USE OF EXTRACELLULAR CYSTEINE PROTEASE TO INHIBIT CELL PROLIFERATION

Federally Sponsored Research

This work was supported in part by National Institutes of Health grants CA-16672 and AI-33119. The government may have rights to this invention.

INTRODUCTION Technical Field

The present invention relates generally to the field of cell biology. More specifically, the present invention relates to methods and compositions for inhibiting cell proliferation. Background

The ability to control tumor cell growth without serious side effects to the host organism has long been a goal in the continuing search for improved methods for treating cancer. Drugs or naturally occurring compounds usually have a broad range of activities towards different cells. Moreover, at concentrations that may be effective in inhibiting the growth of neoplastic cells, these treatment formulations frequently demonstrate undesired effects on normal cells. Of particular interest therefore are compositions that selectively inhibit the growth of neoplastic cells while not inhibiting the growth of surrounding or other normal tissue. Peptides such as the interferons (IFN) and lymphocyte-derived tumor necrosis factor- β (TNF-β) as well as TNF-α, derived from myelocytic cells, display

cytostatic and/or cytocidal activity against transformed cell lines, but do not affect normal cells. However, even with compounds such as the interferons which show inhibitory activity primarily towards tumor cells, not all tumor cells are affected by the compound, and reliable methods have not been developed to identify tumors which have a high probability of responding to a given compound. Furthermore, there may be a fine line between a therapeutic dose of a given compound and that which is physiologically unacceptable or toxic to the host.

Streptococcus pyogenes is a Gram-positive bacterium which is β-hemolytic and is the etiological agent of several diseases in humans, including pharyngitis and/or tonsillitis, skin infections (impetigo, erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), and a toxic-shock-like syndrome (TSLS). S. pyogenes expresses group A antigen and is susceptible to bacitracin. Since the late 1800s, it has been recognized that some cancer patients who develop S. pyogenes infections undergo remission of their malignant tumors, however the molecular basis for this observation is unknown. In 1881, Coley reported the curative effect of experimental inoculation of Streptococcus pyogenes in patients with inoperable sarcoma. Since management of S. pyogenes infections in the pre-antibiotic era was difficult, Coley at first used heat-killed cultures of this organism, but achieved only limited success. In order to enhance treatment efficacy, heat-killed cultures of Serratia marcescens, a Gram- negative bacterium that synthesizes a lipopolysaccharide (LPS), were incorporated into the preparation. The combination of these two heat- inactivated bacterial cultures, now known as "Coley toxins," received considerable research interest and was used to treat some cancer patients. However, Coley and others before him had observed that some patients who contracted Group A streptococcal infections naturally, or were purposely inoculated with streptococci without co-inoculation with Gram- negative LPS producing organisms, also underwent remission of their

malignant tumors. This observation suggested that streptococcal products alone elaborated during infection participated in tumor regression in some patients. It therefore is of interest to identify the streptococcal product(s) that are responsible for the observed tumor remission for use as an antiproliferative agent(s).

Relevant Literature

S. pyogenes culture supernatants contain a protease that has fibrinolytic activity. (Elliot J. Exp. Med. 81:573-92 (1945)). The enzyme was purified, shown to be a cysteine protease (Liu et al, J. Biol. Chem. 238:251-56 (1963)) and found to be identical to or an allelic variant of streptococcal pyrogenic exotoxin B (SPE B). (Gerlach et al Zbl. Bakt. Hyg. 255:221-23 (1983); Hauser and Schlievert J. Bacteriol. 172:4536-42 (1990)).

An extracellular protease from Entamoeba histolytica, the cause of amebiasis, has a cytopathic effect on cell culture monolayers, (Keene et al,

Exp. Parasitol. 71:199-206 (1990)) and is involved in production of tissue necrosis in rat models of acute amebiasis. (Becker et al, Exp. Parasitol. 67:268-80 (1988).)

Human interleukin-lβ converting enzyme (ICE), a cysteine protease, has been reported to induce apoptosis under certain conditions.

(Gagliardini et al, Science, 263:826-28 (1994), Miura et al., Cell 75: 653-60 (1993).)

SUMMARY OF THE INVENTION

A composition derived from a cysteine protease is provided together with methods of using it for inhibiting cell proliferation and as a means of screening for cells susceptible to inhibition. The method for inhibiting proliferation of neoplastic cells and other hyperproliferative tissue involves contacting the cells with a proliferation-inhibiting amount of the composition. Also provided is a method of identifying hyperproliferative cells that are susceptible to the inhibition by the compositions. The

method involves the steps of treating cultured hyperproliferative cells with the composition and determining whether they proliferate less rapidly than cells grown in the absence of the composition. Alternative screening methods involve contacting a hyperproliferative tissue with a reagent that specifically interacts with a receptor for the expression product of the spe gene, and detecting the resulting complex. Also provided are methods for preparation, isolation and/or purification of speB gene expression products and use of the products in cleaving and/or degrading extracellular matrix proteins, and inducing apoptosis in susceptible cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not necessarily to scale. Certain features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness.

Figure 1 shows alleles of speB. The polymorphic sites within the 160 bp upstream non-coding region and 1197 bp coding region of the speB gene are shown. The sequence described by Hauser and Schlievert (J. Bacteriol. 172:4536-42 (1990), which is incorporated herein by reference) was arbitrarily designated as speBl, and the numbering of nucleotides and codons is cognate with that sequence. Only those nucleotides in the other alleles that differ from the speBl sequence are shown. The position of each polymorphic nucleotide site is shown above the 39 alleles and is numbered in vertical format. Non-synonymous nucleotide changes are underlined and the positions of the coding changes are designated by an asterisk above of the coding region polymorphic sites. The seven polymorphic nucleotide sites in the upstream noncoding region are shown in the left of the figure, and the asterisk in speB6 denotes a deletion of an adenine residue. The codon (numbered in vertical format) containing the polymorphic nucleotide sites is shown below the 39 alleles. The DNA sequence data for speB2 - speB39 are available from EML/GenBank/DDBJ under accession numbers L26125-L26162.

Figure 2 shows the in vitro cytotoxicity of streptococcal cysteine protease. Figure 2A shows the sensitivity of K1735 (open circles) and CM519 (closed triangles) murine melanoma cells to the cysteine protease. Figure 2B shows streptococcal cysteine protease-induced DNA fragmentation in CM519 melanoma cells. Lane 1: 1 kb ladder marker

DNA. Lane 2: Cells treated for 48 hours with 20 μg/ml of streptococcal cysteine protease. Lane 3: untreated cells. Lane 4: Cells treated for 24 hours with 50 μg/ml of streptococcal cysteine protease.

Figure 3 shows the antitumor activity of streptococcal cysteine protease in immunocompetent C3H mice. Figures 3A and 3C show mean tumor diameter. Figures 3B and 3D show percent tumor-free mice. PBS

(open circles); boiled streptococcal cysteine protease (closed triangles); streptococcal cysteine protease (closed circles).

Figure 4 shows the antitumor activity of streptococcal IL-lβ cysteine protease in athymic (nu/nu) nude mice. Figures 4A and 4C show mean tumor diameter; Figures 4B and 4D show percent tumor-free mice.

PBS (open circles); boiled streptococcal cysteine protease (closed triangles); streptococcal cysteine protease (closed circles).

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENT Methods and compositions are provided for inhibiting cell proliferation and for screening of host physiological samples for cells susceptible to inhibition. The methods and compositions utilize peptides derived from all or part of the extracellular cysteine protease from the Group A streptococci, such as Streptococcus pyogenes. The use of peptides of the current invention provides for methods of inhibiting cellular proliferation, particularly of malignant tissue. A host can be treated by administration of the antiproliferative agent to the host in an amount sufficient to inhibit proliferation of the hyperproliferative target tissue. The host is any organism, particularly an animal, which has proliferative tissue, including humans, where it is desirable to inhibit the proliferative

process. The methods and compositions of the invention are also useful as a prophylaxis against metastasis. Metastasis is a process by which tumor cells spread from a tumor to other parts of the body. An antimetastasis therapy is a therapy that reduces or eliminates metastases, or can limit metastases. Also provided are methods for evaluating the sensitivity of target cells to Streptococcus extracellular cysteine protease. Evidence of sensitivity includes detection of binding of a Streptococcus cysteine protease to the extracellular matrix and/or detection of proteolytic products generated following hydrolysis of protein components of the extracellular matrix (ECM).

An antiproliferative agent based upon the streptococcal cysteine protease offers several advantages over other antiproliferative agents. For example, previously known antiproliferative agents often cause undesired side effects such as nausea, vomiting, cytoxicity to normal cells, malaise, etc. The cysteine protease of the invention has no known detrimental effects. The cysteine protease or fragments thereof are not known to cross-react with nonproliferative host tissues.

When used as an antiproliferative agent, the cysteine protease can be any portion of the polypeptide that retains the catalytic nature of the native mature cysteine protease which is derived from the translated portion of the speB gene. For the purpose herein, cysteine protease is defined as a protein or polypeptide which is substantially homologous with the amino acid sequence of the native mature cysteine protease which is expressed from the translated portion of the speB gene. Ordinarily cysteine protease polypeptides will be about from 40 to 100% homologous to the native mature cysteine protease, preferably 80 to 90% homologous, and they will exhibit at least some biological activity in common with the native mature cysteine protease. Biological activity shall include, but is not limited to, cross-reactivity with anti-cysteine protease antibodies raised against cysteine protease from natural (i.e., non-recombinant) sources, catalytic activities, and binding activities. Cysteine protease cleaves

human interleukin lβ precursor to form biologically active IL-lβ, a major cytokine mediating inflammation and shock. Additionally, the purified native protease cleaves fibronectin and rapidly degrades vitronectin. The protein has no substantial activity against laminin. The cysteine protease also cleaves fibronectin from human umbilical vein endothelial cells grown in vitro. Homology is determined by optimizing residue matches, by introducing gaps as required but without considering conservative substitutions as matches. This definition is intended to include natural allelic variations in the cysteine protein sequence. Cysteine protease includes the cysteine proteases of organisms other than S. pyogenes, for example, Pseudomonas aeruginosa, (Morihara Kamp Homma, In: Holder IA, ed. Bacterial enzymes and virulence, FL CRC Press, 1985; 41-75) and Porphyromonas (Bacteriodes) gingivalis, (Lantz et al, J. Bacteriol. 173:495-504 (1991); and Otogoto and Kuramitsu, Infect. Immun. 61:117- 23 (1993)) two bacterial species that produce host tissue destruction by degradation of collagen and fibronectin, respectively. Entamoeba histolytica also produces an extracellular cysteine protease. Like the streptococcal cysteine protease, the E. histolytica enzyme degrades several ECM proteins, including type 1 collagen, fibronectin, and laminin. (Keene, W.E. et al., J. Exp. Med. 163:536-49 (1986).)

The S. pyogenes cysteine protease is synthesized as an extracellular zymogen of 371 amino acids (40,314 kDa) that can be transformed into an enzymatically active protease of 253 amino acids (27,588 kDa) by autocatalytic conversion. Thus, the streptococcal cysteine protease resembles many secreted bacterial extracellular protease virulence factors in having a specific signal peptide and a pro-sequence that is removed in an autocatalytic fashion to generate a fully active enzyme. Accordingly, proteins are provided which enzymatically mimic extracellular proteases encoded by the S. pyogenes bacterium, particularly proteins encoded by the speB region of an S. pyogenes genome. The zymogen contains one or more epitopes not associated with the truncated enzyme. Both the

zymogen and active protease contain a single half-cysteine per molecule that is susceptible to sulfhydryl antagonists. The extracellular zymogen of cysteine protease is a species of cysteine protease included within the foregoing definition of cysteine protease. It is characterized by the presence in the molecule of a signal (or leader) polypeptide, which serves to post-translationally direct the protein to a site inside or outside the cell. Derivatives of cysteine protease included herein are amino acid sequence mutants, glycosylation variants, and covalent or aggregative conjugates with other chemical moieties. Mutant cysteine protease derivatives include predetermined (i.e., site-specific), mutations of cysteine protease or its fragments. A mutant cysteine protease is defined as a polypeptide otherwise falling within the homology definition for cysteine protease as set forth herein, but which has an amino acid sequence different from that of cysteine protease as found in nature, with and by way of deletion, substitution, or insertion.

Preferably, the cysteine protease retains both the catalytic and binding activities of the native cysteine protease. By "derived from" it is meant that the cysteine protease has the amino acid sequence of all or part of a cysteine protease obtained from S. pyogenes. For the purpose of this disclosure, a microorganism is considered to be the same as or equivalent to Streptococcus pyogenes if in its genome is a coding sequence for an extracellular cysteine protease which is involved in the pathogenesis of the microorganism. The sequence can be naturally occurring, or partially or wholly synthetic. The sequence can be truncated as compared to the native protease, or can include additional sequences at the amino and/or carboxy terminals of the protease.

These peptides can be used individually or together for detection of tissues which are susceptible to inhibition of growth and/or in which apoptosis is initiated in response to contact with an antiproliferative peptide. Depending upon the nature of the test protocol, the macromolecules may be labeled or unlabeled, bound to a solid surface,

conjugated to a carrier molecule or other compounds, or the like. The peptide will include at least five, sometimes six, sometimes eight, sometimes about 22 amino acids, but usually greater than 25 amino acids, preferably greater than about 50 amino acids of a cysteine protease. The peptide includes at least the catalytic acid substrate binding region within the amino acid sequence which corresponds to the preproenzyme, the entire sequence of which is as shown below (SE ID NO:l):

310 320 330 340 350 360

SVHQINRSDF SKQDWEAQID KELSQNQPVY YQGVGKVGGH AFVIDGADGR NFYHVN GWG 360 GVSDGFFR D ALNPSALGTG GGAGGFNGYQ SAWGIKP. 399

The peptide will be as small as possible while still maintaining substantially all of the enzymatic activity of the larger peptide, the sequence of which is shown above. Of particular interest as antiproliferative agents are peptides of at least 10 amino acids which include the cysteine residue at position 192 of the cysteine protease.

It should be understood that the polypeptides employed in the subject invention need not be identical to a cysteine protease obtainable from S. pyogenes, so long as the subject compounds are able to provide for enzymatic interaction with a substrate of a cysteine protease obtainable from at least one of the strains of S. pyogenes. It also will be appreciated that the nucleotide sequence to which the polypeptides "correspond" may exhibit strain-to-strain variation and species-to-species variation. Thus, the polypeptides can exhibit various changes such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use. By

"conservative substitution" is intended a substitution of one amino acid in a particular group for another amino acid in the same group. Amino acids are typically placed in groups as follows, based on their side chains: gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Usually, the sequence will not differ by more than 20% from the sequence of at least one strain of S. pyogenes except where additional amino acids may be added at either terminus for the purpose of providing an "arm" by which the peptides of this invention may be immobilized conveniently. The arms usually will be at least 5 amino acids, and may be 50 or more amino acids.

The substrate of the cysteine protease can be a protein that is attached to the membrane of a tumor cell or is found inside a tumor cell. In such case, the cysteine protease is said to "directly" inhibit cellular proliferation. Alternatively, the cysteine protease substrate can be associated with a cell that interacts with a tumor cell to cause or maintain the undesired proliferation, or the cysteine protease substrate can be a

protein such as a hormone or cytokine that triggers or inhibits abnormal proliferation. In this case, the cysteine protease is said to "indirectly" inhibit cellular proliferation by inactivating a protein that would otherwise interact with a tumor cell to cause the proliferation. The peptides can be prepared in a wide variety of ways. In broth cultures of S. pyogenes, inactive precursor accumulates extracellularly during bacterial multiplication and reaches a maximum concentration at the end of logarithmic growth. Some strains yield up to 150 mg/liter of zymogen, and the molecule is a major extracellular protein. Thus, the cysteine protease itself can be purified from natural sources, for example by binding to Red dye, or by other means known to those skilled in the art of protein purification, such as affinity chromatography using an antibody to the peptide.

The peptides, because of their relatively short size, also may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. The peptides are selected based upon the enzymatic activity and antiproliferative activity. To prepare synthetic peptides, overlapping 10- mer peptides are used that overlap two amino acid residues with the previous 10-mer in a consecutive primary sequence corresponding to the 371 amino acids of the mature cysteine protease zymogen (translated product minus leader sequence). Synthetic 10-mers corresponding to each variant amino acid residue are also used. The variant amino acids are positioned in the middle of the 10-mer. Once the 10-mer peptides are synthesized, an ELISA is used to examine the enzymatic activity of each peptide using as substrate component proteins of the ECM. The peptides then are further screened for antiproliferative activity against target hyperproliferative cells, both in vitro and in vivo. Both site-directed and random mutagenesis schemes can be employed to identify residues that disrupt cysteine protease function and

zymogen processing, and to map regions that constitute the catalytic and/or binding domains of the resulting peptides. Of particular interest are regions of the cysteine protease that are exposed to solvent in the testing structure of the molecule and highly conserved in comparison to other cysteine proteases. To create a stable zymogen to facilitate analysis of the enzymatic and/or binding regions (i.e. structure-function studies), mutant forms of the cysteine protease protein are made and characterized. A targeted mutagenesis scheme creates changes that alter protease activity and/or alter substrate binding. Amino acids are changed to structurally- neutral alanine. As an example, a mutant protein that lacks protease activity can be generated by mutagenesis of the single cysteine residue (0 8-192— Ala-192) at the catalytic site of the molecule. Also, His-340 and Gln-I 85 and Asn-356 are mutagenized. These three changes are epistatic to the Cys-192 mutation, but may alone result in a protease which exhibits altered activity. Trp-357, thought to be involved in substrate binding and similarly positioned within papain, is also targeted.

A stable zymogen precursor is created by mutating residues surrounding the protease cleavage site at Lys-145. In addition, mutagenesis of Cys-192 may prevent autoproteolysis, as occurred for a Cys- - Ser mutant of papain, the prototype cysteine protease. Other mutagenesis targets include a putative nucleotide binding domain (GVGKVG) (SE ID NO:2) and a potential collagen docking region (GXX) ₃ within the carboxy terminal portion of the protein. Site-directed mutagenesis is used, by the changed-to-alanine-scanning method, to substitute positively and negatively charged amino acids (often involved in recognition and activity) with alanine. Many of the charged residues (14 lysine, 7 arginine, 12 aspartate, and 7 glutamate residues in the mature peptide) are expected to lie on the surface of the cysteine protease structure, and some are expected to define epitopes on the molecule. Residues in regions identified in the epitope mapping studies described above also are mutated.

In order to create mutant speB proteins, as an example, first, the speB gene is amplified from a S. pyogenes strain, with PCR, and the product is cloned into a multicopy filamid vector such as pBluescript (Stratagene, La Jolla, CA). This vector is chosen because it carries the regulated lac promoter and can be replicated as a single-stranded molecule for site-directed mutagenesis. Cloning is designed to place the ribosome binding site and speB reading frame 3' to an inducible promoter, such as a lac promoter, on the vector so that the protein can be conditionally over- expressed in a bacterial host when an inducer of the inducible promoter is added to the culture broth. For example, with the lac promoter, the lac inducer, isopropyl-β-D-thiogalactopyranoside (IPTG), can be added. The speB promoter is included when expression in S. pyogenes is desired. Whole cell extracts and periplasmic shockates of E. coli cells carrying this primary speB clone are examined for the presence of the cysteine protease protein by SDS-PAGE and by Western blotting with anti-cysteine protease antibody. The resulting plasmid is the target for mutagenesis.

Oligonucleotide-directed mutations, such as substitutions, deletions and small insertions, are created using methods known to those of skill in the art. For example, one can employ uracil-containing single-stranded templates as described by Kunkel (Proc. Natl. Acad. Sci. USA 82: 488-92

(1985)). Kits for performing mutagenesis by this and other methods are commercially available (see, e.g., Amersham Corp. Life Science Catalog, 1994). When possible, mutagenic primers are designed to incorporate a unique restriction site into the speB gene to facilitate mapping and mutant selection. Both single and multiple alanine substitutions are created at the residues indicated above. Once residues critical to function are identified, small regions surrounding them are deleted or substituted, by using the same methods, to further characterize the region and to preclude reversion. When crystallographic data are available, additional amino acids are mutated.

A random mutagenesis scheme also can be employed using methods known to those of skill in the art. By this method, one can recover variant proteins that have altered kinetics and substrate recognition. For example, one can randomize regions of the speB sequence using mixed oligonucleotides in a primed-mutagenesis protocol, or one can create short, in-frame deletions within the gene using a modification of the DNAse 1- linker insertion/deletion protocol of Palzkill and Bostein (Methods: A Companion to Methods in Enzymology 3:155-64 (1991)).

To identify protease mutations with altered proteolytic activity, bacterial host cells producing potential mutant proteins are first screened for protease activity on casein agar plates. Since secretion of cysteine protease to the periplasm is expected, protease activity can be observed on plates. One can examine rapidly thousands of colonies for functional mutations in the cysteine protease. If cysteine protease must be completely secreted from the host cell, such as E. coli, to exhibit activity, then osmotic shockates of each presumptive mutant strain are assayed for protease activity.

Alternatively, one can employ hybrid-DNA technology to prepare a synthetic gene that encodes the cysteine protease. Single DNA strands that code for portions of the polypeptide or substantially complementary strands thereof, where the single strands overlap and can be brought together in an annealing medium so as to hybridize. The hybridized strands can then be ligated to form the complete gene and by choice of appropriate termini, the gene may be inserted into an expression vector. Alternatively, the region of the bacterial genome coding for the peptide may be cloned by conventional recombinant DNA techniques and expressed. The recombinant products may be glycosylated or non- glycosylated, have the wild type glycosylation, or other glycosylation. Thus expression of the product in E. coli cells will result in an unglycosylated product, and expression of the product in insect cells

generally will result in less glycosylation than expression of the product in mammalian cells.

An example of a DNA coding sequence that can be used for expressing the cysteine protease is as shown below (SEQ ID NO:3).

TABLE

SEQ ID NO:3

10 20 30 40 50 atg aat aaa aag aaa tta ggt ate aga tta tta agt ctt tta gca tta ggt gga 54 ttt gtt ctt get aac cca gta ttt gec gat caa aac ttt get cgt aac gaa aaa 108 gaa gca 'aaa gat age get ate aca ttt ate caa aaa tea gca get ate aaa gca 162 ggt gca cga age gca gaa gat att aag ctt gac aaa gtt aac tta ggt gga gaa 216 ctt tct gge tct aat atg tat gtt tac aat att tct act gga gga ttt gtt ate 270 gtt tea gga gat aaa cgt tct cca gaa att eta gga tac tct ace age gga tea 324 ttt gac get aac ggt aaa gaa aac att get tec tte atg gaa agt tat gtc gaa 378 caa ate aaa gaa aac aaa aaa tta gac act act tat get ggt ace get gag att 432 ι aaa caa cca gtt gtt aaa tct etc ctt gat tea aaa gge att cat tac aac caa 486 ^ ggt aac cct tac aac eta ttg aca cct gtt att gaa aaa gta aaa cca ggt gaa 540 caa tct ttt gta ggt caa cat gca get aca gga tgt gtt get act gca act get 594 caa att atg aaa tat cat aat tac cct aac aaa ggg ttg aaa gac tac act tag 648 aca eta age tea aat aac cca tat tte aac cat cct aag aac ttg ttt gca get 702 ate tct act aga caa tac aac tgg aac aac ate eta cct act tat age gga aga 756 gaa tct aac gtt caa aaa atg gcg att tea gaa ttg atg get gat gtt ggt att 810 tea gta gac atg gat tat ggt cca tct agt ggt tct gca ggt age tct cgt gtt 864 caa aga gec ttg aaa gaa aac ttt gge tac aac caa tct gtt cac caa att aac 918 cgt age gac ttt age aaa caa gat tgg gaa gca caa att gac aaa gaa tta tct 972 caa aac gtt ate gga gtc act ggt aaa cct

Fragments from this sequence can be employed for expression of peptide fragments. One can make conservative base changes, where the modified codon(s) code for the same amino acid(s), or non-conservative changes in the coding sequence, where the resulting amino acid substitution can be a conservative or non-conservative change. The coding sequence can be extended at either the 5'- or 3'-terminus, or both termini, to extend the peptide while retaining its catalytic and binding sites. The extension may provide for an arm for linking the peptide to a solid support or to another molecule such as a label, for example an enzyme, an antigen, and the like.

To express the peptides of the invention from the nucleic acid molecules, constructs such as expression cassettes are prepared in which the nucleic acids are typically operably linked to control sequences. The term "control sequence" refers to a DNA sequence or sequences that are capable, when properly attached to a desired coding sequence, of causing expression of the coding sequence. Such control sequences include at least promoters and, optionally, transcription termination signals. Additional factors necessary or helpful for expression can also be included. As used herein, "control sequences" simply refers to whatever DNA sequence signal that is useful to result in expression in the particular host used. The term

"operably linked" as used herein refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence. These constructs are typically inserted into a vector to facilitate insertion of the constructs into a host organism. The term "vector" refers to nucleic acids that are capable of replicating in the selected host organism. The vector can replicate as an autonomous structure, or alternatively can integrate into the host cell chromosome(s) and thus replicate along with the host cell genome. Vectors include viral- or bacteriophage-based expression systems, autonomous self-replicating

circular DNA (plasmids), and include both expression and nonexpression vectors. The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types. The sequences by themselves, fragments thereof, or larger sequences, usually at least 15 bases, preferably at least 18 bases, may be used as probes for detection of bacterial DNA in numerous techniques, such as the Southern technique, Northern technique, and improvements thereon, as well as other methodology. These techniques are described, for example, in Sambrook et al. , Molecular Cloning: A Laboratory Manual,

Cold Spring Harbor, New York, 1989.

Conveniently, the polypeptide may be prepared as a fused protein, where the cysteine protease may be the N- or C-terminus of the fused polypeptide. The fused polypeptide can be used directly by itself as the reagent or the subject peptide may be cleaved from all or a portion of the remaining sequence of the fused protein. With a polypeptide where there are no internal methionines, by introducing a methionine at the fusion site, the polypeptide may be cleaved employing cyanogen bromide. Where there is an internal methionine, it will be necessary to provide for a proteolytic cleavage site, for example, poly-lysine, or -arginine, or combinations thereof. A wide variety of proteases, including dipeptidases, are well known and the appropriate processing signal can be introduced at the proper site. The processing signal may have tandem repeats so as to insure cleavage, since the presence of one or more extraneous amino acids will not interfere with the utility of the subject polypeptides.

The subject peptides may be employed linked to a soluble macromolecular (≥ 20 kD) carrier. Conveniently, the carrier may be a poly (amino acid), either naturally occurring or synthetic, to which antibodies are unlikely to be encountered in human serum. Illustrative polypeptides include poly-L-lysine, bovine serum albumin, keyhole limpet hemocyanin, bovine gammaglobulin, etc. The choice is primarily one of convenience

and availability. With such conjugates, there will be at least one molecule of at least one subject peptide per macromolecule, and not more than about one per 0.5 kD, usually not more than one per 2kD of the macromolecule. One or more different peptides may be linked to the same macromolecule.

The manner of linking is conventional, employing such reagents as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid, anhydride, glutaraldehyde, etc. The linkage may occur at the N-terminus, C-terminus, or at a site intermediate the ends of the molecule. The subject peptide may be derivatized for linking, may be linked while bound to a support, or the like.

The compounds can be employed as labeled or unlabeled compounds, depending upon their use. By label it is intended a molecule which provides, directly or indirectly, a detectible signal. Various labels can be employed, such as radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates, cofactors or inhibitors, particles, e.g., magnetic particles or the like. In addition, the polypeptides may be modified in a variety of ways for binding to a surface, e.g., microtiter plate, glass beads, chromatographic surface, e.g., paper, cellulose, silica gel, and the like. The particular manner in which the polypeptides are joined to another compound or surface is conventional and finds example and illustration in the literature.

The subject compounds can be used in a wide variety of ways, both in vivo and in vitro. The subject compounds can be used for making antibodies to the subject compounds, which may also find use in vivo or in vitro. The antibodies can be prepared in conventional ways, either by using the subject polypeptide as an immunogen and injecting the polypeptide into a mammalian host, e.g., mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, e.g., complete Freunds adjuvant, aluminum hydroxide gel, or the like. The host may then be bled and the blood employed for isolation of polyclonal antibodies, or in the case of the

mouse, the peripheral blood lymphocytes or splenic lymphocytes (B-cells) employed for fusion with an appropriate myeloma cell to immortalize the chromosomes for monoclonal expression of antibodies specific for the subject compounds. Either polyclonal or monoclonal antibodies may be prepared, which may then be used for diagnosis or detection of the presence of the subject polypeptide in a sample, such as cells or a physiological fluid, e.g., blood. Detection of the subject polypeptide in a bodily fluid may also be used as an indication of the presence of a tumor cell. The antibodies may also be used in affinity chromatography for purifying the subject polypeptide or isolating it from natural or synthetic sources. The antibodies may also find use in controlling the amount of the subject polypeptide associated with cells in culture or in vivo, whereby growth of the cells may be modified. The subject compound may be used as a ligand for detecting the presence of receptors for the subject compound. In this way, cells may be distinguished in accordance with the presence of and the density of receptors for the subject compound, monitoring the effect of various compounds on the presence of such receptors as well as determining the sensitivity of a given cell to the effects of the subject compound.

Additionally, peptides believed to have cysteine protease activity may be evaluated by comparing their ability to bind to the cysteine protease receptor with that of naturally occurring cysteine protease. Generally, the test peptides can be evaluated by incubating the test peptide together with labeled cysteine protease or another peptide which binds with high affinity to the cysteine protease receptor with a preparation containing cysteine protease receptors, and observing the amount of inhibition of binding of the labeled cysteine protease.

Therapeutic and Diagnostic Applications

The invention includes compositions and methods for use in therapeutic and diagnostic applications, for use both in vitro and in vivo. The cysteine protease peptide compositions and methods can be used in vitro cultures to inhibit the growth of cells or cell lines that are sensitive to the subject polypeptide, as distinguished from cells which are not sensitive. Thus, heterogeneous cell mixtures or cell lines can be freed of undesirable cells, where the undesirable cells are sensitive to the subject polypeptide. For example, one can use the subject compound in vitro to eliminate malignant cells from marrow for autologous marrow transplants or to inhibit proliferation or eliminate malignant cells in other tissue, e.g. blood, prior to reinfusion.

The subject compositions and methods also find use in vivo for the treatment of a wide variety of conditions characterized by undesirable cell proliferation. To determine the response of particular cells to the streptococcal cysteine protease peptide, one can first test the effect of the protease on a sample of the cells in vitro. Samples of the cells being tested are grown in an appropriate medium in the presence or absence of cysteine protease peptides. The concentration of cysteine protease which may be used in culture will generally be in the range of about 1 μg/ml to

100 μg/ml, final concentration. The growth of a cell sample is said to be "inhibited" if, when assayed by means such as radioisotope incorporation into the cells, the cells with the cysteine protease proliferate at a rate that is less than about 80% of the proliferation rate of untreated control cells, preferably less than about 70% of the untreated cell proliferation rate, and more preferably less than about 50% of the untreated cell proliferation rate. Using this method for screening cells in vitro, one can provide for varying degrees of inhibition and can determine the response of the cells in culture to the different concentrations. In this manner, one may predict particular concentrations that one could use in a mammalian host in the modulation of target cell proliferation.

For pharmaceutical applications, the cysteine protease compositions of the invention as described herein are administered to an individual having an undesirable cellular proliferation, such as neoplastic cell growth. The compositions and methods are useful to treat a wide variety of neoplastic conditions, such as carcinomas, sarcomas, melanomas, lymphomas, leukemias, which may affect a wide variety of organs, such as the blood, lungs, mammary organ, prostate, intestine, liver, heart, skin, pancreas, brain, etc.

The compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the undesired proliferation and its symptoms and/or complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose" or an "effective dose." Amounts effective for this use will depend on, for example, the nature of the cysteine protease peptide, the manner of administration, the stage and severity of the neoplasm, the weight and general state of health of the patient, and the judgment of the prescribing physician, but will generally range from about 1 μg/kg to about 5 g/kg of peptide per day, with dosages of from about 1 μg/kg to about 100 μg/kg of peptide per day being more commonly used. To treat neoplastic conditions, the cysteine protease is administered to a mammal in need of treatment. For in vivo use, the cysteine protease is typically formulated as pharmaceutical compositions in which the peptides are incorporated in physiologically acceptable carriers for application to the affected area. The nature of the carriers may vary widely and will depend on the intended location of application.

The compositions and methods are also useful as a prophylaxis against metastasis. By administering the cysteine protease peptides to a mammal diagnosed as having a cancer, the ability of the cancer to metastasize is reduced or eliminated. Administration should commence as soon after diagnosis of the cancer as practicable, and should preferably continue until cancer is no longer detected in the mammal. Metastasis is

said to be "eliminated" when no tumors in addition to those present at initial diagnosis are formed within three months after initial diagnosis. More preferably, no additional tumors will be formed within six months, and most preferably no additional tumors will form within twelve months of the initial diagnosis. Metastasis is said to be "reduced" when fewer metastatic cancers are observed than is typical for the type of cancer in the absence of an antimetastasis therapy. Usually, the number of metastatic tumors will be less than about 70% of the typical number, more preferably less than about 50%, and most preferably less than about 30%. When used as an antimetastasis therapy, the peptides are administered as pharmaceutical compositions in dosages as described below. When used as an antimetastasis therapy, the compositions and methods of the invention will typically result in a statistically significant increase in survival times, as defined by Kaplan-Meier survival curves or other appropriate statistical measures of survival. Preferably, the antimetastasis therapy increases survival times by at least about 30% over the survival times typical for the particular type of cancer in the absence of an antimetastasis therapy. More preferably, survival times are increased by at least about 50%, and most preferably by at least about 70%. The pharmaceutical compositions for therapeutic treatment are intended for various means of administration, including parenteral, intralesional, peritoneal, topical, oral and local administration and the like. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the cysteine protease peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can

be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as Ph adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The cysteine protease peptides of the invention can also be formulated as liposomes, either being contained within the lumen of the liposomes or being present on the liposome outer surfaces. Particularly are liposomes that are bound to homing molecules targeted for particular neoplastic cells, e.g., antibodies, nondegradable particle matrices, or the like. Methods for preparing liposomes for pharmaceutical use are known to those of skill in the art.

For application to the skin, a cream or ointment base is usually preferred, suitable bases include lanolin, Silvadene (Marion) (particularly for the treatment of proliferative disorders such as hypertrophic scars, Aquaphor (Duke Laboratories, South Norwalk, Connecticut), and the like. If desired, it will be possible to incorporate cysteine protease containing compositions in bandages and other dressings to provide for continuous exposure of the target tissue such as a skin cancer or melanoma to the peptide. Aerosol applications also may find use. The concentration of polypeptide in the treatment composition is not critical. The polypeptide will be present in a cell proliferation-inhibiting amount.

The concentration of cysteine protease peptide of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 1%, usually at or at least about 10-15% to as much as 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 5-25 mg of cysteine protease peptide. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example,

Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, PA (1985), which is incorporated herein by reference.

The pharmaceutical compositions can have two or more adjunctive agents, usually having fewer than six components, which have anti- proliferative activity either alone or in conjunction with cysteine protease.

Besides the cysteine protease, one or more transforming growth factors may be employed, as well as one or more interferons or tumor necrosis factor may be employed. Thus in particular situations, it may be desirable to use mixtures of related compositions, rather than an individual compound. As already indicated, besides the native proteases, active fragments can be employed, as well as chimeras in which a portion of one related molecule is joined to a portion of another related molecule.

The composition will vary widely depending upon its intended purpose, the desired ratio of the components, as well as the nature of the components and their activity. Thus, depending upon the nature of the malignancy, its response to the different components and their synergistic activity, the ratios of the various components will vary. For the most part, the amount of cysteine protease will generally be in the range of about 0.5 to 25 % of the amount of Oncostatin M or congener employed by itself.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. The pharmaceutical formulations should provide a quantity of the cysteine protease peptides of the invention sufficient to effectively treat the patient. Administration preferably begins at the first indication of undesirable cellular proliferation or shortly after diagnosis, and

continue until symptoms are substantially abated and for a period thereafter. In well established cases of disease, loading doses followed by maintenance doses will be required.

The invention also provides methods and compositions for using gene therapy to treat cell proliferation disorders. These methods involve inserting a nucleic acid construct that encodes a cysteine protease peptide into an individual in need of treatment for abnormal cell proliferation. Typically, the nucleic acid that encodes the cysteine protease peptide will be operably linked to control sequences, such as promoters and transcriptional terminators, that are functional in the target organism.

These nucleic acid constructs are administered to the patient by techniques that are known in the art for gene therapy. For a review of such techniques, see, e.g., Mulligan, R.C., Science 260: 926-32 (1993), which is incorporated herein by reference. Another embodiment of the invention is an article of manufacture that comprises packaging material and a pharmaceutical agent contained within the packaging material. The pharmaceutical agent is a cysteine protease peptide as described herein. The packaging material can contain the cysteine protease peptide either alone or in the presence of other components such as a solvent or a physiologically acceptable carrier. In one preferred embodiment, the cysteine protease peptide is in lyophilized form, and is reconstituted by dissolving in water or an acceptable carrier prior to administration to a patient. The article of manufacture typically includes a label upon which printed material indicates that the pharmaceutical agent is useful for inhibiting proliferation of neoplastic cells. The label can also include written instructions for the reconstitution and/or use of the pharmaceutical agent.

The following examples are given for the purpose of illustrating various embodiments of the methods of the present invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Bacterial Isolates Table 1 shows the 68 strains of S. pyogenes studied. MGAS 1719 is identical to strain B220, the designation assigned by Dr. R. Lancefield to strain 5797. The strain expresses type 8 T antigen but is serologically nontypeable for M protein.

Table 1 Properties of 68 5. pyogenes strains representing 50 ETs"

to

C oO

CO

ET Serotype* MGAS no/ Disease or site'' Country and year speB opacity M allele factor protein phenotype ass

4 M2 327 TSLS USA - 1980s speB3 +

60 M9 796 unknown USA - 1970 speB19 +

61 M i l 650 NP Trinidad - 1972 speB23 + M i l 2075 invasive Canada - 1980s speB + null

+

CO

+ CO

32 NT 317 invasive USA - 1980s speB3

- ET, electrophoretic type.

NT, nontypeable for M protein serotype.

- MGAS, Musser group A Streptococcus reference number. Strain sources and original co designations are as follows: J. C. Huang, Laboratory Centre for Disease Control, Ottawa, Canada, MGAS 579 (11111), 587 (9378), 590 (11078), 2075 (DC 11435); J. E. Peters, Wilford Hall Medical Center, San Antonio, Texas, MGAS 1991 (BB6672-3), 1990 (BA9812-4) , P.M. Schlievert, University of Minnesota, Minneapolis, Minnesota, MGAS 1253 (119/6. also known as SF130/13) , MGAS 1251 (C203S) , 166 (Reineke) , 285 (195) , 325 (89.5.5612), 157 (Zinke), 315 (Soldier 1), 282 (192), 289 (199) , 262 (Cal 17), 168 (Reinary) , 302 (Lambert) , 321 (Weck uller) , 156 (Wilson), 300 (Kluss) , 303 (Lundeen) , 162 (Cygan) , 165 (Wicks) , 317 (Timmers) ;

E. L. Kaplan, University of Minnesota, MGAS 480 (90-441) ; M.A. Kehoe, University of Newcastle upon Tyne, Newcastle upon Tyne, England, MGAS 1841 (M41) , 1871 (PT5757) , 1893 (PT4854), 1882 (M59) , 1842 (M43) , 1901 (M23), 1898 (M15) , 1864 (M56) , 1896 (M10) , 1911 (M75), 1881 (M62) _; 1870 (PT4931), 1872 (TR2612), 1838 (M27), 1914A (TR2233); D. LeBlanc, University of Texas Health Science Center at San Antonio, Texas, MGAS 1222 (Cole 36XA87) , 1226 (Cole 40XF1) ,

1233 (Cole 45XA9) , K. H. Johnston, Louisiana State University Medical Center, New Orleans, Louisiana, MGAS 1719 (B220) ; D. E. Bessen, Yale University, New Haven, Connecticut, MGAS 1832 (CS110) , 1294 (1RP232), 1289 (1RP144); S. K. Hollingshead, Department of Microbiology,

University of Alabama School of Medicine, Birmingham, Alabama, MGAS 660 (D469) , 789 (IGLIOO) , 807 (D323) , 429 (C256/86/3) , 684 (1RP284) , 694 (D470) , 427 (J137/69/1) , 366 (AGL130) , 719 (D938) , 686 (D316) , 800 (A724), 758 (86-809), 796 (D339) , 650 (D691) , 659 (D474). All other strains are from the collection of J.M.M. ^ cn

A- TSLS, toxic-shock-like syndrome; SID, severe invasive disease; ARF, acute rheumatic fever; NP, nasopharynx.

EXAMPLE 2

Purification of the Cysteine Protease The streptococcal cysteine protease was purified from strain MGAS 1719 with a combination of ultrafiltration of the cell culture supernatant and dye-ligand affinity chromatography (Kapur et al., Proc. Nat'l. Acad.

Sci. USA 90:7676-80 (1993).

Briefly, bacteria were grown overnight at 37° C and 5% C0 ₂ on brain-heart infusion (BHI) agar (Difco). The overnight culture was used to inoculate 200 ml of BHI liquid medium, and the culture was incubated for 12-14 hours at 37° C in 5% C0 ₂ . A 50 ml aliquot of the overnight culture was added to 2 liters of chemically defined medium (CDM) for group A streptococcal (JRH Bioscience, Lenexa, KS), Ph 6.0, and the culture was incubated at 37° C in 5% C0 ₂ . The broth was maintained at Ph 5.5 - 6.0 by the addition of sterile sodium bicarbonate (10% w/v). After 8-9 hours, the cells were removed by centrifugation and the supernatant was concentrated to 250 ml by passage through a 10 kDa cutoff spiral ultrafiltration cartridge (Amicon). Buffer exchange (> 99%) by diafiltration was conducted with 1.5 liters of 20% ethanol - 20 Mm Tris- Hcl, Ph 7.0 (buffer A) at 4°C, and the resulting material was stored overnight at 4°C. The diafiltered solution was passed through a matrix gel red A (Amicon) column (1.5 cm x 15 cm, Bio-Rad) equilibrated with buffer A. The column was washed with buffer A until the adsorption (280 nm) returned to baseline, and the protein was eluted with buffer A containing 2M NaCl. The eluted material was collected as one fraction, and concentrated to 3 ml by ultrafiltration (Centriprep 10, Amicon). The buffer was then exchanged with PBS, Ph 7.2, by gel-filtration chromatography (BioRad). SDS polyacrylamide gel electrophoresis and Coomassie blue staining of the resulting proteolytically active material revealed a single major band with M _r 30 kDa. The purified protein derived from dye-ligand affinity chromatography was blotted to Problott (ABI) as per manufacturer's

instructions, stained with Coomassie blue, excised from the gel, and applied to a AB1477A protein sequencer. Aminoterminal sequencing of the purified protein derived from dye-ligand affinity chromatography reveals a sequence of -QPWKSLLDSK- (SEQ ID NO:4), corresponding to amino acids 146 - 156, thereby confirming the identity of the purified material as the truncated mature active form of streptococcal cysteine proteinase. The enzyme is stable for at least several months at 20° C. Three distinctive speB allelic variants (identified by sequencing studies) have been purified. The zymogen form can be purified with a closely similar protocol, except cysteine is omitted from the medium and the culture is incubated in the absence of supplemental C0 ₂ .

The published amino acid sequence for cysteine proteinase, including the configuration of the presumed active site, is incorrect. The predicted amino acid sequence encoded by this sequence is not cognate with the published cysteine protease sequence. Instead, the nucleotide sequence resembles, but is distinct from, the allele described by Hauser and Schlievert, supra. However, the configuration of amino acids around the active cysteine residue is identical in strain B220 and all strains characterized thus far. Therefore, the proposition of Hauser and Schlievert (1990) supra, that the lack of protease activity associated with speB purified from their M12 strain 86-858 is a consequence of the difference in amino acid sequence around the Cys residue, is incorrect.

EXAMPLE 3

Cleavage of PIL-lβ by Streptococcal Cysteine Protease An assay employing radiolabeled PIL-lβ made in a rabbit reticulocyte transcription-translation system was used. The cysteine protease produced a cleavage product of approximately 18 kDa, a size very similar to the apparent molecular weight of MIL-lβ. Western blot analysis of the cleavage products generated from recombinant PIL-lβ made in E. coli confirmed this result.

The cysteine protease cleaved a human PIL-lβ mutant (Asp 116— Ala 116, creating an Ala 116 - Ala 117 linkage) that is not degraded by interleukin- lβ converting enzyme (ICE). As observed with wild type PIL- lβ, cysteine protease cleaved the mutant substrate to form a product with an apparent molecular weight of N18 kDa. Thus, the primary cleavage site for the cysteine protease was not the ICE proteolytic site.

To determine exactly where the cysteine protease cleaved P1L-1, the aminoterminal 10 amino acid residues of the N18 Kda product made by degradation of recombinant PIL-lβ was sequenced. The cysteine protease cleaved PIL-lβ between His 115 - Asp 116 to create a molecule one amino acid residue longer than MIL-lβ.

EXAMPLE 4

Normal Biological Activity of the Mature IL-lβ Cleavage Product A highly active form of MIL-1 β with Asp-116 at the amino terminus was described in the course of characterization of a metalloprotease found in human peripheral blood mononuclear cells. The data suggested that the cysteine protease was processing inactive PIL- lβ to biologically active IL-lβ. Mature IL-lβ is a potent inducer of nitric oxide synthase (NOS) activity in vascular smooth muscle cells (SMC). Cysteine protease was added in the presence or absence of PIL-lβ to confluent cultures of SMC and NOS activity was assayed by measuring nitrite anion levels in the medium after 24 hours. (Kapur et al., Proc. Nat'l. Acad. Sci. USA (1993) 90:7676-7680.) Neither cysteine protease nor PIL-lβ alone produced a significant increase in nitrite levels. In contrast, addition of cysteine protease and PIL- lβ together caused approximately a 60-fold increase in nitrite accumulation.

IL-lβ generated by cysteine protease cleavage of PIL-lβ. was also found to be active in the A375 cell line assay (Nakai et al., Biodichem. Biophys. Res. Commun. 154:1189-1196 (1988)). In an assay in which approximately 500 ng/ml of intact PIL-1 β was inactive, a cysteine

protease digest of this material yielded 6.1 x IO ⁴ units/ml of activity; 500 ng/ml of authentic IL-lβ corresponded to 1.1 x 10° units in this assay.

EXAMPLE 5

Cleavage Activity of Variant Cysteine Protease Enzymes Two additional naturally occurring cysteine protease allelic variants

(speB2 and speBl 1) also produced an IL-lβ fragment with an apparent molecular weight identical to that made by speBl purified from MGAS 1719.

EXAMPLE 6 Sequencing of speB

Basically, the sequencing of the cysteine protease structural gene was performed as follows. The cysteine protease structural gene was amplified by the polymerase chain reaction (PCR) using synthetic oligonucleotides. The oligonucleotide primers used to amplify speB and flanking regions were as follows:

S P E B -X ( S E Q I D N O : 5 ) , 5 ' GTTGTCAGTGTCAACTAACCGT 3'; and SPEB-2 (SEQ ID NO:6), 5' = ATCTGTGTCTGATGGATAGCTT - 3'. The following four oligonucleotides were used as internal sequencing primers:

S P E B - 1 ( S E Q I D N O : 7 ) , 5 ' CTTTCTGGCTCTAATATGTATGT - 3'; SPEB-3 (SEQ ID NO:8), 5' - GTTATTGAAAAAGTAAAACC - 3'; SPEB-4 (SEQ ID NO:9), 5' - TTTTCAATAACAGGTGTCAA - 3'; and SPEB-Y (SEQ ID NO: 10), 5' -

TCTCCTGAAACGATAACAAA - 3'. PCR amplification of 1 μl of chromosomal DNA was performed in 100 μl of a mixture containing 50 Mm KC1, 10 Mm Tris-Hcl, pH 8.3, 1.5 mM MgCl ₂ , 0.001% gelatin, 200 μM each of dATP, dCTP, dGTP, and

dTTP, 200 nM each of SPEB-X and SPEB-2, and 2.5 units of AmpHTaq DNA polymerase (Per kin Elmer). The thermocycling parameters were: denaturation at 94° C for 1 minute, annealing at 55° C for 2 minutes, and extension at 72° C for 2.5 minutes for a total of 30 cycles. A final extension at 72° C for 15 minutes was used.

The amplified DNA fragment (1,437 bp) represents the entire coding region (1,197 bp) and 160 bp of upstream and 80 bp of downstream sequence. For about one-third of the strains, single-stranded DNA was prepared by the lambda exonuclease method and sequenced in both orientations with Sequenase version 2.0 (US Biochemicals). Variant alleles were sequenced again to confirm the nucleotide changes.

The protease gene in approximately two-thirds of the strains was characterized by automated DNA sequencing with an Applied Biosystems, Inc., Model 373A instrument. For the automated approach, the gene was amplified with PCR (10 mM Tris-HCl, pH 8.3; 50 mM KC1; 1.5 mM

MgCL;; 2.5 units of Taq polymerase; 20 picomoles of each primer; 1 μL of chromosomal DNA template), with the following thermocycler parameters: denaturation at 94° C for 4 minutes, 30 cycles of denaturation at 94° C for 1 minute, primer annealing at 55° C for 2 minutes, extension at 72° C for 2 minutes, and a final extension at 72° C for 5 minutes. The unincorporated nucleotides and primers were removed by filtration through Microcon 100 microconcentrators (Amicon Inc., MA). Sequencing reactions with the Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Inc., CA) were performed with 7 μL of PCR amplified DNA as template and 3.2 picomoles of primer. The unincorporated dye terminators and primers were separated from the extension products by spin column purification (Centri-Sep, Princeton Separations, Inc., NJ). The sample was dried in a vacuum centrifuge. Prior to gel loading, the sample was resuspended in 4 μL of sample loading buffer (5:1 deionized formamide; 50 mM EDTA, pH 8.0) and heat denatured for 2 minutes at

90° C. The data were assembled and edited with EDITSEQ, ALIGN, and SEQMAN programs (DNASTAR, WI).

EXAMPLE 7

Estimates of Genetic Relationships Among Clones Methods of estimating genetic relationships among S. pyogenes clones by multilocus enzyme electrophoresis were as described by Musser et al., Proc. Natl. Acad. Sci. USA 88:2668-72 (1991). Thirty-six ETs not identified previously were arbitrarily numbered ET 34 - ET 53.

EXAMPLE 8 speB Allele in Strain B220 (Elliott 5797)

The speB gene (speB7) in strain MGAS 1719 does not encode a protein with the amino acid sequence presented previously. There are discrepancies between the protein sequence from strain B220 and a speB allele (herein designated speBl) in a serotype M12 strain (86-858).

EXAMPLE 9 speB Alleles and Disease Type The present invention demonstrates that streptococcal clones with the same speB 25 allele, and speB allele - M protein combination are associated with several different diseases. For example, strains of ET 1 - Ml - speB2 were cultured from patients with pharyngitis, scarlet fever, cellulitis, and TSLS: and ET 2 -M3 - speB3 organisms were recovered from cases of pharyngitis, scarlet fever, and TSLS. Similarly, strains cultured from individuals with acute rheumatic fever had six distinct speB alleles. Hence, there was no apparent preferential association of speB allele and disease type.

The identification of the speB allele in a strain (MGAS 789) recovered in the 1940s expressing Ml protein, but assigned to ET36 rather than ET 1 like contemporary MI strains suggests that variation in speB allele - multilocus enzyme genotype - M protein associations made by a contributing factor in temporal changes in streptococcal disease frequency and severity.

EXAMPLE 10 speB Variation. M Protein Class. Opacity Factor Phenotype. and vir Regulon Architecture The present invention found no compelling evidence for an analogous differentiation of speB allelic variants. Strains assigned to either of two distinct classes based on reactivity with a panel of monoclonal antibodies to M protein did not have consistent sequence differences, and in several instances the identical speB allele was found in strains of two M protein classes. For example, the speB3 allele occurred in strains of both class I (M3 and M12) and class II (M2), and similarly, the speB5 allele was identified in strains expressing Ml and M4 assigned to class I and class II, respectively (Table 1). Similarly, there was no simple congruent relationship between speB allele and vir regulon architecture or opacity factor phenotype. M2, M3, and M12 strains all had the speB3 allele, but, M3 and M123 are opacity factor-negative and M2 is opacity factor-positive. The lack of a significant correlation between M serotype class and speB phylogeny could also be caused by relatively frequent lateral transfer events involving part or all of the emm and speB genes.

EXAMPLE 11

Cleavage of purified extracellular matrix (ECM) proteins Streptococcal cysteine protease rapidly degrades purified vitronectin (VN). After five minutes of protease incubation with VN, degradation

Streptococcal cysteine protease rapidly degrades purified vitronectin (VN). After five minutes of protease incubation with VN, degradation products could not be identified by either Coomassie blue staining or immunoblotting with polyclonal anti-VN antibodies. Similarly, the streptococcal protease cleaved fibronectin (FN) immediately, as shown by the rapid appearance of lower molecular weight products. However, in contrast to VN degradation, FN cleavage apparently occurred at a limited number of specific sites of manuscript). Incubation of FN with the protease for up to 12 hours did not result in formation of additional degradation products.

No significant cleavage of human laminin (LN) was observed under the experimental conditions assayed, or when 10 μg of protease and 2 μg of LN substrate were used.

EXAMPLE 12 Induction of cytopathic effect and fibronectin cleavage in human umbilical vein endothelial cell HUVEC) cultures Patients with invasive S. pyogenes episodes frequently have bacterial sepsis with endothelial cell damage, therefore the ability of the streptococcal cysteine protease to cleave FN directly from HUVECs grown in culture was examined. Western immunoblot analysis of cells in the absence of protease, or treated with boiled protease for up to 8 hours, showed no detectable FN degradation. In contrast, cells incubated with as little as 6 μ/ml of streptococcal protease per well for 2 hours retained only a small fraction of intact native FN. Thus, the streptococcal protease cleaves FN in a dose and time dependent manner in the complex environment of cells growing in tissue culture.

Interestingly, treatment of HUVECs with the streptococcal protease rapidly induced striking cytopathic effects. By 3 hours after protease addition, zones of clearing occurred in the cell monolayer. This effect was followed by loss of cell adherence to the matrix and ablation of the

characteristic cobblestone morphology. FN cleavage was detectable by immunoblot analysis prior to the onset of cytopathic effect. Bands that correspond to native human VN in either the solubilized control or treated HUVECs by western immunoblot analysis were not seen, presumably due to low level or lack of VN expression by these cells.

EXAMPLE 13

Cysteine protease production by S. pyogenes strains

Virtually all clinical isolates of Group A streptococci produce

SPE B/cysteine protease, and patients infected with Group A streptococci develop antiproteinase antibodies. Immunoblot analysis of culture supernatants was used to assess production of speB/streptococcal protease by strains of S. pyogenes, and one naturally occurring serotype M il isolate (MGAS 2075) reported to lack speB. With the exception of the three strains, all 64 other isolates examined produced cysteine protease, a result consistent with the notion that virtually all & pyogenes strains express the molecule extracellularly (Table 1). The three strains had alleles speB3, speB13, and speBlβ, but other isolates with these same alleles produced the protease. Therefore, all 39 speB alleles can be expressed by Group A streptococcal strains under appropriate conditions. The polymorphic site of the 39 alleles within the 160 bp upstream noncoding region and 1197 bp coding region of the speB gene are shown in Figure 1.

EXAMPLE 14

Specific antisera raised against the active cysteine protease Purified protease (100 μg) mixed with Freund's complete adjuvant was injected subcutaneously at multiple sites into two rabbits. Subsequent immunizations with the purified protease mixed with Freund's incomplete adjuvant were conducted at bi-weekly intervals for a total of five

injections. Serum was collected and immunoglobulin purified by FPLC with a protein G-Sepharose column (Pharmacia). Western immunoblot analysis revealed the presence of specific anti-protease reactivity in the post-immunization samples but not in the pre-immunization sera. These rabbits are being maintained and bled at regular intervals to collect large quantities of specific antiserum. This procedure has been described in Kapur, et al., Microb. Pathog. (1993), 15:327-46.

EXAMPLE 15 Mouse monoclonal antibodies against the purified mature cysteine protease Monoclonal antibodies against the purified mature cysteine protease were prepared. A dose of 10 μg of SDS-PAGE purified mature protease has been injected a total of five times intraperitoneally into five mice (Balb/c background). Western immunoblot analysis demonstrated that all mice have serocoverted. The spleens were harvested and fusions performed by standard protocols. Characterization of protease-specific monoclonal antibodies is by standard procedures.

EXAMPLE 16 Measurement of antibody levels

An ELISA has been developed to measure antibody levels against the cysteine protease. Briefly, 10 μg of protease in carbonate-bicarbonate buffer (pH 9.6) was added to each well of a 96- well microtiter plate and incubated overnight at 40° C. The wells were rinsed three times with washing buffer (PBS (pH 7.4) - Tween 20 (0.05%)) and blocked with 200 μL of 0.5% BSA in PBS, pH 7.4, for 2 hours at 37° C. After washing, the wells were charged with 100 μL of a serial dilution of test antisera (1:100 through 1:1600 of rabbit serum). The plate was incubated for 1 hour at 37° C, washed again, and 100 μL of a 1:5000 dilution of extravidin-alkaline

phosphatase was added to each of the test wells and incubated at 37° C for 30 minutes. After washing, 100 μL of alkaline phosphatase substrate (pNPP) was added to each well and reacted for 1 hour at room temperature. The O.D. (405 nm) was read with a microtiter plate reader.

EXAMPLE 17 lmmunodot-blot assay for cysteine protease expression A dot-blot assay was developed that detects as little as 1 nanogram of cysteine protease. Briefly, test material (usually protein precipitates of culture supernatants from bacteria grown in chemically defined medium) was spotted onto a nylon membrane, and unabsorbed sites were blocked by incubation with 0.5 % blocking agent (Amersham) for 1 hour at room temperature. The membrane was rinsed with PBS (pH 7.4) - Tween 20 (0.05%) and incubated for 30 minutes with purified polyclonal rabbit antiserum (1:500 dilution) directed against the cysteine protease. The membrane was rinsed with PBS, a secondary antibody (goat anti-rabbit-

HRP conjugate, 1:2000 dilution) was added and incubated for 30 minutes at room temperature. The blot was visualized with chemiluminescence (ECL developing reagents, Amersham). With this technique, many isolates previously reported to lack speB production based on less sensitive conventional immunologic assays express the cysteine protease.

EXAMPLE 18

Antibody directed against cysteine protease The immunoprophylactic protection of cysteine protease is seen by the use of two models. First, the intranasal immunization model is used as developed by Bessen and Fischetti (Infect. Imτnun. 56: 2666-72 (1988)) to evaluate the effect of cysteine protease immunization on mucosal colonization by S. pyogenes. Second, a mouse cutaneous infection model (Bunce et al., Infect. Immun. 60: 2636-40 (1992)) is used against a subcutaneous bacterial challenge. Briefly, the animals are injected with

protease s.c. on the flank and observed daily, including weight measurements. Abscess volumes and area of dermonecrosis is calculated and lesion size curves are determined.

EXAMPLE 19 Preparation of Synthetic Peptides of Cysteine Protease

Synthetic peptides based on cysteine protease may also be used as immunogens in the preparation of a vaccine against Group A streptococcal infections. Several synthetic peptides are selected based on the location of allelic variation and conservation and the cysteine protease antigenic index generated with a Jameson- Wolf plot. First, each of the following three peptides are used. These peptides correspond to the variable region

(amino acids 308 to 317) in mature streptococcal cysteine protease containing two of the six major calculated antigenic peaks.

Peptide 1 (SEQ ID NO: 11): H-Q-l-N-R-S(308)-D-F-S-K-Q-D-W-E-A(317)-Q-I-D-K-E

Peptide 2 (SEQ ID NO: 12):

H-Q-I-N-G(308)-D-F-S-K-Q-D-W-E-A(317)-Q-I-D-K-E Peptide 3 (SEQ ID NO: 13):

H-Q-l-N-S(308)-D-F-S-K-Q-D-W-E-A(317)-Q-l-D-K-E Subsequently, each of the following four peptides, which correspond to four invariant calculated antigenic peaks are used for immunization: Peptide 4 (SEQ ID NO: 14):

P( 171)-V-I-E-K-V-K-P-G-E-Q-S-F-V-G-Q Peptide 5 (SEQ ID NO: 15): Y(203)-H-N-Y-P-N-K-G-L-K-D-Y-T-Y-T-L

Peptide 6 (SEQ ID NO: 16):

P(247)-T-Y-S-G-R-E-S-N-V-Q-K-M-A-I

Peptide 7 (SEQ ID NO: 17):

I(344)-D-G-A-D-G-R-N-F-Y-H Naturally occurring variant zymogens and cysteine protease display unique linear B-cell epitopes. Overlapping 10-mer peptides are used which overlap 2 amino acid residues with the previous one in the consecutive primary sequence corresponding to 371 amino acids of the mature cysteine protease zymogen (translated product minus leader sequence). Synthetic 10-mers corresponding to the 10 variant amino acid residues are also used. The variant amino acids are positioned in the middle of the 10-mer. For example, if the sequence of a 10-mer corresponding to one region of the speBl variant is position 304-QINRSDFSKQ-313 (SEQ ID NO: 18), then 304-QINRGDFSKQ-313 (SEQ ID NO: 19) is also examined, a 10-mer that incorporates a variant amino acid found in the speB2 variant. Once the 10-mer peptides are synthesized, an EL1SA is used to examine the reactivity of all peptides with the following materials: (i) rabbit polyclonal hyperimmune antiserum made against purified cysteine protease (positive control), (ii) rabbit pre-immune serum (negative control), (iii) our panel of 28 murine monoclonal antibodies raised against purified cysteine protease, (iv) acute and convalescent sera obtained from 20 patients with necrotizing fascitis and/or TSLS in Canada (obtained from D. Low, Mount Sinai Hospital, Ontario, Canada), five USA patients with TSLS characterized by extensive soft tissue destruction (obtained from D. Stevens, V.A. Hospital, Boise, Idaho), and five patients with ARF (obtained from A. Bisno, University of Miami Medical School). The great majority of the synthetic peptides usually are not reactive with each sera and there are a large number of internal redundant negative control peptides. Sera dilutions are used in these assays (1:1000 for hyperimmune rabbit antiserum, 1:500 for human serum, and 1:5 - 1:10 for MAb culture supernatants).

To determine the linear B-cell epitopes, for each sera and MAb tested, OD^ is plotted versus 10-mer peptide number. The linear B-cell epitopes are displayed as a peak in the OD^ values. In general, a peak is composed of several contiguous overlapping peptides, and the 10-mer peptide with the highest Ou _t value is defined as the parent peptide. The pro region contains at least one unique linear B cell epitope. The same linear B cell epitopes will most likely be recognized by all 15 human convalescent sera specimens.

EXAMPLE 20 Creation of Mutant speB Proteins

Both site-directed and random mutagenesis schemes are employed to identify residues that disrupt cysteine protease function and zymogen processing, and to map regions that constitute antigenic domains of the protein. Targets for functional amino acid replacement are based on biochemical analysis of cysteine protease (Tai, et al, J. Biol. Chem. 251;

1955-59 (1976)) and by analogy with similar residues in eukaryotic cysteine protease.

To create a stable zymogen to facilitate crystallographic studies and generate enzymatically deficient or inactive protease for structure- unction studies, mutant forms of the cysteine protease protein are made and characterized. A target mutagenesis scheme creates changes that: (i) disrupt protease activity; (ii) prevent zymogen processing; (iii) prevent substrate binding; and (iv) alter immunoreactivity. Amino acids are changed to structurally neural alanine. A mutant protein that lacks protease activity, but which retains antigenicity, is generated by mutagenesis of the single cysteine residue (Cys-192— Ala-192) at the catalytic site of the molecule. Also, His-340 and Gin-185 and Asn-356 are mutagenized. These three changes are epistatic to the Cys-192 mutation, but may alone exhibit altered activity. Trp-357, thought to be involved in substrate binding and similarly positions within papain, is also to be

targeted. A stable zymogen precursor is also created by mutating residues surrounding the protease cleavage site at Lys-145. In addition, mutagenesis of Cys-192 may prevent autoproteolysis, as occurred for a Cys— Ser mutant of papain, the prototype cysteine protease. Other mutagenesis targets include a putative nucleotide binding domain

(GVGKVG) (SEQ ID NO:2) and a potential collagen docking region (GXX) ₃ within carboxy terminal portion of the protein. Site-directed mutagenesis is used, by the charged-to-alanine-scanning method, to substitute positively and negatively charged amino acids (often involved in recognition and activity) with alanine. Many of the charged residues

(14 lysine, arginine, 12 aspartate, and 76 glutamate residues in the mature peptide) are expected to lie on the surface of the cysteine protease structure, and some are expected to define epitopes on the molecule. In particular, a region of charged amino acids, from 307 to 321 (8/15 charged), is examined; this region includes the site of speB and speB4 amino acid substitutions. Residues in antigenic regions identified in the epitope mapping studies are also mutated.

First, the speB gene is amplified from an ET1/MI S. pyogenes strain, with PCR, and the product is cloned into a multicopy plasmid vector such as Pbluescript (Stratagene, La Jolla, Ca). This vector is chosen because it carries the regulated lac promoter and can be replicated as a single- stranded molecule for site-directed mutagenesis. Cloning is designed to place the promoter, ribosome binding site and speB reading frame 3 ¹ to the inducible lac promoter on the vector so that the protein can be conditionally over-expressed in E. coli when the lac inducer, IPTG, is added. The speB promoter is included requiring expressing in <S. pyogenes. Whole cell extracts and periplasmic shockates of E. coli cells carrying this primary speB clone are examined for the presence of the cysteine protease protein by SDS-PAGE and by Western blotting with anti-cysteine protease antibody. The resulting plasmid is the target for mutagenesis.

Oligonucleotide-directed mutations, such as substitutions, deletions and small insertions, are created on uracil-containing single-stranded templates by the method of Kunkel (Kunkel, 1985, supra.). When possible, mutagenic primers are designed to incorporate a unique restriction site into the speB gene for mapping and mutant selection. Both single and multiple alanine substitutions are created at the residues indicated above. Once residues critical to function are identified, small regions surrounding them are deleted or substituted, by using the same methods, to further characterize the region and to preclude reversion. When crystallographic data is available, additional amino acids are mutated.

A random mutagenesis scheme is also employed. Variant proteins created by this method are most useful for epitope screening, although molecules with altered kinetics and substrate recognitions may also be recovered. Regions of the speB sequence are randomized with mixed oligonucleotides in the primed-mutagenesis protocol, or short, in-frame deletions within the gene are created with a modification of the DNAse 1- linker insertion/deletion protocol of Palzkill and Bostein, supra. Here, synthetic "excision linkers" are first ligated to randomly linearized target DNA, then excised with flanking nucleotides to create small substitutions or deletions. For example, a linker with two copies of the recognition sequence for the enzyme <SqpI (GCTCTTC) is used to create six base deletions (three bases on each end of the linker), or random two amino acid deletions, across the speB gene. Flanking bases are also randomized by filling the ends of the target sequence after linker excision, then inserting a second blunt end linker that includes a random sequence in place of the Ns. The second linker is then removed by digestion with Sαpl and the target sequence is ligated to generate substitutions.

To identify protease minus mutations, E. coli cells producing potential mutant proteins are first screened for protease activity on casein agar plates. Since secretion of cysteine protease to the periplasm is

expected, it is possible that protease activity can be observed on plates. If this screening strategy is successful, then thousands of colonies rapidly are examined for functional mutations in cysteine protease. If cysteine protease must be completed secreted from E. coli to exhibit activity, then osmotic shockates of each presumptive mutant strain is assayed for proteolytic activity.

EXAMPLE 21

In Vitro Cvtotoxicitv of Streptococcal lL-lβ Convertase. Cells were plated at a density of 5 x IO ³ cells/well in 96-well microtiter plates in 200 μl of DMEM with 10% fetal calf serum, and incubated overnight at 37° C. The medium was removed and replaced with fresh medium containing varying concentrations of the purified streptococcal enzyme. The plates were then incubated for 24 h and cell viability was determined by a colorimetric assay (Green et al.,

J. Immunol. Methods (1984), 70:257). Briefly, 10 μl of a solution of (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) (5 μ/ml) was added to the wells to a final concentration of 0.5 μ/ml. The plates were incubated at 37° C for 2 h, the medium was removed, DMSO (100 μg/well) was added, and the plates were kept on an orbital shaker for 10 min. The OD was measured at 495 nm with an EL1SA reader. Percent cytotoxicity was calculated as follows: (OD in presence of streptococcal IL- lβ convertase ÷ OD in the absence of streptococcal IL-lβ convertase) x 100 (Green et al. (1984), supra.) The results demonstrate that murine melanoma cell lines K1735 and CM519 were sensitive to killing by streptococcal cysteine protease in a dose-dependent manner (Fig. 2A). In contrast, boiled (proteolytically inactive) streptococcal IL-lβ convertase lacked cytotoxic activity against these two tumor cell lines (data not shown).

EXAMPLE 22

Streptococcal IL-lβ Convertase-Induced DNA

Fragmentation in CM519 Melanoma Cells.

Cells (1 x 10 ₆ ) were treated with streptococcal IL-lβ convertase (20 μ/ml for 48 h or 50 μ/ml for 24 h) and then harvested by treatment with trypsin. Cells were centrifuged, washed with PBS, resuspended in

0.5 ml of lysis buffer (10 mM Tris, lmM EDTA, 0.2% Triton X-100, pH

7.5), and incubated for 20 minutes on ice. After centrifugation at 13,000 x g for 10 minutes, DNA was precipitated from the supernatants by the addition of 5.0 M NaCl (to a final concentration 0.5 M) and 0.5 volumes of 100% isopropanol. After storage at -20° C, the samples were centrifuged at 13,000 x g for 10 minutes. The pellets were resuspended in 50 μl of TE buffer containing proteinase K (300 μ/ml) and RNAse A (100 μg/ml) and incubated for 30 min at 50° C. The samples were electrophoresed through a 1.5% agarose gel in 0.6x TBE buffer at 120 V for 2 h. The DNA bands were visualized by staining with ethidium bromide and photographed under UV light with a transilluminator.

To investigate the mechanism of streptococcal enzyme-induced cytotoxicity, K1735 and CM519 cells were treated with the protease for 24 h and checked for induction of DNA fragmentation. The streptococcal enzyme caused DNA ladder formation characteristic of apoptotic cell death (Fig. 2B). The streptococcal enzyme also exhibited a varying degree of cytotoxicity to six other tumor cell lines (Table 2).

-54-

Table 2 Sensitivity of various miirine tumor cell lines to streptococcal cvsteine protease.

Cell Line Tumor Type Survival (%)' Mean Std. Error

'Percent survival and standard error were calculated as follows: (viable cell number in the presence of the streptococcal enzyme ÷ viable cell number in untreated control) x 100.

EXAMPLE 23

Antitumor Cell Activity of Streptococcal IL-lβ Convertase in Immunocompetent C3H/ΗeNCr Mice. Subconfluent cultures of K1735 and CMS 19 cells were harvested by trypsinization and washed with serum-free RPMI 1640 medium

(GIBCO). Then, 1 x 10° cells suspended in 0.2 ml of serum-free RPMI 1640 were injected S.C. into groups of five immunocompetent six-to-eight- week old specific pathogen-free female C3H HeNCr (mouse mammary tumor virus-negative) mice (Frederick Cancer Research Center Animal Protection Area, Frederick, MD). Twenty-four hours later, the experimental groups of mice were given 100 μg of purified streptococcal IL-lβ convertase intraperintoneally, while control groups received 100 μl of either boiled streptococcal IL-lβ convertase or PBS. The treatments were administered twice per week for two weeks and the animals were checked at weekly intervals for tumor growth for 12 weeks.

Next, it was investigated whether streptococcal IL-lβ convertase had antitumor cell activity 15 in vivo. Immunocompetent C3H mice were injected subcutaneously with tumorigenic doses of K1735 or CM519 melanoma cells, and 24 hours later, injected intraperitoneally with either native enzyme, boiled enzyme, or PBS (Fig. 3). All animals injected with

K1735 or CMS 19 cells followed by intraperitoneal administration of boiled streptococcal enzyme or PBS developed tumors (Fig. 3). In striking contrast, administration of the active streptococcal IL-1B convertase protected mice from developing tumors after injection with K1735 and CM519 cells, in 100% and 80% of animals, respectively (Fig. 3).

Moreover, mice given streptococcal Il-Iβ convertase did not exhibit observable detrimental side effects such as weight loss, lethargy, or behavioral changes (Fig. 3).

EXAMPLE 24

Antitumor Cell Activity of Streptococcal IL-lβ Convertase in Athymic (nu/nu) Nude Mice. K1735 and CM519 cells were injected sub-cutaneously into groups of five six-to eight-week old nu/nu mice (Frederick Cancer Research

Center Animal Production Area, Frederick, MD) at a dose of 1 x IO ⁴ cells/mouse followed by intraperitoneal injection of streptococcal IL-lβ convertase, as described in the schedule above. Mice were checked at twice-weekly intervals for tumor growth for 12 weeks. The proteolytically inactive precursor of the streptococcal protease is thought to be a member of the family of superantigenic streptococcal and staphylococcal exotoxins (Marrack and Kappler, Science, 248:705-11 (1990); Tomai et al, Infec. Immun., 60:701-05 (1992), Abe et al., J. Immunol., 146:3747-50 (1991). Since superantigens stimulate T-cells to release tumor necrosis factor (Miethke et al., J. Exp. Med., 175:91-98

(1992)), we investigated if tumor cell rejection caused by the streptococcal enzyme is T-cell dependent by use of athymic nu/nu mice. The results indicate that treatment with streptococcal IL-lβ convertase completely protected athymic mice against growth of transplanted K1735 melanomas, and protected 60% of the mice from developing CM519 melanomas

(Fig. 4). All mice in the control groups injected with PBS or boiled streptococcal IL-lβ convertase developed tumors. Hence, it is likely that streptococcal IL-lβ convertase-induced tumor rejection is T-cell independent. As the results described above show, a highly conserved streptococcal extracellular cysteine protease (interleukin- lβ convertase) is cytotoxic for murine melanoma (n = 3), fibrosarcoma (n = 2), undifferentiated (n = 1), and squamous cell carcinoma (n = 1) cell lines, and induces apoptosis. Intraperitoneal administration of purified streptococcal IL-lβ convertase to immunocompetent mice resulted in the rejection of subcutaneously transplanted murine melanoma cell lines

CM519 and K1735 in 80% and 100% of animals, respectively. Moreover, intraperitoneal injection of the streptococcal enzyme completely protected athymic mice against growth of transplanted K 1735 melanomas, and protected 60% of the mice from developing CM519 melanomas. These data imply that the streptococcal cysteine protease plays an important role in the inhibition of murine tumor outgrowth in a T-cell independent manner.

All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications cited are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Musser M.D., James M. Kapur M.D., Vivek Ananthas amy, H. N. Fernandez, A.

(ii) TITLE OF INVENTION: Use of extracellular cysteine protease to inhibit cell proliferation

(iii) NUMBER OF SEQUENCES: 58

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: WEIL, GOTSHAL & MANGES

(B) STREET: 2882 Sand Hill Road, Suite 280

(C) CITY: Menlo Park

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 94025-7022

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US not yet assigned

(B) FILING DATE: 01-MAY-1995

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/306,542

(B) FILING DATE: 14-SEP-1994

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/160,965

(B) FILING DATE: 02-DEC-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Rae-Venter Ph.D., Barbara

(B) REGISTRATION NUMBER: 32,750

(C) REFERENCE/DOCKET NUMBER: BAYL-004/02US

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 926-6200

(B) TELEFAX: (415) 854-3713

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 398 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(B) STRAIN: MGAS 1719

(vii) IMMEDIATE SOURCE: (B) CLONE: speB7

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

Met Asn Lys Lys Lys Leu Gly lie Arg Leu Leu Ser Leu Leu Ala Leu 1 5 10 15

Gly Gly Phe Val Leu Ala Asn Pro Val Phe Ala Asp Gin Asn Phe Ala 20 25 30

Arg Asn Glu Lys Glu Ala Lys Asp Ser Ala lie Thr Phe lie Gin Lys 35 40 45

Ser Ala Ala lie Lys Ala Gly Ala Arg Ser Ala Glu Asp lie Lys Leu 50 55 60

Asp Lys Val Asn Leu Gly Gly Glu Leu Ser Gly Ser Asn Met Tyr Val 65 70 75 80

Tyr Asn lie Ser Thr Gly Gly Phe Val lie Val Ser Gly Asp Lys Arg 85 90 95

Ser Pro Glu lie Leu Gly Tyr Ser Thr Ser Gly Ser Phe Asp Ala Asn 100 105 110

Gly Lys Glu Asn lie Ala Ser Phe Met Glu Ser Tyr Val Glu Gin lie 115 120 125

Lys Glu Asn Lys Lys Leu Asp Thr Thr Tyr Ala Gly Thr Ala Glu lie 130 135 140

Lys Gin Pro Val Val Lys Ser Leu Leu Asp Ser Lys Gly lie His Tyr 145 150 155 160

Asn Gin Gly Asn Pro Tyr Asn Leu Leu Thr Pro Val lie Glu Lys Val 165 170 175

Lys Pro Gly Glu Gin Ser Phe Val Gly Gin His Ala Ala Thr Gly Cys 180 185 190 "

Val Ala Thr Ala Thr Ala Gin lie Met Lys Tyr His Asn Tyr Pro Asn 195 200 205

Lys Gly Leu Lys Asp Tyr Thr Tyr Thr Leu Ser Ser Asn Asn Pro Tyr 210 215 220

Phe Asn His Pro Lys Asn Leu Phe Ala Ala lie Ser Thr Arg Gin Tyr 225 230 235 240

Asn Trp Asn Asn lie Leu Pro Thr Tyr Ser Gly Arg Glu Ser Asn Val 245 250 255

Gin Lys Met Ala lie Ser Glu Leu Met Ala Asp Val Gly lie Ser Val 260 265 270

Asp Met Asp Tyr Gly Pro Ser Ser Gly Ser Ala Gly Ser Ser Arg Val 275 280 285

Gin Arg Ala Leu Lys Glu Asn Phe Gly Tyr Asn Gin Ser Val His Gin 290 295 300

lie Asn Arg Ser Asp Phe Ser Lys Gin Asp Trp Glu Ala Gin lie Asp 305 310 315 320

Lys Glu Leu Ser Gin Asn Gin Pro Val Tyr Tyr Gin Gly Val Gly Lys 325 330 335

Val Gly Gly His Ala Phe Val lie Asp Gly Ala Asp Gly Arg Asn Phe 340 345 350

Tyr His Val Asn Trp Gly Trp Gly Gly Val Ser Asp Gly Phe Phe Arg 355 360 365

Leu Asp Ala Leu Asn Pro Ser Ala Leu Gly Thr Gly Gly Gly Ala Gly 370 375 380

Gly Phe Asn Gly Tyr Gin Ser Ala Val Val Gly lie Lys Pro 385 390 395

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: cysteine protease nucleotide binding domain

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2;

Gly Val Gly Lys Val Gly 1 5

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1197 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(B) STRAIN: MGAS 1719

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB7 (cysteine protease)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATGAATAAAA AGAAATTAGG TATCAGATTA TTAAGTCTTT TAGCATTAGG TGGATTTGTT 60

CTTGCTAACC CAGTATTTGC CGATCAAAAC TTTGCTCGTA ACGAAAAAGA AGCAAAAGAT 120

AGCGCTATCA CATTTATCCA AAAATCAGCA GCTATCAAAG CAGGTGCACG AAGCGCAGAA 180

GATATTAAGC TTGACAAAGT TAACTTAGGT GGAGAACTTT CTGGCTCTAA TATGTATGTT 240

TACAATATTT CTACTGGAGG ATTTGTTATC GTTTCAGGAG ATAAACGTTC TCCAGAAATT 300

CTAGGATACT CTACCAGCGG ATCATTTGAC GCTAACGGTA AAGAAAACAT TGCTTCCTTC 360

ATGGAAAGTT ATGTCGAACA AATCAAAGAA AACAAAAAAT TAGACACTAC TTATGCTGGT 420

ACCGCTGAGA TTAAACAACC AGTTGTTAAA TCTCTCCTTG ATTCAAAAGG CATTCATTAC 480

AACCAAGGTA ACCCTTACAA CCTATTGACA CCTGTTATTG AAAAAGTAAA ACCAGGTGAA 540

CAATCTTTTG TAGGTCAACA TGCAGCTACA GGATGTGTTG CTACTGCAAC TGCTCAAATT 600

ATGAAATATC ATAATTACCC TAACAAAGGG TTGAAAGACT ACACTTAGAC ACTAAGCTCA 660

AATAACCCAT ATTTCAACCA TCCTAAGAAC TTGTTTGCAG CTATCTCTAC TAGACAATAC 720

AACTGGAACA ACATCCTACC TACTTATAGC GGAAGAGAAT CTAACGTTCA AAAAATGGCG 780

ATTTCAGAAT TGATGGCTGA TGTTGGTATT TCAGTAGACA TGGATTATGG TCCATCTAGT 840

GGTTCTGCAG GTAGCTCTCG TGTTCAAAGA GCCTTGAAAG AAAACTTTGG CTACAACCAA 900

TCTGTTCACC AAATTAACCG TAGCGACTTT AGCAAACAAG ATTGGGAAGC ACAAATTGAC 960

AAAGAATTAT CTCAAAACCA ACCAGTATAC TACCAAGGTG TCGGTAAAGT AGGCGGACAT 1020

GCCTTTGTTA TCGATGGTGC TGACGGACGT AACTTCTACC ATGTTAACTG GGGTTGGGGT 1080

GGAGTCTCTG ACGGCTTCTT CCGTCTTGAC GCACTAAACC CTTCAGCTCT TGGTACTGGT 1140

GGCGGCGCAG GCGGCTTCAA CGGTTACCAA AGTGCTGTTG TAGGCACTAA ACCTTAG 1197

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(B) STRAIN: MGAS 1719

(vii) IMMEDIATE SOURCE:

(B) CLONE: cysteine protease

(viii) POSITION IN GENOME:

(B) MAP POSITION: 146-156

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Gin Pro Val Val Lys Ser Leu Leu Asp Ser Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

( B ) CLONE : SPEB-X

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GTTGTCAGTG TCAACTAACC GT 22

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide'

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: SPEB-2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

ATCTGTGTCT GATGGATAGC TT 22

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide ¹

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: SPEB-1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7i

CTTTCTGGCT CTAATATGTA TGT 23

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: SPEB-3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GTTATTGAAA AAGTAAAACC 20

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: SPEB-4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

TTTTCAATAA CAGGTGTCAA 20

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Oligonucleotide"

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: SPEB-Y

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TCTCCTGAAA CGATAACAAA 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: Peptide 1

(viii) POSITION IN GENOME:

(B) MAP POSITION: 303

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

His Gin lie Asn Arg Ser Asp Phe Ser Lys Gin Asp Trp Glu Ala Gin 1 5 10 15

lie Asp Lys Glu

20 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: Peptide 2

(viii) POSITION IN GENOME:

(B) MAP POSITION: 304

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

His Gin lie Asn Gly Asp Phe Ser Lys Gin Asp Trp Glu Ala Gin lie 1 5 10 15

Asp Lys Glu

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: Peptide 3

(viii) POSITION IN GENOME:

(B) MAP POSITION: 304

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

His Gin lie Asn Ser Asp Phe Ser Lys Gin Asp Trp Glu Ala Gin lie 1 5 10 15

Asp Lys Glu

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 171

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Pro Val lie Glu Lys Val Lys Pro Gly Glu Gin Ser Phe Val Gly Gin 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 203

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Tyr His Asn Tyr Pro Asn Lys Gly Leu Lys Asp Tyr Thr Tyr Thr Leu 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 247

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Pro Thr Tyr Ser Gly Arg Glu Ser Asn Val Gin Lys Met Ala lie 1 5 10 15

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 344

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: lie Asp Gly Ala Asp Gly Arg Asn Phe Tyr His 1 5 10

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 ^' amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 304

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Gin lie Asn Arg Ser Asp Phe Ser Lys Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(viii) POSITION IN GENOME:

(B) MAP POSITION: 304

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Gin lie Asn Arg Gly Asp Phe Ser Lys Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speBl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

ACAGCAAAGT GCCCCCGCCC CTCCCCAATA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

ACAGCAAGGT GCCCCCGCCT CTCTCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

ACAGCAAAGT GCCCCCGCCT CTCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB4

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

ACAGCAAAGT GCTCCCGCCT CTCCCCAACA CTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ACAGCAAAGT GCCCCCGCCT CTCCCCAATA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ACAGCAAAGT GCCCCCGCCC CTCCCTAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

ACAGCAAAGT GCTCCCGCCC CTCTCCAACG CGACTACTAT CAGGA 45

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speBβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

ACAGCAAAGT GCTCCCGCCT CTCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE: (B) CLONE: speB9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

ACGGCAAAGT GCCCCCGCCT CTCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

ACAGCAAAGC GCCCCCGCCT CTCCCCAACA CGACCACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speBll

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

ACGGTAAAGT GCCCTCGCCC CTCCCCAACA TTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB12

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

ACAACAAAGT GCCCCCACCC CTCCCCAATA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB13

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

ACAGCAAAGT GCCCCCGCCT CGCCCCAACA CGACTACTAC CAGAA 45

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

ACAGCAAAGT GCCCCCGCCC CTCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

ACAGCAAAGT GCCCCCGCCT CTCCCCAACG CGACTACTCC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB16

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

ACAGCAAAGT GCCCCCGCCT CGCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB17

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

ACAGCAAAGT GCCCCCGCCC CTCCCCAACA CTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speBlβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

ACGGTAAAGT GCCCTCGCCT CTCCCCCACA TTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB19

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

ACAGCAAAGT GTTCCCGCCC TTCCCCAACA TGACTACTAC TAGGA 45

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

ACAGCAAAGT GCTCCCGCCC TTCTCCAACA CGACCACTAC CAGGC 45

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB21

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 0:

ACAGCAAAGT GCCCCCGTCC CTCCCCAACA CTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB22

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

ACAGCAAAGT GCCCCCGCCC CTCCCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB23

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

ACGGCAAAGT GCCCCTGCCT CTCCCCAACA CGGCTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB24

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

ATAGCAAAGT GCTCCCGCCC TTCTCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

GCAGCAAAGT ACTCCCGCCC CTCCCCAACA TTACTACCAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB26

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

ACAGCAAAGT GCCCCCGCCT CGCCCCAACA CGACTATTAC CGGGA 45

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB27

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

ACAGCAAAGT GCCCCCGCCC CTCCTCAATA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB28

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

ACAGCGAAGT GCCCCCGCCT CTCCCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB29

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

ACAGCAAGTG CCCCCGCCTT TCCCCAACAT GACTACTACC AGGA 44

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB30

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

ACAGCAAAGT GCTCCCGCCC CTCTCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB31

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

ACGGCAAAGT GCCCTCGCCT CTCCCCAACA TGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB32

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

ACAGCAAAGT GCCCCCGCCT TTCCCCAACA TTACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB33

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

ACAGCAAAGT GCCCCCGCCT CTCTCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE: (B) CLONE: speB34

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

ACAGCAAAGT GCCCCCGCCT TTCCCCAACG CGACTACTCC CAAGA 45

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB35

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

ACAGCAAAGT GCCCCCGCCC CTCCCCAGTA CGATTACTAT CAGGA 45

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB36

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

ACAGCAAAGT GCCCCCGCCT CTTTCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB37

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

ACAGCAAAGT GCTCCCGCTC CTCTCCAACA CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB38

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

ACAGCAAAGT GCCTCCGCCT CTCTCCAACG CGACTACTAC CAGGA 45

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Streptococcus pyogenes

(vii) IMMEDIATE SOURCE:

(B) CLONE: speB39

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

ACAGCAAAAT GCCCCCGCCC CTCTCCAACA CGACTACTAC CAGGA 45