NOVEL PROTEASES WITH DNA FRAGMENTING ACTIVITY

Abstract: Granule proteases having DNA fragmenting activity and useful in targeting tumor cells to cause nuclear collapse and apoptosis in the tumor cells have been isolated and purified. In particular, these proteases, termed fragmentin 1, fragmentin 2 and fragmentin 3, have non-reduced apparent molecular weights of 47 kD, 31 kD and 27 kD respectively, have been isolated from a rat natural killer tumor cell line by solubilization of rat RNK cell granules and purification of the granular proteases.

Description: 3 NOVEL PROTEASES WITH DNA FRAGMENTING ACTIVITY FIELD OF INVENTION The present invention relates to certain proteases isolated and purified from granule protein produced by rat natural killer cells which have DNA fragmenting activity. BACKGROUND TO THE INVENTION The molecular . --mediators of lymphocyte-induced cytolysis are thougfrfcrto reside within the cytcυlasmic granules of cytotoxic T lymphocytes and natural killer cells (refs. 1 to 4 - the references are identified at the end of the disclosure. Reference 1 was published after the priority date of this application) . The delivery of these molecules to the plasma membrane of the target cells occurs by exocytosis (ref. 5) and has been detected by the release of granule constituents into the intercellular space between the killer cell and target (refs. 6 , 1 ) . The destruction of the target cells appears to be of two distinct forms, the dissolution of the plasma membrane and apoptosis including the characteristic cleavage of DNA into oligonucleosomal fragments (for review see ref. 8) . One of the constituents of the cytoplasmic granules is the pore- forming protein called either perforin in the T cell (ref. 3) or cytolysin in the NK cell (ref. 2) which is capable of disrupting the target cell membrane but is unable to produce the DNA damage identified following CTL and NK cell-mediated killing (refs. 1, 8, 9). In addition to perforin/cytolysin, a subfamily of serine proteases has been identified in lymphocytes (ref. 10, 11) which is stored in cytoplasmic granules (ref. 12, 13) . Exocytosis of granules results in the release of serine proteases into the extracellular environment (ref. 14) . The lymphocyte serine proteases, sometimes called 'granzymes', have been identified in human (refs. 15, 16, 17), murine (refs. 10, 18, 19) and rat (ref. 20) homologues, with as many as seven distinct genes now identified in mice and three in humans. The specificity of only two gene products has been determined. Granzyme A/Hanukah. Factor (HF)1 was originally discovered by its ability to cleave the 'BLT' substrate (ref. 14), and was subsequently identified as a tryptase, while CCPI/granzyme B was found to be an asp-ase (refs. 21 to 23) . Other protease activities have been detected in the rat NK granules, although the proteins mediating the hydrolysis have not been identified (ref. 24) . SUMMARY OF INVENTION In accordance with the present invention, there have been isolated, purified and characterized lymphocyte serine granule proteases which have DNA-fragmenting and apoptosis-inducing activity. Specifically, these proteases include materials having an unreduced apparent molecular weight of 47 kD ("fragmentin-l") , 31 kD ("fragmentin-2") and 29 kD ("fragmentin-3") . The corresponding reduced apparent molecular weights are 30, 32 and 29 kD respectively. This family of lymphocyte granule protease fragmentins induces apoptosis through unique substrate and target cell interactions. Fragmentin-2 rapidly induces DNA fragmentation and apoptosis in intact target cells in the presence of cytolysis through asp-ase activity. Amino acid sequencing of internal peptides of this protease indicated the protein to be similar to the rat natural killer protease-1 (RNKP-1) . Fragmentin-1 and Fragmen in-3 also induce DNA fragmentation in target cells with slower kinetics than fragmentin-2 through tryptase activity. Amino acid sequencing of fragmentin-1 indicates that the protein resembled HF/granzyme A and that of fragmentin-3 indicates that the protease resembled human granzyme 3. The proteases and recombinant homologues thereof are useful for targeting tumor cells in order to induce rapid DNA fragmentation of the tumor cell, with nuclear collapse and apoptosis in the tumor cells. Accordingly, in a further aspect, the present invention provides a cytotoxic composition having DNA fragmentation activity, comprising at least one of the lymphocyte serine granule proteases as the active component. The isolation, purification and characterization of the granule proteases enables recombinant products corresponding to the proteases to be formed using standard recombinant techniques. Accordingly, the present invention also includes a recombinant molecule which has an amino acid sequence which is identical or substantially homologous to that of the isolated and purified granule protease as well as a recombinant molecule exhibiting the DNA fragmentation and apoptosis- inducing activity of the isolated and purified granule protease. BRIEF DESCRIPTION OF DRAWINGS Figures 1 (a) to (d) contain graphical representations of a series of chromatographic operations leading to isolation of fragmentins 2 and 3. In this regard: Figure 1 (a) depicts phenyl superose chromatography. Granules (7.5 x 10 9 RNK cell equivalents) were solubilized in 2 M NaCl, centrifuged and filtered to remove debris, then applied on a FPLC Phenyl-Superose column and eluted with a reducing linear gradient of 2 M to 0 NaCl. Fractions were adjusted to 0.15 M NaCl, then assayed against Cr- and 125 IUdR-double labelled YAC-l cells. The first protein peak exhibited DNA fragmentation activity, but this was seen only in the presence of cytolysin. Fractions, therefore, were assayed for DNA damage with an equal amount of cytolysin added to each sample; Figure 1 (b) depicts mono Q chromatography. Active fractions from two runs of Phenyl Superose chromatography (Figure 1 (a)) were concentrated and desalted and then applied to Mono Q beads. Fractions were eluted with a gradient of 0 to 1 M NaCl. DNA fragmenting activity was detected in elutes between 0.3 to 0.5 M NaCl in the presence of cytolysin; Figure 1 (c) depicts heparin-agarose chromatography. Active fractions from the Mono Q column (Figure 1 (b)) were pooled, concentrated and applied to a heparin- agarose column. Fractions were eluted with a gradient of 0 to 1 M NaCl. DNA fragmenting activity was eluted at 0.5 to 0.6 M NaCl in a single protein peak; and Figure 1 (d) depicts mono S Chromatography. Active fractions fromthe Heparin-agarose chromatography (Figure 1 (c)) were pooled, concentrated and buffer changed to 50 mM NaCl, and applied to Mono S beads. Fractions were eluted with a linear gradient of 0 to 1 M NaCl. DNA fragmenting activity was detected in the first protein peak (fragmentin 2) at 0.75 M NaCl. The second peak was a BLT esterase (fragmentin 3) . Figure 1 (e) is a depiction of SDS- AGE of purified lymopocyte granule proteases. FPLC-purified proteases were loaded on a 12% gel run according to Laemmli (ref. 30) under reducing (lanes A to C) and non-reducing conditions (lanes D to K) , then sivler stained. Lanes A and D contain 0.6 μg of fragmentin-2, lanes B and E contain 0.6 μg of fragmentin-3 and lanes C and F contain 0.16 μg of fragmentin-1. Molecular mass markers are shown to the left of the stained gels. Figure 2 illustrates cytolysin purification. Figure 2 (a) depicts heparin agarose chromatography. Cytolysin containing fractions from the phenyl-superose column (Figure la) were pooled, concentrated, applied to the heparin agarose column and eluted with three gradients 0 to 0.3M NaCl, 0.3 to 0.5M NaCl and 0.5 to 1.0M NaCl. The first protein peak was cytolysin and two subsequent peaks had BLT esterase activity. Figure 2 (b) a depiction of SDS-PAGE of cytolysin. Samples (8 μl) of cytolysin eluted from heparin agarose column were loaded on a 12% gel run according to Laemmli under non-reducing conditions. Lanes 5 to 9 were heparin agarose fraction 25 to 29 containing cytolysin activity. The protein concentration of the peak at fraction 27 was 25 μg/ml, and 0.2 μg was loaded in lane 7 of the SDS PAGE. Molecular weight standards are given on the left. Gels were silver stained according to manufacturers instructions. Figure 3 (a) to 3 (c) depict purification of an RNK granule BLT-esterase. After an initial step of phenyl superose chromatography (Figure 1 (a) ) to remove cytolysin, fractions containing BLT-esterase activity were applied to a mono-Q column (Figure 3 (a) ) . The BLT- esterase peaks were identified. The fractions containing the minor peak (arrow) were pooled, concentrated and applied to a heparin agarose column (Figure 3 (b) ) . Fractions containing the peak of BLT-esterase were pooled, concentrated and buffer changed to 10 mM bis- tris, pH6.0, containing 50 mM, NaCl, a;d applied to mono- S beads (Figure 1 (c) ) . Fractions were elected and the BLT-esterase peak was concentrated, desalted and run on SDS-PAGE (Figure 1 (e) , lanes C and F) . Figure 4 depicts DNA fragmenting of YAC-I cells by purified granule proteases. Fragmentin 1 (A) , 2 (•) and 3 (■) were examined in a 2-hr (Figure 4 (a)) and 20-hr (Figure 4 (b) ) cytotoxicity assay. None of the proteases was active in the absence of cytolysin (open symbols) . Figure 5 depicts fragmentin-2 and cytolysin dose responses. Figure 5A shows the effect of increasing fragmentin-2 dose alone or with a constant cytolysin concentration of 20 haemolytic units /ml. Figure 5B shows the effect of increasing cytolysin dose alone or with a constant fragmentin concentration of 100 ng/ml. Figure 5C shows the time course of fragmentin-2 induced DNA and membrane damage. Fragmentin-2 (100 ng/ml) and cytolysin (100 haemolytic units /ml) induced rapid DNA and membrane damage of YAC-1 cells in less than 15 min. and reached a maximum in 60 ins. Figure 6 shows that target cell susceptibility to fragmentin-2-induced damage is cytolysin dependent (see also Figure 4) . A variable degree of DNA damage was produced by 1 μg fragmentin-2 at increasing cytolysin doses in various cell lines as identified in the Figure. About a 10-fold difference was detected between the SL2 and EL4 lymphomas. Figure 7 depicts inhibition of fragmentin-2 activity by cytochalasin B, sodium azide and 2-deoxyglucose. In Figure 7(a), cytochalasin B produced a dose-dependent inhibition of fragmentin-induced DNA and membrane damage while having no effect on cytolyεin activity. Specific 12 IUdR release was 28% and 51 Cr release was 40% in untreated controls. In Figure 7(b), sodium azide and 2- deoxyglucose had only limited effect individually, but when combined produced a significant reduction in fragmenting activity. Specific 125 IUdR release was 40% and 51 Cr release was 66% in untreated controls. Figure 8 depicts the requirement for protein synthesis to induce DNA damage by the fragmen ins. DNA damage induced by fragmentins after treatment of YAC-1 cells with 50 μg/ml of cycloheximide (CHX) (filled symbols) or control medium (open symbols) in a 2-h (circles) or 14-18 h (triangles) cytotoxicity assay is shown. Inhibition of [ 3 H] methionine incorporation was 92% wt 50 μg/ml CHX. In two experiments, the ID 40 for fragmentin-1 was 37% and 32% and fragmentin-3 was 44% and 31% respectively. Figure 9 depicts the differential susceptibility of target cells to fragmentins. Using a 20-h assay, fragmentin 1 (•) , 2 (■) and 3 (▲) induced DNA damage in YAC-1 lymphoma, Al-1 thymoma and B16 melanoma with varying efficiency. Figure 10 depicts synergistic amplification of fragmentin-2 activity by fragmentin-3. YAC-1 target cells were incubated with increasing doses of fragmentin- 2 and a constant amount of cytolysin (68 ng/ml) with or without a constant amount of the fragmentin-3 (640 ng/ml) . 125 IUdR release was assayed without (A) or with (B) Triton-X added immediately prior to assay termination after 2 hours incubation. GENERAL DESCRIPTION OF INVENTION As noted earlier, the present invention relates to certain isolated and purified rat lymphocyte granule proteases which have defined molecular weights. A 31kD protease (fragmentin-2) rapidly induces DNA fragmentation and apoptosis in target tumor cells in the presence of cytolysin. This molecule is homologous to the rat NK protease 1 and its activity can be blocked by the general serine protease inhibitor, 34-dichloroisocoumarin. This protease is an asp-ase. A 47kD (fragmentin-1) an 27 kD (fragmentin-3) protease are tryptases and L er from the asp-ase fragmentin-2 in substrate t , cificity, inhibitory activity by chloromethyl ketones, kinetics of action, requirement for protein synthesis to mediate DNA fragmentation an target cell preference. These properties and differences are illustrated in the Examples below. A summary of the properties and differences appears in Table 7 below. The 47 kD protease (fragmentin-1) is in the form of a dimer under non-reducing conditions and a 30 kD monomer under reducing conditions. This molecule size is similar to the mass described for human granzyme A (ref. 15) , but differs from the 60-kD dineric BLT-esterase affinity purified from the RNK leukemia cell line, which was identified as granzyme A (ref. 4) . The second BLT-esterase (fragmentin-2) has a 29 kD molecular size under non-reducing conditions and has NH 2 - terminal sequence homology to human granzyme 3 (ref. 17) , the only other BLT-esterase which has been identified in lymphocyte granules. The 29 kD BLT-esterase is much more abundant than the 47-kD granzyme A- like BLT-esterase, at least in the RNK-16 granules tested herein. This situation contrasts with murine and human CTL granules, where granzyme A is the major BLT esterase (refs. 15, 17) . Fragmentin-1 and fragmentin-3 are tryptases which preferentially hydrolyze Z-Lys-SBzl and Z-Arg-SBzl substrates, similar to granzyme A (refs. 21 to 23) while fragmentin-2 is an asp-ase, cleaving most efficiently Boc-Ala-Ala-Met-SBzl substrate and, to a lesser extent, the Boc-Ala-Ala-Met-SBzl substrate. The asp-ase and tryptase fragmentins differ considerably in their kinetics and specific activity. Fragmentin-2 is very fast acting and produces DNA damage in less " than 2 hour, while fragmentin-1 and -3 require much longer assays and much higher protein concentration to be detected. At least some of the fragmentin-1 and -3 activity requires protein synthesis. Fragmentin-3 is able to synergistically amplify DNA damage of target tumor cells by fragmentin-2, suggesting that the proteases cleav distinct proteins and each is capable of activiting the apoptotic pathway independently. In addition, unique patterns of DNA damage are induced by the three fragmentins in different target cells, again indicating that each fragmentin is capable of acting as an effecter molecule. The differential ability to induce target cell damage for the fragmentins has potential implications for specificity of killing granulated cytotoxic lymphocytes, since the susceptibility to killing may be determined not only by membrane susceptor intereactions, but by the susceptibility of the target cell to and the abundance of a given fragmentin in the cylotoxic cell granules. For the purpose of administration of the proteases or their recombinant homologues or analogues, a composition is provided containing a cytotoxic effective amount of the protease in a suitable carrier vehicle. EXAMPLES In the Examples that follow and in the preceding text, certain abbreviations are used which have their normal meaning in this art. Some of these abbreviations are defined as follows: BLT, Nα-CBZ-L-lysine thiobenzyl ester; [Ca 2+ ] , calcium concentration; DCI, 3,4-dichlorosiocoumarin; PMSF, phenylmethylsulfonyl fluoride; DTNB, 5,5-dithiobis- (2-nitrobenzoic acid) ; FPLC, fast protein liquid chromatography; 125 IUdR, 125 I-iododeoxyuridine; TEB, buffer with 20 mM Tris-HCl, 0.1 mM EGTA and 0.02% NaN 3 ; HF, Hanukah Factor; CCPI, murine cytotoxic protease I; HBSS, Hank's balanced salt solution; PDS, 4,4'- dithiodipyridine; DNTB, 5,5 , -dithio-bis(2-nitrobenzoic acid) . Example 1: This Example describes the various materials and procedures used in the experimentation performed to produce the results set forth in the suceeding Examples. Cells. The RNK-16 tumor cell line, used as a source of granules has been described previously (ref. 15) . The cell line was serially passaged intraperitoneally in pristine-primed Fischer (F344) rats. The murine T cell lymphoma target cells, YAC-1, SL2, EL4 and L5178Y, and A 1.1 thymona target cell were maintained in RPMI 1640 and E16 melanoma in α-MEM supplemented with 10% fetal calf serum, penicillin-streptomycin, and 2 mM L-glutamine. Isolation of Granules. The method for isolation and solubilization of granules of rat RNK cells has been described in detail (refs. 2, 26). In brief, RNK cells were washed out from the peritoneum using cold HBSS with heparin, pelleted by centrifugation and resuspended at 1 x 10 8 cells/ml in disruption buffer (0.25 M sucrose, 10 MM: HEPES, 4 MM EGTA and 160 U/ml heparin, at pH 7.4). They were equilibrated at 0°C and at 450 psi in a nitrogen cavitation bomb for 20 min. , then lysed by decompression. The resulting homogenate was made up to 8.8 mM MgCl 2 and incubated at room temperature for 25 min. with 1000 U/ml DNase I (Sigma) . Nuclei were removed by successive filtration through 5 and 3 pm nucleopore filters (Nucleopore Corp., Pleasonton, CA) . The resulting homogenate was cooled at 4°C. Five to six ml aliquots were layered on 19 ml of 48% (w/v) Percoll in disruption buffer without heparin and centrifuged at 20,000 rpm at 4°C for 10 min. with 0 deceleration using a 70 Ti rotor in a Beckman L8-70M centrifuge (Beckman Instruments, Fullerton, CA) . The bottom-most 5 ml from the resulting gradients were harvested. This granule- containing fraction was centrifuged at 34,000 rpm for 16 hr. at 4°C using the same rotor and the granules, a loose white layer over a hard Percoll pellet, were collected by Pasteur pipette and stored at -80°C. The granules were solubilized in 2 M Nacl with 3 freeze/thaw cycles. This material was centrifuged at 34,000 rpm for 1 hr. in a Beckman 70 Ti rotor. The supernatant was harvested and filtered through a sterile Millex GV 4 0.22 μm filter (Millipore) to remove membrane fragments. Purification of Granule Proteins. The filtered solubilized granule preparation (7.5 x 10 9 RNK cells equivalents in a 4.5 ml volume) were loaded on a 1 ml Phenyl Superose column (HR 5/5 Pharmacia) connected to a FPLC system. All buffers and fractions were kept on ice. Fractions were eluted with a linear gradient of 2 to 0 M NaCl in 20 mm Tris-HCl, pH 7.2, 0.1 MM EGTA and 0.02% NaN 3 buffer (TEB) as follows: Sample was loaded in 4.5 ml for 9 minutes, then the column was washed for 24 min. A 2 to 0 M NaCl linear gradient in TEB was then run for 20 min. The elution rate was 0.5 ml/min., one ml fractions were collected and the eluate was monitored by measurement of OD 20 . The column was run twice (1.5 x 10 10 RNK cell equivalents total) , and identical fractions were pooled. DNA fragmenting and cytolysin activities were screened in 125 IUdR and 51 Cr release assays using double- labelled YAC-1 cells as described below. All 125 IUdR assays contained a constant amount of cytolysin, which was necessary to detect activity. A) DNA Fragmenting Protein. Fractions containing activity, which were eluted from first protein peak of the Phenyl Superose column, were pooled and concentrated using a Centriprep 10 concentrator (Amicon) to a final volume of 2.4 ml. This material was diluted to 36 ml in TEB to give a final [NaClI of about 0.145 M and applied on a Mono Q column (HR 5/5, Pharmacia). The sample was loaded for 36 min and washed for 6 min in TEB. Fractions were eluted from the Mono Q column with a linear gradient of from 0.2 to 0.6 M NaCl in TEB for 20 min then a steeper gradient from 0.6 to 2 M NaCl in TEB for 6 min. The flow rate was 1 ml/min and one ml fractions were collected and screened for both 125 I-UdR release activity and for BLT-esterase activity. Fractions containing cytotoxic activity were pooled, and concentrated with a Centricon 10 microconcentrator (Amicon) to a final volume of 1 ml and then diluted to 5 ml in TEB to give a final NaCl of about 0.1 M for subsequent heparin chromatography. We used an HR 5/5 column (Pharmacia) that was packed with 1 ml of heparinagarose gel (Pierce Chemical Co. , Rockford, IL) and connected to the FPLC system. Samples were loaded for 25 minutes and the column washed for 25 min with TEB. Fractions were eluted with a linear gradient of from 0 to 1 M NaCl in TEB for 100 minutes. The flow rate was 0.2 ml/min and 1 ml fractions were collected and screened for activity. Active fractions from the heparin-agarose chromatography were pooled, concentrated and buffer changed to 10 mM bis-Tris pH 6.0, containing 50 mM M NaCl, then 12 ml applied to Mono S beads (HR 5/5) for 12 minutes then the column washed with 10 MM bis-Tris for 8 min. Fractions were eluted with a linear gradient of 10 mM bis-Tris pH 6.0 containing 0 to 1 M NaCl with a flow rate of 1 ml/min for 20 min. DNA fragmenting activity was detected in the first protein peak at 0.75 M NaCl. The complete purification protocol was continuously run over a 2 to 3 day period, storing samples at 40C between chromatographic steps. The remaining fractions containing BLT-esterase activity were pooled and concentrated with a Centricon 30 microconcentrator (Amicon Corp., Denvers, MA) to a final volume of 1 ml, then diluted to 5 ml in 20 mM Tris-HCl, pH 7.4, 0.1 mM EGTA, and 0.02% NaN 3 (TEB) to give a final NaCl concentration of 0.1 M. This was applied to a 1-ml Heparin Hi-trap (Pharmacia LKB Biotechnology, Piscataway, NJ) connected to the FPLC system. Fractions were eluted with a linear gradient of 0-1 m NaCl in TBE for 100 min, at a flow rate of 0.2 ml/min, and 1-ml fractions were collected and screened in the BLT-esterase assay. The fractions containing BLT-esterase activity were pooled, concentrated, and buffer changed to 10 mM bis-Tris, pH 6.0, containing 50 mM NaCl (BT) , using a Centricon 30 microconcentrator (Amicon Corp.). This pool in a 12-ml volume was applied to mono-S beads (HR 5/5) for 12 min. and the column was washed with BT for 8 min. Fractions were eluted with two gradients, 5 min of 0-0.5 m NaCl and 20 min of 0.5-1 M NaCl in BT. The flow rate was 1 ml/min, and 1-ml fractions were collected and screened with the BLT-esteras assay. The purity of each fraction from the peak of BLT-esterase was assessed by SDS-PAGE under reducing and nonreducing conditions. B) Cytolysin. A simple and rapid method for purifying cytolysin was developed. The cytolysin- containing fractions eluted from the Phenyl Superose column were usually stored at -80°C. Samples were then thawed, pooled and concentrated with Centricon 30 miσroconcentrators (Amicon) to a final volume of 1.0 ml. This material was diluted to 10 ml with TEB to give a final [NaCl] of about C.l M and loaded on the FPLC heparin agarose column. Fractions were eluted with three gradients, 6 min of 0 to 0.3 M NaCl, 40 min of 0.30.5 M NaCl and 30 min of 0.5 to 1.0 M NaCl in TBE. The flow rate was 0.5 ml/min and 1 ml fractions were collected and screened with a 51 Cr-release assay and BLT esterase assay. The purity of each fraction from the peak of cytolytic activity was assessed by SDS-PAGE. Experimentally useful amounts of pure cytolysin could be obtained from an initial loading on the Phenyl Superose column of as little as 4 x 10 9 cell equivalents of solubilized granule preparation. πa. Trvptic Peotide Sequencing Tryptic peptide cleavage fragments for internal sequence analysis were prepared as described (27) . Separated proteins were electroblotted onto nitrocellulose and detected on the membrane by Amido Black staining. The region where the 32 RD protein was bound was sliced out and the protein was digested on the matrix with trypsin. The resulting peptides were separated by reverse-phase HPLC on a Vydac C 4 column (150 x 2. 1 mm) in a Waters peptide analyzer using a trifluoroacetic acid/acetonitrile buffer system. Prominent peptides were collected manually and frozen imediately. Sequence analysis was performed on an Applied Biosystems Model 477 sequenator, using standard protocols. NH j .-terminal Amino Acid Sequencing of Fragmentin 3. For H z — erminal sequence analysis, proteins were separated by PAGE, electroblotted onto Immobilone P membrane (Millipore Continental Water Systems, Bedford, MA) , and detected by coomassic blue staining as described (Ref. 39) . Selected bands were cut out andsequenced in a protein sequence (477A; Applied Biosystems, Inc., Forster City, CA) using standard protocols. Resulting PTH's were separated in a HPLC system (120A; Applied Biosystems, Inc.) and identified by visual inspection. 51 Cr and 125 IUdR Release Assay. Target cells in the log phase of growth (2.5 x 105/ml) were labelled as described by Russell and colleagues (ref. 28) with some modifications. Briefly, 5 to 40 μCi 125 IUdR and in some experiments also 100 μCi sodium 51 Cr-chromate were " added to 2 to 5 x 10° target cells in 0.5 ml of supplemented RPMI, the cells were incubated at 37°C in a 5% C0 2 atmosphere for 90 min., washed twice in HBSS, incubated an additional 45 min. , then washed once and resuspended at 1.25 x 10 5 cells/ml in HBSS containing 2 MM CaCl2 10 MM HEPES, 4 mg/ml BSA and at pH = 7.2 (HH-BSA) . There were only used double-labelled cells for which 125 IUdR cpm and 51 Cr cpm did not differ by more than a factor of 2. Aliquots of 104 cells in 80 μl in HH-BSA were added to 80 μl of granulederived material in HE (with NaCl adjusted to 140 MM) which had been diluted in normal saline with 10 mM HEPES, 1 mM EGTA and at pH = 7.2 in 96-well, V- bottommed or flat-bottomed microtiter plates at 37°C for 2 hrs or as indicated. Assays in which the effect of various chemicals were analysed utilized a similar procedure except 10 4 cells were delivered in 50 μl and the chemical in 50 μl. Cytochalasin B (Sigma) was dissolved in DMSO (Mallinckrodt, Point-Claire, Quebec) for a stock solution of 20 mm and stored at 4°C. Sodium azide (Sigma) and 2-deoxyglucose (Sigma) were made fresh in assay medium. Target cells were incubated 6 to 8 hours at 37°C in cytochalasin B, and 5 hours with sodium azide or 2-deoxyglucose before addition of granule proteins. Cycloheximide (Sigma) was prepared in assay medium then incubated for 2 hours at 37°C with target cells before the cytotoxicity assay. The plates then were incubated at 37°C for the times indicated and subsequently centrifuged at 800 x g for 5 min. In the 125 IUdR release experiments, 80 μl of target cells and 80 μl of test protein were incubated, then 40 μl of 1% Triton X-100, 5 MM EGTA, 50 MM TrisHCl, pH = 7.2 was added to reaction wells, the well contents were mixed and the plates were centrifuged. In both sets of experiments, 100 μl of supernatant were harvested from each well and the corresponding 51Cr cpm and 125 IUdR cpm were determined in an LKB 1282 Compugamma universal gamma counter, correcting for overlapping energy spectra. The specific isotope release was determined as described previously (ref. 26). In assays using protease inhibitors, inhibitors were dissolved in DMSO at a concentration of 200 mM and stored at 4°C. Inhibitors were diluted in HE medium and incubated with a constant amount of fragmentin or cytolysin at room temperature for 15 min in the assay wells, then target cells were added. The tripeptide chloromethyl ketones were synthesized as described previously (ref. 25) . In assays using chloheximide (Sigma Chemical Co., St. Louis, MO) , the drug was freshly prepared in HH-BSA at a concentration of 50 μg/ml and incubated with 125 I- UdR-labeled YAC-1 cells for 2 h at 37°C before the cytotoxicity assay. Calculation of Lvtic Units. For 125 IUdR and 51 Cr release assays, we defined one lytic unit as the amount of protein that gave 50% specific 125 IUdR or 51 Cr release from 10 4 YAC-1 cells. For calculations of specific activity in lytic units/mg protein concentrations were determined using a Micro BCA protein assay kit (Pierce, Rockford, IL) adapted to 96-well plates. Readings were made in a Titertek ELISA reader with a 540 n filter. For the hemolysis assay, we defined one hemolytic unit of cytolysin as the amount of material (μl) that gave 5 0 % hemoglobin release. Hemolytic units were used as a measure of activity when cytolysin protein concentration was below detection limits. BLT Esterase Microassay. We determined the serine esterase activity of individual fractions from a Percoll gradient of RNK cell homogenate using a modification of Green and Shaw's colorimetric BLT thioesterase assay (ref. 29) . The stock reagents were: 10 MM BLT (Nα-BCZ-Llysine thiobenzyl ester) (Sigma) in H 2 0, and 10 mM DTNB (5,5- dithiobis-(2-nitrobenzoic acid)] (Sigma) in dimethyl formamide. These were further diluted in HBSSHEPES (10 MM) to 1 MM BLT and to 0.5 MM DTNB, respectively. Each well of a 96 well flat- or roundbottomed microtiter plate received dilute DTNB solution. To the reaction wells, we added 50 μl of dilute BLT solution, while to their "blank" counterparts we added 50 μl of HBSS-HEPES. We added 100 μl of appropriately diluted (in HBSS-HEPES) Percoll gradient fractions to both sets of wells, mixed well contents on a plate shaker for 10 min and incubated a further 30 min at room temperature. The BLT esterase activity in each well was determined by measuring the absorbance at 414 on a Titertek Multiskan plate reader. It is assumed that OD is linear with enzyme dose. SDS-Polyacrylamide Gel Electroohoresis. Proteins were run on 6 cm long 12% gel slabs according to Laemmli (ref. 30) . An additional 1 or 2 cm of 4% gel was added over the running gel to resolve high molecular mars bands. Gels were developed using a commercial silver stain kit (Bio-Rad, Richmond, CA) . Substrate Kinetics. The enzymatic hydrolysis of peptide thioester substrates by fragmentins 2 (batches 1 and 2) and 3 were measured in 0.1 M Hepes, 0.5 M NaCl, pH 7.5, buffer containing 8% Me 2 SO and at 25°C in the presence of 4,4-dithiodipyridine (PDS) (27) , and hydrolysis rates of thioester substrates by fragmentins 1 and 2 (batch 3) were measured in 0.1 M Hepes, 0.5 M NaCl, 1 mM EDTA, pH 7.5, buffer containing 13% Me 3 S0 and at 21°C in the presence of 5,5'-dithio-bis(2-nitrobenzoic acid) (DNTE (28, 29). Stock solution of substrate was prepared in Me 2 SO and stored at -20°C. For fragmentins 2 and 3, the initial rates were measured at 324 nM ( 324 = 19800 M/cm) using a spectrophotometer (Beckman Instruments, Inc., Palo Alto, CA) when 5 or 10 μl of an enzyme stock solution was added to a cuvette containing 2.0 ml of buffer, 0.15 ml of 5 mM PDS, and 25 μl of substrate. The same volumes of substrate and PDS were added to the reference cell to compensate for the background hydrolysis rate of the substrates. For fragmentins 1 and 2, the initial rates wer emeasured at 405 nm (9,39 .406 = 14,000 M/cm) using a microplate reader (Termomax) when 2 μl of an enzyme stock solution was added to a well containing 0.2 ml of buffer, 0.02 ml of 5 mM DTNB, and 10 μl of substrate. The same volumes of substrate and DTNB were added to the blank well to compensate for the background hydrolysis rate of the substrates. Initial rates were measured in duplicate for each substrate concentration and were averaged in each case. Inhibition Kinetics Incubation Method. The inactivation reaction was initiated at 21°C by adding a 10 μl aliguot of inhibitor in Me 2 S0, concentration was 8%. Aliquots were removed at various time intervals, diluted into substrate solution (90-fold dilution) , and the residual activity was measured spectrophotometrically with a microplate reader. Fragmentins 1 and 3 were assayed with Z-Arg-SBzl, and fragmentin 2 was assayed with Boc-Ala- Ala-Asp-SBzl. First order inactivation rate constants (K ob .) were obtained from plots in In vs/vs vs, time. Several inhibitors did not show any inhibition toward fragmentins after a 10 min incubation of enzyme with inhibitor. Example 2: This Example describes the isolation and identification of the granule proteases with DNA fragmenting activity. To identify and purify the putative granule DNA fragmenting molecule, there were isolated cytoplasmic granules from rat RNK cells as described previously (7, 16) and two aliquots of 0.75 x 10 10 cell equivalents were solubilized in 2 M NaCl and subjected to FPLC chromatography (see Example 1) . Following an initial step of phenyl superose chromatography we found two protein peaks (Figure la) . The second peak had cytolysin activity but caused no DNA damage in double labelled ( 51 Cr and 125 IUdR) YAC-1 target cells. The first protein peak induced 125 IUdR release only after combining it with the cytolysin containing fractions (Figure la) . The possibility that the DNA fragmentation required the presence of cytolysin from the second peak was confirmed utilizing purified cytolysin prepared by sequential Ultragel AcA54 and heparin-agarose chromatography as described earlier (ref. 26) or phenyl superose and heparin-agarose as described in Example 1. In all subsequent chromatographic steps, DNA fragmenting activity was screened for in the presence of cytolysin. The first protein peak from the phenyl superose chromatography was pooled, concentrated and desalted, then applied to a mono-Q column. The fractions containing the DNA fragmenting activity (Figure lb) were pooled and concentrated then applied to a heparinagarose affinity column. Following heparin chromatography, a single peak of protein containing DNA damaging activity was observed (Figure lc) which ran as two major bands of 30 and 32 RD and a minor 31 RD band on SDS-PAGE after reduction (Figure le) . The active fractions of the heparin-agarose chromatography were pooled, concentrated and subjected to Mono S chromatography. Two protein peaks were identified (Figure Id) . The first contained DNA fragmenting activity (fragmentin 2) and the second was a BLT esterase (fragmentin 3) . The first peak contained the major 32 RD protein which ran at 31 RD under non-reducing conditions (Figure le) . The second protein peak which had BLT esterase activity was 30 kD under reducing and 27 RD under non-reducing conditions (Figure le) . The 31 kD protein eluted between the two peaks. Recovery and purification rate for each step is presented in Table 1 (The Tables referred to in these Examples appear at the end of the description) . The 32 kD protein was name > fragmentin 2 while thesecond protein was named fragm* " .n 3 after their ability to induce DNA fragmentation. Cytolysin was purified by a new two-step method using phenyl superose and heparin agarose chromatography (see method described above) . The initial phenyl superose step separated the cytolysin from most granules proteins including fragmentins 2 and 3 (Fig. la) . The heparin agarose step removed a contaminating BLT esterase (Fig. 2a) and yielded a single band of 65 kD on SDS-PAGE under reducing conditions (Fig. 2b) with a recovery of 12 to 15% and purification rate over 300-fold (Table 1) . After removal of cytolysis, fractions containing BTL-esterase activity were applied to a Mono-Q column and two BTL-esterase peaks were identified (Figure 3(a)). the fractions containing the minor peak (arrow) were pooled, concentrated and applied to a hepar. n agarose column (Figure 3(b)). Fractions containing the peak of BTL-esterase were pooled, concentrated and buffer changed to 10 mM bis-Tris, pH 6.0, containing 50 mM NaCl, and applied to Mono-S bends (Figure 3(c)). Fractions were eluted and the BTL-esterase peak was concentrated, desalted, and run on SDS-PAGE (Figure 1(e), C and F) . The Mono-S protein peak containing the second BTL- esterase ran as a single band at about 47 kD under non- reducing conditions on SDS-PAGE and at 30 kD after reduction. The protease is termed fragmentin 1. The molecular mass of the protease is similar to that of human HF (granzyme A (refs. 15, 17) , which is also a BLT- esterase that runs as a 43 kD homodimer and a 30 kD monomer under non-reducing and reducing conditions, respectively. Example 3: This Example described partial characterization of fragmentin 2. Four tryptic peptides of fragmentin 2 were prepared and sequenced (ref. 27) . The amino acid sequence is illustrated in Table 2 and compared to the deduced amino acid sequences of three granule proteases; the rat natural killer cell protease RNKP-l which was cloned from the same RNK-16 tumor used to purify fragmentin (ref. 20) , the murine T cell granule proteases CCPI/Granzyme B (ref. 18) and Hanukah Factor (HF)/granzyme A (ref. 19). Fragmentin was highly homologous to RNKP-l differing in only two of forty-one identifiable amino acids. CCPI/Granzyme B was also closely related as it differed in seven amino acids, while 21 amino acids did not match the corresponding HF/Granzyme A sequence. Peptide 3 (Table 2) and a conserved peptide from the cytotoxic cell protease or granzyme family (S-R-P-Y-M-A- SEQ ID No: 17) were used to prepare degenerate oligonucleotide primers. Using these primers and a cDNA library from the RNK-16 cell line; a 118 bp fragment has been generated by PCR and sequenced. This cDNA and its deduced amino acid sequence were compared to known proteases. The deduced sequence was found to be identical to the RNKP-l * teir. and the PCR fragment sequence as identical to 11 ->ut of 118 nucleotide „o the RNKP-l cDNA sequence. On this basis, there may be identity between fragmentin-2 and the RNKP-l protein. The NH 2 -terminal amino acids of the 27 kD BLT- esterase fragmentin 3 was sequenced to further identify this protein. The sequence contained the conserved regions of the granzyme family and was most homologous to human granzyme 3 (ref. 17) , which is one of only two known granule BLT-esterase (Table 3) . It differed significantly from the other BLT-esterase, human and murine HF/granzyτe A (Table 3) , particularly in position 14, which is a conserved tyrosine, and positions 17, 18 and 20, which are conserved leucises. Example 4: This Example illustrates induction of DNA fragmentation by fragmentins 1, 2 and 3. -The three purified granule proteases were tested for DNA "fragmentation activity in YAC-1 cells. The results obtained are illustrated in Figure 4. Time-lapse morphological examination of YAC-1 over the 2 hour period following incubation with fragmentin-2 and cytolysin revealed extensive blebbing of the cell surface and convolution of the nuclear membrane, which are typical of CTL induced damage and apoptosis (refs. 32, 33, 34). Cell death was associated with nuclear collapse and severe chromatin condensation within 1 hour which was visualized as apoptotic bodies (refs. 33, 34) by transmission electron microscopy. Cytolysin alone produced membrane damage without chromatin condensation, while the fragmentin 2 by itself was without effect. As may be seen in Figure 4, all three proteases were able to induce DNA fragmentation of YAC-1 cells. However, after a 2-hr incubation, DNA damage of YAC-l cells was only observed with the 31-kD fragmentin in the presence of a constant amount of cytolysin (Figure 4(a) ) . By 20 hr, both the 7-kD and 27-kD BTL-esterases induced significant DNA damage, but only when cylolysin was present and at a specific activity that was lower than the 31-kD fragmentin (Figure 4(b)) . Fragmentins 1 and 3, therefore, possess considerably slower kinetics and lower specific activity than the fragmentin 2 protease. Example 5 This Example illustrate the cytolysin dependence of fragmentin-2 induced DNA damage. In the presence of a constant amount of cytolysin, fragmentin 2 produced a dose-dependent increase in DNA damage as measured by 125 IUdR release and membrane damage as measured by 51 Cr release, but was inactive when presented to target cells on its own (Fig. 5A) . Fragmentin 2 could induce DNA damage at a dose of cytolysin that produced no detectable membrane damage (Figure 5A) . Over the dose range of fragmentin 2, there was first, observed an increase in membrane damage at lower doses and this was followed by 125 IUdR release at higher fragmentin concentrations. Figure 5b shows that fragmentin 2 activity was also cytolysin dose-dependent with both DNA and membrane damage increasing with cytolysin dose. However, membrane damage in the presence of fragmentin 2 was always higher than that induced by cytolysin alone. DNA and membrane damage were inhibited by EGTA, indicating that the process was Ca 2+ -dependent (Fig. 5A) . In a kinetic analysis, DNA and membrane damage were seen as early as fifteen minutes after treatment with fragmentin and cytolysin and were maximal by sixty minutes (Fig. 5C) . Again, membrane damage preceded the DNA damage induced by fragmentin 2. When examining fragmentin 2 activity against other lymphoma targets, activity was detected using EL4, L5178Y and SL2 tumor cells. DNA damage was found to be dependent on the dose of cytolysin in each tumor although the range of sensitivity varied by 10-fold (Figure 6) . Although fragmentin 2 and also fragmentins 1 and 3 have an absolute requirement for cytolysin, even at sublytic doses, the mechanism by which it promotes fragmentin activity is not immediately apparent. One possibility is that cytolysin insertion into the plasma membrane might stimulate endocytosis and thus facilitate fragmentin movement from the extracellular space to the cytoplasm. To test this hypothesis, the effect of cytochalasin B, an inhibitor of microfilament assembly, and of sodium azide and 2-deoxyglucose, which indirectly inhibit itrochondrial respiration, were examined. Both cytochalasin B and metabolic inhibitors block receptor- mediated endocytosis (ref. 35) . It was found that cytochalasin B completely blocked DNA and membrane damage by fragmentin without affecting cytolysin activity (Fig. 7a) . Furthermore, sodium azide and 2-deoxyglucose when combined also effectively inhibited fragmentin 2 activity (Fig. 7b) . Example 6 This Example illustrates the effect of the fragmentins on substrates and inhibition of the fragmentins. To identify the specificity of peptide hydrolysis by fragmentins 1, 2, and 3, thiobenzyl ester substrates were utilized (Table 4) . Fragmentin 1 had high enzymatic activity toward Z-Arg-SBzl, but had very low or no activity toward other substrates. Fragmentin 2 had primarily Boc-Ala-Ala-Asp-SBzl esterase activity, but contained Boc-Ala-Ala-Met-SBzl activity in some enzyme batches. Fragmentin 2 also slowly hydrolyzed Ser-, Asn-, Phe-, and Arg-containing substrates. Fragmentin 3, like fragmentin 1, was active toward Z-Arg-SBzl, but had no activity against other thioester substrates. The asp-ase activity of fragmentin 2 was not enhanced by fragmentin 3. Fragmentin 2 (23 nM) was incubated with fragmentin 3 (52 nM) for 0.5, 10, and 30 min; the asp-ase activity was not increased when compared to fragmentin 2 in buffer for the same period of time. Similarly, the tryptase activity of fragmentin 3 was not enhanced by fragmentin 2. Fragmentin 3 (52 nM) was incubated with fragmentin 2 (23 nM) for 0.5, 10, and 30 min, and the tryptase activity was not greater than the sum of fragmentins 3 and 2 alone in buffer for the same period of time. To identify the substrates that were important for DNA fragmentation, a series of tripeptide chloromethyl ketones were tested as inhibitors. These compounds are able to form a tetrahedral complex with protease-active site residues and permanently inactivate serine proteases (ref. 36) . By comparing chloromethyl ketones with various amino acid sequences, unique inhibitors were identified that have allowed us to distinguish the DNA fragmentation induced by these three fragmentin proteins (Table 5) . The fast-acting fragmentin 2 was completely blocked by Boc-Ala-Ala-Asp-CH 2 Cl (ID 50 , 0.3 μM) but was poorly inhibited by the other chloromethyl ketones (ID 50 , 12-155 μM) . In constrast, the slow-acting fragmentins 1 and 3 were both highly inhibited by D-Phe-Pro-Arg-CH 2 Cl (ID 50 >250 μM) . Fragmentin 1 was not inhibited by Nle-, Phe-, or Trp-containing chloromethyl ketones, but fragmentin 3 was moderately inhibited by Z-Trp-CH 2 C1. The inhibition of enzymatic activities of fragmentins by peptide chloromethyl ketones was also examined (Table 6) . Fragmentins 1 and 3, which have trypsin-like activity, were only inhibited by a potent thrombin inhibitor, D- Ph3-Pro-Arg-CH 2 Cl, but not inhibited by the other four chloromethyl ketones. Fragmentin 2, which contained asp- ase and met-ase activities, was inhibited by Boc-Ala-Ala- Asp-CH 2 C1 and Z-trp-CH 2 Cl. Generally, the inhibition of enzymatic activities of these fragmentins by peptide chloromethyl ketones is consistent with the inhibition results of the DNA fragmentation. Example 7 This Example shows the effect of pretreatment of the tumor cells with cycloheximide on DNA damage produced by the fragmentins. The slow kinetics of DNA damage produced by the tryptase fragmentins 1 and 3 contrast sharply with the rapid action of the asp-ase fragmentin 2. Induction of DNA fragmentation and apoptosis by many agents is slow and requires protein synthesis (refs. 37, 38). The T relative DNA damage produced by the fragmentins, with or without pretreatment of YAC-1 cells with 50 μg/ml cycloheximide, was examined. The DNA damage produced by fragmentin 2 was unaffected by cycloheximide in either a rapid 2-h assay or after a long 1-h incubation (Figs 8B and D) . In constrast, fragmentin 1 and 3 activity in 14 to 18-h assays was reduced by 30 to 40% after cycloheximide treatment (Figs. 8A and C) , indicating that at least part of their activity was dependent upon protein synthesis. Example 8 This Example illustrates the specificity of the fragmentins towards DNA fragmentation in various target cells. The existence of at least three granule fragmentins with unique substrate specificities as demonstrated herein suggested that they may induce DNA fragmentation at varying rates in different target cells. Several target cells, including the YAC-1 lymphoms, Al.l thymoma, and B16 melanoma have been examined and unique patterns of DNA fragmentation have been found in each cell line when assessed after a 20-h assay (Fig. 9) . YAC-1 tumor cells are preferentially damaged by fragmentin 2, while the Al.l thymoma is barely affected by this protease. In contrast, fragmentin 1 efficiently induces DNA damage in the Al.l thymoma, while being the least effective against the B16 melanoma. Fragmentin 3 is active against all of the tumor cell lines. Example 9 This Example illustrates enhancement of the DNA fragmentation activity of fragmentin-2 by fragmentin-3. YAC-l target cells were incubated with increasing doses of fragmentin-2 and a constant amount of cytolysin (68 ng/ml) , with or without a constant amount of fragmentin-3 (640 ng/ml) . The presence of the fragmentin-3 enhanced fragmentin-2 activity in a synergistic manner (Figure 9) SUMMARY OF THE DISCLOSURE In summary of this disclosure, the present invention provides certain proteases, newly isolated and identified from RNK large granule lymypocyte leukemia, which induce DNA fragmentation and hence are potentially useful in tumor therapy. Modifications are possible within the scope of this invention. TABLE 1 PURIFICATION OF FRAGMENTIN AND CYTOLYSIN FROM RNK GRANULES A unit of fragmentin activity was defined as the amount required to produce 50% release of 125 IUdR from 10 4 YAC-l cells. A unit of cytolysin activity was the amount producing 50% release of 51 Cr from 10 4 YAC-l cells. TABLE 2 AMINO ACID SEQUENCE OF TRYPTIC PEPTIDES OF FRAGMENTIN Peptide 1 Fragmentin* Tyr-Ser-Asp-Thr-Leu-Gln-Glu-Val-Glu-Leu-Thr-Val-Gln-Glu-Asp-Gln RNKP-1 + CCPI/Granzyme B° * — * —Asn— * — * — * — * — * — * — * — * — * — * — ys— * —Arg HF/Granzyme A Pro- * -Glu- * - * -Arg- * - * -Asn-Ile- * - * -Ile-Asp-Arg-Lys Peptide 2 Fragmentin Ala-Asn-Glu-Ile-Xxx-Ala-Gly-Asp - SEQ ID NO: 5 RNKP-l * _ * _ * _ * -cys- * - * - * - SEQ ID NO: 6 CCPI/Granzyme B Tyr- * -Gin- * -Cys- * - * - * - SEQ ID NO: 7 HF/Granzyme A Leu- * -Met- * -Cys- * - * - * - SEQ ID NO: 8 Peptide 3 Fragmentin Pro-phe-Pro-Ala-Tyr-Asn-Ser - SEQ ID NO: 9 RNKP-l * -His- * - * - * - * - * - SEQ ID NO: 10 CCPI/Granzyme B * -His- * -Asp- * - * -Pro - SEQ ID NO: 11 HF/Granzyme A * -Tyr- * -Cys- * -Asp-Glu - SEQ ID NO: 12 Val-Asp-Pro-Gly-Asp-Val-Xxx-Tyr-Val-Ala-Gly-Xxx-Gly - SEQ ID NO: 13 * -Lys- * - * - * - * -cys- * - * - * - * -Trp- * - SEQ ID NO: 14 * -Lys- * - * - * - * -cys- * - * - * - * -Trp— * - SEQ ID NO: 15 * -Lys- * - * -Thr-Arg-Cys-Arg-Ala-Met- * -Trp- * - SEQ ID NO: 16 SEQ ID NO: 4 TABLE 3 1 2 3 4 5 6 7 8 9 10 11 Fragmentin 3* Xxx-Ile-Gly-Gly-Xxx-Glu-Val-Gln-Pro-His-Ser HUGRANZYME3 + Xe- * - * - * -Lys- * - * -Ser- * - * - * HUGRANZYMEA 0 lie- * - * - * -Asn- * - * -Thr- * - * - * MuGRANZYMEA* He- * - * - * -Asp-Thr- * -Val- * - * - * 12 13 14 15 16 17 18 19 20 21 22 Fragmentin 3 Xxx-Pro-Phe-Met-Ala-Ala-Ile-Gln-Tyr-Xxx-Gly HUGRANZYME3 Arg- * - * - * - * HUGRANZYMEA Arg- * -Tyr- * -Val-Leu-Leu-Ser-Leu-Asp-Arg MuGRANZYMEA Arg- * -Tyr- * - * -Leu-Leu-Lys-Leu-Leu-Ser * SEQ ID NO: 18 + SEQ ID NO: 19 0 SEQ ID NO: 20 SEQ ID NO: 21 TABLE 4 Enzymatic Hydrolysis Rate of Peptide Thiobenzyl Esters by Fragmentins * Hydrolysis rates were measured in 0.1 M Hepes, 0.5 M NaCl, 1 mM EDTA, pH 7.5 and 13% Me 2 S0 at 21°C. An aliquot of 2 μl of the enzyme was added to the assay mixture containing 0.2 ml of buffer, 0.02 ml of 5 mM DTNE, and 10 μl of substrate. Enzyme concentrations were: fragmentin 1, 1.7 nM; fragmentin 2 , 8.4 nM (batch 2) . ++ Hydrolysis rates were measured in 0.1 M Hepes, 0.5 M NaCl, pH 7.5, and 8% Me 2 S0 at 25°C. An aliquot of 5 or 10 μl of the enzyme was added to the assay mixture containing 2 ml of buffer, 0.15 ml of 5 mM PDS, and 25 μl of substrate. Enzyme concentrations were fragmentin 2, 7.3 nM (batch 1): fragmentin 3, 6.2 nM. TABLE 5 Inhibition of DNA Fragmentation (ID 50 ) by Chloromethyl Ketones * Amino acid refers to the third position in a tripeptide chloromethyl ketone. Asp, Boc-Ala-Ala-Asp-CH 2 Cl; Nle, Boc-Ala-Ala-Nle-CH 2 Cl; Phe, Boc-Gly-Leu-Phe- CH 2 C1; Arg, D-Phe-Pro-Arg-CH 2 Cl; Trp, Z-Trp-CH 2 C1. ++ NT, not tested. position 14, which is a conserved tyrosine, and positions 17, 18, and 20, which are conserved lcucines. TABLE 6 Inhibition of Fragmentins by Peptide Chloromethyl Ketones Inhibition was measured in 0.1 M Hepes, 0.5 M NaCl, 1 mM EDTA, pH 7.5, and 8% Me 2 SO at 21°C, Z-Arg-SBzl (120 μM) , Boc-Ala-Ala-Asp-SBzl (210 μM) , and Z-Arg- SBzl (300 μM) were used as the substrates for fragments 1, 2, and 3, respectively. * No inhibition after 10 min of incubation of enzyme and inhibitor. Characteristics of Apoptosis-inducing Granule Proteases REFERENCES 1. Shi, ., R.P. Kraut, R. Aebersold, and A.H. Greenberg. 1992. A natural killer cell granule protein that induces DNA fragmentation and apoptosis. J. Exτ>. Med. 175:553-556. 2. Henkart, P.A., P.J. Millard, C.W. Reynolds, M.P. Henkart. 1984. Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocyte tumors. J. EXP. Med. 160:75-93. 3. Podack, E.R. , J.D.E. Young, and Z.A. Cohn 1985. Isolation and biochemical and functional characterization of perforin I for cytolytic T-cell granules. Proc. Natl. Acad. Sci. 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Claims: CLAIMS What I claim is: 1. An isolated and purified lymphocyte serine granule protease having DNA fragmenting and apoptosis-inducing activity. 2. The protease of claim 1 wherein said protease has a non-reduced apparent molecular weight of 47 kD and a reduced apparent molecular weight of 30 kD. 3. The protease of claim 2 wherein said protease has a amino acid sequence exhibiting homology to HF/granzyme A. 4. The protease of claim 1 wherein said protease has a non-reduced apparent molecular weight of 31 kD and a reduced apparent molecular weight of 32 kD. 5. The protease of claim 4 wherein said protease has an amino acid sequence exhibiting homology to rat NK protease-1. 6. The protease of claim 4 which includes peptide 1, peptide 2, peptide 3 and peptide 4 having the amino acid sequence set forth in Table 2. 7. The protease of claim 1 wherein said protease has a non-reduced apparent molecular weight of 27 kD and a reduced apparent molecular weight of 29 kD. 8. The protease of claim 7 wherein said protease has an amino acid sequence exhibits homology to human granzyme 3. 9. The protease of claim 7 wherein said protease has an NH 2 -terminal amino acid sequence as identified for fragmentin-3 in Table 3. 10. A recombinant molecule having an amino acid sequence which is identical to that of a protease as claimed in any one of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9 or which is substantially homologous thereto. 11. A recombinant molecule exhibiting the DNA fragmentation and apoptosis-inducing activity of a protease as claimed in any one of claims 1, 2 , 3, 4, 5, 6, 7, 8 or 9. 12. A cytotoxic composition for the treatment of tumor cells to effect DNA fragmentation and apoptosis therein, which comprises a cytotoxic amount of at least one isolated and purified lymphocyte serine granule protease having DNA fragmenting and apoptosis-inducing activity, a recombinant molecule having an amino acid sequence which is identical or substantially homologous to that of said granule protease or a recombinant molecule exhibiting the DNA fragmentation and apoptosis-inducing activity of said granule protease, as the active component thereof, and a physiologically-acceptable carrier therefor. 13. The composition of claim 12 wherein said protease has a non-reduced apparent molecular weight of 47 kD and a reduced apparent molecular weight of 30 kD. 14. The composition of claim 13 wherein said protease has an amino acid sequence exhibiting homology to HF/granzyme A. 15. The composition of claim 12 wherein said protease has a non-reduced apparent molecular weight of 31 kD and a reduced apparent molecular weight of 32 kD. 16. The composition of claim 15 wherein said protease has an amino acid sequence exhibiting homology to rat NK protease-1. 17. The composition of claim 15 wherein said protease includes peptide l, peptide 2, peptide 3 and peptide 4 having the amino acid sequence set forth in Table 2. 18. The composition of claim 12 wherein said protease has a non-reduced apparent molecular weight of 27 kD and a reduced apparent molecular weight of 29 kD. 19. The composition of claim 18 wherein said protease has an amino acid sequence exhibits homology to human granzyme 3. 20. The composition of claim 18 wherein said protease has an NH 2 -terminal amino acid sequence as identified for fragmentin-3 in Table 3.

Inventors: GREENBERG ARNOLD H, GREENBERG, ARNOLD, H.
Applicants: UNIV MANITOBA, UNIVERSITY OF MANITOBA
Application year: 1992
Main classification: A61K37
IPC codes: A61K38, A61P35, C12N9, C12N15, C12R1