WO2009105681A1 - Neutralization of staphylococcal and streptococcal toxins - Google Patents

Abstract

Staphyloccocus and Streptococcus bacteria secrete various toxins that act as superantigens by stimulating a large fraction of the host's T cells. The binding of toxins to the variable domains of T cell receptor ß chains (Vß) leads to massive release of inflammatory molecules and, potentially, toxic shock syndrome (TSS). Soluble forms of different Vß domains able to bind superantigens with high-affinity have previously been generated. However, to neutralize unknown toxins or multiple toxins a broader spectrum antagonist is required. The present invention expresses multiple Vß domains in tandem as a single-chain protein and neutralizes the clinically important superantigens, particularly staphylococcal enterotoxin B and TSS toxin-1, with a single agent.

Description

NEUTRALIZATION OF STAPHYLOCOCCAL AND STREPTOCOCCAL

TOXINS

STATEMENT REGARDING FEDERAL FUNDING

[0001] This invention was made with Government support under Grant Number AI06461 1 , AI55882 and T32 GM07283 awarded by the National Institutes of Health. The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Staphylococcus and Streptococcus bacteria secrete families of structurally- related toxins that bind to the variable region of T cell receptor β chains (Vβ domain) and to a major histocompatibility complex (MHC) class Il molecule. Toxicity results from stimulation of a large fraction of the host's T cell repertoire and subsequent release of massive levels of inflammatory cytokines that can lead to toxic shock syndrome and organ failure. The term superantigen (SAg) was given to this class of molecules because these toxins stimulate a large fraction of T cells bearing the same Vβ domain. As up to 20% of the T cell repertoire can bear the same Vβ domain, SAgs are capable of stimulating thousands of times more T cells than conventional antigens. Since soluble monovalent ligands for the T cell receptor (TCR) cannot themselves stimulate T cells, SAgs act by cell-to-cell cross-linking TCRs and class Il major histocompatibility complex (MHC) molecules on antigen presenting cells. Two of the most commonly expressed superantigens that have each been associated with significant mortality are staphylococcal enterotoxin B (SEB) and TSS toxin-1 (TSST-1 ) (McCormick et al. "Toxic shock syndrome and bacterial superantigens: an update," Annu Rev Microbiol 2001 ;55:77-104).

[0003] Toxic shock syndrome (TSS) was characterized as a disease associated with staphylococci infection over 25 years ago. Subsequently, TSST-1 from Staphylococcus aureus was identified as the protein responsible for the disease in most cases. TSST-1 is a member of SAgs secreted by Staphylococcus aureus and Streptococcus pyogenes bacteria that cause elevated systemic cytokine levels, including tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1 ), leading to fever, TSS, and ultimately organ failure.

[0004] The Staphylococcus bacterial SAg family contains over 20 known members, including the S. aureus enterotoxins TSST-1 , A (SEA) to E, and G to Q. The Streptococcus SAg family further contains S. pyogenes exotoxins A (SpeA), C, G to M, and the mitogenic exotoxins called SMEZ. Sequence based phylogenetic relationships among the S. aureus enterotoxins indicated that they represent five groups, in which one group contains TSST-1 as the only known member. The general structures of SAgs, including TSST-1 , have been shown to be similar. A smaller N-terminal domain contains two β-sheets and a larger C-terminal domain consists of a central α-helix and a five-stranded β-sheet. Although all bacterial SAgs share a common three-dimensional structure, they exhibit diversity in their specificities for TCR Vβ domains and class Il MHC molecules, as well as in the molecular architecture of the respective MHC-SAg-TCR signaling complexes that they form.

[0005] These superantigens cause or exacerbate many diseases, including pneumonia, mastitis, phlebitis, meningitis, urinary tract infections; osteomyelitis, endocarditis, nosocomial infection, severe atopic dermatitis, staphylococcal food poisoning and toxic shock syndrome. Current treatments include supportive care, antibiotics, and intraveneous immune globulin. However, there are several strains of S. aureus that are antibiotic resistant, such as the methicillin resistant S. aureus (MRSA) strains.

[0006] To develop potential neutralizing agents against individual exotoxins, single Vβ domains were previously used as a platform for engineering picomolar affinity toxin-binding agents. Recently, it has been shown that these 12-15 kDa proteins can be generated against TSST-1 or SEB (Buonpane et al. "Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1 ," J MoI Biol 2005;353:308-21 ; and Buonpane et al. "Neutralization of staphylococcal enterotoxin B by soluble, high-affinity receptor antagonists;" Nat Med 2007; 13:725- 9). Soluble forms of the SEB-reactive Vβ proteins were effective in rabbit models of SEB-induced disease. [0007] In order to develop potential treatments of S. aureus toxin-mediated diseases, efforts have explored whether toxins could elicit antibodies that are capable of neutralizing multiple members of the toxin family (Boles et al. "Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity," Clin Immunol 2003;108:51-9; and Burnett et al. "The evolving field of biodefense: therapeutic developments and diagnostics," Nat Rev Drug Discov 2005;4:281-97). In addition, there have been some studies to generate mouse monoclonal antibodies that cross-react with more than one exotoxin (Pang et al. "Inhibition of staphylococcal enterotoxin B-induced lymphocyte proliferation and tumor necrosis factor alpha secretion by MAb5, an anti-toxic shock syndrome toxin 1 monoclonal antibody," Infect lmmun 2000;68:3261-8). Despite some limited success, there remains a problem in generating a broad spectrum neutralizing approach, due to the structural diversity represented by these toxins (McCormick et al. "Toxic shock syndrome and bacterial superantigens: an update," Annu Rev Microbiol 2001 ;55:77- 104). For example, SEB and TSST-1 share only 22.3% amino acid identity. Furthermore, many of the toxins are known to exert their toxic effects by binding to different Vβ region subfamily members (Fleischer et al. "Reactivity of mouse T-cell hybridomas expressing human Vbeta gene segments with staphylococcal and streptococcal superantigens," Infect lmmun 1996;64:987-94). TSST-1 binds almost exclusively to human Vβ2⁺ T cells, whereas SEB binds to various human Vβ regions, as well as mouse Vβ8 (Choi et al. "Selective expansion of T cells expressing V beta 2 in toxic shock syndrome," J Exp Med 1990;172:981-4; and Marrack et al. "The staphylococcal entertoxins and their relatives," Science 1990;248:705-71 1 ).

[0008] There is a need in the art for therapeutic agents to treat superantigen- mediated disease. In particular, there is a need for a single agent that can neutralize multiple bacterial superantigens that bind to different Vβ regions.

SUMMARY OF THE INVENTION

[0009] The present invention provides a single chain polypeptide comprising a first Vβ domain and a second Vβ domain, wherein the first Vβ domain is capable of binding to a bacterial SAg and the second Vβ domain is capable of binding to a different bacterial SAg. The Vβ domains are covalently linked and, in some embodiments, the polypeptide comprises a linker between the first and second Vβ domains. In one embodiment, the first or second Vβ domain is a domain capable of binding to SEB. In a further embodiment, the first or second Vβ domain is S4-8 Vbeta. In another embodiment, the first or second Vβ domain is a domain capable of binding to TSST-1. In a further embodiment, the first or second Vβ domain is D10 Vbeta. The two-domain fusion proteins serve as a potential drug for use in bacterial infections, such as caused by S. aureus, where multiple toxins are involved in disease. The single chain polypeptide may optionally comprise more than two covalently linked Vβ domains, where each Vβ domain is capable of binding to a bacterial SAg, preferably to a different SAg. This is useful where more than two toxins need to be neutralized or where multiple Vβ domains are desired to neutralize an unidentified toxin. Because of their small size (less than one-tenth the size of an IgG molecule) and their modular nature, different Vβ region domains can be cloned in tandem to generate a single protein capable of neutralizing multiple toxins. A further embodiment of the invention provides a pharmaceutical composition comprising the single chain polynucleotide and a pharmaceutically acceptable carrier.

[0010] Another embodiment of the present invention provides a polynucleotide encoding the single chain polypeptide. Preferably, a single chain polynucleotide is produced by expressing a coding sequence for the single chain polypeptide in a host cell. A nucleotide sequence with suitable promoter sequences capable of expressing each desired Vβ domain is cloned into a vector which is inserted into a host cell where the polynucleotide is expressed. The host cell can be any cell known in the art for expressing genetically modified or engineered proteins, including but not limited to E. coli. In particular, the present invention provides a fusion protein of two Vβ domains, expressed in high quantities from E. coli, capable of fully or partially neutralizing the activity of SEB and TSST-1.

[0011] In one embodiment, the present invention provides a method of neutralizing the toxic effects of a bacterial SAg in an individual by administrating a single chain polypeptide comprising a first and second Vβ domain, wherein either the first or second Vβ domain is capable of binding with high affinity to the SAg. Preferably, the first and/or second Vβ domain of the fusion protein have the same or higher binding affinity to the SAg than natural (wild-type) T cell receptors. In a further embodiment, the first and/or second Vβ domain is able to bind to SEB and/or TSST-1 .

[0012] Another embodiment of the invention provides a method of treating bacterial toxicity caused by one or more SAgs comprising producing a single chain polypeptide having a first Vβ domain and second Vβ domain, wherein the first Vβ domain is capable of binding to a bacterial SAg and the second Vβ domain is capable of binding to a different bacterial SAg. An effective amount of the single chain polypeptide is administered to an individual diagnosed with, or suspected of having toxicity caused by one or more bacterial SAgs. The SAg responsible for causing the toxicity may not be known, thus more than one single chain polypeptide of the present invention or a single chain polypeptide having more than two Vβ domains may be administered, where each Vβ domain binds to a different known bacterial SAg. In a further embodiment, the toxicity is caused by multiple bacterial SAgs, including, but not limited to Staphyloccocus aureus or Streptococcus pyogenes. Preferably, the first and/or second Vβ domains have a binding affinity to a bacterial SAg the same as or greater than natural T cell receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows a single chain protein of the present invention designed to neutralize multiple different exotoxins. This protein contains a Vβ domain (S4-8 in the present example) designed to bind to SEB and a second Vβ domain (D10 in the present example) designed to bind to TSST-1 .

[0014] Figure 2 illustrates the cloning of a multiple Vβ domain construct containing a S4-8 Vβ domain gene and a D10 Vβ domain gene linked together with a (Gly₄Ser)₄ linker. The genes are cloned into a pET28a vector and the single chain protein expressed in E coli.

[0015] Figure 3 shows a gel filtration profile of a two-domain protein (S4-8/D10 Vβ) expressed from the construct of Figure 2. The protein eluted in a peak corresponding to a size of approximately 30,000 daltons (peak 2). [0016] Figure 4 shows a SDS-Polyacrylamide gel of proteins having one or more Vβ domains, including a single domain S4-8 protein (Vβ8), single domain D10 protein (Vβ2) and a two-domain fusion protein (S4-8/D10 Vβ) of Figure 3.

[0017] Figure 5 shows in vitro binding of TSST- 1 by a protein having a single Vβ domain (D10) and a protein containing both S4-8 and D10 Vβ domains, using a competition enzyme-linked immunoassay (ELISA).

[0018] Figure 6 shows in vitro binding and inhibition of SEB by a protein having a single Vβ domain (S4-8) and a protein containing both S4-8 and D10 Vβ domains, using a competition ELISA.

[0019] Figure 7 shows binding to both SEB and TSST-1 by the same S4-8/D10 fusion protein. Neither the S4-8 protein alone or the D10 alone were positive in this assay.

[0020] Figure 8 shows surface plasmon resonance (SPR) analyses for the S4- 8/D10 fusion protein against SEB and TSST-1 .

[0021] Figure 9 shows the thermal stability of the individual Vβ domains and the S4- 8/D10 fusion protein as determined using circular dichroism.

[0022] Figures 10, 1 1 , and 12 show the ability of the S4-8/D10 fusion protein to neutralize 25 nM, 250 pM, and 2.5 pM TSST-1 , respectively, in an interleukin-2 secretion assay with a T cell line transfected with the human Vβ2.1 T cell receptor. Soluble high-affinity Vβ2 D10 (open square) or the fusion protein S4-8/D10 (solid triangle) were added at the indicated concentrations to wells that contained the toxin, T cells, and antigen presenting cells, as described below. IL-2 released after 18 hours was measured by ELISA and percent inhibition in the presence of the Vβ preparation was calculated as: 100 X ((A450-A570)_no inhibitor - (A450-

A570)_ιnh,bιtor)/(A450-A570)no inhibitor)-

[0023] Figures 13 shows the ability of the S4-8/D10 fusion protein to neutralize 50 nM SEB in an interleukin-2 secretion assay with a T cell line transfected with the Vβ8.2 T cell receptor. Soluble high-affinity Vβ2 D10 (open square), Vβ8 S4-8 (open diamond), or the fusion protein S4-8/D10 (solid triangle) were added at the indicated concentrations to wells that contained the toxin, T cells, and antigen presenting cells, as described below. IL-2 released after 26 hours was measured by ELISA and percent inhibition in the presence of the Vβ preparation was calculated as: 100 X

((A450-A570)no inhibitor - (A450-A570)_lnh,b_ltor)/(A450-A570)no inhibitor).

[0024] Figure 14 shows the ability of the S4-8/D10 fusion protein to neutralize TSST-1 , SEB, or a combination of TSST- 1 and SEB in a proliferation assay involving fresh peripheral blood lymphocytes taken from normal human donors. Human peripheral blood mononuclear cells (PBMCs) were stimulated in 96-well microtiter plates (2 x 10⁵ cells/well) in the presence of TSST-1 , SEB, or both toxins (each at 80 nM), and various concentrations of the Vβ proteins. After three days, ³H-thymidine was added for an additional 24 hr to measure proliferation. Concentrations of the Vβ proteins just above the concentration of the toxins (80 nM) were capable of effective inhibition.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The following non-limiting description provides further illustration of some embodiments of the present invention. Applicant does not wish to be bound by any theory presented here. The present description discloses generation of a single chain polypeptide having two or more Vβ domains, where each Vβ domain is capable of binding to a different bacterial SAg. In particular, a single chain polypeptide able to bind to both SEB and TSST-1 is demonstrated here. However, one of ordinary skill in the art would be able to produce polypeptides able to bind to SAgs other than or in addition to SEB and TSST-1 using the methods described herein and methods known in the art without undue experimentation.

Definitions

[0026] In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. [0027] "Vβ domain" refers to the variable region of a T cell receptor beta chain, or a fragment thereof, able to bind to a bacterial SAg. Currently, there are over 20 known Vβ domains able to bind to bacterial SAgs. As described herein, a polypeptide may be synthesized or expressed to contain one or more Vβ domains, or contain amino acid sequences functionally equivalent to one or more Vβ domains. Preferably, the polypeptide contains two or more different Vβ domains, or amino acid sequences functionally equivalent to two or more Vβ domains, where each Vβ domain is able to bind to a bacterial SAg.

[0028] A "coding sequence" is the part of a gene or cDNA which codes for the amino acid sequence of a protein or polypeptide, or for a functional RNA such as a tRNA or rRNA.

[0029] "Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of a mRNA into a protein or polypeptide.

[0030] A "polypeptide" is a linear polymer of amino acids that are linked by peptide bonds. The polypeptides described herein can comprise naturally occurring amino acids, modified amino acids, synthetic amino acids and combinations thereof.

[0031] An amino acid sequence that is functionally equivalent to a specifically exemplified peptide sequence is an amino acid sequence that has been modified by single or multiple amino acid substitutions, by addition and/or deletion of amino acids, or where one or more amino acids have been chemically modified, but which nevertheless retains the binding specificity and high affinity binding activity of a cell- bound or a soluble protein of the present invention. Functionally equivalent nucleotide sequences are those that encode polypeptides having substantially the same biological activity as a specifically exemplified cell-bound or soluble protein.

[0032] A "linker" is polypeptide that operably links two functional or structural domains of a protein. The linker can be any polypeptide having two or more amino acids, preferably having between at least two amino acids and twenty amino acids.

[0033] A "nucleic acid construct" is a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. An "expression construct" is a nucleic acid construct containing DNA or RNA capable of being expressed in a host cell. In particular, the expression constructs used in the present invention contain a nucleic acid encoding a polypeptide having two or more Vβ domains. Introduction into the host cell is typically achieved by inserting the expression construct into a vector.

[0034] A "recombinant nucleic acid molecule", for instance a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro through the ligation of two or more nonhomologous DNA molecules (for example a recombinant plasmid containing one or more inserts of foreign DNA cloned into at least one cloning site).

[0035] "Transformation" means the directed modification of the genome of a cell by the external application of purified recombinant DNA or an expression construct from another cell of different genotype, leading to its uptake and integration into the subject cell's genome. In bacteria, the recombinant DNA is not typically integrated into the bacterial chromosome, but instead replicates autonomously as a plasmid.

[0036] A "vector" is a nucleic acid molecule that is able to replicate autonomously in a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g. promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0037] It will be appreciated by those of skill in the art that, due to the degeneracy of the genetic code, numerous functionally equivalent nucleotide sequences encode the same amino acid sequence. Additionally, those of skill in the art, through standard mutagenesis techniques, in conjunction with the assays described herein, can obtain altered peptide sequences and test them for the expression of polypeptides having particular binding affinity. Useful mutagenesis techniques known in the art include, without limitation, oligonucleotide-directed mutagenesis, region-specific mutagenesis, linker-scanning mutagenesis, and site-directed mutagenesis by PCR (see e.g. Sambrook et al. (1989) and Ausubel et al. (1999)). [0038] A "promoter" means a cis-acting DNA sequence, generally 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription. There can be associated additional transcription regulatory sequences which provide on/off regulation of transcription and/or which enhance (increase) expression of the downstream coding sequence.

[0039] Various promoters (transcriptional initiation regulatory region) may be used according to the invention. The selection of the appropriate promoter is dependent upon the proposed expression host. Promoters from heterologous sources may be used as long as they are functional in the chosen host.

[0040] Promoter selection is also dependent upon the desired efficiency and level of peptide or protein production. Inducible promoters such as tac are often employed in order to dramatically increase the level of protein expression in E. coli. Overexpression of proteins may be harmful to the host cells. Consequently, host cell growth may be limited. The use of inducible promoter systems allows the host cells to be cultivated to acceptable densities prior to induction of gene expression, thereby facilitating higher product yields.

Expression construct and transformation

[0041] Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and IQJ.; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose (1981 ) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning \/o\. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, VoIs. 1 -4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.

[0042] The expression construct is assembled by employing known recombinant DNA techniques (Sambrook et al., 1989; Ausubel et ai, 1999). Restriction enzyme digestion and ligation are the basic steps employed to join two fragments of DNA. The ends of the DNA fragment may require modification prior to ligation, and this may be accomplished by filling in overhangs, deleting terminal portions of the fragment(s) with nucleases (e.g., Exolll), site directed mutagenesis, or by adding new base pairs by PCR. Polylinkers and adaptors may be employed to facilitate joining of selected fragments. The expression construct is typically assembled in stages employing rounds of restriction, ligation, and transformation of E. coli. Numerous cloning vectors suitable for construction of the expression construct are known in the art (λZAP and pBLUESCRIPT SK-1 , Stratagene, LaJoIIa, CA; pET, Novagen Inc., Madison, Wl - cited in Ausubel et ai, 1999) and the particular choice is not critical to the invention. The selection of cloning vector will be influenced by the gene transfer system selected for introduction of the expression construct into the host cell. At the end of each stage, the resulting construct may be analyzed by restriction, DNA sequence, hybridization and PCR analyses.

[0043] The expression construct may be transformed into the host as the cloning vector construct, either linear or circular, or may be removed from the cloning vector and used as is or introduced onto a delivery vector. The delivery vector facilitates the introduction and maintenance of the expression construct in the selected host cell type. The expression construct is introduced into the host cells by any of a number of known gene transfer systems (e.g., natural competence, chemically mediated transformation, protoplast transformation, electroporation, biolistic transformation, transfection, or conjugation) (Ausubel et al., 1999; Sambrook et al., 1989). The gene transfer system selected depends upon the host cells and vector systems used. [0044] For instance, the expression construct can be introduced into S. cerevisiae cells by protoplast transformation or electroporation. Electroporation of S. cerevisiae is readily accomplished, and yields transformation efficiencies comparable to spheroplast transformation. Industrial strains of microorganisms (e.g., Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Mucor miehei, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis or Bacillus licheniformis) or plant species (e.g., canola, soybean, corn, potato, barley, rye, wheat) may be used as host cells for the recombinant production of the fusion peptides of the present invention.

[0045] Elements for enhancing transcription and translation have been identified for eukaryotic protein expression systems. For example, positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp on either side of a heterologous promoter may elevate transcriptional levels by 10- to 400-fold in plant cells. The expression construct should also include the appropriate translational initiation sequences. Modification of the expression construct to include a Kozak consensus sequence for proper translational initiation may increase the level of translation by 10 fold.

Pharmaceutical compositions

[0046] Pharmaceutical compositions containing polypeptides of the present invention can be formulated by any of the means known in the art. They can be typically prepared as injectables, especially for intravenous, intraperitoneal or synovial administration (with the route determined by the particular disease) or as formulations for intranasal, oral, or topical administration, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection or other administration may also be prepared. The preparation may also, for example, be emulsified, or the protein(s)/peptide(s) encapsulated in liposomes.

[0047] The active ingredients are often mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the polypeptide of the present invention in injectable, aerosol, nasal, or topical formulations is usually in the range of 0.05 to 5 mg/ml. The selection of the particular effective dosages is known and performed without undue experimentation by one of ordinary skill in the art. Similar dosages can be administered to other mucosal surfaces.

Examples

[0048] The present invention provides methods for neutralizing multiple different bacterial exotoxins with a single polypeptide comprising two or more covalently linked Vβ domains. Each Vβ domain is independently engineered to have high- affinity binding against a different SAg exotoxin, preferably SEB and/or TSST-1 , allowing the polypeptide to bind multiple different toxins and neutralize the effects of each exotoxin. Preferably, the multiple Vβ domain polypeptide neutralizes the toxic effects, such as toxic shock syndrome, caused by Staphyloccocus aureus and Streptococcus pyogenes exotoxins.

[0049] The Staphylococcus bacterial SAg family contains over 20 known members, including the S. aureus exotoxins A (SEA) to E, G to Q, and TSST-1. The Streptococcus SAg family further contains S. pyogenes exotoxins A (SpeA), C, G to M, and the mitogenic exotoxins called SMEZ. A large number of the S. aureus and S. pyogenes SAgs bind to one or more known Vβ domains of human and animal T cell receptors. Polypeptides of the present invention contain one or more Vβ domains, or fragments thereof, able to bind a particular SAg or multiple different SAgs. Tables 1 and 2 provide Vβ domains of T cell receptors able to bind to various S. aureus and S. pyogenes SAgs. It understood that these Tables are not exhaustive and are not meant to exclude Vβ domains known in the art to bind with known SAgs.

Table 1. Staphylococcal SAgs and Vβ domains of human and mouse T cell receptors reactive with them.

Table 2. Streptococcal SAgs and Vβ domains of human and mouse T cell receptors reactive with them.

Example 1

[0050] A two-domain single chain fusion peptide was formed by fusing the S4-8 Vbeta (Vβ 8.2) anti-SEB domain and D10 Vbeta (Vβ 2.1 ) anti-TSST-1 domain (Figure 1 ). The resulting peptide, approximately 30,000 daltons, was purified and tested in various binding assays where it bound to both SEB and TSST-1. Both the single D10 domain and the D10/S4-8 Vbeta fusion peptide were able to neutralize TSST-1 in vitro. In particular the fused peptide was able to inhibit stimulation of T cells by TSST-1 , indicating that the fused protein would have potent neutralizing activity in vivo.

[0051] The two Vβ domain genes were cloned as a single-chain gene with a (Gly₄Ser)₄ linker (Figure 2), expressed in E. coli, and refolded from inclusion bodies. The fusion protein and each of the individual Vβ region proteins were purified by nickel affinity chromatography and gel filtration chromatography. The gel filtration profile (S100) of the two-domain fusion protein is shown in Figure 3. The proteins migrated with the expected monomeric sizes under non-denaturing conditions and by SDS-PAGE (Figure 4). Accordingly, the Vβ proteins migrated at approximately 14-17 kDa and the fusion protein migrated at approximately 30 kDa.

[0052] To determine if the fusion protein contained properly folded Vβ domains, competition ELISAs were performed with immobilized individual domains and biotinylated TSST-1 (Figure 5) or SEB (Figure 6). The fusion protein bound to SEB and TSST-1 with similar effectiveness as the single Vβ domains. In addition, the fusion could bind simultaneously to TSST-1- and SEB, as determined in an ELISA with immobilized SEB and biotinylated TSST-1 as detecting agent (Figure 7).

Example 2

[0053] Binding affinities of the two-domain fused polypeptide for SEB and TSST-1 were shown to be very high-affinity, similar to the individual domains. The fusion protein also had excellent thermal stability as would be advantageous as a therapeutic agent for in vivo use and in vitro storage.

[0054] To measure the binding affinities of the two Vβ domains in the fusion, surface plasmon resonance (SPR) analysis with immobilized TSST-1 and SEB was performed (Figure 8). The results showed that both Vβ domains in the fusion protein bound to their respective ligands with picomolar affinities. The fusion protein exhibited a K₀ value for TSST-1 of ~190 pM, similar to that measured for the individual Vβ2.1 D10 domain (K₀ = 180 pM). The fusion protein exhibited a K₀ value for SEB of -340 pM, only 7-fold lower than the affinity of the individual Vβ8 S4-8 domain (K₀ = 50 pM). Thus, the fusion retains the affinities that have been shown to be effective as neutralizing agents for these two toxins.

[0055] Thermal denaturation experiments showed that all three of the proteins, including the fusion protein, exhibited excellent thermal stability, with T_M values (temperature at one-half maximal denaturation) of 61⁰C (Vβ8 S4-8 and fusion protein) and 68°C (Vβ2 D10 protein) (Figure 9).

Example 3

[0056] The multi-domain Vβ fusion protein showed potent neutralization of TSST-1 and SEB toxicity, as measured by use of T cell lines that have been transfected with the TCRs that are specific for these toxins. The effectiveness of the fusion protein for each toxin was nearly equivalent to that of the individual Vβ proteins.

[0057] Figures 10, 1 1 , and 12 show in vitro neutralization of 25 nM, 250 pM, and 2.5 pM of TSTT- 1 by the S4-8/D10 fusion protein and the D10 domain protein, in the presence of a class ll-positive MHC cell line. Activity (and its inhibition) is measured by the secretion of IL-2 released into the surrounding media. No detectable IL-2 is released in the absence the toxin. The S4-8/D10 fusion protein and the individual Vβ2 domain (D10) were each capable of almost 100% inhibition of TSST-1 activity at every concentration of TSST-1 tested.

[0058] Figure 13 shows in vitro neutralization of 50 nM of SEB by the S4-8/D10 fusion protein and the Vβ8 S4-8 domain, in the presence of a class ll-positive MHC cell line. Activity (and its inhibition) is measured by the secretion of IL-2 released into the surrounding media. No detectable IL-2 is released in the absence the toxin. The S4-8/D10 fusion protein and the individual Vβ8 domain (S4-8), but not the TSST-1 -specific Vβ2 domain (D10), were each capable of almost 100% inhibition of SEB activity. Example 4

[0059] Figure 14 shows the effectiveness of the fusion protein in another in vitro assay, in this case using human peripheral blood mononuclear cells that were stimulated with TSST-1 (upper panel), SEB (middle panel), or both SEB and TSST-1 (lower panel). The Vβ proteins were examined for their ability to inhibit T cell proliferation. In each assay, the individual Vβ domains against the specific toxin and the fusion were capable of inhibiting the polyclonal T cell activity stimulated by the toxins. The neutralization of toxin activity with the fusion occurred at low concentrations of the fusion, just above the stochiomethc level of toxin (e.g. 130 nM of fusion and 80 nM toxin in each panel of Figure 14).

MATERIALS AND METHODS

[0060] Toxins and monoclonal antibodies. SEB, TSST-1 , and their biotinylated forms were obtained from Toxin Technology (Sarasota, FL). Monoclonal antibodies against human IL-2 and mouse IL-2 were obtained from BD Biosciences (Pharmingen, CA).

[0061] Cloning, expression, and purification of Vβ proteins. S4-8 (sometimes also referred to as G5-8), a mouse Vβ domain (Vβ8.2) engineered to neutralize SEB with 48pM affinity (Buonpane et al. "Neutralization of staphylococcal enterotoxin B by soluble, high-affinity receptor antagonists," Nat Med 2007;13:725-9), was PCR amplified and cloned into the Λ/col and EcoRI sites of the bacterial expression vector pET28a as a His-Thrombin-S4-8 fragment without a stop codon (Figure 2). A (Gly₄Ser)₄ linker followed by the D10 gene, a human Vβ domain(Vβ2.1 ) engineered to neutralize TSST-1 with 18OpM affinity (Buonpane et al. "Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1 ," J MoI Biol 2005;353:308-21 ), was subcloned at the C-terminus of S4-8 after PCR amplification and cloning into the EcoRI and Xho\ sites (Figure 2).

[0062] The hybrid and individual Vβ proteins were expressed as inclusion bodies from the pET28a expression vector in E. coli BL21 (DE3). Inclusion bodies were solubilized with 1 .1 g guanidine-HCI and 2.5μL β-mercaptoethanol for 1 hr at 37°C. Solubilized inclusion bodies (400 mg in one ml) were added drop wise into 400 mL denaturant buffer (7.5M Urea, 5OmM Tris pH8.0, 2nM reduced glutathione, 0.2mM oxidized glutathione). After 4 hr, dilution buffer (20OmM NaCI, 5OmM Tris pH8.0) was added drop wise until the total volume reached 2500ml. After stirring for one day, proteins were purified using a nickel affinity resin and run through an S-100 gel filtration column by High Pressure Liquid Chromatography (Figure 3). Fractions were monitored for absorbance at 220 nm (left axis) and 280 nm (right axis). The two-domain fusion protein eluted at in a peak labeled as peak 2 in Figure 3. 400 mg of inclusion bodies typically yielded approximately 1 mg of purified soluble protein.

[0063] The purified single domain Vβ proteins and the two-domain fusion protein were run on a polyacrylamide gel electrophoresis, and the gel stained with Coomaise Blue to visualize the proteins. The single domain Vβ proteins migrated at approximately 14-17 kDa and the two-domain fusion protein migrated at approximately 30 kDa by SDS-PAGE (Figure 4).

[0064] Thermal denaturation analysis. The thermal stabilities of the individual Vβ proteins and the fusion protein were determined by circular dichroism by monitoring changes in ellipticity at 215 nm every one degree from 20 - 80 ⁰C, each at a concentration of 9 μM in 10 mM sodium cacodylate, pH 7.4. An equilibration time of 1 minute between each temperature point and an averaging time of 15 seconds was used.

[0065] Thermal denaturation experiments showed that all three of the proteins exhibited excellent thermal stability, with T_M values (temperature at one-half maximal denaturation) of 61⁰C (S4-8 and fusion) and 68°C (D10) (Figure 9).

[0066] Enzyme-linked immunoassays (ELISAs) for Vβ binding to TSST-1 and SEB. High-affinity D10 Vβ2 or S4-8 Vβ8 (also called G5-8) proteins at 1 μg/ml in PBS were absorbed on ELISA plates at 4°C. After blocking with PBS containing 0.5% BSA, various concentrations of soluble Vβ proteins (D10 or S4-8) or the S4- 8/D10 fusion protein were added to the wells followed by 20 nM biotinylated TSST-1 or 20 nM biotinylated SEB. After washing, streptavidin/horse radish peroxidase (SA- HRP) was added for 45 minutes, wells were washed, and substrate was added. Plates were read at A450. Percent inhibition was calculated as (A450_no inhibitor - A450_ιrihιbιtor/ A450_no inhibitor) x 100 (Figure 5 and Figure 6). [0067] To determine if fusion protein had both domains folded on the same molecules (i.e. could bind to TSST-1 and SEB simultaneously), 20 nM SEB was absorbed to an ELISA plate and various concentrations of soluble S4-8, D10 and S4- 8/D10 fusion protein were added to the wells. After washing, and biotinylated TSST- 1 was added. After additional washing, SA-HRP was added, followed by substrate, and A450 was measured (Figure 7). Only the measured A450 for the fusion protein is shown in Figure 7 as the individual S4-8 protein and D10 protein were not positive for this assay.

[0068] Surface plasmon resonance analysis. The binding parameters of the individual Vβ proteins and the fusion protein were determined by surface plasmon resonance analysis. Briefly, SEB, TSST-1 and streptococcal exotoxin I (Spel; as a negative control) were immobilized to a density of 500 resonance units (RU) by standard amine coupling to a CM5 sensor chip (Biacore, Piscataway, NJ). Concentration gradients of the individual Vβ proteins and the fusion protein in Hepes buffered saline, pH 7.4 were injected over all of the surfaces and the net responses were for kinetic analysis using the BiaEvaluation 4.1 software package (Biacore, Piscataway, NJ) and affinities calculated as the ratio of the on- and off-rates.

[0069] The results showed that both Vβ domains in the fusion bound to their respective ligands with picomolar affinities (Figure 8). The fusion protein exhibited a K₀ value for TSST-1 of ~190 pM, similar to that measured for the individual Vβ2.1 D10 domain (K₀ = 180 pM). The fusion exhibited a K₀ value for SEB of -340 pM, approximately 7-fold lower than the affinity of the individual Vβ8 S4-8 domain (K₀ = 50 pM). It is unclear what caused this modest reduction in affinity of the Vβ8 domain, but it is possible that Vβ2:Vβ8 interactions might influence the affinity of the domains and that shortening the length of the linker would minimize this effect (similar to the strategy used for scFv "diabodies" to prevent intrachain V_H:V_L associations) (Holliger et al. "Diabodies: small bivalent and bispecific antibody fragments," Proc Natl Acad Sci U S A 1993;90:6444-8).

[0070] In vitro toxin activity assays with T cell receptor transfected T cell lines. To measure TSST-1 activity, the Jurkat T cell line JRT3-2.1 , transfected with the human Vβ2.1 gene (Rahman et al. "Molecular Basis of TCR Selectivity, Cross- Reactivity, and Allelic Discrimination by a Bacterial Superantigen: Integrative Functional and Energetic Mapping of the SpeC-Vbeta2.1 Molecular Interface," J Immunol 2006;177:8595-603). The cell line was maintained in R-10 medium (RPMI 1640, 10% fetal bovine serum, 100ug/ml streptomycin, 100units/ml penicillin, 2mM L- glutamine, 1 mM MEM sodium pyruvate, 10OuM non-essential amino acid, 25mM HEPES, pH7.2) and 0.8mg/ml hygromycin B. The MHC class ll-positive B cell line LG-2 was also maintained in R-10 medium. JRT3- 2.1 cells (10⁶ cells/well) were stimulated with various concentrations of TSST- 1 in the presence of LG-2 cells (2 x 10⁵ cells/well). Soluble, high-affinity Vβ domains D10 or the S4-8/D10 fusion protein were added at various concentrations. After 18 hr, plates were centrifuged, supernatant was collected, and IL-2 was measured by ELISA using the BD OptEIA™ Human IL-2 ELISA Kit (BD Biosciences, Pharmingen).

[0071] To measure SEB activity, the mouse T cell hybridoma 58-/-, transfected with the mouse Vβ8.2 gene (the 2C TCR mutant called m6, co-transfected with CD8αβ genes) was maintained in RPMI 1640, 10% FCS, 5mM HEPES, 2mM L-glutamine, 100U penicillin, 0.1 mg/ml streptomycin and 4 μM β-mercaptoethanol, with 1 mg/ml G418, 0.5mg/ml hygromycin B, and 1 ug/ml puromycin. LG-2 cells (2 x 10⁵ cells/well) and 50 nM SEB were added to the Vβ8.2⁺ T cell line (2 x 10⁶ cells per well). Soluble, high-affinity Vβ domains S4-8, D10, or the S4-8/D10 fusion protein were added at various concentrations. After 26 hr, plates were centrifuged, supernatants were collected, and IL-2 was measured by ELISA using rat-anti-mouse-IL-2 Ab and a detecting biotinylated rat-anti-mouse-IL-2 Ab.

[0072] Since the fusion protein exhibited SEB and TSST-1 binding activity, in vitro T cell assays were performed to determine if the single-chain protein could neutralize both SEB and TSST-1. In these assays, two transfected T cell lines, expressing either the mouse Vβ8.2 domain, or the human Vβ2.1 domain, were used to assay activity mediated by the toxins SEB and TSST-1 , respectively. In the presence of the human class N⁺ antigen-presenting cell LG-2 and various concentrations of either SEB or TSST-1 , both cell lines secrete IL-2, which was measured by a capture ELISA. SEB was assayed at a concentration of 50 nM, in the presence of various concentrations of the three Vβ proteins, Vβ8.2 S4-8, Vβ2.1 D10, or the fusion protein. Both S4-8 and the fusion protein completely inhibited the activity of SEB, with nearly equal effectiveness (Figure 13). As expected, the TSST-1 reactive Vβ D10 was unable to inhibit SEB-mediated activity at any concentration.

[0073] A similar experiment was performed with TSST-1 and the Vβ2.1 T cells, except that the concentrations of TSST-1 used were analyzed over a broader range (25 nm, 250 pM and 2.5 pM) due to the potency of this toxin in the in vitro T cell assay (Figures 10, 1 1 , and 12, respectively). Even at the highest concentration of TSST-1 (25 nM), both the soluble Vβ2.1 D10 domain and the fusion were capable of complete inhibition. As expected, higher concentrations of Vβ proteins were required for complete neutralization at 250 pM and 25 nM TSST-1 , because the toxin is active even under conditions where there are very low levels of unbound TSST-1 (e.g. at a concentration of 25 nM, even 0.01 % of free toxin would be capable of stimulating T cells). The high-affinity of the Vβ2.1 D10 domain no doubt accounts for such potency over a range of TSST-1 concentrations.

[0074] In vitro toxin activity assays with fresh peripheral blood T lymphocytes from human donors. T cells from normal humans express a diverse repertoire of T cell receptors, as opposed to a single TCR as described in the transfected T cell lines. To assess whether this diverse population of T cells could be inhibited by the fusion protein, T cell proliferation assays were performed. For the proliferation assays, human peripheral blood mononuclear cells (PBMCs) were stimulated in 96- well microtiter plates (2 x 10⁵ cells/well) in the presence of TSST-1 , SEB, or both toxins (each at 80 nM), and various concentrations of the Vβ proteins. After three days, ³H-thymidine was added for an additional 24 hr to measure proliferation. The amount of ³H-thymidine incorporated into DNA was determined by lysis of the cells, capture of DNA on filter disks, followed by liquid scintillation counting. Less than 3000 cpm was incorporated in the absence of toxin, whereas 50,000 to 100,000 cpm was incorporated in the presence of the toxins (Figure 14).

[0075] Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

[0076] One of ordinary skill in the art will appreciate that starting materials, reagents, purification methods, materials, substrates, device elements, analytical methods, assay methods, mixtures and combinations of components other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0077] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms.

[0078] When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and/or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0079] All references cited herein are hereby incorporated by reference in their entirety to the extent that there is no inconsistency with the disclosure of this specification. Some references provided herein are incorporated by reference to provide details concerning sources of starting materials, additional starting materials, additional reagents, additional methods of synthesis, additional methods of analysis, additional biological materials, additional nucleic acids, chemically modified nucleic acids, additional cells, and additional uses of the invention. All headings used herein are for convenience only. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.

Claims

Claims

1. A single chain polypeptide comprising a first variable region of a T cell receptor beta chain (Vβ domain), and a second Vβ domain, wherein said first Vβ domain is capable of binding to a bacterial superantigen (SAg) and said second Vβ domain is capable of binding to a different bacterial SAg.

2. The polypeptide of claim 1 wherein the first Vβ domain is covalently linked to the second Vβ domain.

3. The polypeptide of claim 1 further comprising a linker between the first and second Vβ domains.

4. The polypeptide of claim 1 wherein the first or second Vβ domain is able to bind to staphylococcal enterotoxin B (SEB).

5. The polypeptide of claim 1 wherein the first or second Vβ domain is able to bind to toxic shock syndrome toxin-1 (TSST-1 ).

6. The polypeptide of claim 1 wherein the first Vβ domain has a binding affinity to a bacterial SAg equal to or greater than natural T cell receptors.

7. The polypeptide of claim 1 wherein the second Vβ domain has a binding affinity to a bacterial SAg equal to greater than natural T cell receptors.

8. The polypeptide of claim 1 wherein the first or second Vβ domain contains the amino acid sequence of the S4-8 Vbeta protein.

9. The polypeptide of claim 1 wherein the first or second Vβ domain contains the amino acid sequence of the D10 Vbeta protein.

10. The polypeptide of claim 1 wherein said polypeptide comprises more than two Vβ domains wherein each Vβ domain is capable of binding to a different bacterial SAg.

1 1 . A polynucleotide encoding a single chain polypeptide comprising a first Vβ domain and a second Vβ domain, wherein said first Vβ domain is capable of binding to a bacterial SAg and said second Vβ domain is capable of binding to a different bacterial SAg.

12. A vector comprising the polynucleotide of claim 1 1.

13. A host cell comprising the vector of claim 12.

14. A pharmaceutical composition comprising the single chain polynucleotide of claim 1 and a pharmaceutically acceptable carrier.

15. A method of neutralizing the toxic effects of a bacterial SAg in an individual comprising the steps of: a) producing a single chain polypeptide comprising a first and second Vβ domain, wherein either the first or second Vβ domain is capable of binding with high affinity to the SAg; b) administering an effective amount of the single chain polypeptide to the individual.

16. The method of claim 15 wherein said SAg is staphylococcal enterotoxin B or TSS toxin- 1.

17. The method of claim 15 wherein said single chain polypeptide is produced by expressing a polynucleotide encoding the single chain polypeptide in a host cell.

18. A method of treating bacterial toxicity caused by one or more SAgs comprising the step of producing a single chain polypeptide comprising a first Vβ domain and second Vβ domain, wherein said first Vβ domain is capable of binding to a bacterial SAg and said second Vβ domain is capable of binding to a different bacterial SAg.

19. The method of claim 18 further comprising administering an effective amount of the single chain polypeptide to an individual diagnosed with or suspected of having toxicity caused by one or more bacterial SAgs.

20. The method of claim 18 wherein said toxicity is caused by Staphyloccocus aureus or Streptococcus pyogenes.

21. The method of claim 18 wherein the first or second Vβ domain is able to bind to staphylococcal enterotoxin B.

22. The method of claim 18 wherein the first or second Vβ domain is able to bind to TSS toxin-1.

23. The method of claim 18 wherein the first and/or second Vβ domains have a binding affinity to a bacterial SAg equal to or greater than natural T cell receptors.

24. The method of claim 18 wherein the first or second Vβ domain contains the amino acid sequence of the S4-8 Vbeta protein.

25. The method of claim 18 wherein the first or second Vβ domain contains the amino acid sequence of the D10 Vbeta protein.

26. The method of claim 18 wherein said toxicity is caused by multiple bacterial SAgs.

27. The method of claim 18 wherein said polypeptide comprises more than two Vβ domains wherein each Vβ domain is capable of binding to a different bacterial SAg.