogen neutralziation and other protetive humoral immune responses. (Reddy, “Monoclonal antibodies isolated without screwewning by analyzing the variable-gene repertoire of plasma cells” (2010) Nature Biotechnology, 28(9); 965-969.

A first consideration is the source of B cells. Most human antibody sequencing studies have used B cells from peripheral blood becazeu the blood is one of the few readily accesible sources of B cells in human (tonsils is the other one). However, it is estimated that only 2% of B cells in the human are present in periopheral blood, compared with almost 28% in lymph nodes, 23% in the spleen and on mucosal surfaces adn 17% in the red bone marrow (medulla ossium rubra). Second, it is important co consider wehther to sue genomic DNA or mRNA for immunolgoubilin sequencing analyses. This depends on the quesiton being asked. Sequencing gDNA vaciliates estimation of the clonality of a given Ig sequence (in other words, the number of B cells expressing that antibody) becasue the number of sequence reads will, in gerenal, be proportional to the number of gDNA template molecules. On the other hand, using mRNA as a template can provide an estimate of the relative epxression level of vairous immunogloublin sequences in the repertoires. (Georgiou, Nature biotechnology, 32(2) Feb 2014). 

Density-Gradient centrifugation-Flow cytommetry-FACS-rTPCR-PCR-sequence/clone:

Perhaps the most well-defined use of antibody sequencing after vaccination is in the generation of mAbs. mAbs can be used for passive immunization agaisnt certain diseases and have important research applications, including verification of methods for identificaiton of antigen-specific sequences from NGS repertoire data. After antigen adminsitration, plasma cells (or antigen-enriched cells) can be isolated form peripheral blood by FACS and sequenced to identify the sequences of the antibodies that are produced in response to the antigen. Generally, the most abundant sequences are then cloned into an expresison vector for functional characterization. (Galson, Trends in Immunology, July 2014, 35 &).)

Galson (Ebiomedicine 2 (2015) 2070-2079) discloses generating a deverse B cell immunogloublin reperotire. Bllood wsa taken immediately before vaccination as well as 7, 14, 21 and 28 days after vaccination. PBMCs were isolated by density-gradient centrifugaiton, B cells were enriched form PBMCs using CD19 microbeads by FACS, RNA was extracted using the RNeasy Mini Kit (Quiagen) and reverse transcription was performed followed by PCR using primers for the VH family, amplicons were gel-extracted and purified and sequenced. 

Smith, (Nat Protoc, 2009, 4(3), 372-384) disclsoes a detailed protocal for the production of antigen specific human monoclonal antibodies using antibody-secreting cells isolated form whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antigeby genes of teh ASCs are then amplified by RT-PCR and neted PCR, cloned into expression vectors and transfected into a human cell line. 

Next-generation Sequencing (see outline)

Phage Display Methods: see outline

Antibody production in plants: 

Antibody production in plants has gained acceptance. However, some potential problems include differences in glycan usage between mammalian systems and plants. Some advantages include freedom from human pathogens and savings on cost of goods (Hood, Current Opinion in biotechnology, 2002, 13: 630-635). 

In vitro production

 In vitro production of mAbs offers several important advantages: (1) only a few micrograms of antigen are needed, (2) defined amounts of antigens and lymphokines are present during the immunization, (3) it is possible to follow visually the induction of blast cells (4) the immunization takes only 4-5 days, (5) the normal cellular control of the immune response to self-antigens seems not to function in cutre, as a result, hihgly ocnserved antigen, giving rise to poor or no immune response in vivo, may induce antibody rpoduction in vitro. Borrebaeck (Scad J. Immunol. 18, 9-12, 1983)

Borrebaeck (Scad J. Immunol. 18, 9-12, 1983) discloses antigen-specific lymphocyte hybridomas produced by a simple in vitor immunization procedure. Non-immune spleen cells were activated in vitro and the B cell balsts subsequently used for somatic cell hybridization. Hybrids secreted mAbs to human myobloin, pig insuland and beno(a) pyrene were obtained. MAbs of both IgG and IgM isotypes could be isolated.

Grasso (US2004/0214288) discloses a method for generating high titers of high affinity antibodies by fusing myeloma cells with in vitro immunized donor cells. According to the method, immunoglobulin proudcing cells are combined with an immunogenic antigen in vitro, the immunoglobulin producing cells are fused with myeloma cells to form parental hybridoma cells and the cells are screened for antibodies to antigen produced form the cells.

Poptic (US2014/0273031) discloses an in vitro method for the production of monoclonal antibodies, of the IgG type, making use of Act1 deficient B cells. An Act1 deficient mouse spleen cell exposed to an antigen in vitro can be fused to an immortal myeloma cell, thereby producing a hybridoma capable of expressing antigen specific IgG monoclonal antibodies. 

Retroviral vector mediated transduction of vertebrate host cells (e.g., B cells):

McConnel ((“High affinity humanized antibodeis without making hybridomas; immunizaiton paired with mammalian cell display and in vitro somatic hypermutation” PLOS ONE, 7(11) 2012) disclsoes a method for generation of high affinity humanized antibodies which includes immunization of a mouse with an antigen such as C5, isolating and amplifying rearrange IgHD(J) regions from the cells and fusing the regions with human germline H chain V genes to form intact humanized H chains and paried with H light chain library. The complete H and L chains were assembed and transfected into HEK 293 cells corexpressing activaiton-induced cytidine deaminase (AID) and clones were isolated by fluorescence activated cell sorting and affinity maturation, initated by AID. 

Sturken, (EP 2098536) discloses a method of producing antibodies and other bind proteins using retroviral vector mediated transduction of vertebrate host cells. Recombinant retroviral constructs stably integrate into the host cell genome and thereby confer a stable and sustained expression phenotype of the binding protein. The constructs can additional include cis regulatory elements in target cells endogenously or ectopically expressing the enzyme activiton induced cytidine deaminase (AID) which is known to strongly enhance somatic hypermutation. With the constructions, all molecular and genetic events occurring in the adaptive immune system are replicated, namely the generation of a primary antibody repertoire from one or a limited number constructs comprising a limited number of V, D and J gene segmetns and the additional AID mediated somatic hypermutation of the coding regions for antigen binding variable domains of antibodies. Additionally repetive cycls fo antigen slection/FACS-sorting/expansion of antigen reactive cells in the presence of mutagenizing conditions, specifically targeting utations to the coding regions of variable antibody binding domains can be performed. By this approach higher affintiy mutants, which are generated in situ, are generated in each round of cell amplication. A high mutation rate targeted to antibody variable region domains can be aheived by overexpressing the AiD enzyme in the antibody expressing cells, in particular, when the epxression constructs contain cis regulatory promoter and enhancer elements. 

Su (US14/769,294, US2015/0377887) discloses a controllable activation-induced cytidine deaminase (AID) expression system to induce somatic hypermutation in hybridomas. Selection of high affinity antibodies was then achieved by fluorescence-activated cell sorting of hybridomas that preferentially bind fluorescence-labeled antigens. First, lentiviral particles expressing inducible  (e.g., by addition of doxycycline) or constitutive AID were infected into target hybridomas and selected in puromycin. Second, epxression of AID induced somatic hypermutation of the antibody variable region genes, generating a population of hybridomas with a distribution of affinities. Third, limiting amounts of fluorescence labeled antigen such as PEG was added and hybridomas were sorted on a fluorescence activated cell sorter to collect hybridomas expressing membrane anchored high affinity antibodies. Hypermutation was terminated by removal of doxycylcine or by CRE recombination to remove the sAID cassette. 

Repetitive, multiple site immunisation strategy (RIMMS):  See immunology and ways to increase immunogenicity

Multiple antigen peptide system (MAPs):  See immunology and ways to increase immunogenicity

MPS system is a novel approach for antibody geenration using B eptiopes and T helper epitopes which are linked tandemly on a multi-branched lysine core. ((Yuang, Vaccine, 15(4), pp. 377-386, 1997).