System for production of antibodies and their derivatives

The present disclosure provides methods and compositions for the production of chimeric antibodies that specifically bind an antigen of interest.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2014, is named 099523_0105_SL.txt and is 270,890 bytes in size.

TECHNICAL FIELD

The present disclosure relates to methods and compositions for producing chimeric antibodies that specifically bind an antigen of interest.

BACKGROUND

Prior to Sep. 11, 2001 the list of pathogens that humanity was threatened by on a day-to-day basis was relatively short and people had found means of decreasing the threat from these pathogens by developing corresponding vaccines. Nowadays this list has swelled many times from its pre-September 11 size and the threat of exposure of populations to agents from this list has grown immensely. Many vaccines are so old that they have lost their potency, while vaccines for other agents simply do not exist. The situation with the anti-BoNT vaccine is a perfect example of the former situation. As a result, the traditional vaccination approach can no longer be used to the full extent to protect society from such threats.

BoNTs are classified as Category A agents, one of the 6 highest risk threat agents for bioterrorism (2). These homologous, but serologically distinct toxins (serotypes A, B, C, D, E, F and G), specifically target neurons and, through interruption of neurotransmission, cause muscle paralysis, which leads to death from asphyxiation. It has been estimated that aerosol exposure of 100,000 individuals to the toxin, as could occur with an aerosol release over a metropolitan area, would result in 50,000 cases of illness with 30,000 fatalities (3). Such an exposure would result in 4.2 million hospital days and an estimated cost of $8.6 billion.

Pentavalentbotulinumtoxoid was generated over 30 years ago via chemical inactivation of native toxins of five different serotypes. This vaccine received Investigational New Drug status from the CDC (for at-risk workers), and from the United States Army's Office of the Surgeon General (for military deployment). It was stockpiled and over years was used more than 20,000 times (4). However, it was also losing its potency over the years and the CDC recently issued a notice of its discontinuation (5). The first reports of efforts to generate a new recombinant substitute for pentavalent toxoid were published almost 17 years ago (6). However, no new anti-BoNT vaccines have been approved yet. BoNTs of serotypes A and B are currently used under trade names BOTOX® and MIOBLOCK® in medicine as potent drugs and rejuvenation agents in cosmetics. Thus, it is unlikely that many people would be willing to undergo vaccination and give up the current benefits of these “miracle” drugs even if new anti-BoNT vaccines were to be developed. A more realistic strategy for raising preparedness against the threat of a bioterrorist attack would include stockpiling pathogen-specific antibodies and using them in case of an immediate threat of bioterrorist attack or soon after it.

The injection of heterologous antibodies, however, causes acute or delayed hypersensitivity reactions in 9% of cases, including serum sickness (3.7%) and anaphylactic shock (1.9%) (7). Further, application of non-human antibodies might trigger the development of an immunologic response, which will reduce or eliminate the benefit of repeating applications of such antibodies. Securing substantial quantities of human antigen-specific serums, however, may be an extremely expensive endeavor. For example, Orphan Drug human Botulism Immune Globulin has been approved by the FDA for treatment of infant botulism. It was formulated on the basis of serum obtained from human volunteers vaccinated with pentavalentbotulinumtoxoid. The price of this drug for treatment of one patient is $45,300.

SUMMARY

In one aspect, the present disclosure provides a method for producing a chimeric immunoglobulin-G (IgG) antibody that specifically binds an antigen of interest comprising: a) isolating nucleic acid sequences encoding IgG heavy and light chain variable regions from a single immune cell producing an IgG that specifically binds the antigen of interest; b) cloning the nucleic acid sequences of part a) into separate expression vectors comprising the IgG heavy or light chain constant regions, or into a single expression vector comprising both the IgG heavy and light chain constant regions; c) introducing the expression vector(s) of part b) into a host cell; d) establishing a stable cell line from the host cell of part c); and e) isolating the IgG produced by the stable cell line of part d), wherein the method comprises simultaneous cloning of the IgG heavy and light chain variable regions isolated from the immune cell of part a), and wherein the expression vector of part b) allows for (i) unidirectional insertion of the IgG heavy and light chain variable regions into the vector, and (i) positive selection of expression vectors comprising cloned sequences.

In some embodiments, the antigen of interest is derived from a pathogen. In some embodiments, the antigen of interest is aClostridium botulinumneurotoxin.

In some embodiments, the expression vector is selected from the group consisting of pVLentry-Hyg10, pVHentry-Cm5, pVHentry-GFP1, pVHentry-MLuc7, pVHentry-Hisbio1, and pVHentry-CBD1.

In some embodiments, the stable cell line of part d) is established through expression of an antibiotic resistance gene present in the expression vector of part b). In some embodiments, the level of expression of the antibiotic resistance gene by the stable cell line correlates to the level of IgG production by the stable cell line.

In some embodiments, parts a) and b) comprise the steps of: i) reverse-transcription of mRNA released from the immune cell upon exposure to perfingolysin O; ii) simultaneous amplification of cDNAs produced in part i) encoding the IgG heavy chain variable region (VH) and the IgG light chain variable region (VL); iii) separate re-amplification of the VHand VLsequences of part ii), and iv) insertion of the re-amplified sequences of part iii) into the expression vector of part b).

In some embodiments, the reverse transcription is performed using a primer selected from the group consisting of IgG-CHH, Cm1, and Clv-3.

In some embodiments, the method further comprises formulating the chimeric IgG into a therapeutic composition. In some embodiments, the method further comprises formulating the chimeric IgG into an antigen-specific resin or system for detecting corresponding antigens.

In some embodiments, the immune cell is selected from the group consisting of a plasma cell, a B-cell, or any other cell that secretes or displays on the cell surface immunoglobulins specific for the antigen of interest.

In some embodiments, the host cell is selected from the group consisting of a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK), a mouse NS1/1-Ag 4-1 cell, a NSO/u cell, an X63/Ag 8.653 cell, an SP2/0 Ag14 cell, a rat Y3 (210.RCY3.Ag 1.2.3) cell, a YB213.0Ag3 (Y0) cell, and any other mammalian secondary cell line capable of producing immunoglobulins.

In some embodiments, the method allows for high-throughput production of antibodies against the antigen of interest.

In one aspect, the present disclosure provides a method for detecting an antigen of interest in a sample, comprising the steps of (a) contacting the sample with an antibody that specifically binds the antigen under conditions that promote the formation of an antibody-antigen complex, (b) contacting the antibody-antigen complex with a fusion protein comprising (i) the immunoglobulin-binding domains of staphylococcal protein A and streptococcal protein G, and (ii)Metridia longaluciferase or a derivative lacking the N-terminal region, under conditions that promote binding of the fusion protein to the antibody-antigen complex, and (c) detecting theMetridia longaluciferase.

In some embodiments, the fusion protein is encoded by a vector selected from the group consisting of pS14L-spAG-MLuc16, pETspAG-ΔN-MLuc1, and pS14L-spAG-ΔN-MLuc15. In some embodiments, the fusion protein is encoded by pS14L-spAG-MLuc16 or pETspAG-ΔN-MLuc1. In some embodiments, the fusion protein is encoded by pS14L-spAG-ΔN-MLuc15.

In one aspect, the present disclosure provides an IgG fusion protein comprising IgG heavy chains fused with a peptide or polypeptide selected from the group consisting of green fluorescent protein (GFP),Metridia longaluciferase, cellulose binding domain, 6× histidine (SEQ ID NO: 1), or a biotinylatable peptide.

DETAILED DESCRIPTION

The present disclosure provides methods and compositions for robust generation of human monoclonal antibodies targeted at pathogens of interest.

In addition to the set of products that address existing needs, this technology advances our understanding of structure-function relationships in the neurotoxin molecule and provides information about mechanisms of inactivation of this molecule by antibodies.

In practicing the present disclosure, many conventional techniques in cell biology, molecular biology, protein biochemistry, immunology, and bacteriology are used. These techniques are well-known in the art and are provided in any number of available publications, including Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Certain terms used herein are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same general meaning as commonly understood by one skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. All references cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference in its entirety for all purposes.

As used herein, “administration” of a composition to a subject includes any route of delivering the compound to the subject to perform its intended function. Administration can be carried out by any suitable route including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another.

As used herein, the terms “antigen” and “antigenic” refer to molecules with the capacity to be recognized by an antibody or otherwise act as a member of an antibody-ligand pair. “Specific binding” refers to the interaction of an antigen with the variable regions of immunoglobulin heavy and light chains. Antibody-antigen binding may occur in vivo or in vitro. The skilled artisan will understand that macromolecules, including proteins, nucleic acids, fatty acids, lipids, lipopolysaccharides and polysaccharides have the potential to act as an antigen. The skilled artisan will further understand that nucleic acids encoding a protein with the potential to act as an antibody ligand necessarily encodes an antigen. The artisan will further understand that antigens are not limited to full-length proteins, but can also include partial amino acid sequences. Moreover, sequences from different sources may be combined to generate mosaic antigens, depending on the specific intended use. In some embodiments, the mosaic antigen will include epitopes derived from different proteins. In some embodiments, the mosaic antigen will include epitopes derived from the same protein. The term “antigenic” is an adjectival reference to molecules having the properties of an antigen. In some embodiments, the antigen of interest is a bacterial toxin. In some embodiments the antigen of interest is abotulinumneurotoxin.

As used herein, the term “epitope” refers to that portion of a molecule that forms a site specifically recognized by an antibody or immune cell. A protein epitope may comprise amino acid residues directly involved in antibody binding, as well as residues not directly involved in binding that are nonetheless included in the antibody-epitope footprint and excluded from the solvent surface. Epitopes may derive from a variety of physical characteristics of a protein, including primary, secondary, and tertiary amino acid structure, and amino acid/protein charge. Epitopes present within a molecule are referred to as “real epitopes.” Real epitopes encompass wild-type sequences and variants of wild-type sequences. Real epitopes may exist within a wild-type protein, a naturally occurring variant of a wild-type protein, or an engineered variant of a wild-type protein. The term “mimetic epitope” refers to a molecule whose primary structure is unrelated to the primary structure of a given real epitope that nonetheless specifically binds to antibodies that recognize the real epitope. Epitopes may be isolated, purified, or otherwise prepared by those skilled in the art. They may be obtained from natural sources including cells and tissues, or they may be isolated from host cells expressing a recombinant form of the epitope.

As used herein, “effective amount” refers to a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and on the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. In the context of an antigenic composition, in some embodiments, an effective amount is an amount sufficient to result in a protective response against a pathogen. In other embodiments, an effective amount of an antigenic composition is an amount sufficient to result in antibody generation against the antigen. With respect to antigenic compositions, in some embodiments, an effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject's immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of a biochemical application, in some embodiments, an effective amount will depend on the size and nature of the sample in question. It will also depend on the nature and sensitivity of the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations.

As used herein, the term “polymer resin” refers to resins, such as, but not limited to polysaccharide polymers such as agarose, cellulose, and Sepharose™. The skilled artisan will understand that proteins may be covalently attached to the resin using methods well known in the art, including but not limited to cyanogen bromide activation, reductive animation of aldehydes, and the addition of iodoacetyl functional groups. The skilled artisan will further understand that functional equivalents of polysaccharide polymers may also be to immobilize proteins.

As used herein, the term “BoNT” refers to any of the seven serologically distinctbotulinumneurotoxins produced byClostridium botulinum, Clostridium argentiensis, andClostridium baratti. Individual serotypes are referred to as BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, and BoNT/G. Exemplary, non-limiting nucleic acid sequences of BoNT/A, /B, /C, /D, /E, /F, and /G are found in GenBank Accession numbers DQ409059, FM865705, AB200364, NZ ACSJ01000015, AM695754, X81714, and X74162, respectively. Exemplary, non-limiting amino acid sequences of BoNT/A, /B, /C, /D, /E, /F, and /G are found in GenBank Accession numbers ABD65472, CAR97779, BAD90572, ZP 04863672, CAM91137, CAA57358, and CAA52275, respectively. Exemplary, non-limiting nucleic and amino acid sequences ofC. tetanitetanus toxin are found in GenBank Accession numbers AF154828 and AAF73267, respectively. As used herein, the term “BoNT/A-L” refers to the full-lengthbotulinumneurotoxin A light chain. As used herein, the term “BoNT/B-L” refers to the full-lengthbotulinumneurotoxin B light chain.

As used herein, the term “anti-BoNT antibody” refers to an antibody capable of specifically binding to BoNT. As used herein, an antibody includes a polyclonal antibody, a monoclonal antibody, and also refers to functional fragments (e.g., fragments which bind an antigen/epitope), such as Fv, Fab, Fc and CDRs.

As used herein, the terms “immunogen” and “immunogenic” refer to molecules with the capacity to elicit an immune response. The response may involve antibody production or the activation of immune cells. The response may occur in vivo or in vitro. The skilled artisan will understand that a variety of macromolecule, including proteins, have the potential to be immunogenic. The skilled artisan will further understand that nucleic acids encoding a molecule capable of eliciting an immune response necessarily encodes an immunogen. The artisan will further understand that immunogens are not limited to full-length molecules, but may include partial amino acid sequences (e.g., epitopes). Moreover, sequences from different sources may be combined to generate mosaic immunogens, depending on the specific intended use.

As used herein, the terms “isolate” and “purify” refer to processes of obtaining a biological substance that is substantially free of material and/or contaminants normally found in its natural environment (e.g., from the cells or tissues from which a protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized).

As used herein, the term the terms “polypeptide,” “peptide,” and “protein” are used interchangeable to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). Polypeptides may include amino acids other than the naturally-occurring amino acids, as well as amino acid analogs and mimetics prepared by techniques that are well known in the art. The skilled artisan will understand that polypeptides, peptides, and proteins may be obtained in a variety of ways including isolation from cells and tissues expressing the protein endogenously, isolation from cell or tissues expressing a recombinant form of the molecule, or synthesized chemically.

As used herein, the term “subject” refers to a member of any vertebrate species. In some embodiments, the subject is avian and includes domestic (e.g., chicken, turkey) and wild bird species. In some embodiments, subjects include mammals such as humans, as well as those mammals of importance due to being endangered, of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans. In particular embodiments, the subject is a human. In other embodiments, the subject is not human.

As used herein, the term “pathogen” refers to any entity that causes disease, including, for example, but not limited to, mycoplasma, fungi, bacteria, viruses, viroids, virus-like organisms, protozoa, and nematodes, toxins, and prions. In some embodiments, the pathogen is aClostridium. In some embodiments, the pathogen isClostridium botulinum.

As used herein, the term “chimera” and “chimeric” refers to biological molecules comprising materials derived from two or more organisms of the same or different species. For example, the terms “chimeric antibody,” and “chimeric IgG” refer to antibodies comprising amino acid sequences derived from two or more organisms of the same or different species. In some embodiments, the organisms are both of the same species. In some embodiments, the organisms are both human. In some embodiments, the organisms are from different species. In some embodiments, the terms refer to nucleic acid sequences encoding chimeric polypeptide sequences.

The present disclosure provides methods and compositions for high-throughput production of chimeric antibodies that specifically bind to an antigen of interest. The methods combine three procedures into one streamlined process: 1) isolation of lymphocytes producing antibodies of interest from the blood of immunized individuals, 2) amplification of sequences encoding variable domains of light and heavy chains of immunoglobulin from individual isolated cells, and 3) assembly of amplified sequences into specially designed vectors and construction of cells encoding human/human chimeras targeted at antigens of interest. The uniqueness of this process is its ability to generate multiple (up to 100) immunoglobulin-producing clones within a very short time (one-two months). Each such clone encodes an IgG whose variable domains of light and heavy chains originate from the same lymphocyte.

Since the required antibody-producing blood cells could come from a patient recovered from the infection, this system does not depend on the availability of a developed vaccine. Consequently, this system could be used to develop protective entities against rare and even new natural and engineered pathogens at very early signs of appearance. Additionally, the system does not involve use of viruses and, consequently, is safe to use.

The methods allow for rapid generation of IgGs whose heavy chains carry additional polypeptides at the C-termini. This grants the opportunity to produce derivatives of antibodies that can be used to monitor corresponding antigens (IgGs fused with reporter molecules) or to immobilize those pathogens (IgGs fused with polypeptides like Cellulose Binding Domain). Among other fusions, the system allows creation of fusions withMetridia longaluciferase, which allows fast and inexpensive examination of conditions to identify those for optimal production of antibodies. Also, the methods allow for the use of fluorescence activated cell sorting (FACS) for fast selection of clones producing increased levels of IgGs.

The present disclosure provides methods and compositions for robust development of human antibodies targeted at specific antigens of interest. The chosen approach required the ability to 1) isolate individual human lymphocytes specific to the chosen antigen, 2) isolate immunoglobulin-encoding sequences from a single selected cell, and 3) assemble immunoglobulin-encoding constructs that can be introduced into chosen cell cultures for production of corresponding antibodies. Prior to this work, it was unknown whether the dynamics of antibody secretion and the limited number of antigen-specific lymphocytes in the peripheral blood would permit efficient separation of these specific cells from all others. It was unclear whether protocols for rtPCR at the single cell level would be robust enough to allow their application in a high throughput format. Finally, described procedures for assembling expression vectors carrying IgG-encoding sequences were suitable for manipulation with just a very small number of IgG-encoding sequences at a time. By contrast, suitable methods for high throughput production must be capable of simultaneous handling of tens and even hundreds of different sequences.

In some embodiments, the compositions comprise expression vectors encoding constant regions of either light or heavy chains of human IgG. In some embodiments, the compositions comprise an expression vector encoding the constant regions of both the IgG heavy and light chains.

In some embodiments, the methods comprise isolating sequences encoding variable domains of light and heavy chains of IgG from single cells and assembly of Ig-encoding vectors.

In some embodiments, the methods comprise introducing designed IgG-encoding constructs into mammalian cells and evaluation of conditions for efficient IgG production. In some embodiments, the methods comprise producing and characterizing chimeric IgGs. In some embodiments, the chimeric IgGs are specific forbotulinumneurotoxin serotype A (BoNT/A).

Embodiments described herein are set forth in the following non-limiting examples.

EXAMPLES

Development of Expression Vectors

This Example demonstrates the construction of expression vectors for the cloning and production of chimeric IgG antibodies that specifically bind an antigen of interest.

In order to create a system for generation of human antibodies that is capable of working in a high throughput format, vectors were necessary that would allow 1) a 100%-certain assembly of sequences encoding light and heavy chains of immunoglobulins, 2) simple assembly of such sequences into one plasmid, and 3) robust selection of cells carrying such plasmids and expressing both chains of immunoglobulins. Plasmids pVLentry-Hyg10 and pVHentry-Cm5 are designed for assembly of expression-competent sequences for light and heavy chains of IgG, respectively, meet all of these requirements (FIG. 1). Specifically, both of these plasmids possess two recognition sites for restriction endonuclease Esp3I per plasmid and these sites flank the sequence encoding protein 10b of bacteriophage T7. These two features ensure that practically 100% of colonies growing after cloning experiments utilizing vectors pVLentry-Hyg10 and pVHentry-Cm5 carry inserts of interest in a pre-determined orientation.

Restriction endonuclease Esp3I cuts DNA outside of its recognition sequence and generates four nucleotide-long cohesive 5′-overhanging ends. As depicted inFIG. 1, each Esp3I cleavage site in plasmids pVLentry-Hyg10 and pVHentry-Cm5 is unique. Therefore, fragments generated as a result of treatment of these plasmids with Esp3I and removal of the protein 10b-encoding sequence are not able to form a viable circular DNA unless the reaction is supplemented with a DNA fragment carrying appropriate sticky ends. As demonstrated inFIG. 2, the insertion of such a DNA fragment will occur only in one orientation, thus eliminating the need for following analysis of recombinant clones. The sequence encoding protein 10b of bacteriophage T7 functions as a safeguard, preventing re-assembly of the original vector.

In our vectors, its expression is controlled by the lactose promoter. Expression of this sequence is lethal to F plasmid-containingE. coli(17). Therefore, while our vectors are maintained in F-negative cells, cloning experiments require strains carrying F factor and, after transformation, cells are grown in the presence of IPTG and the corresponding antibiotic (ampicillin in the case of plasmid pVLentry-Hyg10 and chloramphenicol in the case of plasmid pVHentry-Cm5). Under these conditions, only cells carrying plasmids in which the protein 10b-encoding fragment has been substituted with a new insert survive.

Another important element of our vectors is a strong promoter that can direct transcription of the inserted sequence in mammalian cells. In vectors pVLentry-Hyg10 and pVHentry-Cm5, this role is served by the sequence from cytomegalovirus (CMV). However, we also designed plasmids in which a sequence from Rouse Sarcoma virus is used for this purpose. Plasmids pVLentry-Hyg10 and pVHentry-Cm5 are designed in such a way that transcripts initiated from the CMV promoter incorporate not only a sequence lying immediately downstream of the promoter, but also an Internal Ribosome Entry Site (IRES) and sequence for antibiotic resistance. In the case of plasmid pVLentry-Hyg10, this is resistance to Hygromycin B and, in the case of plasmid pVHentry-Cm5, this sequence confers resistance to G418. Presence of IRES makes synthesis of antibiotic-inactivating protein proportional to synthesis of protein encoded by the preceding portion of the transcript (immunoglobulin chain in the derivatives of these plasmids). This feature is not absolutely necessary for selection of stable transfectants (in some of our plasmids it is not present), however, it makes further maintenance of selected clones easier and opens opportunities for their further improvement.

In addition, design of our vectors allows simple combination of sequences encoding light and heavy chains of IgG in the same plasmid, which, in turn, ensures equal amounts of IgG chain-encoding sequences to be introduced into the cell during transfection. I-SceI recognition sites are one of elements enabling such combination.

I-SceI is a site-specific homing endonuclease that recognizes an 18 nucleotide-long sequence and generates DNAs with cohesive ends that can be used for cloning. Due to the length of the target sequence, its occurrence in the sequence encoding a variable domain of Ig is practically impossible. Therefore, using this enzyme enabled transfer of entire IgG-encoding sequences from one plasmid into another without destroying the integrity of these sequences. Nonsymmetrical cohesive ends generated by the I-SceI 1 ensure that, in all generated plasmids, relative orientation of IgG-encoding sequences is the same. This feature allows further improvement of the reproducibility of IgG production experiments. As shown inFIG. 1, plasmids pVLentry-Hyg10 and pVHentry-Cm5 possess two I-SceI sites each. However, in plasmid pVLentry-Hyg10, I-SceI sites flank the Ig-encoding cassette, while in plasmid pVHentry-Cm5, both I-SceI sites are located on one side of the Ig-encoding cassette and flank the gene of the alpha peptide of beta-galactosidase (lacZ′).

In addition to differences in location of I-SceI sites, both plasmids possess different antibiotic-resistance markers. Both of these plasmids use the same origin of replication for propagation inE. colicells and therefore are not be able to coexist in the same cell. All of these features allow us to speed up the process of assembly and identification of the plasmid carrying both L- and H-chain encoding sequences. Indeed, a simple treatment of the mixture of L- and H-chain encoding plasmids with I-SceI and ligase generates the required hybrid plasmid. Similarly to one of its parents, this plasmid inherits the chloramphenicol-resistance gene, while, unlike this parent, it will not be able to produce the alpha-peptide of beta-galactosidase. As a result, only cells carrying the required plasmid and not the three others present in the mixture are able to form white colonies on the media supplemented with chloramphenicol, X-Gal and isopropyl-β-D-thiogalactopyranoside (IPTG).

Also disclosed are four derivatives of plasmid pVHentry-Cm5. These derivatives have all elements described above. However, instead of the sequence encoding the constant part of IgG heavy chain alone, all these plasmids contain sequences that encode fusions of the same part of IgG heavy chain with different polypeptides. One of them encodes a fusion with green fluorescent protein (GFP), the second—a fusion with luciferase fromMetridia longa(MLuc) (18, 19), the third—a fusion with His-tag and a peptide that can be biotinylated by biotin ligase, and the fourth—a fusion with a polypeptide that specifically binds cellulose (20, 21).

Isolation of Sequences Encoding Variable Domains of Light and Heavy Chains of IgG

A single individual who was vaccinated with pentavalentbotulinumtoxoid vaccine six years prior received several boosts and served as a donor of blood cells. These cells were subject to fractionation on Ficoll gradient, enrichment on BD IMag™ Anti-human CD19 Particles-DM, and, finally, cell sorting. As a marker for cells producing anti-BoNT/A, we used a fusion between Green Fluorescent Protein and the receptor-recognizing domain of BoNT/A (gfpBoNT/A-CH5). This protein was constructed in our lab and, prior to use in cell sorting experiments, was tested for the ability to recognize specific receptors present in neuroblastoma cells (FIG. 3).

Cells simultaneously binding APC-Mouse-anti-human CD19 and gfpBoNT/A-CH5 were sorted into wells of a 96-well plate, one cell per well.

Isolated cells were used as a source of sequences encoding VH- and VL-regions. We have developed a procedure for rtPCR of these sequences that includes three steps: 1) reverse transcription of mRNA released from the cell by perfringolysin 0, 2) simultaneous amplification of cDNAs encoding VH- and VL-regions in the same tube by PCR and 3) re-amplification of sequences encoding each region in its own tube. Each step has its own set of primers. The whole procedure takes less than 8 hours. The number of cells that can be processed during this time is mostly limited by the capacity of the available thermo-cycler. Primers were designed based on the analysis of available human Ig-encoding sequences known in the art (8, 22). Primers used during each step are summarized in Table 1. Primers used in the re-amplification step were designed to introduce unique sequences, which can be converted into four-nucleotide-long cohesive ends compatible with ends generated by Esp3I restriction endonuclease in the corresponding vectors (see previous section), into the ends of amplified fragments. The conversion occurs as a result of treatment of purified DNA fragments by DNA polymerase T4 in the presence of dCTP as demonstrated inFIG. 2. The lack of restriction endonucleases at this stage guarantees that none of the sequences is lost due to the presence of sites for corresponding restriction endonucleases in some of them.

In the end, only 24% of originally sorted cells produced sequences for both VH- and VL-regions. This may sound like a relatively low success rate. However, given the potential of collecting hundreds of cells and the ability to process them in just few days, this allows the accumulation of tens of pairs of sequences for further antibody assembly. In the future, we expect to increase this rate by including anti-CD27 or anti-B220 monoclonal antibodies in the cell sorting protocol and thus increase the number of those among selected cells that produce antibodies versus those that may just absorb them.

Sequencing of 11 pairs of isolated DNA fragments revealed that practically all pairs were unique. Even when two pairs had one identical chain, the second chains were different (Sequences of variable domains of light and heavy chains are listed in Appendix 2 and 3).

Introduction of Designed IgG-Encoding Constructs into Mammalian Cells and Evaluation of Conditions for Efficient IgG Production

Eight pairs of isolated sequences were incorporated into the previously-described vectors and the resulting plasmids were introduced into CHO and HEK cells. ELISA registered accumulation of human antibodies in media of both of these cultures. In isolated stable cell lines, the level of production varied but did not exceed 1-2 μg/ml (the level of production was determined on the basis of the amount of anti-BoNT/A purified from 100 ml of culture media—will be described below). In our experience, HEK cells proved to be more robust and capable of producing more antibodies from the same volume of media. Also, these cells were easier to adapt to grow and produce IgGs in the serum-free media. This is why, in most of our later analyses, we preferred to use HEK cells.

To select clones with higher production, we decided to use correlation between translations of sequences encoding light and heavy chains of IgGs and those encoding antibiotic-inactivating proteins, built into our system and discussed earlier. Specifically, by gradually increasing amounts of antibiotics in the culture media, we were able to select cell lines whose resistance to antibiotics is 3-4 times higher than resistance of originally selected cultures. As demonstrated inFIG. 4, ELISA revealed that cells with increased resistance to antibiotics did not produce substantially more immunoglobulins than cells possessing a lower level of resistance to these antibiotics.

This data suggest that the bottleneck of production lies somewhere at the post-translational level. The conventional way for identifying cells with increased production of IgGs is a limiting dilution cloning. The low throughput nature of this method significantly limits the number of clones that can feasibly be screened. We tested whether fluorescence activated cell sorting (FACS) can be used to increase throughput. As a marker for IgG-producing cells, we used previously mentioned gfpBoNT/A-CH5. Cells were released from the solid support via treatment with trypsin and washed two times with fresh RPMI media to remove trypsin. Then, cells were incubated in RPMI media for 1 hour, co-incubated with gfpBoNT/A-CH5 for 10 minutes and subject to FACS. Out of the 1% of cells with the highest fluorescence intensity, corresponding to the highest antibody production rates, single cells were sorted directly into 96-well plates at one cell per well. One plate was assembled per each IgG-producing cell line. Table 2 demonstrates that we were able to find clones with increased production of IgG-luciferase hybrids for five cell lines out of seven used in the experiment. These results clearly demonstrate the potential of FACS for further development of cell lines producing high quantities of IgGs.

TABLE 2Production of IgG-MLuc by original cultures and individualclones selected from these culturesOriginal cultureLuminescenceCloneLuminescenceHEK-1HL-MLuc657,1481E71,641,522HEK-7HL-MLuc1,387,9807B88,013,339HEK-8HL-MLuc981,7028E83,783,486HEK-9HL-MLuc1,991,5129F62,778,794HEK-14HL-MLuc951,13214G11721,576HEK-15HL-MLuc104,46615F2594,677HEK-41HL-MLuc3,274,11941C93,163,750

Production of the Chimera IgGs and their Characterization.

As result of the reasons mentioned in the previous section, most of the IgG constructs were purified from culture media of HEK cells. Our analysis of accumulation of luciferase activity in the culture media of two cell lines encoding IgG-MLuc fusions revealed that the accumulation in both continued for seven days. Therefore, all HEK cultures were grown for seven days in the same media, which was then passed through a column containing the hybrid between staphylococcal protein A and streptococcal protein G. In the case of CHO cells, the media was collected after three days. Elution of absorbed IgGs was achieved by a buffer change to 0.1 M glycine HCl (pH 2.3). Immediately after elution, the pH of collected fractions was increased by addition of 1 M Tris-Base. Then, fractions were subjected to buffer exchange and concentrated by ultrafiltration.

In addition to IgGs alone, we purified fusions of these IgGs with luciferase, GFP, and His-tag connected to the peptide that serves as a target for biotin ligase (BirA). Analysis confirmed the presence of polypeptides with expected molecular weights and recognized by anti-human antibodies in isolated fractions (FIG. 5).

Fractions with IgG-MLuc fusions produced light in the presence of luciferase's substrate-coelenterazine. The IgG-GFP fusion emitted the green light characteristic of GFP upon illumination with UV light. Finally, the IgG fusion with His-tag and BirA substrate interacted with Ni-column and, after treatment with BirA in the presence of biotin and ATP, was recognized by streptavidin-alkaline phosphatase substrate (data not presented).

ELISA revealed that out of eight different IgGs that we purified, all eight recognize the receptor-recognizing domain of BoNT/A (FIG. 6). This data suggests that practically all isolated cells from which we were able to recover IgG-encoding sequences produced BoNT/A-specific antibodies.

IgGs were recognized by hybrid proteins composed of staphylococcal protein A, streptococcal protein G andMetridia longaluciferase (spAG-MLuc and spAG-ΔN-MLuc) and developed in our lab (sequences of plasmids encoding these proteins are presented in Appendix 4). These hybrids allowed quantitative monitoring of IgG present in wells of 96-well plate. Hybrid spAG-MLuc possessed luciferase activity only when it was purified from culture media of mammalian cells. Hybrid spAG-ΔN-MLuc possesses luciferase activity irrespective to where it was expressed,E. colior mammalian cells.

Examples 1-3 demonstrate 1) the number of peripheral blood cells encoding specific IgGs in blood and the efficiency of cell sorting protocols used are sufficient to produce hundreds of cells that can serve as a source of Ig-encoding sequences; 2) the methods disclosed herein permit reliable isolation of cDNA encoding variable domains of both Ig-chains from ⅕ of all isolated individual lymphocytes; 3) practically all isolated cDNA pairs encode IgG specific to the antigen used in the cell sorting procedure; 4) the expression vectors described herein are suitable for high throughput assembly of plasmids encoding both full size human IgGs, as well as their derivatives carrying polypeptides that allow monitoring or/and specific binding of these IgGs to other molecules; 5) the vectors allow efficient selection of cells producing both IgG chains; and 6) FACS can be used as an efficient tool allowing selection of clones producing increased quantities of IgGs and their derivatives.

Accordingly, the compositions and methods described herein are useful in methods comprising one or more of these aspects.

Construction and Expression of Libraries of Anti-BotulinumChimeras that Recognize Regions of BoNT/a

This example demonstrates the construction and use of libraries of anti-botulinumchimeras that recognize regions of BoNT/A.

First, we will use conventional methods of gene engineering to create fusions of corresponding domains with GFP. Similar to previously-mentioned gfpBoNT/A-CH5, these fusions will be used as markers for lymphocytes producing antibodies specific for catalytic and transport domains of BoNT/A. As a source of lymphocytes, we will use white blood cells from the blood of an immunized individual that were generated and tested previously, and preserved under liquid nitrogen. It has been demonstrated that such cells can be used as a source of immunoglobulin-encoding sequences (25). These cells will be subjected to enrichment on BD IMag™ Anti-human CD19 Particles-DM and then sorted into wells of a 96-well plate, one cell per well. Prior to FACS, cells will be labeled with APC Mouse Anti-Human CD19 (BD Biosciences) and the corresponding GFP-BoNT/A fusion. To increase the level of discrimination of IgG-producing cells from those that do not produce, but instead absorb them from serum, we will include an additional marker—memory B cell marker. Bleesing and Fleisher reported that human B cells expose either B220 or CD27 on their surface [30]. Therefore, as the third component of the cell labeling mixture, we will use anti-CD27 (Ancell Co.) and/or anti-B220 (Beckman Coulter) monoclonal antibodies, each conjugated to R-Phycoerythrin.

Isolated cells will be used as a source of sequences encoding VH- and VL-regions. Isolation and further handling of these sequences will be done according to protocols described above. At this stage, the goal will be to isolate 10-20 VH- and VL-encoding pairs that have unique sequences per each BoNT/A domain.

Unique VH- and VL-encoding pairs will be used to assemble and produce human/human IgG chimeras as described above.

Identification of IgGs and their Combinations that can Neutralize Toxic Activity of BoNT/A

This Example demonstrates the identification of chimeric IgG antibodies with the capacity to neutralize toxicity of BoNT/A using phage display.

Choosing VH- and VL-encoding pairs with unique sequences does not guarantee that they will recognize different epitopes. Therefore, prior to conducting expensive toxin neutralizing experiments, we will sort developed IgGs according to their epitope specificities. For this, we will use phage display known in the art. This technology involves a library of random peptides. Sequences of these peptides are incorporated in the region of the phage genome that encodes the capsid protein. As a result, each phage particle in the library encodes and exposes on its surface only one type of peptide. We previously demonstrated that incubation of such a library with immobilized polyclonal antibodies raised against BoNT/A allows isolation of phage particles that encode peptides mimicking BoNT/A epitopes (mimetics).

We will use a similar approach to sort developed IgGs according to their epitope specificities. Specifically, each developed IgG will be purified and immobilized on a solid support. Then, each immobilized IgG will be co-incubated with the phage display library MD-12™ (Alpha Universe, LLC). Phages that do not bind to IgG will be removed by washing and those bound to IgG will be released and grown on appropriate host cells. Following this amplification, phages will be subjected to two additional cycles of the above-described screening procedure. According to our previous experience, practically all phages released after the third cycle will possess affinity to the IgG used in selection. To ensure that selected phages carry mimetics of BoNT/A, we have to prevent isolation of phages that interact with IgG parts other than the antigen-binding region. In order to do this, phages will be subject to depletion with human naïve serum every time prior to incubation with immobilized developed IgG. After mixing with phages, components of human naïve serum, as well as phage particles bound to them, will be removed by addition of magnetic beads with immobilized staphylococcal protein A-streptococcal protein G hybrid to the mixture.

Individual phages carrying BoNT/A mimetics will be used for characterization of developed IgGs. Specifically, each IgG will be immobilized on wells of a 96-well plate and each immobilized IgG will be incubated with all chosen mimetic-exposing phages. Wells with bound phages will be identified using M13 phage-specific antibodies conjugated with horse radish peroxidase (GE Healthcare) and 1-Step™ Slow TMB-ELISA (PIERCE). IgGs interacting with the same phage will be considered as recognizing the same epitope.

In addition to classification of developed IgGs according to their epitope (actually, mimetic) specificity, we will characterize these IgGs according to the nature of recognized epitopes (linear or structural). In these experiments, we will compare interaction of developed IgGs with corresponding recombinant domains subjected or not subjected to denaturing treatment. For this, corresponding BoNT/A fragments will be subjected to native or SDS polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane and probed with each chosen IgG separately. Then, filters will be treated with biotinylated anti-human IgGs, followed by treatment with streptavidin-horse radish conjugate and Metal Enhanced DAB Substrate Kit (Pierce, Inc.). IgGs recognizing both forms of BoNT/A fragment will be considered as recognizing linear epitopes. Those that recognize only BoNT/A fragments not subjected to denaturing conditions will be considered as recognizing structural epitopes.

The information about the nature of the recognized epitope will not only be used to verify epitope-based grouping of IgGs, but also to gain information about locations of corresponding epitopes on the BoNT/A molecule. Specifically, our previous experience suggests that, in the case of mimetics of linear epitopes, some similarities between sequences of these mimetics and the BoNT/A sequence can be observed. Such similarities may be used as indicators of the location of the corresponding epitope in the structure of the molecule.

After developed IgGs are classified and grouped, representatives from each group will be tested for the ability to neutralize BoNT/A.

It has been demonstrated that even when individual monoclonal antibodies do not have substantial protective activity, their combination may have such activity (24). This is why the analysis will include testing of the BoNT/A-neutralization potential of each chosen IgG separately and, then, testing of such potential for selected groups of IgGs.

The goal of this analysis will be to identify IgGs or their combinations that will be able to protect mice from at least 1000 minimal doses that are lethal to a fifty percentage of mouse (MLD50) of BoNT/A. In addition, the aim will be to determine which among three regions of the BoNT/A molecule (catalytic, transport, or receptor-recognizing) contains the highest number of protective epitopes. This information will be instrumental for development of antibodies capable of neutralizing other serotypes of BoNTs.

Development of Human/Human IgG Chimeras Capable of Neutralizing BoNT/B

This Example demonstrates the development of human/human IgG chimeras capable of neutralizing BoNT/B.

Previously, we demonstrated that different serotypes of BoNTs have similar epitopes and information about locations of epitopes in one serotype can be used to predict locations of epitopes in other serotypes (26). We will use this phenomenon to speed up the process of development of IgGs capable of neutralizing BoNT serotype B. Specifically, instead of developing IgGs to the whole molecule of BoNT/B, we will focus on just one region. This region will be the same one as that revealed in BoNT/A as possessing the most potent protective epitopes. We will create a fusion between GFP and a fragment of BoNT/B after the targeted region of BoNT/B is determined. This fusion will be used to isolate corresponding lymphocytes from the same cryopreserved fractions of blood cells mentioned earlier. FACS and following isolation of cDNAs, their PCR, cloning, expression of assembled sequences, purification of IgGs, and analysis of their protective properties will be done the same way as described in the previous two sections.

As in case with BoNT/A, our goal will be to identify IgGs or their combinations that will ensure protection of mice from at least 1000 MLD50.

Optimization of Protocols for Production of Chosen Chimeras.

The ability to efficiently produce developed protective IgGs is a key element for the system to become a commercially viable. Earlier analysis of different monoclonal antibody-producing cell lines conducted by O'Callaghan and coauthors revealed that each cell line had its own bottleneck, limiting production of antibodies (27). This research supports the approach for selection of high producers from population of cells already producing IgG. This approach has been successfully used by many groups including ourselves. However, such selection often requires multiple cycles and is very lengthy. Development of a strain with bottlenecks that are widened or even removed will substantially increase the potential for high throughput development of cells producing high quantities of IgGs. Recent reports of successful increase of antibody production via introduction of specific DNA sequences into the cells suggest the possibility of such an approach (28-30).

To create a cell line originally capable of producing increased quantities of IgGs, we will produce IgG derivatives carrying different polypeptides on the C-termini of heavy chains. Specifically, we will engineer a plasmid encoding one of the anti-BoNT/A IgGs fused with the trans-membrane domain of platelet derived growth factor receptor (31). This plasmid will allow generation of transiently transfected cells expressing IgG anchored in the cell membrane. Such cells will be stained with gfpBoNT/A-CH5 and subjected to FACS. Individual cells carrying the highest levels of fluorescent label will be sorted into wells of a 96-well plate and allowed to grow. We anticipate that the majority of such cells will lose IgG-encoding plasmids. As a result, such cells will stop producing the corresponding IgG derivative and antibiotic-inactivating enzymes encoded by the plasmid. Cell lines grown from such cells will be transfected again. This time, we will use the plasmid encoding IgG-luciferase hybrid formed by different VH- and VL-pair that was used in the previous transfection. Parental cell lines for those transient transfectants whose culture media contains the highest amounts of luciferase will be tested further for the ability to produce high quantities of other types of IgG-luciferase fusions. Eventually, we expect to be able to isolate a cell line that will produce increased quantities if IgGs irrespective of sequences of their VH- and VL-regions.

To increase the success rate of the above-described selection, we will use a cell line whose diversity will be increased by chemical mutagenesis. Further, to eliminate difficulties associated with sorting originally adherent cells, we will use FREESTYLE™ CHO-S® cells (Invitrogen, Inc.). This cell line has been adapted to grow in suspension in serum-free media. The latter feature will beneficial for future production of antibodies.

Even with a developed host cell line capable of increased production of IgGs, we do not exclude the need for additional selection of super-producers among created IgG-producing cells. Traditionally, such selection is done by Limiting dilution cloning, which is a very labor-intensive process. We will use FACS protocols for the isolation of cells that bind the highest amounts of the label after a very short exposure to it from the population, followed by isolation of cells that lose this label faster than others.

As a result of these activities, we will not only generate cell lines producing high quantities of chosen IgGs, but will also determine the best way to efficiently develop new IgG-producing cell lines.

REFERENCES

APPENDIX 2Sequences of cloned light chains (SEQ ID NOs: 59-75, in order of appearance).

APPENDIX 3Alignment of sequences of cloned variable domains of heavy chains(SEQ ID NOs: 76-87,in order of appearance).