# Patent

## Title
Methods and systems for multi-antibody therapies

## Abstract

The present invention relates to methods and systems for administering antibody therapeutic agents. The methods include administering one or more (e.g., two or three) binding agents, wherein each of the binding agents has a binding region that is specific to a portion of a disease agent and one or more copies of a tag. The binding agents can be specific to one or more portions of the same or different disease agents. The tag is the same for each of the binding agents. The methods include administering an anti-tag antibody, wherein the anti-tag antibody has an anti-tag region that is specific to the tag, and can have an immunoglobulin (e.g., IgA, IgD, IgE, IgG, and IgM.). Disease agents include bacterial proteins, viral proteins, cancer cells, and proteins or toxins produced therefrom. In particular, the present invention includes methods and systems for binding agents that are specific to neurotoxins that cause botulism.

## Background
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 60/890,626, filed Feb. 20, 2007.


GOVERNMENT SUPPORT
This invention was made with government support under grant AI030050 awarded by the National Institutes of Health. The government has certain rights in the invention.


The entire teachings of the above application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
The production of antibodies and their storage is often a costly and lengthy process. In fact, development of a single antibody therapeutic agent can take years. Yet the use of multiple, different therapeutic antibodies are often necessary or desirable for the effective treatment of patients following a disease, outbreak or a bio-terrorist assault. The threat of a pandemic attack is real, and efforts to stockpile agents to combat such an outbreak have been attempted. However, producing and stockpiling enough antibody to protect large populations is challenging. The shelf life of antibodies is often relatively short, and so antibodies have to be periodically replaced. As a result, developing and producing multiple antibodies that can bind to different targets (e.g. microbial and viral pathogens, toxins, cancer cells) for improved therapeutic effect is often a difficult task because it involves storing multiple antibodies for each pathogen or toxin.
Hence, a need exists for a cost effective and efficient way to provide antibody treatments to a large quantity of people. A further need exists for antibody therapeutics that are easier to develop and produce, and have a longer shelf life. Yet, a further need exists for antibody therapeutics that bind to multiple targets on the same disease agent, as well as different disease agents.
SUMMARY OF THE INVENTION
The methods of the present invention include methods for administering antibody therapeutic agents to treat one or more disease agents. The steps involve administering at least one binding agent, wherein the binding agent has a binding region and one or more copies of a tag; and administering an anti-tag antibody (e.g., an anti-tag antibody), wherein the anti-tag antibody has an anti-tag region that is specific to the tag (e.g., to the one or more tags). The binding region of the binding agent is specific to a portion of the disease agent. The binding agent binds to a portion of the disease agent (e.g., the target) and the anti-tag antibody binds to the tag of the binding agent. Accordingly, the anti-tag antibody, which is directed to the disease agent by the binding agent, provides the effector activity that leads to a therapeutic effect. In fact, anti-tag antibody further embodies a polyclonal, antibody mixture, or an immunoglobulin such as IgA, IgD, IgE, IgG, or IgM. An embodiment of the methods includes administering two or more binding agents, each having a binding region that binds to different portions of the disease agent, but each have the same tag or multiple copies of the same tag. The binding region of the binding agent can be an antibody fragment, a microprotein, peptide, a synthetic molecule, or an aptamer. The disease agent, in an embodiment, can be a virus, bacteria, cancer cell, parasite, or a molecule, protein or toxin produced therefrom. The tag can be an antibody epitope, including a polypeptide, sugar or DNA molecule. In the case of a polypeptide, the tag encompasses the epitope and generally includes between about 8 to about 15 amino acids (e.g., having an amino acid sequence of SEQ ID NO: 15).
The methods of the present invention further include methods of treating an individual having a viral infection, parasitic infection, bacterial infection, cancer, or a molecule, protein or toxin produced therefrom (e.g., a pathogenic molecule, protein, or toxin). The method relates to administering at least one binding agent, as described herein, and administering an anti-tag antibody, also as described herein. The administration of the binding agent and the anti-tag antibody can occur at the same time or at different times (e.g., sequentially). The methods include reducing one or more symptoms associated with the viral infection, parasitic infection, bacterial infection, cancer or protein or toxin produced therefrom.
The present invention further embodies an antibody therapeutic system or kit that has at least one binding agent, and an anti-tag antibody, described herein.
Lastly, the present invention pertains to isolated polypeptide and nucleic acid molecules relating to the binding agents specific to a botulism neurotoxin disease agent. Specifically, the present invention includes binding agents with amino acid sequence encoded by a nucleic acid molecule having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or combination thereof an amino acid sequence encoded by a complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combination thereof an amino acid sequence encoded by a nucleic acid molecule that hybridizes to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combination thereof and an amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or combination thereof. Similarly, the present invention includes nucleic acid molecules having one of the following sequences: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combination thereof a complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combination thereof that hybridizes to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or combination thereof and that encodes SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, or combination thereof. The present invention further includes binding agents having one or more copies of a tag with an amino acid sequence of SEQ ID NO: 15. Vectors, plasmids, or host cells having the nucleic acid molecules or that express the amino acid molecules of the present invention are further included herein. The present invention includes compositions that have the binding agents and an anti-tag antibody of the present invention and a physiologically acceptable carrier.
The present invention has a number of advantages. The systems and methods of the present invention allow for a more efficient system of treating disease agents and for storing quantities of antibodies for a large number of people. Binding agents of the present invention are compositions that can be stored for a longer period of time, as compared to antibodies with heavy and light chains. Binding agents are also generally easier to make than antibodies. The invention involves multiple (e.g., two or more) binding agents for use against multiple targets of the same disease, which often results in greater effectiveness. The binding agents in the system have the same tag, and an anti-tag antibody binds to all of the binding agents, even those that are specific to different portions of the disease agent. To make and store multiple antibodies for a number of targets is costly and inefficient, in comparison. Hence, a number of binding agents can more easily be stored, e.g., to protect against a potential biological threat, along with a single anti-tag antibody.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-E show the nucleic acid sequences of scFv#2 (SEQ ID NO: 1), scFv#3 (SEQ ID NO: 3), scFv#7 (SEQ ID NO: 5), scFv#8 (SEQ ID NO: 7), scFv#21 (SEQ ID NO: 9), scFv#E (SEQ ID NO: 11), and amino acid sequences of scFv#2 (SEQ ID NO: 2), scFv#3 (SEQ ID NO: 4), scFv#7 (SEQ ID NO: 6), scFv#8 (SEQ ID NO: 8), scFv#21 (SEQ ID NO: 10), scFv#E (SEQ ID NO: 12).
FIG. 2 shows the nucleic acid sequence of scFv#7-2E (SEQ ID NO: 13) and amino acid sequence of scFv#7-2E (SEQ ID NO: 14).



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods and systems that include one or more binding agents having a binding region and an epitopic tag; and an anti-tag antibody that binds the tag. The anti-tag antibody has a tag-specific binding region, also referred to as “anti-tag region.” The methods of the present invention use, in an embodiment, two or more binding agents that are specific for different targets of the same disease agent, but have the same epitopic tag. Since the anti-tag antibody is specific for the tag, it can bind to each of the different binding agents. Additionally, in certain instances in which a single binding agent has more than one copy of the same tag, the overall efficacy of the antibody therapy increases.
Accordingly, in vivo, the binding agents bind to the disease agent (e.g., a polypeptide toxin produced by a bacterium). Using two or more binding agents has shown to be particularly effective in ridding the subject of the disease agent and improving symptoms caused by it. See Exemplification. The anti-tag antibody then binds to all of the binding agents, even binding agents that are specific for different portions of the disease agent. In the case in which the binding agents have multiple, identical tags, the anti-tag antibody has more available sites to which it can bind. As such, the anti-tag antibody allows for effector functions to occur (e.g., phagocytosis, neutralization, clearance of the disease agent), and in certain instances, an increase in the effector functions occurs when more than one copy of the tag is present on the binding agent. Binding agents are generally easier to make and have longer shelf-lives than antibodies. Rather than having multiple antibodies, which are more difficult to engineer and store, multiple binding agents along with a single anti-tag antibody can be used to treat individuals.
Binding Agent
The binding agent is a molecule that binds to a portion of a disease agent, and has a tag. The antibody therapeutic agents of the present invention allow the disease agent to be cleared, undergo phagocytosis, undergo neutralization, be inhibited, or otherwise mitigated by immune activity.
Binding agents include molecules such as antibody fragments (e.g., single chain antibodies, and nanobodies), microproteins (also referred to as cysteine knot proteins or knottins), darpins, anticalins, adnectins, peptide mimetic molecules, aptamers, synthetic molecules, and any molecule that binds to a disease agent and can elicit immune effector activity against the disease agent when the binding agent is bound by an anti-tag antibody. The binding agent, along with the anti-tag antibody, results in various effector functions such as phagocytosis and/or clearance, which is further described herein.
In certain embodiments, the binding agents can neutralize or inhibit the disease agent. In the case that the binding agents themselves are neutralizing, they can be advantageous in that they neutralize at the same time as triggering antibody mediated effector activity. In an example, a binding agent, referred to as scFvs (#2) in the Exemplification, is a neutralizing agent and it works well, as compared to similar neutralizing agents. However, in certain experiments performed in the exemplification, a neutralizing binding agent is not always necessary to protect mice from the toxin. Additionally, another factor is that the anti-tag antibody can increase the serum half-life of the binding agents.
Binding Agents that Include Antibodies Fragments, Microproteins and Other Molecules that Bind the Disease Agent
The term “antibody fragment” refers to portion of an immunoglobulin having specificity to the disease agent, or a molecule involved in the interaction or binding of the disease agent. The term, “antibody fragment”, is intended to encompass fragments from both polyclonal and monoclonal antibodies including transgenically produced antibodies, single-chain antibodies (scFvs), recombinant Fabs, and recombinant camelid heavy-chain-only antibodies (VHHs). VHHs are also referred to as nanobodies.
Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. The Nanobody technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide harboring the antigen-binding capacity of the original heavy-chain antibody. (Ablynx, Ghent, Belgium; http://www.ablynx.com/02_understanding.htm.)
Suitable methods of producing or isolating antibody fragments of the requisite specificity are known in the art and include for example, methods which select recombinant antibody from a library, by PCR.
Functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies, can also be produced. Functional fragments or portions of the foregoing antibodies include those which are reactive with the disease agent. For example, antibody fragments capable of binding to the disease agent or portion thereof, including, but not limited to scFvs, Fabs, VHHs, Fv, Fab, Fab′ and F(ab′)2 are encompassed by the invention. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab′)2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab′)2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. Accordingly, the present invention encompasses a polynucleic acid that encodes the binding agent described herein (e.g., a binding fragment with a tag).
Antagonist includes proteins or polypeptides that bind to the disease agent, inhibit function of the disease agent, and can form the binding region of the binding agent. Known antagonists, or those developed in the future, can be used with the present invention.
Binding agents include any molecule that binds to the disease agent including those that have scaffolds. Other examples of molecules include DARPins, Anticalins, and AdNectins. DARPins are derived from natural ankyrin repeat proteins and bind to proteins including e.g., human receptors, cytokines, kinases, human proteases, viruses and membrane proteins (Molecular Partners AG Zurich Switzerland). Anticalins are derived from lipocalins, and comprise a hypervariable loops supported by a conserved β-sheet framework, which acts as a binding agent. (Pieris AG, Germany). The scaffold for anticalins are lipocalins. AdNectins are derived from human fibronectin (e.g., the scaffold), and bind to targets of various medical conditions. (Adnexus, Waltham Mass.).
Binding Agent for Botulinum neurotoxin (BoNT) serotype A (BoNT/A)
In particular, the present invention relates to binding agents that are specific to the microbial neurotoxin that causes botulism. There are at least seven different botulinum toxin serotypes (A to G), sometimes with various isotypes, and many of these different toxins can cause human disease. As described in the Exemplification section, several binding agents specific to one of the botulinum neurotoxins, serotype A (BoNT/A) were made. Hence, the methods and systems of the present invention include binding agents that have binding regions specific to one or more target areas of one or more neurotoxins involved with botulism. Sequences engineered to bind to this neurotoxin are shown in FIG. 1. Specifically, the present invention relates to binding agents having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or combination thereof. Similarly, the present invention also includes binding agents that are encoded by a nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combination thereof. A tag was engineered and has amino acid sequence of SEQ ID NO: 15, and is genetically fused to the carboxyl end of these binding agents.
The present invention embodies multiple binding agents, each to target different areas of one or more disease agents. In an embodiment, two or three binding agents specific to different targets of a disease agent can be used. In a case in which a number of disease agents can be involved in causing a disease or condition, such as botulism, multiple disease agents can be targeted. In the case of botulism, since any one of at least seven neurotoxin serotypes could be responsible, a pool of binding agents can be prepared that contain binding agents for all of the known serotypes that cause human disease. Botulism is often caused by exposure to a single BoNT serotype, but it is generally difficult to quickly determine which serotype is the cause. Thus, the standard of care in treating botulism includes administration of a number of antibodies to protect against most if not all of the serotypes that cause the disease in human. Hence, to protect against such a disease, an embodiment includes having or stockpiling binding agents that bind to several or preferably all of the serotypes that cause botulism. Certain studies have shown that effective antibody neutralization of a single BoNT serotype requires three different anti-BoNT monoclonal antibodies. Thus, to protect patients against all seven BoNT serotypes would require a pool of 21 different mAbs, a very formidable task. In contrast, preparation of a pool of small binding agents, each with a common tag, together with a single anti-tag mAb is a much more achievable task.
Similarly, the targeted disease agents can be agents from different classes of pathogens. For example, a multi-target approach further includes binding agents that bind to viral disease agents, bacterial disease agents, parasite disease agents, cancer cells, proteins produced therefrom and any combination therefrom.
Tags of Binding Agents
The binding agent is modified, for example, by incorporation of or attachment (directly or indirectly (e.g., via a linker)) of one or more tags. A tag is a molecule or antibody epitope that is attached or genetically fused to the binding agent and to which the anti-tag antibody binds. Genetic fusion refers to a recombinant protein that is engineered to contain extra amino acid sequences that constitute the tag. Thus, the DNA encoding the tag is joined (in reading frame) with the DNA encoding the binding agent. An embodiment of the invention includes more than one binding agents, with the same tag. Hence, in a system, the anti-tag antibody binds to all of the binding agents via the tag.
The tag can be attached to a portion of the binding agent so long as the tag does not interfere with the agent's ability to bind to the disease agent. The tag, for example, can be a polypeptide, sugar, or DNA molecule.
In an embodiment, the tag is incorporated by genetic fusion at the carboxyl end of the binding agent. The tag, itself, can also be a polypeptide joined at the amino terminal end or within the binding agent as long as the tag does not affect binding of the binding agent to the target and the tag remains accessible to the anti-tag mAb. The tag itself does not interact or bind with the disease agent. Preferably, the tag is an uncommon or unique molecule or peptide in nature. In an aspect, the tag is a polypteptide that ranges from about 5 amino acids to about 20 amino acids, and preferably between about 8 and about 15. In the exemplification, the tag used consisted of the following 13 amino acids: GAPVPYPDPLEPR (SEQ ID NO: 15). Examples of such tags also include c-myc and haemagglutinin protein, biotin, avidin, hapten (e.g., a carbohydrate or nucleotide) and the like.
The tag can be incorporated into the binding agent using recombinant technology in which the DNA encodes the binding agent genetically fused with the tag. Specifically, the coding sequence for the tag can be cloned into an expression vector and transfected into cells for recombinant expression.
Once the tag is incorporated into the binding agent, the binding agent, like an antibody, can be evaluated for its ability and affinity to bind to the disease agent.
Optionally, a linker or spacer can be used to attach the binding region of the binding agent with the tag. A linker can be used to indirectly attach a tag to the binding region. In one embodiment of the invention, the binding agent includes the binding region, a linker and a tag. The spacer/linker can be any compound, now known or later developed, that can attach the binding region with the tag.
Inclusion of more than one copy of the tag on a binding agent, in certain aspects, has additional advantages, such as by increasing the number of anti-tag antibodies that can bind to the binding agent. This would be the case, for example, where increasing the number of antibody Fc effector domains bound to the pathogenic protein through the tagged binding agent increased the therapeutic efficacy. Such an increase in efficacy is demonstrated and described in the Exemplification.
Disease Agent
The disease agent to which the binding agent binds can be any disease-causing target including those to be inhibited, or whose activity can be altered (e.g., neutralized, reduced or ceased), or that can be recognized by immune effectors and lead to clearance, opsonization, killing, etc. The disease agent can be a portion of a pathogen or a molecule released or secreted by the pathogen (e.g. toxin). A pathogen is an agent that causes a disease or condition, and includes a virus, cancer cell, bacterium, parasite or pathogenic protein. The disease agents include pathogenic proteins that are derived from normal cells, such as prions. Proteins or other molecules that are disease agents can be either independent of the pathogen or associated with or produced by a pathogen.
A virus is a microscopic particle that can infect the cells of a biological organism and replicate themselves in the host cell. With respect to the present invention, viral antigens, usually proteins, are targeted by the binding agent. Binding agents can be made to bind to such molecules on the virus, using the processes described herein. Example of viruses include Influenza, Rhinovirus, Rubeola, Rubella, Herpes, Smallpox, Chickenpox, Human Papilloma, Rabies, and Human Immunodeficiency viruses.
A parasite is an organism which lives on or in a different organism. Parasites have or express molecules that can be used as a target by the binding agent. Types of parasites include endoparasites (e.g., parasites that live inside the body of the host) and ectoparasites (e.g., parasites that live on the outside of the host's body). Examples of parasites include protozoans (e.g., plasmodium, cryptosporidium, microsporidia, and isospora), ticks, lice and parasitic worms.
Molecules on cancer cells can also be targets of the binding agent. In one embodiment, the target is a protein on the cancer cell or proteins that are characteristic to the cells of the cancer in question. Examples of proteins associated with cancer cells include CD33 (i.e., to a cytotoxic agent expressed in most leukemic blast cells), and the HER2/neu receptor for breast cancer.
Bacteria that can also be a target for the present invention include any bacteria including gram negative and gram positive bacteria. Examples of pathogenic bacteria belong to the genuses such as Clostridium, Staphylococcus, Neisseria, Streptococcus, Moraxella, Listeria, Enterobacteriaceae, Escherichia coli, Corynebacterium, Klebsiella, Salmonella, Shigella, Proteus, Pseudomonas, Haemophilus, Bordetella, Legionella, Campylobacter, Helicobacter, and Bacteroides. Methods for ascertaining the target are known in the art and will depend on the type of molecule being inhibited. For example, in the case where a class or group of bacteria are to be inhibited, conserved regions of a bacteria can be targeted, and binding agents that bind to these targets can be made. In other cases, if a specific bacterium is to be inhibited, then a non-conserved region of the bacteria can be targeted with the binding agents. The binding of the agents can be measured using standard assays, such as ELISAs, western blots and radioimmunoassays.
Pathogenic molecules including polypeptides or toxins are also disease agents to which binding agents can target. Pathogenic proteins refers to proteins that can cause, directly or indirectly, a disease, or condition in an individual. Proteins or toxins produced by bacteria, virus, or cancer cells are often such examples. In the Exemplification section, binding agents were made to a toxin produced by a Clostridium bacterium. 
As shown in the Exemplification, using binding agents that target more than one area of the disease agent are shown more effective at protecting animals from the pathology of the disease agent, and symptoms caused by the disease agent are reduced or alleviated all together, as further described herein.
Anti-Tag Antibody
The anti-tag antibody is, in an embodiment, an antibody having an anti-tag region. The anti-tag region of the anti-tag antibody binds to the tag of each of the binding agents. Since the same tag is used for each of the binding agents, the anti-tag antibody essentially binds to the binding agents via the tag.
To generate an anti-tag antibody, processes known for making monoclonal antibodies, which are described herein, can be used. In the process of making monoclonal antibodies, for example, a mouse is injected with a disease agent along with an adjuvant. After a series of injections, the spleen of the mouse is removed, and the cells that make the antibody are fused with myeloma SP2/0 cells. The fused cells are grown and tested for their ability to make an antibody that binds to the disease agent, e.g., with ELISAs. To elicit an antibody that is specific to the tag, the tag, rather than the disease agent, is injected into the mouse during this process. Since the tags are generally small molecules (e.g. haptens) or peptides, they may need to be chemically coupled to a larger antigen for immunization or, if peptides, expressed as a genetic fusion to a larger protein, to make them more immunogenic. Humanized or chimeric antibodies can be made once the variable regions are determined.
Antibodies are already described herein, and can be used to carry out or facilitate effector functions. The anti-tag antibody further includes an immunoglobulin such as IgA, IgD, IgE, IgG, and IgM, including subtypes thereof. In addition to monoclonal antibodies, polyclonal antibodies specific to the tag can also be used with the present invention. Effector functions are generally carried out by the Fc portion of the immunoglobulin. Depending on the type of immunoglobulin chosen, the effector functions results in clearance of the disease agent (e.g., excretion, degradation, lysis or phagocytosis). Other molecules, now known or developed in the future, can be used as an anti-tag antibody so long as they bind to the tag and contain one or more of the antibody effector functions. For example, an anti-tag antibody may be an engineered protein consisting only of a domain that binds to the tag (e.g. an anti-tag scFv or VHH) fused to a minimal functional antibody Fc domain.
In mammals there are at least five types of antibody: IgA, IgD, IgE, IgG, and IgM, with 4 IgG and 2 IgA subtypes present in humans. These are classified according to differences in their heavy chain constant domains. Each immunoglobulin class differs in its biological properties. IgA can be found in areas containing mucus (e.g. in the gut, in the respiratory tract or in the urogenital tract) and prevents the colonization of mucosal areas by pathogens. IgD functions mainly as a disease agent receptor on B cells. IgE binds to allergens and triggers histamine release from mast cells and also provides protection against helminths (worms). IgG, in its four forms, provides the majority of antibody-based immunity against invading pathogens. IgM is expressed on the surface of B cells and also in a secreted form with very high affinity for eliminating pathogens in the early stages of B cell mediated immunity.
Some cells such as mast cells and phagocytes have specific receptors on their cell surface for binding antibodies. These are called Fc receptors and they interact with the Fc region of some antibodies (e.g. IgA, IgG, IgE). The engagement of a particular antibody with the Fc receptor on a particular cell will trigger the effector function of that cell. For example, phagocytes will phagocytose, and mast cells will degranulate. Effector functions generally result in destruction of the invading microbe. Hence, the type of immunoglobulin can be chosen depending on the type of effector function desired.
Methods and Systems of Administering Antibody Therapeutic Agents
The present invention includes methods of administering one or more binding agents and an anti-tag antibody, as described herein, to an individual. The binding agents having a binding region that is specific to the disease agent and one or more tags are administered. The binding agents bind to the target region on the disease agent. Administration of two or more binding agents, in an embodiment, increases the effectiveness of the antibody therapy, and better reduces the severity of one or more symptoms. Similarly, when the binding agents have more than one copy of the tag, an increase in efficacy occurs in certain embodiments. A single anti-tag antibody type binds to all binding agents with the tag. In the case in which the binding agents have multiple copies (e.g., two or more) of the same tag, the anti-tag antibody can bind to each copy of the tag on the binding agent. The phrase, “antibody therapeutic agents” or “antibody therapeutic preparation” refers to one or more compositions that include at least one binding agent and at least one anti-tag antibody, as described herein. The preparation can have additional elements including carriers, as described herein.
The administration of the binding agent and anti-tag antibody can occur simultaneously or sequentially in time. The binding agents can be administered before, after or at the same time as the anti-tag antibody, so long as they are administered close enough in time to have the desired effect (e.g., before the binding agents have been cleared by the body). Thus, the term “co-administration” is used herein to mean that the binding agents and the anti-tag antibody will be administered at times to achieve treatment of the disease, or reduction in the level of the pathogen (e.g., virus, bacteria, cancer cell, proteins associated therewith, or combination thereof) and/or symptoms associated with it. The methods of the present invention are not limited to the sequence in which the binding agents and anti-tag antibody are administered, so long as the compositions are administered close enough in time to produce the desired effect. In an embodiment, the binding agent and anti-tag antibody can be premixed and administered together. The binding agent and anti-tag antibody can also be co-administered with other medications or compositions normally administered when treating the disease agent.
The methods of the present invention include treating a bacterial disease, a parasitic infection, viral diseases, cancer, molecules, proteins or toxins associated therewith. This is accomplished by administering the binding agents and anti-tag antibodies described herein to the infected individual. Administration ameliorates or reduces the severity of one or more the symptoms of the disease or condition. The presence, absence or severity of symptoms can be measured using tests and diagnostic procedures known in the art. Similarly the presence, absence and/or level of the disease agent can be measured using methods known in the art. Symptoms or levels of the disease agent can be measured at one or more time points (e.g., before, during and after treatment, or any combination thereof) during the course of treatment to determine if the treatment is effective. A decrease or no change in the level of the disease agent, or severity of symptoms associated therewith indicates that treatment is working, and an increase in the level of the disease agent, or severity of symptoms indicates that treatment is not working. Symptoms and levels of disease agents are measured using methods known in the art.
For example, where toxin is the disease agent, five mice per treatment group are injected intravenously or intraperitoneally with a lethal dose of toxin (10 LD50, 10× the dose that is lethal to 50% of the mice) and the binding agents to be tested are co-administered. The mice are regularly monitored for symptoms and survival. Symptoms that are monitored include difficulty breathing, lethargy, mobility, appetite and responsiveness. Toxin protection is assessed as increased survival and reduction of symptoms. The steps of the present invention led to a decrease or alleviation of the symptoms, and increase in survival. See Exemplification Section.
The antibody therapeutic agents including one or more binding agents and an anti-tag antibody can be administered in one or more pharmaceutical carriers. The terms “pharmaceutically acceptable carrier” or a “carrier” refer to any generally acceptable excipient or drug delivery device that is relatively inert and non-toxic. The binding agents and anti-tag antibody can be administered with or without a carrier. Exemplary carriers include calcium carbonate, sucrose, dextrose, mannose, albumin, starch, cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, rice flour, magnesium stearate, and the like. Suitable formulations and additional carriers are described in Remington's Pharmaceutical Sciences, (17th Ed., Mack Pub. Co., Easton, Pa.), the teachings of which are incorporated herein by reference in their entirety. The binding agents and anti-tag antibody can be administered systemically or locally (e.g., by injection or diffusion).
Suitable carriers (e.g., pharmaceutical carriers) also include, but are not limited to sterile water, salt solutions (such as Ringer's solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. Such preparations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, e.g., enzyme inhibitors, to reduce metabolic degradation. A carrier (e.g., a pharmaceutically acceptable carrier) is preferred, but not necessary to administer one or more binding agents and an anti-tag antibody.
The binding agents and anti-tag antibody of the present invention can be administered intravenously, parenterally, orally, nasally, by inhalation, by implant, by injection, or by suppository. The composition can be administered in a single dose or in more than one dose over a period of time to confer the desired effect.
The actual effective amounts of compositions of the present invention can vary according to the binding agent being utilized, the particular composition formulated, the mode of administration and the age, weight and condition of the patient, for example. As used herein, an effective amount of the binding agents and anti-tag antibody is an amount which is capable of reducing one or more symptoms of the disease or conditions caused by the disease agent. Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol).
Systems or kits of the present invention include one or more binding agents having a binding region and one or more tags, and an anti-tag antibody having an anti-tag region (e.g., an anti-tag antibody), as described herein.
Polypeptides, Nucleic Acid Sequences, Vectors, Host Cells of the Binding Agents Engineered and Specific to a Botulism Neurotoxin
The present invention relates to isolated polypeptide molecules that have been engineered or isolated to act as binding agents. In particular, the present invention includes polypeptide molecules that contain the sequence of any one of the binding agents (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or combinations thereof). See FIG. 1. The present invention also pertains to polypeptide molecules that are encoded by nucleic acid sequences, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combinations thereof). A tag having the sequence set forth in SEQ ID NO:15 was used with these sequences.
As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full length proteins (i.e., disease agents), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide can comprise a portion of the binding agent, the entire binding agent, or it can contain additional sequences. The polypeptides of the binding agents of the present invention referred to herein as “isolated” are polypeptides that are separated away from other proteins and cellular material of their source of origin. The compositions and methods of the present invention also encompass variants of the above polypeptides and DNA molecules. A polypeptide “variant,” as used herein, is a polypeptide that differs from the recited polypeptide only in conservative substitutions and/or modifications, such that the ability of the binding agent to bind to the disease agent target is retained.
The present invention also encompasses proteins and polypeptides, variants thereof, or those having amino acid sequences analogous to the amino acid sequences of binding agents described herein. Such polypeptides are defined herein as analogs (e.g., homologues), or mutants or derivatives. “Analogous” or “homologous” amino acid sequences refer to amino acid sequences with sufficient identity of any one of the amino acid sequences of the present invention so as to possess the biological activity (e.g., the ability to bind to the disease agent target). For example, an analog polypeptide can be produced with “silent” changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of any one of the sequence, yet still possesses the function or biological activity of the polypeptide. In particular, the present invention relates to homologous polypeptide molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or combination thereof. Percent “identity” refers to the amount of identical nucleotides or amino acids between two nucleotides or amino acid sequences, respectfully. As used herein, “percent similarity” refers to the amount of similar or conservative amino acids between two amino acid sequences.
Homologous polypeptides can be determined using methods known to those of skill in the art. Initial homology searches can be performed at NCBI against the GenBank, EMBL and SwissProt databases using, for example, the BLAST network service. Altschuler, S. F., et al., J. Mol. Biol., 215:403 (1990), Altschuler, S. F., Nucleic Acids Res., 25:3389-3402 (1998). Computer analysis of nucleotide sequences can be performed using the MOTIFS and the FindPatterns subroutines of the Genetics Computing Group (GCG, version 8.0) software. Protein and/or nucleotide comparisons were performed according to Higgins and Sharp (Higgins, D. G. and Sharp, P. M., Gene, 73:237-244 (1988) e.g., using default parameters).
The present invention, in one embodiment, includes an isolated nucleic acid molecule having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combinations thereof. See FIG. 1. As used herein, the terms “DNA molecule” or “nucleic acid molecule” include both sense and anti-sense strands, cDNA, genomic DNA, recombinant DNA, RNA, and wholly or partially synthesized nucleic acid molecules. A nucleotide “variant” is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications can be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA 2:183, 1983). Nucleotide variants can be naturally occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences preferably exhibit at least about 70%, more preferably at least about 80% and most preferably at least about 90% homology to the recited sequence. Such variant nucleotide sequences will generally hybridize to the recited nucleotide sequence under stringent conditions. In one embodiment, “stringent conditions” refers to prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° Celsius, 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
The present invention also encompasses isolated nucleic acid sequences that encode the binding agents and in particular, those which encode a polypeptide molecule having an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or combinations thereof.
As used herein, an “isolated” nucleotide sequence is a sequence that is not flanked by nucleotide sequences which normally (e.g., in nature) flank the gene or nucleotide sequence (e.g., as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in a cDNA or RNA library). Thus, an isolated gene or nucleotide sequence can include a gene or nucleotide sequence which is synthesized chemically or by recombinant means. Nucleic acid constructs contained in a vector are included in the definition of “isolated” as used herein. Also, isolated nucleotide sequences include recombinant nucleic acid molecules and heterologous host cells, as well as partially or substantially or purified nucleic acid molecules in solution. The nucleic acid sequences of the binding agents of the present invention include homologous nucleic acid sequences. “Analogous” or “homologous” nucleic acid sequences refer to nucleic acid sequences with sufficient identity of any one of the nucleic acid sequences described herein, such that once encoded into polypeptides, they possess the biological activity of any one of the binding agents described herein. In particular, the present invention is directed to nucleic acid molecules having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or combinations thereof.
Also encompassed by the present invention are nucleic acid sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the polypeptides of the present invention, and which specifically hybridize with their DNA sequences under conditions of stringency known to those of skill in the art. As defined herein, substantially complementary means that the nucleic acid need not reflect the exact sequence of the sequences, but must be sufficiently similar in sequence to permit hybridization with nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the nucleic acid sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization therewith. Conditions for stringency are described in e.g., Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et al., Nature, 366:575 (1993); and further defined in conjunction with certain assays.
The invention also provides vectors, plasmids or viruses containing one or more of the nucleic acid molecules having the sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or combinations thereof). Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989).
A description of preferred embodiments of the invention follows.
EXEMPLIFICATION
The concept is to improve therapies that involve multiple monoclonal antibodies (mAbs) by using small recombinant peptide, protein or polynucleotide agents that have the same binding specificity as the mAbs. Each of the recombinant binding agents is produced containing the same epitopic tag. A single mAb that recognizes the epitopic tag is co-administered to patients with the binding agents. The different agents bind to the same targets as the multiple mAbs and the anti-tag mAb binds to these agents through the epitopic tag. This permits delivery of the same therapeutic effect that is achieved with multiple mAb therapy, but requires only a single mAb. If desired, mAbs of different isotypes, or polyclonal anti-tag antibodies, could be used therapeutically to deliver different immune effector activities.
A number of small recombinant protein agents were generated. They were called single-chain Fvs (scFvs) and recognize botulinum neurotoxin serotype A (BoNT/A). These scFvs are essentially recombinant proteins that represent the antigen combining region of an immunoglobulin. Several anti-BoNT/A scFvs were produced and purified at laboratory scale. Each of the scFvs contains the amino acid sequence (GAPVPYPDPLEPR—SEQ ID NO: 15) near the carboxyl terminus which is an epitopic tag referred to herein as “E-tag.” One of the scFvs (scFv#2) was shown to neutralize BoNT/A in a cell-based toxin assay (IC50 ̃7 nM). A second scFv (scFv#7) had little or no neutralization activity in the assay, but was found to bind to BoNT/A with high affinity (Kd ̃1 nM). The scFvs were tested for their ability to protect mice BoNT/A against intoxication following intravenous administration of the agents and toxin. The two scFvs were administered individually or together, and were given +/− a mouse anti-E-tag mAb by intravenous administration. Each mouse received 10 LD50s of BoNT/A, 5 mice per group. The results were as follows:







TABLE 1







Results from Experiment #1









Agents Administered
Survival
Comments












None
0%
Death in less than a day


20 ug scFv#2
0%
Death delayed about a day


20 ug scFv#7
0%
Death delayed less than a day


20 ug scFv#2 + 25 ug
100%
Symptoms severe


anti-E-tag mAb


20 ug scFv#7 + 25 ug
0%
Death delayed several days


anti-E-tag mAb


10 ug scFv#2 + 10 ug scFv#7 +
100%
No symptoms


25 ug anti-E-tag mAb









These results clearly show that a BoNT/A neutralizing scFv (scFv#2) does not significantly protect mice from the toxin unless it is accompanied by a mAb that recognizes an epitopic tag (E-tag) on the scFv. More importantly, combining this mAb with two scFvs, each with E-tag, dramatically improves the protective effect. In this case, the different scFv binding agents provide the additive effect and only the single anti E-tag mAb is needed.
A second study was performed using the same toxin challenge (10 LD50) and lower doses of the scFvs and the anti-E-tag mAb. In addition, two other non-neutralizing anti-BoNT/A scFvs (#3 and #21) were tested in combination with the neutralizing scFv#2. Whether the anti-E-tag mAb would function if administered at a different site and time than the toxin was also tested.







TABLE 2







Results from Experiment #2









Agents Administered
Survival
Comments












none
0%
Death in about a day


10 ug scFv#2
0%
Death delayed about




2 days


10 ug scFv#2 + 10 ug anti-E-tag mAb
100%
Symptoms moderate


(mAb administered intraperitoneally)


10 ug scFv#2 + 10 ug anti-E-tag mAb
100%
Symptoms mild


10 ug scFv#2 + 2 ug anti-E-tag mAb
100%
Symptoms mild


2 ug scFv#2 + 10 ug anti-E-tag mAb
100%
Symptoms moderate


5 ug scFv#2 + 3 ug scFv#7 +
100%
No symptoms


10 ug anti-E-tag mAb


1 ug scFv#2 + 1 ug scFv#7 +
100%
No symptoms


10 ug anti-E-tag mAb


5 ug scFv#2 + 4 ug scFv#3 +
100%
No symptoms


10 ug anti-E-tag mAb


5 ug scFv#2 + 3 ug scFv#21 +
100%
No symptoms


10 ug anti-E-tag mAb









These results entirely confirm those from the first experiment and extend them as follows. The mAb against the epitopic tag does not have to be pre-mixed with the scFv containing the epitopic tag to be effective (it can be administered at a different site and time). Combinations of two scFvs (each with E-tags) and the single anti-E-tag mAb, provide greater protection than with one scFv alone. This synergistic protective effect occurs using different scFvs and at significantly lower doses of the scFvs or mAb than used previously.
In the third experiment, we tested combinations of three and four scFvs with anti-tag mAb to protect against 100, 1000 and 10,000 LD50 doses of BoNT/A.







TABLE 3







Results from Experiment #3











Agents Administered





All received


BoNT/A
10 μg of anti-E-tag mAb
Survival
Comments













100 LD50
None
0%
Death in less than a





day


100 LD50
2 μg scFv#2 + scFv#3 +
100%
No symptoms



scFv#21


1000 LD50
None
0%
Death in less than a





day


1000 LD50
2 μg scFv#2 + scFv#3 +
100%
No symptoms



scFv#21


1000 LD50
2 μg scFv#2 + scFv#3 +
100%
No symptoms



scFv#7 + scFv#21


10,000 LD50
None
0%
Death in a few hours


10,000 LD50
2 μg scFv#2 + scFv#3 +
0%
Death delayed one day



scFv#21


10,000 LD50
2 μg scFv#2 + scFv#3 +
100%
Moderate symptoms



scFv#7 + scFv#21









The results clearly demonstrate the excellent potency of the tagged binding agent approach as antitoxins. Specifically, we find that we can completely protect mice against even mild symptoms of intoxication by 1000 LD50s using combinations of three or four scFvs with anti-E-tag mAb. We can protect mice against lethality from a 10,000 LD50 dose with a combination of four scFvs, although the mice did develop moderate symptoms. The ability to protect mice receiving up to 10,000 LD50s of BoNT/A is equivalent to the highest level of protection reported with pools of different anti-BoNT/A mAbs (Nowakowski et al, Proc Natl Acad Sci USA, 99:11346-50).
The next experiment tested whether a binding agent containing two copies of the epitopic tag would improve efficacy. To perform this experiment, the anti-BoNT/A binding agent, scFv#7, was engineered to contain another copy of the E-tag peptide. In all previous studies, the E-tag peptide was present on the carboxyl terminus of each scFvs. A new version of scFv#7 (called scFv#7-2E) was engineered, identical to scFv#7 except for an additional copy of the E-tag peptide fused to the amino terminus.







TABLE 4







Results from Experiment #4










BoNT/A
Agents Administered




LD50
All received 10 μg of anti-E-tag mAb
Survival
Comments













100
None
0%
Death in less than 6 hours


100
1 μg scFv#2 + scFv#3 + scFv#7
100%
No symptoms


100
1 μg scFv#2 + scFv#3 + scFv#7-2E
100%
No symptoms


1000
None
0%
Death in less than 2 hours


1000
1 μg scFv#2 + scFv#3 + scFv#7
0%
Death delayed 2 days


1000
1 μg scFv#2 + scFv#3 + scFv#7-2E
100%
No symptoms


10,000
None
0%
Death in less than 2 hours


10,000
1 μg scFv#2 + scFv#3 + scFv#7
0%
Death delayed less than a





day


10,000
1 μg scFv#2 + scFv#3 + scFv#7-2E
20%
Death delayed many days


10,000
1 μg scFv#2 + scFv#3 + scFv #21 +
0%
Death delayed 2 days



scFv#7


10,000
1 μg scFv#2 + scFv#3 + scFv #21 +
100%
Moderate symptoms



scFv#7-2E









The results demonstrate that the binding agent with two epitope tags dramatically improved the in vivo antitoxin efficacy of the tagged binding agent. With a combination of three scFvs, including scFvs#2, scFvs#3 and scFvs#7 or scFvs#7-2E, clearly the use of scFvs#7-2E was substantially superior in protection of mice to the use of scFvs#7 with only one E-tag. This was particularly evident in the groups of mice challenged with 1000 LD50. In these groups, the triple combination of scFv#2+scFv#3+scFv#7 was insufficient to allow survival of the mice. When scFv#7 was replaced with scFv#7-2E, all the mice survived without symptoms. Furthermore, use of a pool of scFv#2+scFv#3+scFv#7-2E permitted the survival of one of five mice challenged with 10,000 LD50 and delayed the death of the other mice by several days. The equivalent pool with scFv#7 having only one E-tag only delayed death for one day in mice challenged with 10,000 LD50. Finally, an identical combination of four scFvs (#2, #3, #21 and #7) in which the efficacy using scFv#7 was compared with scFv#7-2E. With only one μg of each scFv, the use of scFv#7 did not permit survival of mice challenged with 10,000 LD50 while the same combination using scFv#7-2E was protective. The implication of these results is that it is possible to protect mice against high doses of toxin simply by administering a smaller number high affinity binding agents, each containing two or more epitope tags together with an anti-tag mAb.
The new approach improves therapeutic agent flexibility, permits the use of highly stable binding agents with long shelf life, substantially reduces the cost of production, and permits therapeutic applications that involve multiple target agents to be more commercially feasible. Furthermore, the new strategy will permit much more rapid development of new antitoxins. The binding agents should be much quicker to develop to commercialization than mAbs. The single anti-tag mAb needed for co-administration is the same for therapies requiring different tagged binding agents and thus can be pre-selected for its commercial scale up properties and stockpiled in advance of the development of the binding agents.
An immediate application is in anti-toxin therapy, an area of high interest because of bioterrorist threats. For example, it is now thought that effective prevention of botulinum intoxication using toxin neutralizing mAbs will require administration of three different mAbs each targeting the same toxin. Since there are at least seven different botulinum toxins, this suggests that 21 different mAbs (or more) may need to be stockpiled for use in the event of a major botulism outbreak as might occur through bioterror. Monoclonal antibodies are very expensive to produce and have relatively short shelf lives. This concept would make it possible to produce 21 different recombinant binding agents, each having longer shelf-life and lower production costs, and then stockpile only a single mAb. It is possible that this approach could open up many other mAb therapeutic strategies that involve multiple binding targets, but which have not been pursued because of prohibitive development and production costs and poor product shelf life. It also permits the use of mAbs of different antibody isotypes to be used with the same binding agents to provide greater therapeutic flexibility.
The relevant teachings of all the references, patents and/or patent applications cited herein are incorporated herein by reference in their entirety.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method for administering antibody therapeutic agents to treat an individual for exposure to a Botulinum neurotoxin, the method comprising:
a. administering to the individual at least one binding agent, wherein the binding agent comprises a single-chain antibody (scFv) and has a binding region specific to the Botulinum neurotoxin and has one or more copies of a tag comprising an antibody epitope; and
b. administering to the individual an anti-tag antibody, wherein the anti-tag antibody binds to the one or more copies of the tag of the binding agent and promotes clearance of the Botulinum neurotoxin, thereby treating the individual; and the binding agent binds to the portion of the Botulinum neurotoxin wherein the Botulinum neurotoxin is Botulinum neurotoxin serotype A, and wherein the scFv is encoded by a nucleic acid comprising a nucleotide sequence selected from the group of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13.

2. The method according to claim 1, wherein the disease agent target is Botulinum neurotoxin a serotype A, and wherein the scFv comprises an amino acid sequence selected from the group of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.