HK1164899B - Methods for producing antibodies from plasma cells - Google Patents

Description

Method for producing antibodies from plasma cells

The present application claims priority from: british patent application No.0819376.5 filed on 22/10/2008, U.S. provisional patent application serial No.61/181,582 filed on 27/5/2009, PCT patent application No. PCT/US2009/051851 filed on 27/7/2009, and U.S. patent application No.12/509,731.

Background

Plasma cells are terminally differentiated, non-proliferating cells that secrete antibodies at a very high rate (several thousand molecules per second, corresponding to approximately 30-50pg per cell per day).

Isolation of antibodies, such as monoclonal antibodies, from plasma cells relies on the cloning and expression of immunoglobulin genes. This can be done using a phage display library of promiscuous VH and VL genes isolated from plasma cells, or by isolating pairs of VH and VL genes from single plasma cells using single cell PCR. However, in order to screen antibodies produced by plasma cells, immunoglobulin genes need to be cloned and expressed in recombinant form to determine the specific and functional properties of the encoded antibodies. This method is difficult, expensive, time consuming, not suitable for high throughput and does not efficiently obtain rare antibodies produced from a small fraction of the total repertoire (total reterite) of plasma cells.

Therefore, there is a need to identify more efficient methods suitable for high throughput isolation and screening of antibodies, such as monoclonal antibodies, from plasma cells.

Summary of The Invention

The present invention is based, in part, on the discovery of an efficient and high throughput method for producing antibodies from plasma cells that enables the characterization of antibodies without relying on the cloning and expression of immunoglobulin genes. Antibodies produced using the present invention can be characterized by multiple screens, including binding, functional and/or neutralization assays. The present invention provides methods for identifying rare antibodies produced by plasma cells.

Accordingly, in one aspect of the invention, the invention provides a method of producing an antibody from plasma cells comprising culturing a limited number of plasma cells. In one embodiment, the invention provides a method of producing monoclonal antibodies from plasma cells comprising culturing plasma cells in a single cell culture. The methods of the invention may further comprise characterizing the antibody or antibody fragment. Characterization of an antibody or antibody fragment includes, but is not limited to, performing a functional assay to determine the function of the antibody or antibody fragment, performing a binding assay to determine the binding specificity of the antibody or antibody fragment or an epitope recognized by the antibody or antibody fragment, and/or performing a neutralization assay to determine the ability of the antibody or antibody fragment to neutralize a toxin or pathogen.

In another embodiment, the invention provides a method of producing an antibody or antibody fragment. The method comprises culturing a limited number of plasma cells; identifying a culture that produces an antibody having the desired characteristics; isolating nucleic acid encoding the produced antibody; and expressing the nucleic acid in a host cell.

In another aspect of the invention, the invention provides an isolated antibody or antibody fragment produced by the method of the invention. The invention also provides methods of diagnosing and/or treating various disorders or diseases using the isolated antibodies or antibody fragments of the invention.

Brief Description of Drawings

Figure 1 shows IgG produced by accumulation of CD138+ plasma cells isolated from peripheral blood and cultured on mesenchymal stromal cell monolayers for 50 days.

Fig. 2 shows IgG produced by CD138+ plasma cell accumulation in cultures containing single plasma cells isolated from (a) peripheral blood and (B) bone marrow.

Figure 3 shows the results of testing for the presence of IgG, IgA, IgM, and IgE in 10 day culture supernatants of CD138-high plasma cells isolated from peripheral blood and cultured on mesenchymal stromal cell monolayers.

FIG. 4 shows the plate efficiency (plating efficiency) of Antibody Secreting Cells (ASC) producing IgG, IgA or IgM antibodies when cultured on hMSC-TERT cells and expressed as the percentage of plasma cells that survived long enough to produce detectable amounts of antibody in the supernatant.

FIG. 5 shows the identification of tetanus toxoid-specific IgG secreting plasma cells from plasma cells isolated from pooled peripheral blood 7 days after Tetanus Toxoid (TT) boosting.

Figure 6 shows the binding of recombinant antibodies produced by cloning and expression of VH and VL genes recovered from cultured plasma cells isolated from the blood of donors 10 years after immunization with tetanus toxoid, to tetanus toxoid.

Detailed Description

The present invention is based on the discovery of an efficient and high throughput method for producing antibodies from plasma cells that enables the characterization of antibodies without relying on the cloning and expression of immunoglobulin genes. In one aspect, the invention provides a method of producing an antibody from plasma cells, comprising culturing a limited number of plasma cells. Antibodies produced with the present invention can be conveniently characterized by using a variety of screens, including binding, functional and/or neutralization assays, and can even be performed in situ, i.e., in wells in which plasma cells are cultured.

As used herein, the term "plasma cells" includes all primary Antibody Secreting Cells (ASCs) found in peripheral blood, bone marrow, tissue or body fluids or produced in vitro from B cells. Newly generated plasma cells are referred to as "plasmablasts". Naturally occurring plasmablasts are commonly found in blood, particularly peripheral blood. Plasmablasts can also be generated in vitro by stimulating B cells with various stimuli, including polyclonal activators such as TLR agonists. Herein, the term "plasma cell" shall be taken to include "plasma cell", "plasmablast cell" and ASC.

In theory, any number of plasma cells can be cultured in a culture medium to produce and identify antibodies with desired characteristics. In practice, the number of plasma cells that can be cultured is limited due to existing techniques for cloning and expressing the multiple VH and VL gene sequences and combinations thereof present in polyclonal cell cultures. In one embodiment, "a limited number of plasma cells" means that the number of plasma cells is about 100 or less, e.g., 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

In one embodiment, the invention provides a method of producing an antibody from plasma cells comprising culturing plasma cells in culture at a low plasma cell concentration. Low plasma cell concentrations in culture typically include from about 1 to about 10, or from about 1 to about 15, or from about 1 to about 20, or from about 1 to about 25, or from about 1 to about 30, or from about 1 to about 40, or from about 1 to about 50, or from about 1 to about 60, or from about 1 to about 70, or from about 1 to about 80, or from about 1 to about 90, or from about 1 to about 100 cells per culture.

In another embodiment, the invention provides a method of producing antibodies from plasma cells comprising culturing plasma cells, wherein the plasma cells have been diluted to a low cell concentration in each culture. In another embodiment, the invention provides a method of producing an antibody from plasma cells comprising culturing a reduced number of plasma cells. The number of plasma cells isolated, for example, from a biological source can be reduced as described below. As used herein, "reduced number of plasma cells" is used interchangeably with "limited number of plasma cells" described above.

Techniques for obtaining the desired cell number in culture are well known in the art. Such techniques include, but are not limited to, limiting dilution or cell sorting and deposition. For example, a culture comprising a limited or reduced number of plasma cells can be achieved by single cell deposition using a cell sorter or dilution of a suspension of plasma cells with sufficient media such that there are 1, 2, 3 or more, e.g., 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 cells per well of a microtiter culture plate.

In one embodiment, a single plasma cell is cultured. Given the monoclonal nature of the antibodies produced by individual plasma cells, culturing plasma cells in a single cell culture will produce a population of monoclonal antibodies. Accordingly, in one embodiment, the present invention provides a method of producing monoclonal antibodies from plasma cells comprising culturing the plasma cells in a single cell culture.

As used herein, "single cell culture" is used interchangeably with "culturing a single plasma cell" and refers to a culture that, on average, comprises a single plasma cell. Thus, in a multi-well plate, such as a 96-well plate or a 1536-well plate, most wells contain a single plasma cell, some wells do not contain plasma cells, and some other wells contain more than one plasma cell. In some embodiments, the plasma cells can be cultured in a culture in which there is an average of less than 1 cell per well, e.g., 0.8 cells/well, 0.6 cells/well, 0.5 cells/well, 0.3 cells/well, or 0.1 cells/well. The technique for obtaining single cells in culture is similar to that described above, except that there is an average of 1 or less than 1 cell per well in a microtiter plate.

The invention further provides methods of producing antibodies or antibody fragments. The method comprises culturing a limited number of plasma cells according to any of the methods of the invention, identifying a culture that produces an antibody having the desired characteristics, isolating nucleic acid encoding the produced antibody, and expressing the nucleic acid in a host cell.

Unlike memory B cells (Traggiai et al, 2004, Nat Med 10: 871-) -875; Lanzavecchia et al, 2007, Curr Opin Biotechnol.18: 523-) -528), which can be cloned by immortalization into antibody-producing cells, plasma cells do not differentiate and cannot be stimulated or immortalized. Therefore, in order to utilize these "antibody factories" in any meaningful way, plasma cells must remain viable in culture. The plasma cells produce and secrete antibodies in a continuous manner, so the size of the antibody population increases over time. Although plasma cells survive for very long periods in vivo, they do not survive much longer than one day in vitro (experimental data not shown). Accordingly, the present invention provides methods for producing antibodies by culturing plasma cells (including, but not limited to, single plasma cells) in a medium comprising an exogenous component that prolongs the survival of the cultured cells.

Typically, the survival of the cultured plasma cells is prolonged for a sufficient time whereby the antibodies are produced in the amount required to characterize the antibodies, i.e., the culture medium contains sufficient antibodies so that it can be used in screening assays, including but not limited to binding assays, neutralization assays, or other assays that determine function or otherwise characterize the antibodies. The culture containing the antibodies of the desired specificity can then be isolated and the immunoglobulin genes can be cloned, sequenced and expressed to produce monoclonal antibodies.

The survival of plasma cells in culture, including but not limited to individual plasma cells, can be prolonged, either short-term or long-term. As used herein, "short-term" refers to a period of at least 2 days to about 9 days, i.e., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or about 9 days. As used herein, "long-term" refers to a period of at least 10 days, e.g., 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 130 days, 140 days, or about 150 days.

While short-term prolongation of plasma cell survival cannot produce as much antibody as long-term prolongation of plasma cell survival does, short-term prolongation of plasma cell survival as described herein is simpler, faster and more economical and is particularly useful for screening for antibodies in sensitive assays. Prolonged plasma cell survival over a long period is particularly suitable in situations where antibody characterization requires multiple screening assays or low sensitivity assays.

In one embodiment, the presence of exogenous components in the culture medium extends the survival of cultured plasma cells for a short period of time. In another embodiment, the exogenous component prolongs the survival of cultured plasma cells for an extended period of time. The exogenous component may be one or more ligands for a receptor expressed by the plasma cell or one or more non-plasma cells.

Examples of ligands for receptors expressed by plasma cells for prolonging the survival of cultured plasma cells include, but are not limited to, cytokines, chemokines, and other ligands. In one embodiment, the ligand is a ligand for IL-5, IL-6, stromal cell derived factor-1 (SDF-1), TNF- α, or CD44, such as ialuronic acid. In another embodiment, the exogenous component includes one or more ligands selected from the group consisting of IL-5, IL-6, stromal cell derived factor-1 (SDF-1), TNF- α, a ligand for CD44, such as ialuronic acid, and combinations thereof, and is used to prolong the survival of cultured plasma cells, either short-term or long-term.

Examples of non-plasma cells for prolonging the survival of cultured plasma cells include, but are not limited to, mesenchymal stromal cells, fibroblasts, or osteoclasts. In one embodiment, the non-plasma cells are mesenchymal stromal cells, fibroblasts, or osteoclasts, and are used to prolong the survival of cultured plasma cells, either short-term or long-term. In another embodiment, the non-plasma cells are mesenchymal stromal cells and are used to prolong the survival of cultured plasma cells, either short term or long term. The mesenchymal stromal cells may be mammalian mesenchymal stromal cells, including but not limited to human mesenchymal stromal cells. The mesenchymal stromal cells may optionally be immortalized prior to use in culture.

In one embodiment of the invention, the plasma cells are cultured, e.g., in a single cell culture, in the presence of one or more ligands of a receptor expressed by the plasma cells for about 3 days to about 7 days, or about 5 days to about 9 days. In another embodiment, the plasma cells are cultured, e.g., in a single cell culture, in the presence of one or more types of non-plasma cells for about 5 days to about 7 days, or about 10 days, or about 15 days, or about 20 days, or about 25 days, or about 30 days, or about 35 days, or about 40 days, or about 45 days, or more than 50 days. In another embodiment, the plasma cells are cultured in, for example, a single cell culture in the presence of mesenchymal stromal cells for about 5-7 days, or about 10 days, or about 15 days, or about 20 days, or about 25 days, or about 30 days, or about 35 days, or about 40 days, or about 45 days, or more than 50 days. In another embodiment, the plasma cells are cultured, for example, in a single cell culture, in the presence of one or more types of non-plasma cells and one or more ligands of a receptor expressed by the plasma cells for about 5-7 days, or about 10 days, or about 15 days, or about 20 days, or about 25 days, or about 30 days, or about 35 or about 40 days, or about 45 or about 50 days, or about 55 or about 60 days, or about 65 days, or more than 70 days.

In one embodiment, the plating efficiency of the cultured cells may be at least about 30%, in another embodiment at least about 40%, in another embodiment at least about 50%, in another embodiment at least about 55%, in another embodiment at least about 60% or more.

As used herein, the term "plating efficiency" refers to the percentage of plasma cells that survive long enough to produce detectable amounts of antibody in the supernatant.

The cultured plasma cells may be obtained from any desired species. In one embodiment, the plasma cell is a mouse, rat, camel, or monkey plasma cell. In another embodiment, the plasma cells cultured are human plasma cells and the antibodies produced are human antibodies. In another embodiment, the human monoclonal antibody is produced by culturing human plasma cells in a single cell culture.

Plasma cells such as human plasma cells can be isolated from human peripheral blood. These human plasma cells may be referred to as "peripheral plasma cells" or "circulating plasma cells". Plasma cells, such as human plasma cells, may also be isolated from bone marrow, tissues, or from bodily fluids, including but not limited to human synovial fluid, cerebrospinal fluid, and exudates. The term "tissue" is intended to cover any tissue present in the human body and may include cardiac tissue, neural tissue, muscle tissue, epithelium, connective tissue, and lymphoid organs such as the thymus, spleen, and lymph nodes.

Plasma cells are typically characterized by expression of CD138, optionally by additional expression of CD27, CD38, CD9, CD44, and MHC class II molecules. In one embodiment, the cells may be isolated from peripheral blood, tissue, bone marrow, or body fluids based on the expression of CD 138. Surface markers such as CD27, CD38, CD9, CD44 and MHC class II molecules in addition to CD138 can also be used to improve the isolation procedure and identify subsets of plasma cells (Arce et al, 2004, J Leukoc Biol, 75: 1022-. In another embodiment, plasma cells can be separated using magnetic microbeads. In another embodiment, plasma cells may be separated using magnetic microbeads coated with immobilized anti-CD 138 antibody. In another embodiment, enrichment of plasma cells with magnetic microbeads can be followed by cell sorting.

In one embodiment, the plasma cells may be isolated from peripheral blood of a human donor after immunization. Immunization refers to the administration of any antigen that induces an immune response. The vaccine can be any vaccine now known or later available to those of skill in the art and includes, but is not limited to, tetanus toxoid, influenza, yellow fever, tetanus-diphtheria, hepatitis b, smallpox, and cancer vaccines. In another embodiment, the immunization can be a booster. Plasma cells can be isolated from donors 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more after immunization. In one embodiment, the plasma cells may be isolated from donors responding to known pathogens. In another embodiment, the plasma cells may be isolated from a donor responding to an unknown pathogen. In a further embodiment, the plasma cells may be isolated from a donor with an allergic reaction. In another embodiment, the plasma cells can be isolated from a donor under homeostatic conditions. In another embodiment, the plasma cells may be isolated from a donor having an autoimmune disease.

In another embodiment, plasma cells can be produced in vitro by stimulating B cells. This stimulation can be performed by any method known in the art, including polyclonal or antigen-specific stimulation of naive or memory B cells (Bernasconi et al, 2002, Science, 298: 2199-.

The method of the invention can be used to culture plasma cells that secrete any antibody of any isotype. In one embodiment, the plasma cell may be an IgG plasma cell, in another embodiment, the plasma cell may be an IgA plasma cell, in another embodiment, the plasma cell may be an IgM plasma cell, in another embodiment, the plasma cell may be an IgD plasma cell, and in a further embodiment, the plasma cell may be an IgE plasma cell. In another embodiment, the isolated population of plasma cells can be a mixed population of plasma cells comprising two or more isoforms.

Isolated human plasma cells can be counted by enzyme-linked immunosorbent assay spot (ELISPOT) assay (Bernasconi et al, 2002, Science, 298: 2199-. This assay is performed by visualizing the products secreted by the cells of interest, with each spot generated by the assay representing a single cell.

In one embodiment, the human plasma cells may be seeded as single cells by limiting dilution or by single cell deposition. In one aspect, the human plasma cells may be seeded as single cells in the presence of mesenchymal stromal cells. In another embodiment, the human plasma cells can be inoculated as a polyclonal culture. Plasma cells may be seeded as polyclonal cell cultures in the presence of mesenchymal stromal cells. Alternatively, polyclonal human plasma cell cultures can be isolated as single cell cultures using limiting dilution. In another aspect, a polyclonal human plasma cell culture can be isolated as a single cell culture using single cell deposition.

Mesenchymal stromal cells are fibroblast-like cells, but have a greater differentiation potential than fibroblasts, and are capable of differentiating into osteoblasts, chondrocytes, and adipocytes. Mesenchymal stromal cells are found as heterogeneous populations and form the supporting structure of the tissue in which they are located. In bone marrow, mesenchymal stromal cells are required for the growth and differentiation of hematopoietic cells and for the maintenance of leukemia cells. Primary mesenchymal stromal cells can be isolated and cultured in a suitable medium for several passages, but only after a limited time in culture undergo senescence. Transduction using telomerase reverse transcriptase (TERT) has been used to immortalize mesenchymal stromal cells that expand indefinitely in vitro while maintaining their physiological growth rate and functional characteristics.

The mesenchymal stromal cells used in the culture may be bone marrow-derived mesenchymal stromal cells. The mesenchymal stromal cells may be mammalian mesenchymal stromal cells, such as human mesenchymal stromal cells. Mesenchymal stromal cells for use in the methods of the invention can be isolated from adherent bone marrow cells by culturing in a suitable medium. Such a medium may contain hydrocortisone. Mesenchymal stromal cells may also be derived from other tissues.

For practical reasons, the mesenchymal stromal cells may be immortalized prior to use in the methods of the invention. As used herein, "immortalization" refers to the ability of mesenchymal stromal cells to have improved proliferation capacity while retaining all the characteristics that enable them to maintain plasma cells, including the ability to undergo contact-dependent growth inhibition. In one embodiment, the mesenchymal stromal cells may survive at least about 1 week after reaching confluency. In another embodiment, the immortalized mesenchymal stromal cells may survive at least about 2 weeks after reaching confluency, or at least about 3 weeks after reaching confluency, or at least about 4 weeks after reaching confluency.

The mesenchymal stromal cells may be immortalized by any means known in the art. In one embodiment, the mesenchymal stromal cells are immortalized by transduction with a telomerase reverse transcriptase gene. In another embodiment, the mesenchymal stromal cells may be cultured according to Mihara et al, 2003 BrJ Haematol 120: 846-849 the method was immortalized by transduction with the TERT gene.

As discussed above, the present invention provides methods of producing antibodies or antibody fragments. The method comprises culturing a limited number of plasma cells, identifying a culture that produces an antibody having the desired characteristics, isolating a nucleic acid encoding the produced antibody, and expressing the nucleic acid in a host cell.

As used herein, the terms "fragment" and "antibody" fragment are used interchangeably to refer to any fragment of an antibody of the invention. In one embodiment, the antibody fragment retains the antigen binding activity of the antibody. In another embodiment, an antibody fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more contiguous amino acids. Exemplary antibody fragments may comprise Fc, Fab ', F (ab')₂Fv, scFv fragment, heavy chain, light chain, hinge region, antigen binding site, single chain antibody, or any portion thereof.

As used herein, the term "nucleic acid" includes all forms of nucleic acids, including, but not limited to, genomic DNA, cDNA, and mRNA. Cloning and heterologous expression of the antibody or antibody fragment can be carried out using molecular biology and recombinant DNA routine techniques known in the art (Wrammert et al, 2008Nature453, 667-Biol 358 & Meijer et al, 2006J Mol Biol 358, 764-772). Such techniques are explained in detail in the literature, for example, Sambrook, 1989 Molecular Cloning; a laboratory Manual, Second Edition. For the acquisition and expression of VH/VL sequences Tiller et al, J immunological Methods 2008329: 112, 124.

In one embodiment, the antibody is expressed in a eukaryotic cell using a suitable vector or virus. The eukaryotic cell may be a CHO, 293T, 293F or yeast cell. In another embodiment, the antibody is expressed in prokaryotic cells using a suitable vector or phage. The prokaryotic cell may be a bacterial cell, such as an E.coli cell. In a further embodiment, the heterologous expression system may be a cell-free system.

Antibodies and antibody fragments produced by the methods of the invention can be readily isolated using known methodologies (Coligan et al Eds Current Protocol in Immunology 1: 2.7). In one embodiment, the antibodies or antibody fragments of the invention, including monoclonal antibodies or antibody fragments, may be isolated from the culture supernatant by centrifugation or affinity chromatography. In another embodiment, antibodies or antibody fragments may be isolated according to their binding specificity. For example, antibodies can be isolated by application to a solid support comprising a suitable immobilized antigen. In further embodiments, the antibodies may be isolated with anti-IgG, anti-IgE, anti-IgA, anti-IgD, or anti-IgM antibodies, which in some cases may be immobilized.

Plasma-secreted Ig or specific antibodies can be isolated using immunoaffinity methods. First, the secreted product can be captured on the surface of the secreting cell using a suitable covalently bound capture reagent. The captured product can then be revealed with a fluorescently labeled secondary antibody or antigen (Manz et al, 1998 Int Immunol10, 1703-1711).

Antibody characterisation

Plasma cells do not express surface immunoglobulins and therefore cannot be selected for isotype or antigen specificity. The antibodies produced by plasma cells therefore need to be isolated for characterization. Currently, the prior art uses mainly two methods to prepare monoclonal antibodies from plasma cells. The first method was to screen a display library of antibodies prepared from the entire bone marrow of the immunized donor (Williamson et al, 1993Proc Natl Acad Sci U SA 90, 4141-4145). But this method is limited by the availability of bone marrow samples.

The second method involves the isolation of circulating plasma cells after boosting, followed by recovery of the Ig genes from individual plasma cells using single cell PCR (Wrammert et al, 2008Nature453, 667-Biol 671& Meijer et al, 2006J Mol Biol 358, 764-772). This method is based on the fact that: 6-8 days after the boost, a substantial portion of the circulating plasma cells are specific for the immunizing antigen. However, this approach requires extensive gene cloning and expression before antibody specificity can be assessed and is therefore less suitable when plasma cell responses are directed against multiple antigens such as complex pathogens. The development of a long-term culture system of human plasma cells is therefore particularly useful for obtaining sufficient antibodies for in vitro binding assays, functional assays and further antibody characterization, in order to be able to select plasma cells producing the antibody of interest.

Other Methods for isolating antibodies from plasma cells or other antibody secreting cells are based on micromanipulation and include a first step in which cells are plated in semi-solid media (Harriman WD et al JImmunel Methods 341; 135-. Once identified, plasma cells can be recovered by micromanipulation, and VH and VL genes amplified and sequenced. These methods are based on short-term culture and local detection of secreted antibodies, require specialized equipment for micromanipulation of antibody-secreting cells and are not suitable for testing functional properties of antibodies such as virus or toxin neutralization. The methods of the invention are not based on nor require any micromanipulation and allow the antibodies produced to be screened in a variety of assays, including but not limited to binding assays, functional assays and/or neutralization assays. In one embodiment, the invention provides a method of producing an antibody from plasma cells, comprising culturing a limited number of plasma cells and characterizing the antibody, wherein the method does not comprise micromanipulation of the antibody-secreting plasma cells.

The invention includes characterizing the antibodies or antibody fragments isolated by the methods of the invention. In one embodiment, antibody characterization may include determining the binding specificity of an antibody or antibody fragment. In another embodiment, antibody characterization may include determining the epitope recognized by the antibody or antibody fragment. The binding specificity of an isolated antibody or antibody fragment and/or the epitope recognized by the antibody or antibody fragment can be determined by any method known in the art. In one embodiment, the binding specificity and/or epitope recognized may be determined as follows: labeling the isolated antibody or antibody fragment, presenting the labeled antibody or antibody fragment to an antigen library, and detecting the labeled antibody or antibody fragment bound to its cognate antigen. In another embodiment, the labeled antibody or antibody fragment may be applied to a purification column containing the immobilized antigenic molecule, and the presence or absence of the labeled antibody or antibody fragment on the column may be used as an indication of the epitope specific and/or recognized by the antibody. It is clear to one skilled in the art that plasma cells isolated from peripheral blood of donors who have been immunized with a particular antigen or have been exposed to a particular pathogen will produce antibodies or antibody fragments that bind to the antigen or pathogen. However, pathogens, particularly complex pathogens, are likely to contain many antigens, and the binding specificity and/or recognized epitope of a particular antibody or antibody fragment may still be uncertain.

The single cell culture method of the present invention provides a single plasma cell producing a specific antibody from which nucleic acids encoding the antibody can be readily isolated using well-known methodologies (Wrammert et al, 2008Nature453, 667-. In one embodiment, antibody characterization may involve sequencing nucleic acids encoding the antibody or antibody fragment. Nucleic acid sequencing can be performed by any method known in the art. In one embodiment, nucleic acid sequencing may be performed with chain termination, where radioactive, fluorescent, or other dyes may be used. In another aspect nucleic acid sequencing can be performed using automated sequencing methods.

In another embodiment, the characterization may involve sequencing the antibody protein. Antibody proteins can be sequenced by any method known in the art. In one embodiment, the antibody protein can be sequenced by N-terminal analysis, C-terminal analysis, or Edman degradation. The N-terminal analysis may include: i) reacting the protein with a reagent that selectively labels the amino-terminal amino acid; ii) hydrolysing the protein; and iii) the amino terminal amino acid is determined by chromatography and compared to a standard. In this aspect, any labeling reagent can be used, including but not limited to Sanger's reagent, dansyl derivatives such as dansyl chloride and phenyl isothiocyanate. C-terminal analysis may involve incubating the protein with a carboxypeptidase and sampling at regular intervals to generate a plot of amino acid concentration versus time.

Following antibody protein sequencing, the invention also includes chemical synthesis of binding proteins based on the identified antibody sequences. Chemical synthesis may be performed according to any method known in the art. In one embodiment, chemical synthesis may be performed by attaching the carboxyl group of an amino acid to an insoluble solid support and reacting the amino group of the immobilized antibody with the carboxyl group of the next antibody in sequence. This process can be repeated until the desired amino acid sequence is produced, at which stage the intact protein can be cleaved from the solid support and allowed or induced to adopt the correct protein fold.

The invention also includes antibodies or antibody fragments produced by any of the methods of the invention.

Pharmaceutical use of antibodies

The invention provides antibodies or antibody fragments produced by any of the methods of the invention for use in therapy, e.g., for use in the treatment of allergy, infectious conditions or diseases, cancer, and autoimmune conditions or diseases

The term "allergy" includes all forms of hypersensitivity reactions caused by non-parasitic antigens, including but not limited to allergic dermatitis, allergic rhinitis, angioedema, anaphylaxis, aspirin sensitivity, asthma, allergic dermatitis, avian allergy, canary allergy, cat asthma, atopic dermatitis, bird allergy, canary allergy, cat allergy, chemical sensitivity, chicken allergy, conjunctivitis, chronic fatigue, contact dermatitis, cosmetic allergy, cow allergy, dermatitis, dog allergy, drug reaction, duck allergy, dust mite allergy, eczema, goose allergy, grass allergy, hay fever, headache, arrhythmia, urticaria, childhood hyperkinetic in childhood (hyperkinetic in childhood), hypoglycemia, respiratory and contact allergens, lactose intolerance, migraine, dairy allergy, mite allergy, rubella, parrot allergy, parakeet allergy (parakeet allergy), Perennial rhinitis, pigeon allergy, pollen allergy, rhinitis, lacquer allergy, salicylate allergy, sinusitis, rash, sparrow allergy, turkey allergy, ucaria and yeast allergy.

The term "infectious disease" includes any disease with significant clinical manifestations due to the presence of pathogenic microorganisms, including but not limited to viruses, bacteria, protozoa, parasites, and fungi. The term "infectious disease" includes, but is not limited to, AIDS-related complex, chicken pox, cold, cytomegalovirus infection, Colorado tick fever, dengue fever, Ebola hemorrhagic fever, hand-foot-mouth disease, hepatitis, herpes simplex, herpes zoster, HPV, influenza, lassa fever, measles, Marburg hemorrhagic fever, infectious mononucleosis, mumps, Norovirus (Norovirus), poliomyelitis, progressive multifocal leukoencephalopathy, rabies, rubella, SARS, smallpox, viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, West Nile river fever, yellow fever, anthrax, bacterial meningitis, botulism, Brucella, campylobacteriosis, cat scratch, cholera, diphtheria, epidemic typhus, gonorrhea, pustular disease-Legionella, leprosy, leptospirosis, Lerisonia, Leydia disease, Corynia, Marsdenia, Marble fever, Marble's, Mar, Lyme disease, melioidosis, rheumatic fever; MRSA infection, nocardiosis, pertussis, plague, pneumococcal pneumonia, psittacosis, Q fever, Rocky Mountain Spotted Fever (RMSF), salmonellosis, scarlet fever, Shigellasis, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhus fever-urinary tract infection, African trypanosomiasis, amebiasis, babesiosis, south America trypanosomiasis, bronchoschistosomiasis, cryptosporidiosis, cysticercosis, laceworm disease, longnose taeniasis, echinococcosis, enterobiasis, fascioliasis, fasciolopsiasis, filariasis, autogenous amebiasis, Dirofilariasis, palaemostomiasis, taenia, isosporosis, leiomycosis, leishmaniasis, leiomycosis, malaria, retrozoiasis, pinworm disease, pediculosis, scabies, schistosomiasis, taenia, toxoplasmosis, leprosomiasis, tuberculosis, enterobiasis, ascariasis, schistosomiasis, schistos, Trichinosis (trichellosis), trichinosis (trichinosis), trichinosis, trichomoniasis, trypanosomiasis, aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, tinea pedis, transmissible spongiform encephalopathy, bovine spongiform encephalopathy, creutzfeldt-jakob disease, kuru, fatal familial insomnia, and Alpers syndrome.

The term "autoimmune disease" includes all forms of diseases in which the immune system reacts with autoantigens, including, but not limited to, rheumatoid arthritis, type 1 diabetes, hashimoto's thyroiditis, hyperthyroidism, scleroderma, celiac disease, crohn's disease, ulcerative colitis, sjogren's syndrome, multiple sclerosis, acute infectious polyneuritis, goodpasture's syndrome, addison's disease, wegener's granulomatosis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, rheumatoid arthritis, autoimmune thyroid disease, systemic lupus erythematosus, psoriasis, psoriatic arthritis, sympathetic ophthalmia, autoimmune neuropathy, autoimmune oophoritis, autoimmune orchitis, autoimmune lymphoproliferative syndrome, antiphospholipid syndrome, lupus, autoimmune lymphoproliferative syndrome, lupus, autoimmune diseases, inflammatory bowel disease, inflammatory bowel, Multiple endocrine gland deficiency syndrome, multiple endocrine gland deficiency syndrome type 1, multiple endocrine gland deficiency syndrome type 2, immune thrombocytopenic purpura, pernicious anemia, myasthenia gravis, mixed connective tissue disease, primary glomerulonephritis, vitiligo, autoimmune uveitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, celiac disease, dermatitis herpetiformis, pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, autoimmune myocarditis, autoimmune vasculitis, autoimmune ophthalmopathy, alopecia areata, autoimmune atherosclerosis, Behcet's disease, autoimmune myelopathy, autoimmune hemophilia, autoimmune interstitial cystitis, autoimmune uropathy, autoimmune endometriosis, recurrent polychondritis, diabetes, multiple sclerosis, Ankylosing spondylitis, autoimmune rubella, paraneoplastic autoimmune syndrome, dermatomyositis, Miller Fisher syndrome, and IgA nephropathy.

The invention also provides an antibody or antibody fragment produced by any of the methods of the invention for use in the manufacture of a medicament for the treatment of an allergy, infectious disorder or disease, and autoimmune disorder or disease.

The invention further provides methods of treating allergy, infectious disorders or diseases, and autoimmune disorders or diseases, comprising administering an antibody or antibody fragment produced by any of the methods of the invention.

The invention also includes formulating an antibody or antibody fragment produced by any of the methods of the invention, or a nucleic acid encoding such an antibody or antibody fragment, into a pharmaceutically acceptable composition. In one embodiment, the pharmaceutical composition may comprise one or more isolated antibodies or antibody fragments produced by any of the methods of the invention. In another embodiment, the pharmaceutical composition may comprise 2, 3, 4, 5 or more isolated antibodies or antibody fragments produced by any of the methods of the invention.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier for administration. The carrier should not itself induce the production of antibodies that are detrimental to the individual receiving the composition and should be non-toxic. Suitable carriers may include large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactivated viral particles.

In some embodiments, pharmaceutically acceptable salts may be used, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, organic acid salts such as acetate, propionate, malonate and benzoate.

In some embodiments, the pharmaceutical composition may also contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting agents or pH buffering substances may be present in the composition, and may allow the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

The pharmaceutical composition may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal (transdermal), transdermal (transcutaneous), topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes.

The pH of the pharmaceutical composition may be between 5.5 and 8.5, in some embodiments between 6 and 8, and in further embodiments about 7. The pH may be maintained by using a buffer. The composition may be sterile and/or pyrogen-free. The composition may be isotonic with respect to the human body.

In another embodiment, the antibody or antibody fragment produced by any of the methods of the invention may be combined with a diagnostic excipient to form a diagnostic reagent. In one embodiment, the diagnostic reagent may comprise one or more isolated antibodies produced by any of the methods of the invention. For example, the diagnostic reagent may comprise 2, 3, 4, 5 or more antibodies or antibody fragments produced by any of the methods of the invention.

The diagnostic excipient may comprise a pharmaceutically acceptable carrier so that the diagnostic agent can be administered to a patient. The carrier should not itself induce the production of antibodies that are detrimental to the individual receiving the composition and should be non-toxic. Suitable carriers may include large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactivated viral particles.

In certain embodiments, pharmaceutically acceptable salts may be used, for example, inorganic acid salts such as hydrochloride, hydrobromide, phosphate and sulfate, organic acid salts such as acetate, propionate, malonate and benzoate.

In some embodiments, the pharmaceutical agent may also contain liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present.

The diagnostic reagents may be used in vivo, in vitro or ex vivo diagnostics. For in vivo use, the diagnostic agent may be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal routes.

The pH of the diagnostic reagent may be between 5.5 and 8.5, in some embodiments between 6 and 8, and in further embodiments about 7. The pH may be maintained by using a buffer. The composition may be sterile and/or pyrogen-free. The diagnostic reagent may be isotonic with respect to the human body.

In one embodiment, the diagnostic reagent may comprise a labeled antibody. The label may be selected from fluorescent labels, radiolabels, haptens and biomarkers, including enzyme labels.

The diagnostic reagents can be used to determine the presence or absence of a particular antigen. From this information, it can be inferred to determine the presence or absence of a particular antigen, and thus the presence or absence of a particular condition or disease. In one aspect of the invention, the disease may be an allergy, infectious condition or disease or an autoimmune condition or disease. The information obtained using the diagnostic reagents can be used to determine the appropriate course of treatment for a particular patient. In particular, the diagnostic reagents may be used to determine the presence or absence of an allergen.

The term "allergen" encompasses any non-parasitic antigen that can stimulate a hypersensitivity reaction in an individual, including, but not limited to, cats, fur, dander, cockroach calyx, wool, dust mites, dust mite excreta, penicillin, sulfanilamide, salicylates, anesthetics including local anesthetics, celery, celeriac, grains, corn, wheat, eggs, proteins, fruits, squash, beans, peas, nuts, peanuts, soybeans, milk, seafood, sesame, soybeans, tree nuts (trees nuts), pecans, almonds, insect stings, bee stings, wasp stings, mosquito bites, mold spores, latex, metals, plant pollen, grasses including ryegrass and cattail, weeds including ragweed, plantain, nettle, mugwort, quinoa, and sorrel, and including birch, alder, hazelnut, horntree, quassia, horse chestnut, willow, poplar, cupana, poplar, etc, Sycamore, linden, olive, Ashe juniper.

Use of isolated antibodies in protein purification

The invention also encompasses methods of immobilizing an isolated antibody or antibody fragment produced by any of the methods of the invention on a solid support. The term "solid support" includes solid and semi-solid supports, and encompasses any support that can be used to immobilize the isolated antibody or antibody fragment thereon. The solid support may comprise a gel, a mesh, beads including glass or magnetic beads, a column, a tube, a microtiter plate well, or a plastic sheet. The immobilized antibodies or antibody fragments produced by any of the methods of the invention can be used in protein purification. In one embodiment, the immobilized antibody may be used for immunoaffinity chromatography. A solution comprising a protein of interest can be used in a solid support comprising immobilized antibodies or antibody fragments specific for the protein of interest produced by any of the methods of the invention. The antibody or antibody fragment can be, for example, immobilized on a bead, which in some embodiments can be placed in a column.

General concepts

The term "comprising" encompasses "including" as well as "consisting of … …," e.g., the composition "comprises" X means that it may consist of X alone or may include something else, such as X + Y.

The word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be a composition that is completely free of Y. Where desired, the word "substantially" may be omitted from the definition of the invention.

The term "about" in relation to the numerical value x means, for example, x ± 10%.

As used herein, the term "disease" is generally synonymous with the terms "disorder" and "condition" (as in medical conditions), both reflecting an abnormal condition in which the normal function of the human or animal body or a part thereof is impaired, typically manifested by different signs and symptoms, and resulting in a reduction in the life span or quality of life of the human or animal.

As used herein, "treatment" with respect to a patient is meant to include prevention and prophylaxis as well as therapy. The term "patient" refers to all mammals including humans. Typically, the patient is a human.

Examples

Illustrative embodiments of the invention are provided in the following examples. The following examples are merely illustrative of the invention and serve to assist the skilled person in using the invention. The examples do not limit the scope of the invention in any way.

Example 1: plasma cells in mesenchymal stromal cell cultures

The present inventors observed that primary cultures of human mesenchymal stromal Cells established from normal bone marrow according to standard procedures (Pittenger et al, 1999, Science 284: 143-. These cells were detected by ELISPOT and identified as plasma cells. Plasma cells in mesenchymal stromal cell cultures were still detectable in vitro after 3 weeks (data not shown).

Example 2: human plasma cell cultures up to 50 days

In order to develop a culture system in which individual plasma cells can remain viable so that the produced antibodies can accumulate over the culture time, the present inventors examined primary mesenchymal stromal cells of different origin prepared according to standard methods. Briefly, tissue culture flasks were pre-coated with FCS for 1 hour. Bone marrow cells were supplemented with 30% FCS and 10^-8M dexamethasone was cultured adherently in complete IMDM medium overnight. Nonadherent cells were washed away and adherent cells were cultured in complete DMEM-10% FCS. Of the 7 lines tested, 3 lines supported human plasma cell survival, but terminated proliferation after several passages. In subsequent experiments, immobilized mesenchymal stromal cells were transduced with the telomerase reverse transcriptase gene (MSC-TERT). These cells were those isolated by Mihara et al (Br J Haematol2003, 120, 846-.

Peripheral blood mononuclear cells were stained with PE-labeled anti-CD 138 monoclonal antibody, enriched using anti-PE microbeads (Miltenyi), and further purified by cell sorting to isolate CD 138-positive cells. The number of IgG-secreting plasma cells recovered was determined using isotype-specific ELISPOT. Different numbers of CD 138-positive cells were seeded on mesenchymal stromal cell monolayers in 96-well culture plates with RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone), non-essential amino acids, pyruvate and glutamax (gibco). Based on ELISPOT performed at plating, the cultures shown in fig. 1 contained 7 IgG-secreting cells. Half of the culture supernatant was collected at different time points and replaced with fresh medium. In addition, on day 18 and day 34, the medium was completely removed and replaced with fresh medium. The IgG production rate per culture per day and the estimated IgG production rate per plasma cell per day were determined. As shown in FIG. 1, the amount of IgG produced by the cultures of the invention of approximately 7 cells increased as a linear function with culture time over a period of 50 days, consistent with a production rate of 676 pg/day and an estimated production rate of 96 pg/cell/day.

Example 3: 3 week cultures of individual plasma cells

Plasma cells were isolated from peripheral blood or bone marrow using PE-conjugated anti-CD 138 antibodies followed by anti-PE microbeads and cell sorting and seeded at 0.5 cell/well density on mesenchymal stromal cell monolayers in 96-well plates. Cultures containing IgG were monitored over a 22-23 day period by periodic sampling. The medium was changed on day 16. Throughout the culture period, IgG productivity in the monoclonal cultures was constant at 72-134 pg/cell/day (FIG. 2a, peripheral blood derived (4 cultures); FIG. 2b, bone marrow derived (5 cultures)).

In 5 limiting dilution experiments, the plate efficiency of blood and bone marrow plasma cells was 30% -65% (data not shown). In addition, plasma cells recovered from polyclonal cultures can be replated into single cell cultures, where they maintain a constant Ig secretion rate (data not shown). The linear accumulation of IgG is consistent with the maintenance of individual cells that secrete IgG at a constant high rate. Cells were irradiated at a level that completely abolished proliferation and differentiation of memory B cells stimulated by TLR agonists, which did not affect IgG production by cultured plasma cells (data not shown).

Example 4: culture of IgG, IgA, IgM and IgE producing plasma cells

Peripheral blood CD 138-positive cells producing IgG, IgA, IgM, and IgE were isolated from healthy donors and plated at a density of 5 cells/well in 384-well plates containing monolayers of mesenchymal stromal cells, 617 copies were cultured in duplicate. Culture supernatants from day 10 were tested for the presence of IgG, IgA, IgM, and IgE using isotype specific ELISA. The total amount of 4 isoforms was measured in the culture supernatant (see figure 3). The mean values for IgG, IgA, IgM and IgE productivity of plasma cells were 860, 770, 1100 and 1800pg, respectively, i.e.86, 77, 110 and 180 pg/cell/day, respectively, over 10 days.

Example 5: efficiency of plasma cell survival in vitro

Peripheral blood-derived human plasma cells were isolated from 7 donors based on CD138 expression and seeded at a density of 1 or 25 cells/well. The number of cells secreting IgG-, IgA-and IgM-antibodies at the start of the culture was calculated by isotype specific ELISPOT. The plate efficiency of IgG-antibody, IgA-antibody and IgM-antibody secreting cells was calculated according to Poisson distribution analysis, with IgG ranging from 50% to 74%, IgA ranging from 31% to 78% and IgM ranging from 0 to 26% (see FIG. 4). In addition, plasma cells recovered from polyclonal cultures can be replated into single cell cultures, where they maintain a constant Ig secretion rate (data not shown).

Example 6: isolation of rare IgE monoclonal antibodies

Plasma cells were isolated from peripheral hematomas of allergic individuals and plated at a density of 1 cell/well on hMSC-TERT monolayers in 10 microplates of 384 wells. The 5 culture supernatants were scored positive for IgE production. The IgE-positive cultures were subjected to RT-PCR, and the two paired VH/VL genes were recovered and sequenced (Table 1). The V gene was cloned into an expression vector for expression of the light chain (. kappa.or. lamda.) or the human IgG1 or IgE heavy chain according to the method described by Wardemann et al (Science 301, 1374-1377, 2003). IgG or IgE antibodies were produced by transient transfection of 293T cells. This example illustrates the possibility of recovering rare plasma cells and isolating representative IgE monoclonal antibodies.

TABLE 1 two IgE monoclonal antibodies recovered from circulating plasma cells

SHM nt: somatic hypermutation nucleotides; aa: amino acids

Example 7: isolation of antigen-specific monoclonal antibodies from plasma cells cultured in the presence of mesenchymal stromal cells

Plasma cells were isolated from the donor peripheral blood 7 days after booster inoculation with Tetanus Toxoid (TT) and seeded in clonal conditions onto MSC-TERT monolayers in 384-well microplates. Culture supernatants at day 10 were analyzed for the presence of total IgG (ng/culture) as well as TT-specific IgG antibodies (OD405) and monoclonal cultures producing TT-specific antibodies were identified (see figure 5). This example illustrates the possibility of identifying a large number of antigen-specific plasma cells after boosting.

Example 8: isolation of potent and broadly reactive influenza A neutralizing antibodies from plasma cells cultured in the presence of IL-6

CD 138-positive cells from donors 7 days prior to vaccination with seasonal influenza vaccine were seeded at 0.5 cell/well density in 16 384-well plates in the presence of 10ng/ml IL-6. On day 6 and 8 of the world, culture supernatants were examined in three parallel ELISAs using recombinant H5 or H9 baculovirus-derived recombinant Hemagglutinin (HA) and the unrelated antigen Tetanus Toxoid (TT) as antigens. Of the 4,928 culture supernatants screened, 12 supernatants bound H5HA, 25 supernatants bound H9 HA, and 54 supernatants bound both H5 and H9. Some of the latter with the highest OD signals were subjected to RT-PCR and two pairs of VH/VL genes were recovered. These two monoclonal antibodies FI6 and FI28 share most of the V, D and J gene fragments (IGHV3-30 x 01, IGHD3-9 x 01, IGHJ4 x 02 and IGKV4-1 x 01), but differ in the N region, in IGKJ usage and in the somatic mutation pattern and are therefore not clonally related.

The V genes of FI6 and FI28 were cloned into expression vectors and recombinant antibodies were produced by transfecting 293T cells. The specificity was detected by ELISA using a panel of recombinant HA belonging to different subtypes (table 2). FI6 binds to all influenza a HA subtypes tested, including group 1 (H1, H5 and H9) and group 2 (H3 and H7), but not to influenza B HA. In contrast, FI28 only bound to group 1 HA.

TABLE 2 binding of plasma cell-derived human monoclonal antibodies to influenza virus HA

We next tested FI6 and FI28 for their ability to neutralize group 1 and group 2 influenza a virus subtypes, using pseudoviruses as well as infectious viruses. Clearly, FI6 neutralizes all pseudoviruses tested, including six H5 isolates and two H7 avian isolates belonging to antigenic divergent clades (table 3) 0, 1, 2.1, 2.2 and 2.3. In addition, FI6 neutralized all infectious viruses tested, including two H3N2 viruses and four H1N1 viruses, across the swine-derived H1N1 isolate a/Cal/04/09 (table 4) that was circulating up to the last 70 years. In contrast, FI28 neutralized all H5 pseudoviruses, but failed to neutralize H7 pseudoviruses as well as all infectious viruses detected (table and 4).

TABLE 3 neutralization of H5 and H7 pseudotypes by human monoclonal antibodies

TABLE 4 neutralization of influenza Virus by human monoclonal antibodies (nd, not performed)

nd, do not proceed

It should be noted that the above method administers 50. mu.l of monoclonal antibody in an amount of about 8-16ng/ml within 5-10 days. This volume and antibody concentration is sufficient for multiple assays. These assays include not only binding assays such as ELISA (performed using 5 μ l in standard shallow 384 well plates), but also functional assays such as pseudotype neutralization, which are within the sensitivity range (see table 3). Importantly, the ability to perform multiple parallel assays is necessary to rapidly identify rare plasma cells that secrete antibodies that bind multiple antigen variants.

Example 9: isolation of tetanus toxoid-specific monoclonal antibodies from cultured plasma cells isolated from peripheral blood after 10 years of inoculation

CD138+ HLA-DR + CD62L + plasma cells were isolated by cell sorting from peridonor blood 10 years after Tetanus Toxoid (TT) inoculation. A total of 1,700 cells were seeded at a density of 0.5 cells/well in 384-well microplates and cell culture supernatants were screened by ELISA for the presence of tetanus toxoid specific IgG antibodies on day 7. A tetanus toxoid-specific culture was identified and the VH/VL genes were recovered by RT-PCR and sequenced on day 8 (see Table 5). The genes were cloned into expression vectors and recombinant antibodies were produced by transient transfection of 293T cells (TT 14). Binding of different concentrations of antibody to tetanus toxoid or to an unrelated antigen (negative control) was detected by ELISA (see figure 6).

TABLE 5 tetanus toxoid specific monoclonal antibodies recovered from circulating plasma cells 10 years after inoculation

SHM nt: somatic hypermutation nucleotides; aa: amino acids

It should be noted that there are additional ways of implementing the invention, and that various modifications can be made without departing from the scope and spirit of the invention. Accordingly, the present embodiments are to be considered as illustrative and not restrictive, and the invention is not to be limited to the details given herein, but may be modified within the scope and equivalents of the appended claims.

Claims (10)

1. A method of producing an antibody comprising: culturing a limited number of plasma cells in the presence of IL-6 or mesenchymal stromal cells and obtaining the antibody therefrom,

wherein the number of cultured plasma cells is 25 or less.

2. The method of claim 1, wherein the number of cultured plasma cells is 1.

3. The method of claim 1 or 2, wherein the antibody is a human antibody and the plasma cell is a human plasma cell.

4. The method of claim 1, wherein the plasma cells are cultured in a medium comprising an exogenous component that prolongs the survival of the plasma cells.

5. The method of claim 1, wherein the survival of the cultured plasma cells is extended for a time sufficient to produce the antibody in an amount necessary to characterize the antibody.

6. The method of claim 4 or 5, wherein the survival of the plasma cells is prolonged by a short period, and wherein the short period is from 5 days to 7 days.

7. The method of claim 4 or 5, wherein the survival of the plasma cells is prolonged for a long period, and wherein the long period is at least 10 days.

8. The method of claim 4 or 5, wherein the survival of the plasma cells is prolonged by at least 20 days.

9. A method of producing an antibody or antibody fragment comprising the steps of:

a. culturing a limited number of plasma cells according to the method of claim 1;

b. identifying a culture that produces an antibody having the desired characteristics;

c. isolating nucleic acid encoding the antibody; and

d. expressing the nucleic acid in a host cell.

10. The method of claim 1 or 9, further comprising characterizing the antibody or antibody fragment, wherein the characterization of the antibody or antibody fragment comprises performing a functional assay to determine the function of the antibody or antibody fragment, performing a binding assay to determine the binding specificity of the antibody or antibody fragment or an epitope recognized by said antibody or antibody fragment, and/or performing a neutralization assay to determine the ability of the antibody or antibody fragment to neutralize a toxin or pathogen.