The presence of tumor nodules in organs often results in serious clinical manifestations and the permeation by cancer cells of sheaths surrounding organs often produces clinical manifestations of pleural effusion, ascites or cerebral edema. The present invention addresses this problem by providing a method for treating tumors comprising (a) intratumoral administration of a superantigen and/or (b) intrathecal or intracavitary administration of a superantigen directly into the sheath. Intratumoral superantigen results in significant and sustained reduction of the tumor size. Intrathecal administration produces significant sustained reduction of the fluid accumulation associated with clinical improvement and prolonged survival. Useful superantigen compositions for intrathecal and intratumoral injection include tumoricidally effective homologues, fragments and fusion proteins of native superantigens. Also disclosed is combined therapy that includes intratumoral or intrathecal superantigen compositions in combination with (i) intratumoral low, non-toxic doses of one or more chemotherapeutic drugs or (ii) systemic chemotherapy at reduced and non-toxic doses of chemotherapeutic drugs.

Description of the Invention

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Production and Isolation of Superantigens

The SAgs disclosed herein are prepared by either biochemical isolation, or, preferably by recombinant methods. The following SAgs, including their sequences and biological activities have been known for a number of years. Studies of these SAgs are found throughout the biomedical literature. For, biochemical and recombinant preparation of these SAgs, see the following references: Borst, D W et al., Infect. Immun. 61: 5421-5425 (1993); Couch, J L et al., J. Bacteriol. 170: 2954-2960 (1988); Jones, C L et al., J. Bacteriol. 166: 29-33 (1986); Bayles K W et al., J. Bacteriol. 171: 4799-4806 (1989); Blomster-Hautamaa, D A et al., J. Biol. Chem. 261:15783-15786 (1986); Johnson, L P et al., Mol. Gen. Genet. 203, 354-356 (1986); Bohach G A et al., Infect. Immun. 55: 428-433 (1987); Iandolo J J et al., Methods Enzymol 165:43-52 (1988); Spero L et al., Methods Enzymol 78(Pt A):331-6 (1981); Blomster-Hautamaa D A, Methods Enzymol 165: 37-43 (1988); Iandolo J J Ann. Rev. Microbiol. 43: 375-402 (1989); U.S. Pat. No. 6,126,945 and U.S. provisional patent application 60/389,366 filed Jun. 15, 2002. These references and the references cited therein are hereby incorporated by reference in their entirety.

These SAgs are Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C (SEC--actually three different proteins, SEC1, SEC2 and SEC3)), Staphylococcal enterotoxin D (SED), Staphylococcal enterotoxin E (SEE) and toxic shock syndrome toxin-1 (TSST-1) (U.S. Pat. No. 6,126,945 and U.S. provisional patent application 60/389,366 filed Jun. 15, 2002, and the references cited therein). The amino acids sequences of the above group of native (wild-type) SAgs is provided below -- see Original Patent.

The sections which follow discuss SAgs which have been discovered and characterized more recently.

Staphylococcal Enterotoxins SEG, SEH, SEI, SEJ, SEK, SEL, SEM

New Staphylococcal enterotoxins G, H, I, J, K, L and M (SEG, SEH, SEL, SEJ, SEK, SEL, SEM; abbreviated below as "SEG-SEM") were described in Jarraud, S. et al., J. Immunol. 166: 669-677 (2001); Jarraud S et al., J. Clin. Microbiol. 37: 2446-2449 (1999) and Munson, S H et al., Infect. Immun. 66:3337-3345 (1998). SEG-SEM show SAgic activity and are capable of inducing tumoricidal effects. The homology of these SE's to the better known SE's in the family ranges from 27-64%. Each induces selective expansion of TCR V.beta. subsets Thus, these SEs retain the characteristics of T cell activation and V.beta. usage common to all the other SE's.

SEG and SEH of this group and other enterotoxins including SPEA, SPEC, SPEG, SPEH, SME-Z, SME-Z.sub.2, (see below) utilize zinc as part of high affinity MHC class II receptor. Amino acid substitution(s) at the high-affinity, zinc-dependent class II binding site are created to reduce their affinity for MHC class II molecules.

Jarraud S et al., 2001, supra, discloses methods used to identify and characterize SEG-SEM, and for cloning and recombinant expression of these proteins. These investigators have used a number of TCR-specific mAbs (V.beta. specificity indicated in brackets) for flow cytometric analysis: E2.2E7.2 (V.beta.2), LE89 (V.beta.3), IMMU157 (V.beta.5.1), 3D11 (V.beta.5.3), CRI304.3 (V.beta.6.2), 3G5D15 (V.beta.7), 56C5.2 (V.beta.8.1/8.2), FIN9 (V.beta.9), C21 (V.beta.{umlaut over ({acute over ()}1), S511 (V.beta.12), IMMU1222 (V.beta.13.1), JIJ74 (V.beta.13.6), CAS1.1.13 (V.beta.14), Tamayal.2 (V.beta.16), E17.5F3 (V.beta.17), .beta.A62.6 (V.beta.18), ELL1.4 (V.beta.20), IG125 (V.beta.21.3), IMMU546 (V.beta.22), and HUT78.1 (V.beta.23).

Jarraud S et al., 2001, supra, indicates that the seven genes and pseudogenes composing the egc (enterotoxin gene cluster) operon are co-transcribed. The association of related co-transcribed genes suggested that the resulting peptides might have complementary effects on the host's immune response. One hypothesis was that gene recombination created new SE variants differing by their SAg activity profiles. Purified recombinant SEL, SEM, SEI, SEK, and SEGL29P (a mutant of SEL) were expressed in E. coli and analyzed. Recombinant SEL SEM, SEL and SEK consistently induced selective expansion of distinct subpopulations of T cells expressing particular V.beta. genes. By contrast. SEGL29P failed to trigger expansion of any of 23 V.beta. subsets, and the L29P mutation accounted for the complete loss of SAg activity (although this mutation did not induce a major conformational change). It is believed that this substitution mutation located at a position crucial for proper SAg/MHC II interaction.

Flow cytometry revealed preferential expansion of CD4 T cells in SEI and SEM cultures. By contrast, the CD4/CD8 ratios in SEK-, SEL-, and SEG-stimulated T cell lines were close to those in fresh PBL. Overall, TCR repertoire analysis confirm the SAgic nature of SEG-SEM.

The amino acid sequences of SEG-SEM are shown below -- see Original Patent.

Streptococcal Pyrogenic Exotoxins (SpEs)

The SpE's SPEA, SPEB, SPEC, SPEG, SPEH, SME-Z, SME-Z.sub.2 and SSA are SAgs induce tumoricidal effects. SPEA, SPEB, SPEC have been known for some time and their structures and biological activities described in numerous publications.

SPEG, SPEH, and SPEJ genes were identified from the Streptococcus pyogenes M1 genomic database and described in detail in Proft, T et al., J. Exp. Med. 189: 89-101 (1999) which also describes SMEZ, SMEZ-2. This document also describes the cloning and expression of the genes encoding these proteins.

The smez-2 gene was isolated from the S. pyogenes strain 2035, based on sequence homology to the streptococcal mitogenic exotoxin z (smez) gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ and SPEC, whereas SPEH is most similar to the SEs than to any other streptococcal toxin.

As described by Proft, T et al supra, rSMEZ, rSMEZ-2, rSPE-G, and rSPE-H were mitogenic for human peripheral blood T lymphocytes. SMEZ-2 appears to be the most potent SAg discovered thus far.

All these toxins, except rSPE-G, were active on murine T cells, but with reduced potency.

Binding to a human B-lymphoblastoid line was shown to be zinc dependent with high binding affinity of 15-65 nM. Analysis of competition for binding between toxins of this group revealed overlapping but discrete binding to subsets of class II molecules in the hierarchical order (SMEZ, SPE-C)>SMEZ-2>SPE-H>SPE-G. The most common targets for these SAgs were human V.beta.2.1- and V.beta.4-expressing T cells.

Streptococcus Pyrogenic Exotoxin A (SPEA)

SPEA can be purified from cultures of S. pyogenes as described by Kline et al., Infect. Immun. 64:861-869 (1996). Plasmids that include the spea1 gene which encode SPEA, and the expression and purification of recombinant SPEA ("rSPEA") are described by Kline et al., supra. The native SPEA sequence is shown below -- see Original Patent.

Streptococcus Pyrogenic Exotoxin B (SPEB)

Purification of native SpEB is described by Gubba, S. et al., Infect. Immun. 66: 765-770 (1998). Expression and purification of recombinant SpEB are also described in this reference. The native SPEB sequence is shown below (Kapur, V. et al., Microb. Pathog. 15:327-346 (1993)) -- see Original Patent.

Streptococcus Pyrogenic Exotoxin C(SPEC)

Methods of isolation and characterization of SPEC is carried out by the methods of Li, P L et al., J. Exp. Med. 186: 375-383 (1997). These references also describe T cell proliferation stimulated by this SAg and the analysis of its selectivity for TCR V.beta. regions. The native sequence of SPEC (Kapur, V. et al., Infect. Immun. 60:3513-3517 (1992) is shown below -- see Original Patent.

Streptococcal Superantigen (SSA)

SSA is an .about.28-kDa SAg protein isolated from culture supernatants as described by Moflick J et al., J. Clin. Invest. 92: 710-719 (1993) and Reda K et al., Infect. Immun. 62: 1867-1874 (1994). SSA stimulates proliferation of human T cells bearing V.beta.1, V.beta.3, V.beta.5.2, and V.beta.15 in an MHC class II-dependent manner. The first 24 amino acid residues of SSA are be 62.5% identical to SEB, SEC1, and SEC3. Purification and cloning of SSA is described in Reda K et al., Infect. Immun. 62: 1867-1874 (1994). The native sequence of SSA (Reda, K. B. et al., Infect. Immun. 64: 1161-1165 (1996)) is shown below -- see Original Patent.

Streptococcal Pyrogenic Exotoxins G and H and SMEZ

The sequences of the more recently discovered Streptococcal exotoxin SAgs are provided below -- see Original Patent.

Yersinia pseudotuberculosis Mitogen (Superantigen) (YPM)

Cloning, expression and purification of YPM is described by Miyoshi-Akiyama, T. et al., J. Immunol. 154: 5228-5234 (1995).

The above reference described assays of YPM using lymphoid cells and murine L cells transfected with human HLA genes, including T cell proliferation and cytokine (IL2) secretion. The sequence of YPM is shown below -- see Original Patent.

Staphylococcal Exotoxin like Proteins (SET)

The identification characterization of the SETs (SET-1 and SET-2) and the cloning and purification of SET-1 is described in Williams, R. J. et al., Infect. Immun. 68: 4407-4414 (2000). This reference discloses the distribution of the set1 gene among Staphylococcal species and strains.

The set1 nucleotide sequences are deposited in the GenBank database under accession numbers AF094826 (set gene cluster fragment), AF188835 (NCTC 6571 set1 gene), AF188836 (FRI326 set1 gene), and AF188837 (NCTC 8325-4 set1 gene). Recombinant SET-1 protein stimulates production of the proinflammatory cytokines IL-1, IL-6, and TNF.alpha.

Preferred Form of Superantigen for Therapeutic Use

A preferred construct for intrathecal and intrapleural use comprises a SAg in native form. In contrast, for systemic use the preferred SAg is one to which humans do not make or make only marginal amounts neutralizing antibody fused recombinantly or biochemically to a high affinity tumor specific antibody, Fab or single chain Fv. To this end, SAg epitopes in the conjugate which bind endogenous (to include preexisting) SAg specific antibodies are deleted and/or substituted by alanine or amino acid sequences to which the host does not have preexisting antibodies. For example, a dominant epitope on SEB recognized by anti-SEB antibodies is the sequence 225-234 (Nishi et al., J. Immunol. 158: 247-254 (1997) and an epitope on SEA recognized by anti-SEA antibodies is the sequence 121-149 (Hobieka et al., Biochem. Biophys. Res. Comm. 223: 565-571 (1996). Alternatively, SAgs such as Y. pseudotuberculosis or C. perfringens toxin A or to which humans do not have preexisting antibodies are used. Y. pseudotuberculosis SAg has, in addition, a natural RGD domain which has tumor-localizing properties.

Functional Homologues and Derivatives of Superantigen Proteins of Peptides

The present invention contemplates, in addition to native SAgs, the use of homologues of native SAgs that have the requisite biological activity to be useful in accordance with the invention.

Thus, in addition to native SAg protein and nucleic acid compositions described herein, the present invention encompasses functional derivatives, among which homologues are preferred. Thus, biologically active homologues of staphylococcal enterotoxins, streptococcal exotoxins. Y. pseudotuberculosis SAg YPM, C. perfringens toxin A, M. arthritides SAgs are included herein.

By "functional derivative" is meant a "fragment," "variant," "mutant," "homologue," "analogue," or "chemical derivative". Homologues include fusion proteins, chimeric proteins and conjugates that include a SAg portion fused to or conjugated to a fusion partner polypeptide or peptide. A functional derivative retains at least a portion of the biological activity of the native protein which permits its utility in accordance with the present invention. Such biological activity includes stimulation of T cell proliferation and/or cytokine secretion, stimulation of T cell-mediated cytotoxic activity, as a result of interactions of the SAg composition with T cells preferably via the TCR V.beta. region.

A "fragment" refers to any shorter peptide. A "variant" of refers to a molecule substantially similar to either the entire protein or a peptide fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well-known in the art.

A homologue refers to a natural protein, encoded by a DNA molecule from the same or a different species. Homologues, as used herein, typically share at least about 50% sequence similarity at the DNA level or at least about 18% sequence similarity at the amino acid level, with a native protein.

An "analogue" refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof

A "chemical derivative" contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

A fusion protein comprises a native SAg, a fragment or a homologue fused by recombinant means to another polypeptide fusion partner, optionally including a spacer between the two sequences. Preferred fusion partners are antibodies, Fab fragments, single chain Fv fragments. Other fusion partners are any peptidic receptor ligand, cytokine, extracellular domain ("ECD") of a costimulatory molecule and the like.

The recognition that the biologically active regions of the SEs, for example, are substantially homologous, i.e., that the sequences are substantially similar, enables prediction of the sequences of synthetic peptides which will exhibit similar biological effects in accordance with this invention (Johnson, L. P. et al., Mol. Gen. Genet. 203:354-356 (1986).

The following terms are used in the disclosure of sequences and sequence relationships between two or more nucleic acids or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity"

As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or other polynucleotide sequence, or the complete cDNA or polynucleotide sequence. The same is the case for polypeptides and their amino acid sequences.

As used herein, "comparison window" includes reference to a contiguous and specified segment of a polynucleotide or amino acid sequence, wherein the sequence may be compared to a reference sequence and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides or amino acids in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences for comparison are well-known in the art. For comparison, optimal alignment of sequences may be done using any suitable algorithm, of which the following are examples: (a) the local homology algorithm ("Best Fit") of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); (b) the homology alignment algorithm (GAP) of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); or (c) a search for similarity method (FASTA and TFASTA) of Pearson and Lipman, Proc. Natl. Acad. Sci. 85 2444 (1988);

In a preferred method of alignment, Cys residues are aligned. Computerized implementations of these algorithms, include, but are not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG) (Madison, Wis.). The CLUSTAL program is described by Higgins et al., Gene 73:237-244 (1988); Higgins et al., CABIOS 5:151-153 (1989); Corpet et al., Nuc Acids Res 16:881-90 (1988); Huang et al., CABIOS 8:155-65 (1992), and Pearson et al., Methods in Molecular Biology 24:307-331 (1994).

A preferred program for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, J Mol Evol 25:351-360 (1987) which is similar to the method described by Higgins et al. 1989, supra).

The BLAST family of programs which can be used for database similarity searches includes: NBLAST for nucleotide query sequences against database nucleotide sequences; XBLAST for nucleotide query sequences against database protein sequences; BLASTP for protein query sequences against database protein sequences; TBLASTN for protein query sequences against database nucleotide sequences; and TBLASTX for nucleotide query sequences against database nucleotide sequences. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, Chapter 19, Greene Publishing and Wiley-Interscience, New York (1995) or most recent edition. Unless otherwise stated, stated sequence identity/similarity values provided herein, typically in percentages, are derived using the BLAST 2.0 suite of programs (or updates thereof) using default parameters. Altschul et al., Nuc Acids Res. 25:3389-3402 (1997).

As is known in the art, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequence which may include homopolymeric tracts, short-period repeats, or regions rich in particular amino acids. Alignment of such regions of "low-complexity" regions between unrelated proteins may be performed even though other regions are entirely dissimilar. A number of low-complexity filter programs are known that reduce such low-complexity alignments. For example, the SEG (Wooten et al., Comput. Chem. 17:149-163 (1993)) and XNU (Clayerie et al., Comput. Chem, 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. It is recognized that when using percentages of sequence identity for proteins, a residue position which is not identical often differs by a conservative amino acid substitution, where a substituting residue has similar chemical properties (e.g., charge, hydrophobicity, etc.) and therefore does not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the % sequence identity may be adjusted upwards to correct for the conservative nature of the substitution, and be expressed as "sequence similarity" or "similarity" (combination of identity and differences that are conservative substitutions). Means for making this adjustment are well-known in the art. Typically this involves scoring a conservative substitution as a partial rather than as a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of "1" and a non-conservative substitution is given a score of "0" zero, a conservative substitution is given a score between 0 and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers et al., CABIOS 4:11-17 (1988) as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

As used herein, "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the nucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which lacks such additions or deletions) for optimal alignment, such as by the GAP algorithm (supra). The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing that number by the total number of positions in the window of comparison and multiplying the result by 100, thereby calculating the percentage of sequence identity.

The term "substantial identity" of two sequences means that a polynucleotide or polypeptide comprises a sequence that has at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence identity to a reference sequence using one of the alignment programs described herein using standard parameters. Values can be appropriately adjusted to determine corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

One indication that two nucleotide sequences are substantially identical is if they hybridize to one other under stringent conditions. Because of the degeneracy of the genetic code, a number of different nucleotide codons may encode the same amino acid. Hence, two given DNA sequences could encode the same polypeptide but not hybridize under stringent conditions. Another indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Clearly, then, two peptide or polypeptide sequences are substantially identical if one is immunologically reactive with antibodies raised against the other. A first peptide is substantially identical to a second peptide, if they differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that nonidentical residue positions may differ by conservative substitutions.

Thus, in one embodiment of the present invention, the Lipman-Pearson FASTA or FASTP program packages (Pearson, W. R. et. al., 1988, supra; Lipman, D. J. et al, Science 227:1435-1441 (1985)) in any of its older or newer iterations may be used to determine sequence identity or homology of a given protein, preferably using the BLOSUM 50 or PAM 250 scoring matrix, gap penalties of -12 and -2 and the PIR or SwissPROT databases for comparison and analysis purposes. The results are expressed as z values or E ( ) values. To achieve a more "updated" z value cutoff for statistical significance, preferably corresponding to a z value>10 based on the increase in database size over that of 1988, in a FASTA analysis using the equivalent 2001 database, a significant z value would exceed 13.

A more widely used and preferred methodology determines the percent identity of two amino acid sequences or of two nucleic acid sequences after optimal alignment as discussed above, e.g., using BLAST. In a preferred embodiment of this approach, a polypeptide being analyzed for its homology with native SAg is at least 20%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% as long as the reference sequence. The amino acid residues (or nucleotides) at corresponding positions are then compared. Amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology".

In a preferred comparison of a putative SAg homologue polypeptide and a native SAg protein, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch alignment algorithm (incorporated into the GAP program in the GCG software package (available at the URL www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between the encoding nucleotide sequences is determined using the GAP program in the GCG software package (also available at above URL), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the algorithm of Meyers et al., supra (incorporated into the ALIGN program, version 2.0), is implemented using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The wild-type (or native) SAg-encoding nucleic acid sequence or the SAg protein sequence can further be used as a "query sequence" to search against a public database, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs, supra (see Altschul et al (1990) J. Mol. Biol. 215:403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to identify nucleotide sequences homologous to native SAgs. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to identify amino acid sequences homologous to identify polypeptide molecules homologous to a native SAg. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, supra). Default parameters of XBLAST and NBLAST can be found at the NCBI website (www.ncbi.nlm.nih.gov)

Using the FASTA programs and method of Pearson and Lipman, a preferred SAg homologue is one that has a z value >10. Expressed in terms of sequence identity or similarity, a preferred SAg homologue for use according the present invention has at least about 20% identity or 25% similarity to a native SAg. Preferred identity or similarity is higher. More preferably, the amino acid sequence of a homologue is substantially identical or substantially similar to a native SAg sequence as those terms are defined above.

One group of substitution variants (also homologues) are those in which at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G. E. Principles of Protein Structure Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions which may be made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 (see Original Patent) of Schulz et al. (supra) and FIG. 3-9 (see Original Patent) of Creighton (supra). Based on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His, kg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. Pro, because of its unusual geometry, tightly constrains the chain. Cys can participate in disulfide bond formation which is important in protein folding. Tyr, because of its hydrogen bonding potential, has some kinship with Ser, Thr, etc.

More substantial changes in functional or immunological properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups, which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Examples of such substitutions are (a) substitution of gly and/or pro by another amino acid or deletion or insertion of Gly or Pro; (b) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g., Leu, Ile, Phe, Val or Ala; (c) substitution of a Cys residue for (or by) any other residue; (d) substitution of a residue having an electropositive side chain, e.g., Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g., Glu or Asp; or (e) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.

The deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays, for example direct or competitive immunoassay or biological assay of T cell function as described herein. Modifications of such proteins or peptide properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assessed by methods well known to the ordinarily skilled artisan.

Chemical Derivatives

Covalent modifications of the SAg proteins or peptide fragments thereof, preferably of SEs or peptide fragments thereof, are included herein. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the protein or peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. This may be accomplished before or after polymerization.

Cysteinyl residues most commonly are reacted with a-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing a-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides as noted above. Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

Superantigen Homologues

The variants or homologues of native SAg proteins or peptides including mutants (substitution, deletion and addition types), fusion proteins (or conjugates) with other polypeptides, are characterized by substantial sequence homology to (a) the long-known SE's --SEA, SEB, SEC.sub.1-3, SED, SEE and TSST-1; (b) long-known SpE's; (c) more recently discovered SE's (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SETs 1-5); or (d) non-enterotoxin superantigens (YPM, M. arthritides superantigen). Preferred homologues were disclosed above.

Table 1 (see Original Patent) lists a number of native SEs and exemplary homologues (amino acid substitution, deletion and addition variants (mutants) and fragments) with z values>10 (range: z=16 to z=136) using the Lipman-Pearson algorithm and FASTA. These homologues also induce significant T lymphocyte mitogenic responses that are generally comparable to native SE's.

In addition, as shown in Table 2 (see Original Patent), several of these homologues also promote antigen-nonspecific T lymphocyte killing in vitro by a mechanism termed "superantigen-dependent cellular cytotoxicity" (SDCC) or, in the case of SAg-mAb fusion proteins, "superantigen/antibody dependent cellular cytotoxicity" (SADCC).

According to the present invention, other SE homologues (e.g., z>10 or, in another embodiment, having at least about 20% sequence identity or at least about 25% sequence similarity when compared to native SEs), exhibiting T lymphocyte mitogenicity, SDCC or SADCC, are useful anti-tumor agents when administered to a tumor bearing host via any intrathecal route.

Tumors in Sheaths Encasing Organs

The appearance of tumors in sheaths ("theca") encasing an organ often results in production and accumulation of large volumes of fluid in the organ's sheath. Examples include (1) pleural effusion due to fluid in the pleural sheath surrounding the lung, (2) ascites originating from fluid accumulating in the peritoneal membrane and (3) cerebral edema due to metastatic carcinomatosis of the meninges. Such effusions and fluid accumulations generally develop at an advanced stage of the disease.

Intrathecal Superantigens for Treatment of Malignant Ascites and Malignant Pleural Effusions

The present invention contemplates the use of any SAg or SET in any form. This includes but is not limited to staphylococcal enterotoxins A, B, C, D, E, F, G H, I, J, K, L, M, SpE's, YPM, M. arthritides SAg, C. perfringens exotoxin for direct administration into cavities or spaces, e.g., peritoneum, thecal space, pericardial and pleural space containing tumor.

The present invention contemplates the direct administration of any SAg (SEA, SEB and SEC are preferred) into a fluid space containing tumor cells or adjacent to membranes such as pleural, peritoneal, pericardial and thecal spaces containing tumor. These sites display malignant ascites, pleural and pericardial effusions or meningeal carcinomatosis. The SAg is preferably administered after partial or complete drainage of the fluid (e.g., ascites, pleural or pericardial effusion) but it may also be administered directly into the undrained space containing the effusion, ascites and/or carcinomatosus. In general, the SE dose may vary from 1 picogram to 10 .mu.g and given every 3 to 10 days. It is continued until there is no reaccumulation of the ascites or effusion. Therapeutic responses are considered to be no further accumulation of four weeks after the last intrapleural administration. See Example 1 for further description of treatment.

Fusion Partners for Native SEs or SE Homologues

Antibodies

Fusion protein partners for the SAg include tumor specific antibodies, preferably F(ab').sub.2, Fv or Fd fragments thereof, that are specific for antigens expressed on the tumor. In another embodiment, a fusion partner is a polypeptide ligand f for a receptor expressed on tumor cells. These antibodies, fragments or receptor ligands may be in the form of synthetic polypeptides. The nucleic acid form of the antibody is envisioned which is useful as a fusion construct with the SAg DNA.

One advantage of certain antibody constructs of the present fusion polypeptides is prolonged half-life and enhanced tissue penetration. Intact antibodies in which the Fc fragment of the Ig chain is present will exhibit slower blood clearance than their Fab' fragment counterparts, but a fragment-based fusion polypeptide will generally exhibit better tissue penetrating capability.

Preferentially, the tumor targeting structure in the SAg conjugate (e.g., tumor specific antibody, Fab or single chain Fv fragments or tumor receptor ligand) has a greater affinity for the tumor than the SAg in the conjugate has for the class II molecule thus preventing the SAg from binding all MHC class II receptors and favoring binding of the conjugate to the tumor. In the case of SEB, the dominant epitope for neutralizing antibodies 225-234 is recombinantly or biochemically bound to the tumor targeting molecule e.g., tumor specific antibodies, Fas or Fv fragments. In so doing, it sterically interferes with the recognition of the dominant epitope by preexisting antibodies.

To further enhance the affinity of the tumor specific antibody in the conjugate for tumor cells in vivo, tumor specific antibodies are used which are specific for more than one antigenic structures on the tumor, tumor stroma or tumor vasculature or any combination thereof. The tumor specific antibody or F(ab').sub.2, Fab or single chain Fv fragments are mono or divalent like IgG, polyvalent for maximal affinity like IgM or chimeric with multiple tumor (tumor stroma or tumor vasculature) specificities. Thus, when the SE or SPE-MoAb conjugate is administered in vivo, it will preferentially bind to tumor cells rather than to endogenous SE antibodies or MHC class II receptors.

To reduce affinity of the SE-mAb conjugate for endogenous MHC class II binding sites, the high affinity Zn.sup.++ dependent MHC class II binding sites in SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ, SMEZ2, M. arthritides are deleted or replaced by inert sequence(s) or amino acid(s). These structural alterations in SE or SPEA reduce the affinity for MHC class II receptors from a K.sub.d of 10.sup.-7 or 10.sup.-8 to 10.sup.-5. SEB, SEC and SSA and other SEs or SPEs do not have a high affinity Zn++ dependent MHC class II binding site but have multiple low affinity MHC class II binding sites (K.sub.d 10.sup.-5-10.sup.-7). In these cases, alteration of the MHC class II binding sites is not always necessary to further reduce affinity for MHC class II receptors; at the very least mutation of one or two of the low affinity MHC class II binding sites will suffice in most instances.

Most importantly, tumor specific antibodies, Fab, F(ab').sub.2 or single chain Fab or Fv fragments in the SAg-mAb conjugate have a higher affinity for tumor antigens (K.sub.d 10.sup.-11-10.sup.-14 or lower) than for the SAg has for MHC class II binding sites (K.sub.d 10.sup.-5 to 10.sup.-7) and its dominant epitope has for SAg specific antibodies (K.sub.d 10.sup.-7 to 10.sup.-11). In this way, the conjugate will bind preferentially to the tumor target in vivo rather than preexisting antibodies or MHC class II receptors.

Antibody fragments are obtained using conventional proteolytic methods. Thus, a preferred procedure for preparation of F(ab').sub.2 fragments from IgG of rabbit and human origin is limited proteolysis by the enzyme pepsin. Rates of digestion of an IgG molecule may vary according to isotype; conditions are chosen to avoid significant amounts of completely degraded IgG as is known in the art.

Fab fragments include the constant domain of the light chain (C.sub.L) and the first constant domain (C.sub.H1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the C-terminus of C.sub.H1 domain including one or more cysteine(s) from the antibody hinge region. F(ab').sub.2 fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

An "Fv" fragment is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, con-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V.sub.H-V.sub.L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

"Single-chain Fv" or "scFv" antibody fragments comprise the V.sub.H and V.sub.L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains that enables the scFv to form the desired structure for antigen binding.

The following documents, incorporated by reference, describe the preparation and use of functional, antigen-binding regions of antibodies: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,051,230; 6,004,555; and 5,877,289.

"Diabodies" are small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H and V.sub.L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described in EP 404,097 and WO 93/11161, incorporated herein by reference. "Linear antibodies", which can be bispecific or monospecific, comprise a pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) that form a pair of antigen binding regions.

An antibody fragment may be further modified to increase its half-life by any of a number of known techniques. Conjugation to non-protein polymers, such PEG and the like, is also contemplated

The antibody fusion partner for use in the present invention may be specific for tumor cells, tumor stroma or tumor vasculature. Antigens expressed on tumor cells that are suitable targets for mAb-SAg fusion protein therapy include erb/neu, MUC1, 5T4 and many others. Antibodies specific for tumor vasculature bind to a molecule expressed or localized or accessible at the cell surface of blood vessels, preferably the intratumoral blood vessels, of a vascularized tumor. Such molecules include endoglin (TEC-4 and TEC-11 antibodies), a TGF.beta.. receptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPF receptor, an FGF receptor, a TIE, an .alpha..sub.v.beta..sub.3 integrin, pleiotropin, endosialin and MHC class II proteins. Such antibodies may also bind to cytokine-inducible or coagulant-inducible products of intratumoral blood vessels. Certain preferred agents will bind to aminophospholipids, such as phosphatidylserine or phosphatidylethanolamine.

A tumor cell-targeting antibody, or an antigen-binding fragment thereof, may bind to an intracellular component that is released from a necrotic or dying tumor cell. Preferably such antibodies are mAbs or fragments thereof that bind to insoluble intracellular antigen(s) present in cells that may be induced to be permeable, or in cell ghosts of substantially all neoplastic and normal cells, but are not present or accessible on the exterior of normal living cells of a mammal.

Anti-tumor stroma antibodies bind to a connective tissue component, a basement membrane component or an activated platelet component; as exemplified by binding to fibrin, RIBS (receptor-induced binding site) or LIBS (ligand-induced binding site).

Fusion protein optionally include linkers or spacers. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to fuse the SAg to an antibody or fragment, certain linkers are preferred based on differing pharmacological characteristics and capabilities. For example, linkers that contain a disulfide bond that is sterically "hindered" are preferred, due to their greater stability in vivo, thus preventing release of the SAg moiety prior to binding at the site of action.

Coaguligand

SAgs may be conjugated to, or operatively associated with, polypeptides that are capable of directly or indirectly stimulating coagulation, thus forming a "coaguligand" (Barinaga M et al., Science 275:482-4 (1997); Huang X et al., Science 275:547-50 (1997); Ran S et al., Cancer Res 1998 Oct. 15; 58(20):4646-53; Gottstein C et al., Biotechniques 30:190-4 (2001)).

In coaguligands, the antibody may be directly linked to a direct or indirect coagulation factor, or may be linked to a second binding region that binds and then releases a direct or indirect coagulation factor. The `second binding region` approach generally uses a coagulant-binding antibody as a second binding region, thus resulting in a bispecific antibody construct. The preparation and use of bispecific antibodies in general is well known in the art, and is further disclosed herein.

Coaguligands are prepared by recombinant expression. The nucleic acid sequences encoding the SAg are linked, in-frame, to nucleic acid sequences encoding the chosen coagulant, to create an expression unit or vector. Recombinant expression results in translation of the new nucleic acid, to yield the desired protein product.

Where coagulation factors are used in connection with the present invention, any covalent linkage to the SAg should be made at a site distinct from the functional coagulating site. The compositions are thus "linked" in any operative manner that allows each region to perform its intended function without significant impairment. Thus, the SAg binds to and stimulates T cells, and the coagulation factor promotes blood clotting.

Preferred coagulation factors are Tissue Factor ("TF") compositions, such as truncated TF ("tTF"), dimeric, multimeric and mutant TF molecules. tTF is a truncated TF that is deficient in membrane binding due to removal of sufficient amino acids to result in this loss. "Sufficient" in this context refers to a number of transmembrane amino acids originally sufficient to insert the TF molecule into a cell membrane, or otherwise mediate functional membrane binding of the TF protein. The removal of a "sufficient amount of transmembrane spanning sequence" therefore creates a tTF protein or polypeptide deficient in phospholipid membrane binding capacity, such that the protein is substantially soluble and does not significantly bind to phospholipid membranes. tTF thus substantially fails to convert Factor VII to Factor VIIa in a standard TF assay yet retains so-called catalytic activity including the ability to activate Factor X in the presence of Factor VIIa.

U.S. Pat. No. 5,504,067, specifically incorporated herein by reference, describes tTF proteins. Preferably, the TFs for use herein will generally lack the transmembrane and cytosolic regions (amino acids 220-263) of the protein. However, the tTF molecules are not limited to those having exactly 219 amino acids.

Any of the truncated, mutated or other TF constructs may be prepared in dimeric form employing the standard techniques of molecular biology and recombinant expression, in which two coding regions are arranged in-frame and are expressed from an expression vector. Various chemical conjugation technologies may be employed to prepare TF dimers. Individual TF monomers may be derivatized prior to conjugation.

The tTF constructs may be multimeric or polymeric, which means that they include 3 or more TF monomeric units. A "multimeric or polymeric TF construct" is a construct that comprises a first monomeric TF molecule (or derivative) linked to at least a second and a third monomeric TF molecule (or derivative). The multimers preferably comprise between about 3 and about 20 such monomer units. The constructs may be readily made using either recombinant techniques or conventional synthetic chemistry.

TF mutants deficient in the ability to activate Factor VII are also useful. Such "Factor VII activation mutants" are generally defined herein as TF mutants that bind functional Factor VII/VIIa, proteolytically activate Factor X, but substantially lack the ability to proteolytically activate Factor VII.

The ability of such Factor VII activation mutants to function in promoting tumor-specific coagulation is requires their delivery to the tumor vasculature and the presence of Factor VIIa at low levels in plasma. Upon administration of a conjugate of a Factor VII activation mutant, the mutant will be localize within the vasculature of a vascularized tumor. Prior to localization, the TF mutant would be generally unable to promote coagulation in any other body sites, on the basis of its inability to convert Factor VII to Factor VIIa. However, upon localization and accumulation within the tumor region, the mutant will then encounter sufficient Factor VIIa from the plasma in order to initiate the extrinsic coagulation pathway, leading to tumor-specific thrombosis. Exogenous Factor VIIa could also be administered to the patient to interact with the TF mutant and tumor vasculature.

Any one or more of a variety of Factor VII activation mutants may be prepared and used in connection with the present invention. The Factor VII activation region generally lies between about amino acid 157 and about amino acid 167 of the TF molecule. Residues outside this region may also prove to be relevant to the Factor VII activating activity. Mutations are inserted into any one or more of the residues generally located between about amino acid 106 and about amino acid 209 of the TF sequence (WO 94/07515; WO 94/28017; each incorporated herein by reference).

A variety of other coagulation factors may be used in connection with the present invention, as exemplified by: the agents set forth below. Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa and derivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives, Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, Factor XIII/XIIIa and derivatives, Factor X activator and Factor V activator may be used in the present invention.

These conjugates are administered intrathecally in dosages of 0.01 ng/kg to 100 .mu.g/kg.

Cytokines as Fusion Partners

Cytokines are an effective partner for SAgs. Various cytokines, such as IL-2, IL-3, IL-7, IL-12, and IL-18, may be used.

A preferred fusion polypeptide comprises a SAg fused to anti-apoptotic cytokines. SAg stimulation of T cells can result in activation-driven cell death. Several cytokines and bacterial lipopolysaccharide (LPS) are known to interfere with this process (Vella et al., Proc. Natl. Acad. Sci. 95: 3810-3815 (1998)). IL-3, IL-7, IL-15 and IL-17 prevent SAg-stimulated T cells from undergoing apoptosis in vivo and in vitro. In addition, because of their ability to promote selective proliferation by Th.sub.1 T cells, IL-12 and IL-18 are desirable. IL-18 is preferred for intratumoral injection because it induces tumor suppressive cytokines IFN.gamma. and TNF.alpha. and IL-1.beta., and rescues cytotoxic T cells from apoptosis.

Accordingly, SAg-mAb conjugate as described above is fused recombinantly to the extracellular domains of at least one cytokine from a group consisting of IL-2. IL-7 or IL-3 or IL-12 or IL-15 or IL-17 or IL-18. Other anti-T cell apoptosis agents such as LPS preparations of low virulence or a lipid A component (modified to induce less toxicity) are also effective antiapoptotic agents when conjugated biochemically to the SAg-MoAb (or F(ab').sub.2, Fab, Fd or single chain Fv fragments) conjugate or if administered concomitantly with the SAg. Nucleic acids encoding the cytokine of choice is fused in frame with nucleic acids encoding the SAg. These conjugates are administered parenterally, intrathecally and/or intratumorally by infusion or injection in dosages of 0.01 ng/kg to 100 .mu.g/kg.

Costimulatory Molecules as Fusion Partners

Superantigens Conjugated to OX40L or 4-1BBL

A preferred fusion polypeptide comprises a SAg fused recombinantly to a potent costimulatory molecule, preferably the ECD of a transmembrane costimulatory protein. Examples of such costimulatory molecules are the OX-40 ligand (Godfrey et al., J. Exp. Med. 180: 757-762 (11994); Gramaglia I et al., J. Immunol. 161: 6510-6517 (1998); Maxwell J R et al., J. Immunol. 164: 107-112 (2000) or 4-1BB ligand (Kown B S et al., Proc. Natl. Acad. Sci. USA 86:1963-67 (1989); Shuford W W et al., J. Exp. Med. 186: 47-55 (1997) and CD-38 (Jackson D G et al., J. Immunol. 144: 2811-2817 (1990); Zilber et al., Proc. Nat'l Acad. Sci. USA 97: 2840-2845 (2000). The preparation of such fusion proteins is achieved by recombinant methods in which nucleic acids encoding SAgs are fused in frame to nucleic acids encoding the ECD of the costimulatory molecule such as OX-40L (Godfrey et al., J. Exp. Med 180:757-762 (1994)) or 4-1BBL (Goodwin et al. Eur. J. Immunol. 23: 2631-2641 (1993); Melero I. et al., Eur. J. Immunol. 28: 1116-1121 (1998)).

It is preferred to delete from the conjugates or fusion polypeptides of the present invention any SAg epitope that binds to SAg-specific antibodies, including preexisting or natural antibodies). Such epitopes are deleted or substituted by Ala or by amino acid sequences not recognized by preexisting host antibodies. For example, a dominant epitope of SEB that is recognized by anti-SEB antibodies is the sequence at residues 225-234 (Nishi et al., J. Immunol. 158: 247-254 (1997). An epitope of SEA that is recognized by anti-SEA antibodies is the sequence at residues 121-149 (Hobieka et al., Biochem. Biophys. Res. Comm. 223: 565-571 (1996). Alternatively, to avoid issues with such preexisting immunity. SAgs such as YPM or C. perfringens toxin A to which humans do not have preexisting antibodies are selected. YPM, in addition, a natural RGD domain which gives it tumor localizing properties. The SE may be modified to reduce toxicity by altering its MHC class II binding affinity (e.g., SEA D277A-high affinity Zn++ dependent binding site).

Preferably, the tumor targeting structure in SAg conjugate (e.g., tumor specific antibody or fragment, or a tumor receptor ligand) has greater affinity for the tumor than the affinity of the SAg in the conjugate for the MHC class II molecule thus preventing the SAg from binding "promiscuously" to all MHC class II molecules receptors and favoring binding to the tumor. In the case of SEB, the dominant epitope for neutralizing antibodies, residues 225-234, is recombinantly or biochemically conjugated to the tumor targeting molecule (e.g., tumor specific antibody, etc.) so that it can sterically interfere with the recognition of the dominant epitope by preexisting antibodies in the host.

To further enhance the affinity of the tumor specific antibody in the fusion polypeptide for tumor cells in vivo, one preferably selects a tumor specific antibody that is specific for more than one antigenic structures of the tumor, the tumor stroma or the tumor vasculature (or any combination). The tumor specific antibody or antigen-binding fragment thereof can be made mono or divalent (like IgG), polyvalent like IgM to increase avidity or chimeric with multiple tumor specificities as described above. Thus, when the SAg-mAb conjugate is administered in vivo, it will preferentially bind to tumor cells rather than to endogenous anti-SAg antibodies or MHC class II receptors.

To reduce affinity of the SAg-mAb conjugate for endogenous MHC class II binding sites, the high affinity Zn.sup.++ dependent MHC class II binding site present in a number of SAgs (SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ, SMEZ2, M. arthritides SAg) is deleted or replaced by an "inert" sequence(s) or amino acid. Such structural alterations in SE or SPEA are known to reduce the affinity for MHC class II from a K.sub.d of 10.sup.-7 or 10.sup.-8 to a K.sub.d of 10.sup.-5. SEB, SEC and SSA and other SAgs do not have such a high affinity Zn.sup.++-dependent MHC class II binding site but have multiple low affinity MHC class II binding sites (K.sub.d of 10.sup.-5-10.sup.-7). In these cases, alteration of the MHC class II binding sites is not always necessary to further reduce affinity for MHC class II; mutation of one or two of the low affinity MHC class II binding sites will suffice in most instances.

Most importantly, tumor specific antibodies or their fragments in a SAg-mAb conjugate have higher affinities for tumor antigens (K.sub.d of 10.sup.-11-10.sup.-14 or lower) than (a) the affinity of the SAg for MHC class II binding sites (K.sub.d 10.sup.-5 to 10.sup.-7) or (b) the affinity a dominant SAg epitope for a SAg-specific antibody (K.sub.d 10.sup.-7 to 10.sup.-11). Because of this, the conjugate will bind preferentially to the tumor target in vivo

SAg-OX-40 ligand (OX-40L) or 4-1BB ligand (4-1BBL) are fused to a tumor specific targeting structure using recombinant SAgs. A most preferred construct combines the ECD of OX-40L or 4-1BBL with a high affinity tumor specific Fv antibody fragments. The nucleic acids encoding the ECD of OX-40L (Godfrey et al., supra or 4-1BBL (Goodwin et al., Eur. J. Immunol. 23: 2631-2641 (1993); Melero I. et al., Eur. J. Immunol. 28: 1116-1121 (1998)) are fused in frame with nucleic acids encoding a SAg of any type, although SEA, SEB, SEC and Y. pseudotuberculosis are preferred. The SAg may be modified to reduce antigenicity by modifying a dominant epitope and to reduce toxicity by altering its MHC class II binding affinity as described above. The tumor targeting structure may include but is not limited to a tumor receptor ligand or tumor-specific antibody or a fragment thereof. Preferably, the affinity of the tumor targeting structure is of higher affinity than is the affinity of the modified SAg for MHC class II. High affinity scFv constructs specific for the OX-40 receptor and 4-1BB receptor may be used in place of the OX40L and 4-1BBL in the SAg-tumor targeting construct.

The SE-OX-40L (or 4-1BB) conjugates described above are administered parenterally, intratumorally, intrathecally (e.g., intraperitoneally, intrapleurally) by infusion or injection in conventional or sustained release vehicles in dosages of 0.01 ng/kg to 100 .mu.g/kg using standard protocols or those exemplified herein. Frequency of administration may be every 3-7 days.

Biochemical Cross-linkers

In the above fusion polypeptides or conjugates, the SAgs may be linked directly to a fusion partner or fused/conjugated via certain preferred biochemical linker or spacer groups. For chemical conjugates, cross-linking reagents are preferred and are used to form molecular bridges that bond together functional groups of two different molecules. Heterobifunctional crosslinkers can be used to link two different proteins in a step-wise manner while preventing unwanted homopolymer formation. Such cross-linkers are listed in Table 3 (see Original Patent).

Hetero-bifunctional cross-linkers contain two reactive groups one (e.g., N-hydroxy succinimide) generally reacting with primary amine group and the other (e.g., pyridyl disulfide, maleimides, halogens, etc.) reacting with a thiol group. Compositions to be crosslinked therefore generally have, or are derivatized to have, a functional group available. This requirement is not considered to be limiting in that a wide variety of groups can be used in this manner. For example, primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl, hydroxyl, phosphate, or alkylating groups may be used for binding or cross-linking.

The spacer arm between the two reactive groups of a cross-linker may be of various length and chemical composition. A longer, aliphatic spacer arm allows a more flexible linkage while certain chemical groups (e.g., benzene group) lend extra stability or rigidity to the reactive groups or increased resistance of the chemical link to the action of various agents (e.g., disulfide bond resistant to reducing agents). Peptide spacers, such as Leu-Ala-Leu-Ala, are also contemplated.

It is preferred that a cross-linker have reasonable stability in blood. Numerous known disulfide bond-containing linkers can be used to conjugate two polypeptides. Linkers that contain a disulfide bond that is sterically hindered may give greater stability in vivo, preventing release of the agent prior to binding at the desired site of action.

A most preferred cross-linking reagents for use in with antibody chains is SMPT, a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. Such steric hindrance of the disulfide bond may protect the bond from attack by thiolate anions (e.g., glutathione) which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery to the target, preferably tumor, site. SMPT cross-links functional groups such as --SH or primary amines (e.g., the .epsilon.-amino group of Lys).

Hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond, for example, sulfosuccinimidyl-2-(p-azido salicylamido)-ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane. The use of such cross-linkers is well known in the art.

Once conjugated, the conjugate is separated from unconjugated SAg and fusion partner polypeptides and from other contaminants. A large a number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful. Purification methods based upon size separation, such as gel filtration, gel permeation or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used.

Chemotherapeutic and Other Agents

Chemotherapeutic agents can be used together with intrathecal or intratumoral SAg. They can be administered intrathecally, intratumorally or parenterally by infusion or injection concomitantly with SAg. Preferably they are given together with SAg after 2-7 weeks of treatment with the SAg alone. Anti-cancer chemotherapeutic drugs useful in this invention include but are not limited to antimetabolites, anthracycline, vinca alkaloid, anti-tubulin drugs, antibiotics and alkylating agents. Representative specific drugs that can be used alone or in combination include cisplatin (CDDP), adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, aminopterin, combretastatin(s) and derivatives and prodrugs thereof.

A variety of chemotherapeutic and pharmacological agents may be given separately or conjugated to a therapeutic protein of the invention. Exemplary antineoplastic agents that have been conjugated to proteins include doxorubicin, daunomycin, methotrexate and vinblastine. Moreover, the attachment of other agents such as neocarzinostatin, macromycin, trenimon and .alpha.-amanitin has been described. See U.S. Pat. Nos. 5,660,827; 5,855,866; and 5,965,132; each incorporated by reference herein. Those of ordinary skill in the art will know how to select appropriate agents and doses, although, as disclosed, the doses of chemotherapeutic drugs are preferably reduced when used in combination with SAgs according to the present invention.

Another newer class of drugs also termed "chemotherapeutic agents" comprises inducers of apoptosis. Any one or more of such drugs, including genes, vectors, antisense constructs, siRNA constructs, and ribozymes, as appropriate, may be used in conjunction with SAgs.

Other agents useful herein are anti-angiogenic agents, such as angiostatin, endostatin, vasculostatin, canstatin and maspin.

Chemotherapeutic agents are administered as single agents or multidrug combinations, in full or reduced dosage per treatment cycle. They can be administered with the intrathecal or intratumoral and optionally parenteral SAg composition although, under a preferred schedule, the chemotherapeutic agent is administered within 36 hours of the last of two to four treatments of SAg compositions administered intrathecally or intratumorally.

The combined use of the SAg compositions with low dose, single agent chemotherapeutic drugs is particularly preferred. The choice of chemotherapeutic drug in such combinations is determined by the nature of the underlying malignancy. For lung tumors, cisplatin is preferred. For breast cancer, a microtubule inhibitor such as taxotere is the preferred. For malignant ascites due to gastrointestinal tumors, 5-FU is preferred. "Low dose" as used with a chemotherapeutic drug refers to the dose of single agents that is 10-95% below that of the approved dosage for that agent (by the U.S. Food and Drug Administration, FDA). If the regimen consists of combination chemotherapy, then each drug dose is reduced by the same percentage. A reduction of >50% of the FDA approved dosage is preferred although therapeutic effects are seen with dosages above or below this level, with minimal side effects.

Tumors to treat with SAgs (.+-.chemotherapeutics) using intratumoral injection are preferably at least 6 cm.sup.3 and visible by x-ray, CT, ultrasound, bronchoscopy, laparoscopy, culdoscopy. Intratumoral localization of the agent being delivered is facilitated with fluoroscopic, CT or ultrasound guidance. Representative tumors that are treatable with this approach include but are not limited to hepatocellular carcinoma, lung tumors, brain tumors, head and neck tumors and unresectable breast tumors. Multiple tumors at different sites may be treated by intrathecal or intratumoral SAg.

The chemotherapeutic agent(s) selected for therapy of a particular tumor preferably is one with the highest response rates against that type of tumor. For example, for non-small cell lung cancer (NSCLC), cisplatin-based drugs have been proven effective. Cisplatin may be given parenterally or intratumorally. When given intratumorally, Cisplatin is preferentially in small volume around 1-4 ml although larger volumes can also work. The smaller volume is designed to increase the viscosity of the Cisplatin containing solution in order to minimize or delay the clearance of the drug from the tumor site. Other agents useful in NSCLC include the taxanes (paclitaxel and docetaxel), vinca alkaloids (vinorelbine), antimetabolites (gemcitabine), and camptothecin (irinotecan) both as single agents and in combination with a platinum agent.

The optimal chemotherapeutic agents and combined regimens for all the major human tumors are set forth in Bethesda Handbook of Clinical Oncology, Abraham J et al., Lippincott William & Wilkins, Philadelphia, Pa. (2001); Manual of Clinical Oncology, Fourth Edition, Casciato, D A et al., Lippincott Williaml & Wilkins, Philadelphia, Pa. (2000) both of which are herein incorporated in entirety by reference.

In one embodiment, these recommended chemotherapeutic agents are used alone or combined with other chemotherapeutics in full doses. Alternatively they may be administered parenterally by infusion or injection in doses 10-95% below the FDA recommended therapeutic dose. For intratumoral administration, the dose of a chemotherapeutic drug or biologic agent is preferably reduced 10- to 50-fold below the FDA-recommended dose for parenteral administration.

Cisplatin has been widely used to treat cancer, with effective doses of 20 mg/m.sup.2 for 5 days every three weeks for a total of three courses. Preferred dose per treatment for intratumoral use of Cisplatin is 5-10 mg whereas for intrathecal use 20-80 mg may be administered. Intratumoral cisplatin may be given every 7-14 days for 10-20 treatments whereas intrathecal cisplatin may be given every 2-6 weeks for 10-20 treatments. Cisplatin delivered in small volumes, e.g., 5-10 mg/1-5 ml saline, is extremely viscous and may be retained in a tumor for a sustained period, thereby acting like a controlled release drug being released from an inert surface. This is indeed the preferred mode of administration of Cisplatin when administered intratumorally with or without the SAg. Preferably cisplatin is administered together with the SAg in the same syringe.

Other chemotherapeutic compounds include doxorubicin, etoposide, verapamil, podophyllotoxin, and the like which are administered through intravenous bolus injections at doses ranging from 25-75 mg/m.sup.2 at 21 day intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide intravenously.

Other agents and therapies that are operable together with or after intratumoral SAg include, radiotherapeutic agents, antitumor antibodies with attached anti-tumor drugs such as plant-, fungus-, or bacteria-derived toxin or coagulant, ricin A chain, deglycosylated ricin A chain, ribosome inactivating proteins, sarcins, gelonin, aspergillin, restricticin, a ribonuclease, a epipodophyllotoxin, diphtheria toxin, or Pseudomonas exotoxin. Additional cytotoxic, cytostatic or anti-cellular agents capable of killing or suppressing the growth or division of tumor cells include anti-angiogenic agents, apoptosis-inducing agents, coagulants, prodrugs or tumor targeted forms, tyrosine kinase inhibitors (Siemeister et al., 1998), antisense strategies, RNA aptamers, siRNA and ribozymes against VEGF or VEGF receptors (Saleh et al., 1996; Cheng et al., 1996; Ke et al., 1998; Parry et al., 1999; each incorporated herein by reference).

Any of a number of tyrosine kinase inhibitors are useful when administered together with, or after, intratumoral SAg. These include, for example, the 4-aminopyrrolo[2,3-d]pyrimidines (U.S. Pat. No. 5,639,757). Further examples of small organic molecules capable of modulating tyrosine kinase signal transduction via the VEGF-R2 receptor are the quinazoline compounds and compositions (U.S. Pat. No. 5,792,771).

Other agents which may be employed in combination with SAgs are steroids such as the angiostatic 4,9(11)-steroids and C.sup.21-oxygenated steroids (U.S. Pat. No. 5,972,922).

Thalidomide and related compounds, precursors, analogs, metabolites and hydrolysis products (U.S. Pat. Nos. 5,712,291 and 5,593,990) may also be used in combination with SAgs and other chemotherapeutic drugs agents to inhibit angiogenesis. These thalidomide and related compounds can be administered orally.

Certain anti-angiogenic agents that cause tumor regression may be administered together with, or after, intratumoral SAg. These include the bacterial polysaccharide CM101 (currently in clinical trials as an anti-cancer drug) and the antibody LM609. CM101 has been well characterized for its ability to induce neovascular inflammation in tumors. CM101 binds to and cross-links receptors expressed on dedifferentiated endothelium that stimulate the activation of the complement system. It also initiates a cytokine-driven inflammatory response that selectively targets the tumor. CM101 is a uniquely antiangiogenic agent that downregulates the expression VEGF and its receptors. Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also be used together with or after intratumoral SAg. These are both angiogenesis inhibitors that associate with heparin and are found in platelet .alpha. granules.

Interferons and metalloproteinase inhibitors are two other classes of naturally occurring angiogenic inhibitors that can be used together with or after intratumoral SAg. Vascular tumors in particular are sensitive to interferon; for example, proliferating hemangiomas are successfully treated with IFN.alpha.. Tissue inhibitors of metalloproteinases (TIMPs), a family of naturally occurring inhibitors of matrix metalloproteases (MMPs), can also inhibit angiogenesis and can be used in combination with SAgs.

Pharmaceutical Compositions and Administration

One or more of SAg, SAg homologues, fragments, mutants, fusion proteins and conjugates (SAg agents) are administered by injection, infusion or instillation or implanted intratumorally or subcutaneously in a controlled release formulation. SAg agents are most commonly administered intrathecally in patients with malignant intrathecal fluid accumulation due to primary or metastatic tumors. For example, malignant pleural effusions in patients with lung cancer or metastatic breast, gastric or ovarian cancer. SAg agents may also be administered intrathecally to patients with intrathecal and parenchymal tumor (e.g., involvement of pleura and lung parenchyma) but little or no fluid accumulation in the cavitary space. SAg agents may also be administered intrathecally to patients without malignant involvement or fluid accumulation in the cavitary space or its membranes but with primary or metastatic tumor of the organ (e.g., lung, stomach) and/or lymph nodes. For example, SAg may be administered intrapleurally to patients with primary lung cancer or lung metastases from other primary tumors (e.g., breast, ovary, gastric) without malignant involvement of the pleura or pleural space. In each of the above examples, intrathecal administration of the SAg agents may be administered simultaneously or sequentially with one or more of the SAg agents administered intratumorally, intralymphatically or intravenously.

SAg agents are administered every 3-10 days for up to three months. Dosages of SAg agents used for intrathecal, intratumoral, intralymphatic and intravenous administration range from 0.1 pg-1 ng/kg.

SAg agents are also administered intratumorally to stimulate a T cell-based inflammatory response, including release of tumoricidal cytokines and induction of cytotoxic T cells. The amount of SAg agent administered to a single tumor site ranges from about 0.05-1 ng/kg body weight. The intratumoral dose of a cytotoxic drug administered to the tumor site will generally range from about 0.1 to 500, more usually about 0.5 to 300 mg/kg body weight, depending upon the nature of the drug, size of tumor, and other considerations.

When used to boost the titer of SAg specific antibodies, SAg agents may be incorporated in an adjuvant vehicle such as alum or Freund's incomplete adjuvant. These compositions are administered prior to, during or after intrathecal and/or intratumoral administration of the SAg agent.

They are administered subcutaneously, intramuscularly and intradermally by injection or infusion in doses ranging from 0.1 pg/kg to 1 ng/kg. To induce a maximum immune response, boosters with the SAg agent and vehicle at 1-6 month intervals are given.

The pharmaceutical compositions of the present invention will generally comprise an effective amount of at least a SAg composition dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Combined therapeutics are also contemplated, and the same type of underlying pharmaceutical compositions may be employed for both single and combined medicaments. The intratumoral composition can be administered to the tumor by needle or catheter via percutaneous entry or via endoscopy, bronchoscopy, culdoscopy or other modes of direct vision including directly at the time of surgery. The composition can be localized into the tumor with CT and/or ultrasound guidance.

With each drug in each tumor, experience will provide an optimum level. One or more administrations may be employed, depending upon the lifetime of the drug at the tumor site and the response of the tumor to the drug. Administration may be by syringe, catheter or other convenient means allowing for introduction of a flowable composition into the tumor. Administration may be every three days, weekly, or less frequent, such as biweekly or at monthly intervals.

The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. Veterinary uses are equally included within the invention and "pharmaceutically acceptable" formulations include formulations for both clinical and/or veterinary use.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by U.S. Food and Drug Administration. Supplementary active ingredients can also be incorporated into the compositions.

"Unit dosage" formulations are those containing a dose or sub-dose of the administered ingredient adapted for a particular timed delivery. For example, exemplary "unit dosage" formulations are those containing a daily dose or unit or daily sub-dose or a weekly dose or unit or weekly sub-dose and the like.

Injectable Formulations

The SAg composition of the present invention are preferably formulated for parenteral administration, e.g., introduction by injection, infusion or instillation directly into an affected organ cavity (intrathecal administration) or tumor (intratumorally). Means for preparing aqueous compositions that contain the SAg compositions are known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The SAg compositions can be formulated into a sterile aqueous composition in a neutral or salt form. Solutions as free base or pharmacologically acceptable salts can be prepared in water. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein), and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, trifluoroacetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Suitable carriers include solvents and dispersion media containing, for example, water. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the active agents in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques that yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Suitable pharmaceutical compositions in accordance with the invention will generally include an amount of the SAg composition admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally well known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, or most recent edition, incorporated herein by reference. Endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the U.S. Food and Drug Administration. Upon formulation, the therapeutic compositions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

Once in an acceptable pharmaceutical form, SAg are administered intrathecally including but not limited to intrapleurally, intraperitoneally, intra-pericardially, and/or intratumorally and optionally intra-lymph node and/or parenterally (e.g., intravenously, intramuscularly, subcutaneously) by injection or infusion. SAg are also delivered simultaneously or sequentially via one or more routes, e.g., intrapleurally and intravenously or intrapleurally, intratumorally and intravenously. SAg are also administered simultaneously or sequentially in the same or different vehicles, adjuvants and sustained release formulations.

Sustained Release Formulations

SAg formulations are easily administered in a variety of dosage forms, including "slow release" capsules or "sustained release" preparations or devices. Slow release formulations, generally designed to result in a constant drug level over an extended period, are used to deliver a SAg composition as described herein. Such slow release formulations are implanted intrathecally or intratumorally. Controlled release formulations are prepared using polymers to complex or absorb the therapeutic compositions--SAgs, SAg homologues, chemotherapeutic agents or combined formulations of a SAg/homologue and a chemotherapeutic agent(s). The rate of release is regulated by (1) selection of appropriate macromolecules, for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, and protamine sulfate, (2) the concentration of the macromolecules and (3) the method of incorporation of the active agents into the formulation.

Another method to control the duration of action of the present controlled release preparations is to incorporate the SAgs, SAg homologues and/or chemotherapeutic drugs into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol); polylactides (e.g., U.S. Pat. No. 3,773,919); copolymers of L-glutamic acid and .gamma.-ethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers, such as the Lupron Depot.TM. (injectable microspheres of lactic acid-glycolic acid copolymer and leuprolide acetate); and poly-D-(-)-3-hydroxybutyric acid.

Alternatively, instead of incorporating the bioactive/pharmaceutically active agents into polymeric particles, the active agents may rather be entrapped in microcapsules prepared by interfacial polymerization. Examples include hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, micro emulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980 or most recent edition). Nanoparticles consisting of SAg, SAg homologue and/or chemotherapeutic agents are delivered intrathecally or intratumorally via insufflation using a gas or air propellant.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. For example, it is known that when encapsulated antibodies remain in the body for a prolonged period, they may denature or aggregate as a result of exposure to moisture at 37.degree. C., thus reducing biological activity. Rational strategies are available for stabilization, and they depend on the mechanism involved. For example, if the aggregation mechanism involves intermolecular S--S bond formation through thio-disulfide interchange, stabilization is achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, developing specific polymer matrix compositions, and the like.

A particularly attractive sustained release preparation for use herein comprises collagen and an effective amount of SAg (or homologue) and a cytotoxic drug, as described by Luck et al., RE35,748 and Roskos et al., U.S. Pat. No. 6,077,545. More detail on preparation is given in Example 2.

The collagen composition can be used in the treatment of a wide variety of tumors including carcinomas, sarcomas and melanomas. Specific types of tumors include such basal cell carcinoma, squamous cell carcinoma, melanoma, soft tissue sarcoma, solar keratoses, Kaposi's sarcoma, cutaneous malignant lymphoma, Bowen's disease, Wilm's tumor, hepatomas, colorectal cancer, brain tumors; mycosis fungoides, Hodgkin's lymphoma, polycythemia vera, chronic granulocytic leukemia, lymphomas, oat cell sarcoma, etc. The collagen and other composition will be administered to a tumor to provide a cytotoxic amount of drug at the tumor site. The amount of cytotoxic drug administered to the tumor site will generally range from about 0.1 to 500 mg/kg body weight, more usually about 0.5 to 300 mg/kg, depending upon the nature of the drug, size of tumor, and other considerations. Vasoconstrictive agents will generally be present in from 1 to 50% (w/w) of the therapeutic agent. In view of the wide diversity of tumors, nature of tumors, effective concentrations of drug, relative mobility and the like, a definitive range cannot be specified. With each drug in each tumor, experience will provide an optimum level. One or more rounds of administration may be employed, depending upon the lifetime of the drug at the tumor site and the response of the tumor to the drug. Administration may be by syringe, catheter or other convenient means allowing for introduction of a flowable composition into the tumor. Administration may be every three days, weekly, or less frequent, such as biweekly or at monthly intervals.

Illustrative of the manner of sustained administration would be administration of cis-diaminodichloroplatinum (CDDP). Drug concentrations in the sustained release preparation may vary from 0.01 to 50 mg/ml. Injection may be at one or more sites depending on the size of the lesion. Needles of about 1-2 mm diameter are convenient. For multiple injection, templates with predrilled holes may be employed. The drug dose will normally be less than 100 mg/m.sup.2 body surface area.

The present invention is particularly advantageous against those tumors or lesions that are clinically relevant because of high frequency. The compositions provide therapeutic gain with tumors greater than 100 mm.sup.3, more particularly, greater than 150 mm.sup.3, in volume.

Administration by controlled release of SAg and/or a chemotherapeutic drug may be used advantageously in conjunction with other forms of therapy. The tumors or lesions may be irradiated prior and/or subsequent to SAg administration by controlled release. Dose rates may vary from about 20 to 250 rad/min, usually 50 to 150 rad/min, depending on the lesion, period of exposure, and the like. Hyperthermia (heat) may be used as an adjunctive treatment. Treatment will usually involve heating the tumor and its surrounding tissue to a temperature of about 43.degree. for between about 5 and 100 min.

Intratumoral Administration

A SAgs and SETs or a biologically active homologue, fragment or fusion polypeptide or conjugate as described herein is used for direct intratumoral treatment of a tumor mass. SAgs include Staphylococcal enterotoxins A, B, C, D, E, F, G, H, I, J, K, L, M, SpE's, YPM, M. arthritides, C. perfringens exotoxin for direct intratumoral treatment of tumor masses. Tumor mass may be those appearing in any organ, palpated or visualized on x-ray, CT scan, MRI or ultrasound. Intratumoral administration may be performed with fluoroscopic, CT or ultrasound guidance.

For intratumoral administration, the dose of a chemotherapeutic drug or biologic agent is preferably reduced 10- to 50-fold below the FDA-recommended dose for parenteral administration. As noted above, a preferred dose of intratumoral Cisplatin is 5-10 mg in 1-5 ml every 7-14 days for 10-40 treatments. This regimen for intratumoral Cisplatin (with or without SAg) is preferred. Preferably Cisplatin is administered together with the SAg in one syringe.

The SAg is dissolved in a conventional vehicle such as saline or it may be incorporated into a controlled release formulation (mixture or suspension) preferably biodegradable. All of the biocompatible and biodegradable and controlled release formulations described herein are useful. These formulations also include but are not limited to, ethylene-vinyl acetate (EVAc: Elvax 40W, Dupont), bioerodible polyanhydrides, polyimino carbonate, sodium alginate microspheres and hydrogels. Dosages used range from 1 ng to 10 mg. The poly-(D-, L- or DL-lactic acid/polyglycolide) copolymers are preferred.

For intratumoral administration, the SAg composition is preferably administered once weekly, and this schedule is continued until the tumor has shrunk significantly. Generally 3-10 treatments are sufficient. In some cases the tumor may appear to expand in size during such intratumoral SAg therapy. This is a result of SAg-stimulated accumulation of inflammatory cells and edema. Despite this enlargement, histological examination of such tumors performed during this phase shows evident tumoricidal effects with inflammatory cell infiltrates.

In the case of an enlarging tumor or a slowly regressing tumor when SAg therapy is given alone, conventional chemotherapy may be administered to promote tumor killing. A chemotherapeutic agent is preferably administered intratumorally alone or together with SAg. Importantly, the chemotherapeutic agent should be given in doses well below those prescribed for systemic use of the same agent. Preferably, intratumoral chemotherapy will comprise use of a selected single agent which is known in the art to be effective against a particular tumor. Moreover, intratumoral combination chemotherapy wherein each agent is given in a reduced dose can also be used. Full-dose or reduced-dose systemic chemotherapy can also be used together with, or shortly after, intratumoral SAg therapy. As with intrathecal administration described herein, intratumoral delivery may be carried out in an outpatient setting as it requires no hospitalization.

The intratumoral therapy with a SAg and or a SAg homologue can be used to treat of a wide variety of neoplastic lesions. Indeed, an improvement in 5-year survival from 16% to 26% of small cell lung cancer was produced by increase in local control accomplished by altering the fractionation of radiation therapy (Turisi et al., N. Eng. J. Med. 340: 265-270 (1999)). Illustrative tumors amenable to intratumoral therapy with SAgs include carcinomas, sarcomas and melanomas, including such as basal cell carcinoma, squamous cell carcinoma, soft tissue sarcoma, solar keratosis, Kaposi's sarcoma, cutaneous malignant lymphoma, Bowen's disease, Wilm's tumor, neuroblastoma, gliomas astrocytomas, hepatoma, colorectal cancer, brain tumors, mycosis fungoides, Hodgkin's lymphoma, polycythemia vera, chronic granulocytic leukemia, lymphomas, oat cell sarcoma, breast carcinoma etc. The intratumoral SAg has particular advantage for tumors or lesions which are among the most important clinically because of their frequency. The compositions and methods disclosed herein provide therapeutic gain with tumors exceeding 100 mm.sup.3 in volume, even tumors exceeding 150 mm.sup.3.

Superantigens with Radiation Therapy

Local radiation to tumor sites or the mediastinum using the traditional standard dose of 60-65 gy may be given concomitant with intrathecal or intratumoral SAg. The radiotherapy may be also be given before or after the SAg therapy but in either case there should be a hiatus of no more than 30 days between the start of SAg therapy and the start or conclusion of radiotherapy. The median survival of patients given this type of radiotherapy alone is 5% at one year whereas the combined modality improves the median survival to more than two years.
 

Claim 1 of 4 Claims

1. A method of treating a subject with a carcinoma comprising administering to said subject parenterally by infusion or injection a tumoricidally effective amount of a composition consisting of: (i) a native staphylococcal enterotoxin or streptococcal pyrogenic exotoxin protein which native protein: (a) has the biological activity of stimulating T cell mitogensis via a T cell receptor v.beta. region or (ii) a biologically active homologue or fragment of a native staphylococcal enterotoxin or streptococcal pyrogenic exotoxin, which homologue or fragment: (a) has the biological activity of stimulating T cell mitogenesis via a T cell receptor v.beta. region and (b) has sequence homology characterized as a z value exceeding 13 when the sequence of the homologue or said fragment is compared to the sequence of a native staphylococcal enterotoxin or a native streptococcal progenic exotoxin, determined by FASTA analysis using gap penalties of -12 and -2, Blosum 50 matrix and Swiss-PROT or PIR database; or (iii) a biologically active fusion protein having said biological activity and said sequence homology, comprising (A) said homologue, (B) a native staphylococcal enterotoxin, (C) a native streptococcal pyrogenic exotoxin, or (D) a biologically active fragment of said homologue, said native enterotoxin or said native exotoxin, fused to a peptide or polypeptide fusion partner, wherein a chemotherapeutic drug or drugs is/are administered parenterally by infusion or injection before, together with or after administration of said enterotoxin or exotoxin composition.

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