WO1993024136A1 - Effets tumoricides des enterotoxines, superantigenes et composes apparentes - Google Patents

Effets tumoricides des enterotoxines, superantigenes et composes apparentes Download PDF

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Publication number
WO1993024136A1
WO1993024136A1 PCT/US1993/005213 US9305213W WO9324136A1 WO 1993024136 A1 WO1993024136 A1 WO 1993024136A1 US 9305213 W US9305213 W US 9305213W WO 9324136 A1 WO9324136 A1 WO 9324136A1
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enterotoxins
patient
enterotoxin
cells
staphylococcal
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PCT/US1993/005213
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English (en)
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David S. Terman
Jay L. Stone
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Terman David S
Stone Jay L
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Priority claimed from PCT/US1991/000342 external-priority patent/WO1991010680A1/fr
Application filed by Terman David S, Stone Jay L filed Critical Terman David S
Publication of WO1993024136A1 publication Critical patent/WO1993024136A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]

Definitions

  • This invention relates generally to tumoricidal compositions and methods, and more specifically to superantigens or enterotoxins derived from
  • Staphlococcus aureus Peptides homologous to the enterotoxins including toxic shock syndrome toxin (TSST-1), Streptococcal pyrogenic exotoxins,
  • lymphocyte stimulating antigens heat shock proteins, stress peptides, mammary tumor virus peptides, homologous synthetic polypeptides, biochemically derivatized enterotoxins, genetically engineered enterotoxins and fusion proteins are also described in this application.
  • This invention also relates to enterotoxins and homologous compounds known as superantigens expresses on the surface of lipid droplets (in adjuvant-vehicle formulations) or expressed on biologic cell surfaces as a result of enterotoxin gene transfection and used to produce a tumoricidal response in a tumor bearing host.
  • This invention also relates to enterotoxins and homologous compounds known as superantigens expresses on the surface of lipid droplets (in adjuvant-vehicle formulations) or expressed on biologic cell surfaces as a result of enterotoxin gene transfection and used to produce a tumoricidal response in a tumor bearing host.
  • This invention also relates to
  • enterotoxins and related compounds administered intravenously, subcutaneously, as in adjuvant form, or used extracorporeally in free or bound form to stimulate immunocytes which are subsequently infused into tumor bearing hosts.
  • Protein A was chemically attached. Protein A was prepared by batch fermentation of
  • Staphylococcus It was isolated from the media and partially purified by affinity chromatography.
  • Staphylococcal enterotoxins to be present.
  • various methods of the immobilization of Protein A to solid supports have been used, sometimes resulting in loss of biological activity of the plasma perfusion system.
  • the plasma used for perfusion over the immobilized Protein A has been stored and treated in different ways, sometimes resulting in
  • the system contained an enormous number of biologically active materials, to include Staphylococcal Protein A itself, Staphylococcal proteases, nucleases,
  • exotoxins enterotoxins and leukocidin, as well as the solid support and coating materials. Additional products included several anaphylatoxins generated in plasma after contact with immobilized Protein A.
  • Protein A of immunosuppressive immune complexes capable of blocking the host's antitumor response.
  • the present invention demonstrates that isolated Staphylococcal enterotoxins, identified initially as trace contaminants in commercial Protein A
  • enterotoxins are relatively simple proteins that may be infused after being solubilized in saline. This solubility obviates the need to immobilize Protein A or other biologicals on a solid support, and
  • enterotoxins appear to be far safer and more effective than previously described systems.
  • the system requires no elaborate sterilization and there is no problem with potential leaching of immobilized materials or chemical products from an inert surface as there would be with an extracorporeal column.
  • enterotoxins as being the most active antitumor product in the Staphylococcal Protein A plasma perfusion system.
  • the present invention provides enterotoxins derived from Staphylococcus aureus and superantigens which are useful by themselves for the treatment of cancer.
  • Enterotoxins are known to have molecular weights ranging from 22,000 to 38,000. They are heat stable, and resistant to trypsin digestion.
  • enterotoxins isolated from media which is supporting the growth of various Staphylococcus aureus organisms are used in relatively pure form.
  • the preparation When administered to subjects having tumors, the preparation induces a tumoricidal reaction resulting in tumor regression.
  • tumoricidal reaction means that the material under discussion promotes or assists in the killing of tumor cells.
  • enterotoxin molecule in order to minimize toxieity results in a preparation that also induces
  • tumoricidal reactions and tumor regress on when administered to tumor bearing hosts are tumoricidal reactions and tumor regress on when administered to tumor bearing hosts.
  • Streptococcal pyrogenic exotoxin A which has been shown to have statistically significant
  • enterotoxin B structural homology to enterotoxin B, is also useful for the treatment of cancer.
  • additional superantigens such as minor lymphocyte stimulating loci, mycoplasma and mycobacterial antigens, heat shock proteins, stress peptides, and mammary tumor viruses.
  • enterotoxins or peptides using recombinant DNA technology are also described as useful as tumoricidal therapy. Enterotoxin peptides and homologous amino acid sequences to block or destroy autoreactive T and B lymphocyte
  • Figure 1 shows the alignment of amino acid sequences of Staphylococcal enterotoxins and their relatives.
  • Figure 2 shows the alignment of amino acid sequences of mature Streptococcal pyrogenic exotoxin A and Staphylococcus aureus enterotoxin B.
  • Figure 3 shows the hypothetical structure for the complex of Class II MHC, T cell receptor, and Staphylococcal enterotoxins or Mis. Description Of The Specific Embodiments
  • enterotoxins of Staphylococcus aureus form a group of serologically distinct extracellular proteins, designated A, B, C 1 , C 2 , C 3 , D, E and F. These proteins are recognized as the causative agents of Staphylococcal food poisoning. Enterotoxin F appears to be important in the pathogenesis of the Staphylococcal toxic shock syndrome. Ingestion of preformed enterotoxin in contaminated food leads to the rapid development (within two to six hours) of symptoms of vomiting and diarrhea that are
  • the enterotoxin proteins are of similar amino acids
  • enterotoxins A, B, C 1 , C 2 and E reveal a high content of lysine, aspartic acid and tyrosine. Enterotoxins A and E are similar in methionine, leucine and arginine content,
  • enterotoxins B differing in this regard from enterotoxins B, C 1 and C 2 .
  • the amino acid sequence of enterotoxin B was found to consist of 239 amino acids.
  • a second region at residue 147 also shows a highly conserved sequence. These regions are contained on the peptide fragment of SEC, shown to contain the active sites for emesis and diarrhea. The mitogenic region resides in the C terminal tryptic fragment of SEC, implying that other regions of sequence similarity exist. Amino acid sequence similarities and congruences are given in Tables 2-4.
  • bResidues per mole values are based on a molecular weight of 22,000.
  • staphylococcal enterotoxins and their relatives is shown in Figure 1.
  • the complete primary amino acid sequences of the staphylococcal enterotoxins and related proteins are shown aligned, with the
  • Toxins shown are as follows: SEA to SEE, Staphylococcus aureus
  • enterotoxins A to E enterotoxins A to E
  • SPE A and C Streptococcus pyogenes toxins A and C
  • TSSTl Staphylococcus aureus toxic shock - associated toxin
  • ETA and ETB enterotoxins A to E
  • SPE A and C Streptococcus pyogenes toxins A and C
  • TSSTl Staphylococcus aureus toxic shock - associated toxin
  • ETA and ETB
  • Staphylococcus aureus exfoliating toxins A and B Staphylococcus aureus exfoliating toxins A and B.
  • enterotoxins may contain major cross reactive antigenic sites, while each individual
  • enterotoxin possesses minor specific antigenic regions. Common precipitating antibodies were formed between SEA and SED. In addition, enterotoxins B and C can react immunologically with antisera against either toxin type. Immunologic cross reactivity between Streptococcal pyrogenic exotoxin A and Staphylococcal enterotoxins B and C 1 has been shown. These results suggest a conserved domain present in the three exotoxins. SEA, SEB, SEC, SED, TSST-1 and the pyrogenic exotoxins have also been shown to share considerable DNA and amino acid homology. The enterotoxins, the pyrogenic exotoxins and TSST-1 therefore appear to be evolutionarily related and all belong to a common generic group of proteins.
  • Staphylococcal groups as they are to each other.
  • Exfoliative toxins are of similar size to SEB and SEA with similar modes of action. They share several points of sequence similarity to the Staphylococcal enterotoxins. Overall there are several stretches at which similarities are apparent throughout the total group comprised of Staphylococcal enterotoxins, Streptococcal pyrogenic exotoxins and Staphylococcal exfoliative toxins. The longest of these, located two-thirds of the way through the proteins, is similar to sequences found at the COOH-terminal end of the human and mouse invariant chain.
  • Invariant chain is a polypeptide associated with nascent MHC class II molecules.
  • Class II molecules bind peptides and present them to T cells during immune responses. Indeed, many toxins bind to class II
  • the shared sequences may indicate some or all of the invariant chain and toxin binding sites on class II molecules .
  • tumoricidal agents which are likely candidates based upon structural homology or identity of clinical symptomatology are gram positive bacterial products, cell wall bacterial constituents such as peptidoglycans and various gram negative bacterial components to include meningococcal, pseudomonous and E. Coli products. While presently undemonstrated in animal systems, it is believed that these agents are likely to possess similar tumoricidal utility as those claimed here for the enterotoxins.
  • Figure 2 illustrates the amino acid sequence homology of mature SPEA and Staphylococcus aureus enterotoxin B.
  • the top sequence is the SPEA-derived amino acid sequence.
  • the amino acid sequence of enterotoxin B is on the bottom. Sequences are numbered from the amino acid terminus, with amino acids represented by standard one character
  • synthetic polypeptides useful in tumoricidal therapy and in blocking or destroying autoreactive T and B lymphocyte populations are provided.
  • enterotoxins are presumed to function by affecting emetic receptors in the abdominal viscera which stimulate the emetic and diarrheal response. These toxins also stimulate T lymphocyte
  • Cytokines known to be induced by enterotoxins induce
  • polypeptides would also be expected to demonstrate similar responses.
  • Staphylococcal enterotoxins A, B, C, D, E, toxic shock toxin (TSST-1), a product of mycoplasma arthritidis, mycobacterial species, heat shock peptides and Mls antigens provoke dramatic T cell responses.
  • Staphylococcal enterotoxins are the most powerful T cell mitogens known eliciting strong polyclonal proliferation at concentrations 10 3 lower than such conventional T cell mitogens as
  • SEA is the most potent T cell mitogen, stimulating DNA synthesis at concentrations of 10 -13 to 10 -16 M in the human system. All stimulate a large proportion of both murine and human CD4+ and CD8+ T cells. Activity of these mitogens is tightly restricted by the major histocompatibility complex (MHC) class II antigens. It is proposed that the staphylococcal enterotoxins, streptococcal pyrogenic exotoxins, exfoliative toxins and a product of mycoplasma arthritidis bind directly to the T cell receptor and to class II MHC. These two structures are brought into contact, thus stimulating T cell activation via the V ⁇ region of the T cell receptor mimicking strong alloreactive response.
  • MHC major histocompatibility complex
  • SEA has a Kd for human class II of about 3.2 ⁇ 10 -7 M, SEB of 10 -6 M and TSST-1 of 10 ⁇ 7 M.
  • SEB has a Kd for human class II of about 3.2 ⁇ 10 -7 M
  • SEB of 10 -6 M
  • TSST-1 10 ⁇ 7 M.
  • SEB probably bind to the same site on class II because they cross compete for binding.
  • Exfoliative toxins bind only weakly or not at all to class II.
  • SEB and TSST-1 have different binding sites on class II molecules.
  • the structure of class II consists of two immunoglobulin-like domains located close to the cell membrane which supports a structure constructed from the NH 2 terminal regions of both polypeptides of the protein and comprise an extended ⁇ -pleated sheet supporting two alpha helices separated by a cleft.
  • Peptides derived from foreign materials or from proteolysis of self proteins normally lie in this groove. It is this complex of MHC and peptide that stimulates T cells bearing alpha and beta receptors. Bacterial toxins do not normally bind to MHC
  • Toxins bind to three different class II proteins, namely DR, DP, DQ (or murine I-A, I-E). SEB and TSST-1 bind to DR and DQ alleles but not to DP. Toxin-class II complexes stimulate T cells.
  • DR alleles have different affinities for a few of the toxins most notably SEE.
  • complexes of toxins plus I-E stimulate T cells more efficiently than complexes of toxins with I-A (murine DQ analog).
  • I-A murine DQ analog
  • weak haplotype specificity e.g., toxins bound to I-A k stimulate T cells less well than toxins bound to I-A d or I-A b .
  • Staphylococcus aureus toxins bind more efficiently to human class II proteins than to mouse.
  • a likely location for toxin binding to MHC may be at the sides of class II where 2 wings, the ends of the ⁇ -pleated strands, extend to either side of the proteins.
  • FIG. 3 A hypothetical structure for the complex of class II MHC T cell receptor and Staphylococcal enterotoxins and MHC protein is given in Figure 3.
  • the Figure shows a class II MHC protein, diagrammed according to Bjorkman and co-workers and Brown and co-workers, in contact with a T cell receptor and a staphylococcal enterotoxin or Mls product. Ag is the probable site of binding of a conventional antigenic peptide.
  • Toxins stimulate T cells through V ⁇ binding.
  • T cell receptors for antigenic peptides bound to MHC proteins are made up of 5 clonally variable
  • the toxins stimulate T cells almost exclusively via the V ⁇ region of the T cell receptor. See Table 7 for binding of toxins to T cells bearing various V B receptors.
  • SEA, SED and SEE all stimulate murine T cells bearing V ⁇ 11 and SEE and SED both stimulate human T cells bearing V ⁇ 5.
  • SEB and SECs stimulate mouse T cells bearing members of the V ⁇ 8 family and human T cells positive for V ⁇ 12. The exceptions are as follows:
  • Bacterial toxins and other superantigens do not bind to T cell receptors at those regions involved in binding to conventional antigenic peptides plus MHC.
  • the superantigens engage V ⁇ on an exposed face of V ⁇ or a region predicted to be a ⁇ -pleated sheet and exposed on the side of the T cell receptor. This model predicts that toxins act as clamps engaging the sides of class II and V ⁇ bringing into close proximity the surfaces of the T cell receptor and MHC that would contact each other during T cell recognition of conventional antigens bound in the groove of MHC.
  • T cells from some mice responded well to spleen cells from some other animals even though both responder and stimulator were identical at the MHC.
  • the antigens are called minor lymphocyte stimulating antigens (Mls).
  • Mls minor lymphocyte stimulating antigens
  • a list of the Mls-like products and the V ⁇ s they engage is given in Table 8. Mls products have not yet been found in humans. TABLE 8
  • mice identical and Mls disparate spleen cells in mice.
  • V ⁇ 5 almost regardless of the rest of the structure of the receptor on the T cell. This activity depends on the simultaneous expression by the presenting cell of class II proteins. Some class II products, most notably I-E molecules, present Mls products and bacterial toxins better than others.
  • Mls appear to engage V ⁇ s at the same site on the exposed face of the polypeptide as toxins.
  • Mls products associate with class II and stimulate T cells via V ⁇ much like superantigens but the structure of Mls is unknown.
  • mice expressing Mis products There are consequences for mice expressing Mis products. They cause deletion in the thymus for all prospective T cells bearing V ⁇ S with which they interact. Mice expressing Mls-1 a contain very few T cells bearing V ⁇ 6, 7, 8.2 or 9 and hence are deprived of 20% of their total potential T cell repertoire. Despite this they do not seem to be susceptible to disease.
  • Responding T cells are CD4 + .
  • T cells of many specificities respond. 4. Both elicit responses of T cells expressing receptors having particular V ⁇ gene products.
  • T cells expressing CD8 respond only to proteins degraded within cells; extrinsic proteins are presented by class II MHC to CD4 T cells.
  • T cell responses to protein antigens require all elements of the TCR, whereas those to Mls and SE appear to require only use of certain V ⁇ segments.
  • Mls may represent a protein with homology to Staphylococcal enterotoxins. It has been proposed that the Mls like Staphylococcal enterotoxins directly binds the TCR-CD4 complex via its V p domain and to class II MHC molecules assembling a complex that is highly stimulatory for T cells. Hence, both Mls and Staphylococcal enterotoxins are thought to ligate class II MHC to the TCR:CD4 complex in such a way as to stimulate a large percentage of T cells with restricted V ⁇ usage.
  • Mls antigens such as mycoplasma and mycobacterial antigens.
  • heat shock proteins such as heat shock proteins and the synthetic polypeptides described above.
  • Additional biological properties common to this group include their mitogenic effects, interferon, interleukin and tumor necrosis factor induction activity.
  • enterotoxins contain numerous common steps. On the whole, growth of enterotoxin producing Staphylococcus aureus strains is similar in all cases.
  • the most widely used general medium for the culture of these organisms contains 3% MZ-amine Type A, or MAX, 3% protein hydrolysate powder, 0.00005% thiamine and 0.001% niacin. Optimum yields of the enterotoxins are obtained under controlled fermentation, where pH, temperature and oxygen tension are controlled.
  • enterotoxin B and C 1 and C 2 will be up to several hundred ⁇ grams (toxin)/ml (media), while the yield of other toxins will be only a few ⁇ grams/ml.
  • the producing cells are removed from the medium by centrifugation, and the toxin-containing supernatant is saved. If a large fermentation has been carried out, then the cells and supernatant can be quickly separated using a continuous flow centrifuge.
  • various methods e.g., polyethylene glycol precipitation, or dialysis tubing precipitation, or hollow fiber concentration using membranes with selective
  • molecular weight cutoffs can be used.
  • specific antisera to each of the toxins are used in
  • radioimmunoassays or enzyme labelled immunoassays, hemagglutination, or precipitin reactions.
  • the strain of Staphylococcus aureus, that is used for the production of SEB is e.g., S6 or 10-275.
  • the medium containing the toxin is diluted twice with water adjusted to a pH of 6.4, and AmberLite CG-50 (200 mesh) cation ion-exchange resin is added to the toxin mixture.
  • the toxin is eluted, dialyzed, then reapplied to the CG-50 column again.
  • the eluted toxin is dialyzed, then applied to a column of carboxymethyl cellulose or CM-Sephadex. Unbound proteins are eluted with 0.03 and 0.04 molar sodium phosphate buffer. At this point, the toxin is essentially homogeneous.
  • the SEB may be further subdivided into several isoelectric species using polybuffer 96. Enterotoxin A (SEA.
  • High SEA producers e.g., Staphylococcus aureus 13M-2909 (Source: Dr. John Iandolo, Kansas State University, Manhattan, Kansas) are grown in the general medium that is made 0.2% in glucose.
  • the toxin is eluted and dialyzed.
  • the toxin is then loaded onto a CM-cellulose column and eluted with a linear gradient.
  • the combined fractions are then loaded onto a
  • Enterotoxin D SEP.
  • Staphylococcus aureus 1151M (Source: Dr. John Iandolo, Kansas State University, Manhattan, Kansas) is used for the production of enterotoxin B.
  • the medium is similar to that used for SEA and SEB.
  • Staphylococcus aureus strain FRI-236 (Source: Dr. John landolo, Kansas State University, Manhattan, Kansas) culture supernatant is concentrated and dialyzed. The toxin is then absorbed to a
  • the toxin is eluted in a stepwise fashion and concentrated. It is then chromatographed twice on Sephadex-G-75. To obtain highly purified SEE, it is necessary to chromatograph the toxin once more on G-75 in the presence of 6 molar urea.
  • TSST-la and lb are isolated by one additional electrofocusing step. After focusing TSST-1 on the pH 6-8 gradient, approximately one-half of the
  • Sephadex gel is removed from the anode end.
  • the gel remaining on the cathode end, containing the TSST-1 band is repoured after the addition of two more grams of Sephadex gel and then refocused overnight using the remaining pH gradient.
  • protein bands are located by the zymogen print method. Discrete bands are scraped off the plate and eluted with pyrogen free water from the Sephadex gel.
  • Strain MN8 yields approximately 2 mg of each toxin per liter of culture fluid. For Staphylococcus aureus strains other than MN8, 200 ⁇ g of each toxin is obtained per liter of culture fluid.
  • TSST-1a and lb are proteins which migrate as homogeneous bands in SDS gels to a
  • the removal of enterotoxin from the supernatant is carried out using QAE-Sephadex.
  • the toxin is then eluted batchwise from the ion exchanger and recovered by filtration on a sintered glass funnel. The eluates are
  • This method utilizes fast protein liquid
  • chromatofocusing Mono P column Enterotoxins in media are concentrated and passed over a Sephadex-G- 75 column. The toxin containing fractions are pooled. For C 1 and D, the supernatants are passed over an AmberLite-CG-50 column, as described for SED, and the active fractions pooled. All three toxins are then placed in buffer for chromatofocusing and then separated using the MONO P column FPLC system. Since all of the toxins have isoelectric points in the range of 7 to 9, the polybuffer PBE-96 is used for elution. The purity of SEA, SEC 1 and SED is estimated to be 98, 95 and 80%, respectively. SEA elutes as two peaks at pH 8.8 and 8.6. SEC 1 also elutes as two peaks at pH 8.3 and 7.9, and SED elutes as three peaks at pH 8.6, 8.3 and 8.0.
  • Enterotoxins may also be produced in mutant strains of Staphylococcus aureus by expression of an enterotoxin producing gene in another bacteria or cell. Genetic material which appears to be in the chromosomal plasmid, or phage portion of the bacteria may be used for gene insertion procedures. Complete molecules or fragments with amino acid sequence homology to the parent enterotoxin may be produced with this technology. (Reviewed in landolo, J.J., Annu. Rev. Microbiol., 43, 375, 1989.) Moreover, mutagenic agents such as N-Nitroso compounds are capable of augmenting significantly the production of enterotoxins by some strains of Staphylococcus. Alpha Toxin
  • yeast extract dialysate medium is used and cultured in yeast extract dialysate medium.
  • undialyzed yeast may be used together with casein, glucose, thiamin and nicotinic acid.
  • the organism is incubated in medium for 24h at 37°C.
  • the culture supernatant is applied to a glass-pore bead column and adjusted to pH 6.8.
  • a column of 5 ⁇ 20 cm is used for 3 liter batches and flow rates adjusted to 10-20 ml/min.
  • the column is washed with 0.01M KHPO 4 pH 6.8 and then the alpha toxin is eluted with 1.0M KHPO 4 pH 7.5. Fractions are tested for the presence of alpha hemolysin by a rapid hemolytic assay using rabbit erythrocytes as substrate.
  • Streptococcus NY-5 strain (Source: ATCC 12351) has been the most widely used for toxin production and studies. A list of various strains to produce toxins A, B, and C has been published.
  • the Kalbach S84 type 3 strain (Source: Dr. Joseph E. Alouf, Institute Pasteur-Unite Associee, Paris, France) is cultured and the supernatant is concentrated and stirred in calcium phosphate gel. Fraction S 1 is precipitated with 80% saturated ammonium sulfate. The redissolved pellet is dialyzed and termed
  • Fraction S 2 This fraction is precipitated with 50-80% ammonium sulfate, resuspended in phosphate buffered saline (Fraction S 3 ), and gel filtered on a Bio-Gel P-100 column. The fraction corresponding to the volume eluted between 160 and 240 ml is collected and concentrated by ultrafiltration to about 20 ml in an Amicon PM10 Membrane (Fraction S 4 ). Fraction S4 is then submitted to preparative isoelectric focusing (IEF) performed with a 100 ml column. The material which focuses at around pH 4.8 in a narrow peak is collected and dialyzed in an Amicon cell using PBS to eliminate ampholines and sucrose.
  • IEF isoelectric focusing
  • Fraction (S 5 ) constitutes purified pyrogenic exotoxin.
  • Another electrophoretic form of SPE with a pI of 4.2 is often separated simultaneously with that of pi 4.8. Both forms show total cross reactivity against immune sera raised by rabbit immunization with fraction S 3 .
  • Fraction S 5 shows a single band by SDS-PAGE corresponding to a molecular weight of 28K.
  • Bioassays for determination of activity include erythematosus skin test in rabbits or guinea pigs lymphocyte blast transformation.
  • the toxin may also be detected by enzyme-linked immunoabsorbent assay (ELISA) or hemagglutination inhibition.
  • ELISA enzyme-linked immunoabsorbent assay
  • Staphylococcus aureus strain 110-275 is cultured in NZ-Amine A media supplemented with 10 gm/liter of yeast extract for 18-20 hours in room air at 37° C.
  • the flask is agitated at 300 RPM.
  • the initial pH of the culture is 6.8 and the postincubation pH 8.0.
  • The.culture is filtered through a DC-10 Amicon filter (pore size 0.1 micron).
  • the final filtrate is adjusted to pH 5.6.
  • the filtrate is tested for the presence of SEB in radial immunodiffusion using known antisera to SEB. Eighteen to 20 liters of culture supernatant fluid is diluted with deionized,
  • the column is washed with the same buffer and the enterotoxin eluted by treating the column stepwise with PB 0.03 M pH 6.0, 0.045 M pH 6.25, 0.06 M pH 6.5 and 0.12 M pH 7.2.
  • the fractions containing the enterotoxin are combined, concentrated with polyethylene glycol (200 cc wet volume of packed resin), and dialyzed against 0.5 M NaCl 0.05 M PH pH 7.2.
  • the concentrated enterotoxin solution (5 ml) is placed in a column of Sephacryl S-200 (pretreated with 0.5 M NaCl, 0.05 M PB, pH 7.2).
  • the column is eluted with the same buffer.
  • enterotoxin B concentration is approximately 1 mg/ml.
  • the solution is filter sterilized, frozen and
  • SEB tritiated thymidine mitogenic assay with human and murine immunocytes, SEB showed significant mitogenic activity comparable to that of SEA. SEB was found to be devoid of contaminating alpha hemolysin assessed in a rabbit erythrocyte hemolytic assay.
  • the sterility of the preparation was demonstrated by negative cultures using (a) fluid thioglycollate medium and (b) soybean-casein digest.
  • a sample containing 1 mg/ml of SEB was tested for endotoxin contamination using Sigma E-toxate CAL assay.
  • the final product was found to be free of endotoxin with a standard sensitivity of 0.1 ug endotoxin/mg SEB.
  • Toxieity testing was carried out in two Hartley strain guinea pigs weighing less than 450 grams, and two female C57 black mice (Simonson Laboratories, Watsonville, CA), weighing less than 22 grams. Each animal was observed for 7 days with no significant change in condition or weight after intraperitoneal injection of 0.5 ml of 26 ⁇ g/kg enterotoxin B.
  • SEA, SEC, SED, SEE, TSST-1 and Streptococcal pyrogenic exotoxin in the studies were prepared by the previously described methods. The identity, purity and sterility of these preparations were tested in a fashion similar to that for SEB.
  • CM-SEB carboxymethylated enterotoxin B
  • the vehicle was prepared as follows: To
  • PBS phosphate buffered saline
  • Pluronic 121 5%
  • squalene 5%
  • This mixture was vortexed vigorously to produce a uniform emulsion.
  • One volume of this vehicle mixture was then added to an equal volume of enterotoxin dissolved in PBS and vortexed briefly to ensure complete mixing of components.
  • the final concentrations were (v/v): 0.17% Tween 80, 2.5%
  • Ibuprofen (Sigma, St. Louis, MO) 800 mg was added to solution containing 30 ml of distilled water, 6 ml of 1N N a OH and 50 mg of N a PO 4 . The solution was vortexed vigorously. The pH was adjusted to 7.1-7.8 with 1N HC1 added dropwise. Sterile distilled water was added to a final volume of 40 ml. The solution containing 20 mg/ml of Ibuprofen was stored at -20° C. 6. Animals
  • New Zealand white female rabbits weighing from 2.5 to 5.0 kg, ages 2 to 4 months were used for studies employing purified enterotoxins. Rabbits of higher weight were used in preliminary studies which are discussed in application Serial No. 07/416,530, filed on October 3, 1989. The animals were obtained from the Elkhorn Rabbitry, Watsonville, California.
  • the tumor used for these studies was obtained from the Frederick Cancer Research Facility of the National Cancer Institute. It was stored frozen in the DCT tumor repository.
  • the tumor call lettered G50014 was also known as the VX-2. Stewart, H.L., Snell, K.C., Dunham, L.J. : Transplantable and transmissible tumors of animals. In Atlas of Tumor Pathology. Washington, D.C, Armed Forces Institute of Pathol., pp. 38, 355, 1959.
  • the tumor is a carcinoma indigenous to the New Zealand white rabbit. It was stored as a tissue fragment, and suspended in saline.
  • the tumor was initially induced by Shope virus and derived from a transformed papilloma in a dutch belted rabbit. Kidd and Rous described the tumor in 1937. Histopathologically, the tumor consists of cords and sheets of epithelial cells
  • Tumor fragments for inoculation were obtained from VX-2 growing in rabbit thigh. Fragments were implanted intramuscularly into the right thigh of recipients. Donors were placed under general
  • Tumors were measured by calipers by a certified veterinary oncologist before and at intervals after treatment. Complete remission was present when there was no evident tumor. Partial remission represented a reduction of tumor volume by greater than 50%. Less than partial remission was a 25-50% reduction in tumor volume. 10.
  • enterotoxins Streptococcal pyrogenic exotoxins, carboxymethylated enterotoxin B, or synthetic enterotoxins in lyophilized form were diluted in 0.9% saline or sterile distilled water and then filtered through a 0.45 micron Millipore filter. Aliquots were stored at -20° F. Each aliquot was thawed once, used only for a single injection and then discarded.
  • preparations in appropriate dose were prepared in 1 ml of 0.9% saline and drawn up in a 1 ml syringe. This solution was administered via the central ear vein which was cannulated with a 25 gauge needle and attached infusion tubing
  • tubing and needle were washed with saline using a 3 ml syringe and, with the tubing filled with saline, the toxin infusion was begun using a 1 ml tuberculin syringe (Monoject tuberculin 1.0 cc. Division of Sherwood Medical, St. Louis
  • the tubing and needle were washed with 6 ml of normal saline over an additional 3 minutes using a 3 ml syringe.
  • Purified enterotoxin B in a mean dose of 26 ⁇ g/kg was administered to seven animals on one, two or three occasions (Table 11). Five showed complete remissions while one additional rabbit demonstrated 96% regression. One showed tumor progression. Of the four animals receiving a mean dose of 13 ⁇ g/kg, one had a complete remission while three showed tumor progression. A single animal given a dose of 40 ⁇ g/kg died within 12 hours of injection. Six of eight animals with major regressions showed enduring responses lasting 2 to 6 months without evident tumor recurrence (Table 11).
  • SEA in a dose of 0.9 ⁇ g/kg was given to 5 rabbits on two or three occasions. Two showed complete remissions while three others demonstrated tumor progression (Table 12). and one died acutely after the third injection. SEA in a dose range of 5-12 ⁇ g/kg was administered to 7 animals. Two achieved complete remission while one experienced a 60% remission. Four others died acutely after the first injection.
  • CM-SEB Carboxymethylated Enterotoxin B
  • Magnolia 3.0° -0.14 No anorexia. Normal activity.
  • tumors showed extensive hemorrhagic necrosis in samples obtained 12 to 72 hours after the initial injection.
  • Control untreated tumor showed focal areas of necrosis within the tumor, but no areas of hemorrhagic necrosis Indeed, the areas of necrosis were far more extensive in the treated tumors with few if any areas of viable tumor.
  • small blood vessels In the treated tumors, small blood vessels
  • Tumor bearing rabbits were given two or three injections of SEB, C-SEB, SEA or TSST-1 and showed tumor regressions. It is known that enterotoxins induce production of various cytokines and that one such cytokine namely interferon will in turn
  • lymphocyte activation we may speculate that in the presence of various cytokines induced by the first injection of enterotoxins, upregulated antigen presenting cells are primed to bind
  • enterotoxins might be employed together with various cytokines such as IL-2 in vitro to develop a highly enriched population of T lymphocytes that could subsequently be injected at various intervals to continuously augment the anti-tumor effect in tumor bearing hosts.
  • cytokines such as IL-2
  • the enterotoxins were given intravenously in the present experiments, it is quite conceivable, that the toxins could be administered in adjuvant form bound to vehicles such as aluminum hydroxide, liposomes, water in oil emulsions, pluronic triblock polymers and saponin with similar anti-tumor effects.
  • Ibuprofen (20 mg/ml) given in doses of 0.25 to 0.5 ml subcutaneously when temperatures reached 105°F or greater resulted in reduction in fever by 2 to 5oF.
  • Ibuprofen could be administered every 4 to 6 hours; however, in general, it did not need to be given more than once or twice per 24 hours. The use of this drug did not interfere with the observed tumor reduction or histologic hemorrhagic necrosis.
  • Ibuprofen may inhibit the prostaglandin mediated effects of the inflammatory cytokines including fever and anorexia but does not affect other antitumor immune and inflammatory responses.
  • Ibuprofen is only one of a large group of drugs known as non-steroidal anti-inflammatory agents
  • sticky-ends These sticky-ended fragments can then be ligated to complementary fragments in expression vehicles which have been prepared, e.g., by digestion with the same restriction endonucleases. Having created an expression vector which contains the structural gene of interest in proper orientation with the control elements, one can use this vector to transform host cells and express the desired gene product: with the cellular machinery available. Once expressed, the gene product is generally recovered by lysing the cell culture, if the product is expressed intracellularly, or recovering the product from the medium if it is secreted by the host cell.
  • c .rect expression or the gene product o: interest can be expressed as a fusion protein containing some parts of the amino acid sequence of a homologous protein. This fusion protein is generally processed post-translationally to recover the native gene product. Many of the techniques useful in this technology can be found in Maniatis, T., et al..
  • SEA SEA
  • SEB SEB
  • SEC SEC
  • SEE chromosomal loci
  • the structural gene encoding SED in all strains examined is localized, to a large penicillinase-like plasmid.
  • the enterotoxin A gene has been cloned.
  • SEA was expressed in the E. coli genetic background from a single 2.5 kbp Hind III chromosomal DNA fragment. When sequenced, the DNA was found to contain a single reading frame that generated a protein consistent with the partial sequences of SEA derived by chemical methods. Therefore, it is apparent that the site mapped contained the structural gene for SEA.
  • the enterotoxin A gene was found to be at least 18 kilobases in length and was carried on a mobile element. Enterotoxin A production was linked to the presence of a bacteriophage which integrates into the bacterial chromosome. The enterotoxin A gene is located near the phage attachment. The enterotoxin A gene was mapped between the purine and isoleucine- valine markers in 24 Staphylococcus aureus strains. Conversion to the SEA producing phenotype was induced by lysogenization with a temperate phage purified from staphylococcal aureus strain PS42D. Therefore, a bacteriophage vector was found to be responsible for the toxin phenotype in suitable recipients.
  • the enterotoxin B gene has been cloned and expressed in E. Coli.
  • the DNA of the gene derived from E. Coli has been sequenced and matches the chemically derived sequence with only minor
  • the SEC gene has been cloned from the chromosome of Staphylococcus aureus MN Don.
  • the cloned toxin was expressed in E. coli with a molecular weight comparable to that of the toxin from Staphylococcus aureus.
  • the toxin was biologically active as
  • the enterotoxin D gene has been found to occur on a 27.6 kbp plasmid.
  • the enterotoxin D gene has been cloned an expressed in E. coli and other
  • Staphylococcal strains The enterotoxin D gene in Staphylococcus aureus is under control of the agar locus like most Staphylococcal extracellular protein genes.
  • the DNA sequence was determined encoding a mature protein with amino acid composition and reaction with antibody to SED confirming its identity to the biochemically purified toxin.
  • the enterotoxin E gene has been cloned from S. Aureus FR1918 and was expressed in E. coli encoding an extracellular protein of 26,425 daltons. Its identity to SEE was confirmed immunologically and by correspondence of N terminal and C terminal analysis.
  • TSST-1 gene was not associated with either bacteriophage or plasmid DNA.
  • the gene was cloned on a 10.6 kbp fragment of chromosomal DNA and
  • TSST-1 was expressed in E. Coli, and TSST-1 was secreted into the
  • Hi 555 encodes the tst gene and is a heterologous insertion element that provisionally exhibits some of the characteristics of a transposon. Strains that are Trp- contain Hi555 at regions 17 and 18 (linked to Trp), while strains that are Trp + contain Hi555 elsewhere linked to TryB.
  • Trp- phenotype is not due to insertional inactivation by the unusual element.
  • the sequence and analysis of the tst gene has been described. It codes for a mature protein (TSST-1) of 197 amino acids and a molecular weight of 22,049. Cooney, J., Mulvey, M., Arbuthnott, J.P., Foster, T.J., J. Gen. Microbiol., 134, 2179, 1988.
  • Streptococcal pyrogenic exotoxin (SPEA) is clearly related to the enterotoxins. It has a cysteine loop of 9 amino acids similar to that of SEA and is also encoded by a converting phage. SPEA shows greater amino acid sequence similarity with SEB than SEA. Immunologic studies show that the proteins and antisera to either enterotoxin are cross
  • enterotoxins have been isolated and transfected into other bacteria to obtain selective production. These genes may be used as sources of accelerated
  • Staphylococcus aureus has resulted in a 20 fold increase in enterotoxin production over the amounts produced by the parent Staphylococcus aureus strain. Freedman, M.A., Howard, M.B., J. Bacteriol, 106, 289, 1971.
  • Enterotoxin Genes Genetically Engineered Tumor Cells, Accessory Cells, and Peptides
  • T cell V ⁇ receptors it would be logical to assume that transfection of the enterotoxin gene into tumor cells bearing appropriate HLA-DQ or DR or DP would result in production of a tumor cell bearing the minor lymphocyte stimulating locus capable of
  • the rabbit VX-2 carcinoma cells have been established in tissue culture.
  • the gene for enterotoxins A and B have been isolated and have been made available for these studies by Dr. Marcia Betley and Dr. Saleem Khann, respectively.
  • Plans for transfection of rabbit VX-2 carcinoma cells with both genes have been made with Dr. Susan Faas and Dr. John Mclntyre of Tektagen, Malverne, PA.
  • the transfected cells will then be injected into rabbits bearing the VX-2 carcinoma with appropriate controls consisting of non-transfected rabbit VX-2 carcinoma cells and VX-2 cells transfected with an irrelevant microbial gene. Anti-tumor effects will be assessed in this system.
  • the toxin gene transfected tumor cells could be used for in vitro stimulation of host immunocytes prior to or coordinate with the addition of interleukin 2 to produce an enriched population of tumor specific T cells which could then be reinfused into a tumor bearing host and would be expected to exert tumor killing effects.
  • the enterotoxin gene could be used to transfect various accessory cells resulting in enterotoxin expression on the cell surface which may then induce more potent stimulation and proliferation of
  • T lymphocytes tumoricidal T lymphocytes.
  • the cotransfection of these accessory cells with adhesion molecules and MHC molecules might further augment the mitogenic activity of T lymphocytes induced by these accessory cells.
  • Mutant genes of the toxins could be used to transfect various bacteria such as E. Coli resulting in the production of toxin peptides retaining
  • Such superantigen peptides might have sequences homologous with various naturally occurring viruses such as mammary tumor virus, endogenous proteins such as heat shock proteins, stress proteins and minor lymphocyte stimulating loci, naturally occurring bacteria such as mycoplasma and mycobacterial species.
  • Amino acid sequences in the native toxin molecules associated with toxieity such as emesis, excessive cytokine induction or humoral antibody production would be deleted.
  • histadine residues of SEB may account for emetic responses of the SEB molecule since
  • these genetically engineered molecules might be used to block or eliit nate autoimmune responses induced by proliferation of clones of immunocytes reactive to self constituents such as basic myelin protein in multiple sclerosis or synovial constituents in reheumatoid arthritis.
  • enterotoxin genes would be fused with genes from other bioreacive compounds such as cell poisons to produce molecules with capacity to destroy a selective cell population.
  • fusion peptides might include enterotoxin sequences fused, for example, with peptides of pseudomonas toxin,
  • diphtheria toxin sequences or antibodies yielding complexes retaining the major structural, biologic features of the native proteins.
  • Streptococcal pyrogenic exotoxin or scarlet fever toxin is related to Staphylococcus aureus enterotoxin B.
  • the amino acid sequence of SPE has significant homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library.
  • Figure 2 shows the alignment of amino acid sequences of mature SPEA and Staphylococcus aureus enterotoxin B, as reported in Johnson, L.P., L'ltalien, J.J. and
  • SPE Staphylococcal enterotoxins
  • SPE activates murine T cells mainly V ⁇ 8.2 in physical association with MHC class II molecules expressed on accessory cells. SPE causes deregulation of the immune response in vitro
  • enterotoxin B when administered to rabbits with the VX-2 carcinoma as demonstrated herein.
  • SPE has now been shown to induce a toxic shock like syndrome identical to that associated with various enterotoxins. Given the biological and structural relatedness of these proteins, it would be
  • a hybrid molecule was synthesized representing structures common to both enterotoxins A and B.
  • the molecule contained 26 amino acids and had many structural features as delineated above.
  • This hybrid was administered both intravenously and in adjuvant form to tumor bearing hosts, namely rabbits with VX-2 carcinoma.
  • the adjuvant used for these studies was the pluronic acid triblock
  • copolymer which has been used to boost the immune response to various antigens in animal models and which is under testing at this point in humans with hepatitis and herpe; simplex infections. While we have used this adjuvant specifically, it is
  • adjuvant-vehicle preparations including those prepared in water and oil emulsion and aluminum hydroxide.
  • enterotoxin hybrid molecules containing amino acid sequences homologous to the enterotoxin family would also be effective in this system.
  • mammary tumor virus sequences, heat shock proteins, stress peptides, mycoplasma and mycobacterial antigens and minor lymphocyte stimulating loci bearing tumoricidal structural homology to the enterotoxin family would also be useful in this application as anti-tumor agents.
  • Hybrid enterotoxins and other sequences homologous to the native enterotoxins might be immobilized or polymerized genetically or
  • enterotoxins can activate the autoimmune response.
  • SED is now known to stimulate the production of human rheumatoid factor and mycoplasma arthritidis a well-known superantigen is recognized as the causative agent in murine adjuvant arthritis.
  • various other diseases such as multiple sclerosis are caused by the activation of T lymphocytes (bearing V ⁇ receptors) with specificity for multiple self components.
  • T lymphocytes bearing V ⁇ receptors
  • the receptors for activation of T lymphocytes could be readily blocked by various enterotoxin fragments which retain specificity for the T cell receptor but do not initiate T cell activation or mitogenesis.
  • enterotoxins possess multiple amino acid motifs that are avid for various portions of the T cell V ⁇ repertoire. These sequences on the N or C terminal portion of the molecules would bind to autoreactive T lymphocytes and therefore inactivate these clones by blocking further antigenic stimulation and
  • cellular toxins attached to the enterotoxin fragments could also be used to eliminate such autoreactive clones.
  • enterotoxins are as potent
  • superantigens may be employed for stimulation of protective anti idiotype B and T cell clones
  • a peptide fragment in SEC was shown by Spero and Morlock to contain the active sites for emesis and diarrhea.
  • the mitogenic region resided in the C terminal tryptic fragment of SEC.
  • An immune functional site on Staphylococcal enterotoxin A has been identified corresponding to residues 1-27 of SEA which is responsible for stimulation of T cell proliferation and induction of interferon-y.
  • This SEA (1-27) sequence corresponds to N-Ser-GIv-Lys-Ser-Glu-Glu-Ile-Asn-GFlu-Lys-Asp-Lev.Arg Lys-Lys-Ser-Glu-Leu-Gln-Gly-Thr-Ala-Lev-Gly-Asn-Lev-Ly and blocks SEA induced T cell
  • SEA 28-48 peptide
  • a functional site on SEA responsible for modulation of T cell function involves the N-terminal 27 amino acids. These molecules may interact at either the level of TCR or the binding of SEA to class II MHC antigens.
  • TSST-1 mitogenic activity was shown to be located on a 14,000 dalton cyanogen bromide generated toxin fragment.
  • intramucosal or intradermal ganglion cells and the effect on mast cells is indirectly mediated by neuropeptides.
  • Carboxymethylation of histidine residues of SEB caused a complete loss of emetic and skin sensitizing activity without changing the immunological specificity, e.g., T cell stimulating activity.
  • An anti-idiotype monoclonal antibody against the combining site of an anti-SEB monoclonal antibody had no enterotoxic activity but can inhibit the enterotoxic activity, e.g., emetic response and diarrhea of a 10,000 molar excess of SEB.
  • Anti-idiotype antibody also inhibited immediate-type skin reactions as well.
  • the anti-idiotype antibody and carboxymethylated enterotoxins may be useful tools to protect against the enterotoxin induced intestinal toxieity.
  • Staphylococcal enterotoxins to account for their superantigenic properties.
  • these include the mammary tumor virus, minor lymphyocyte
  • tumoricidal effects could be accomplished with biologically active superantigen peptides, intact enterotoxins or superantigens alone or attached to antigen presenting cells (class II MHC, HLA-DR) and incubated ex vivo with a random T cell population or one which may have been pre- enriched for the appropriate V ⁇ receptor.
  • the activated T cell population with bound enterotoxin might then be reinfused into the host. Similar tumoricidal effects would be anticipated with
  • enterotoxins or biologically active fragments infused into a host who has had an "organoid" an enriched T lymphocyte organ
  • an "organoid" an enriched T lymphocyte organ
  • Antibodies specific for various enterotoxins have been documented to be present in the plasma of humans. Theoretically, these naturally occurring antibodies could neutralize injected enterotoxins and accelerate their removal from the circulation.
  • antibodies could combine with injected enterotoxins and create immunogenic antigen-antibody complexes.
  • enterotoxins To circumvent the presence of antibodies in the circulation, we have explored several methods of administering enterotoxins as follows: First, we have administered enterotoxins to several VX-2 bearing rabbits in adjuvant-vehicle form with slow release properties. Second, we have initiated a collaboration with Dr. Suyu Schu to evaluate the use of enterotoxins in an ex vivo mode, e.g., incubation of entertoxins with T lymphocytes in the presence of IL-2 with resultant enrichment and expansion of T cells and subsequent reinfusion into the tumor bearing host. Such studies are presently underway.
  • a state of tolerance could be induced.
  • the plasma could be cleared of antibodies in advance of intravenous administration of the native toxins.
  • Non-immunogenic hybrid molecules or fragments of enterotoxins could be injected into antibody bearing hosts to neutralize existing circulating antibodies to the enterotoxins prior to administration of the native molecule. Such an approach is presently being tested in tumor bearing hosts.

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Abstract

Des entérotoxines staphylococciques obtenues par la secrétion du staphylocoque doré, par l'expression d'entérotoxines dans d'autres bactéries ou cellules, ou par traitement mutagène chimique de souches du staphylocoque doré, sont utilisées dans le traitement du cancer comme agents tumoricides. Les entérotoxines A, B, C, D, E et la toxine de choc toxique (TSST-1) peuvent être administrées par de simples injections intraveineuses ou sous la forme d'adjuvants tels que des copolymères tribloc pluroniques. Les entérotoxines peuvent également être utilisées ex-vivo pour provoquer la mitogénèse, et pour agrandir et enrichir une population tumoricide de lymphocytes T. Des agents non stéroïdiens, anti-inflammatoires tels que l'ibuprofène peuvent être simultanément administrés pour atténuer les réactions toxiques provenant des entérotoxines. L'exotoxine pyrogène de Streptococcus et l'hémolysine alpha, qui ont une homologie structurelle et fonctionnelle par rapport aux entérotoxines, sont également utiles dans le traitement tumoricide. On décrit également dans cette invention le gène de l'entérotoxine qui vient transfecter des cellules tumorales, produisant ainsi une cellule tumorale dont l'expression de surface du petit locus de stimulation des lymphocytes entraîne à un niveau élevé l'activation et la prolifération des lymphocytes T, en particulier ceux à spécificité V-bêta.
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