EP0360840A1 - Facteur cytolytique - Google Patents

Facteur cytolytique

Info

Publication number
EP0360840A1
EP0360840A1 EP88908641A EP88908641A EP0360840A1 EP 0360840 A1 EP0360840 A1 EP 0360840A1 EP 88908641 A EP88908641 A EP 88908641A EP 88908641 A EP88908641 A EP 88908641A EP 0360840 A1 EP0360840 A1 EP 0360840A1
Authority
EP
European Patent Office
Prior art keywords
macrophages
factor
composition
cytolytic
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP88908641A
Other languages
German (de)
English (en)
Inventor
Jim Klostergaard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
Original Assignee
University of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP0360840A1 publication Critical patent/EP0360840A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to preparations which are useful in inhibiting the proliferation of human tumor cells and more particularly is directed to proteinaceous factors which are cytolytic for such cells.
  • Cancer is a very wide spread and severe health problem which affects millions of people yearly, resultin in debilitating symptoms and often death. Numerous approaches, often fruitless, have been taken by medical scientists in an attempt to identify substances which may be of some usefulness in slowing or stopping the growth o human tumors.
  • One avenue which has shown some promise is through the stimulation of the afflicted patient's immune system, thereby inducing the patient's immune system to produce substances capable of reducing the growth rate of tumor cells or, hopefully, killing them outright.
  • Unfor ⁇ tunately, such an approach is often of little use in that the immune systems of cancer patients are either over burdened already or are simply incapable of responding to such immuno-stimulation.
  • cytostatic mechanism of activated macrophages i mediated through lesions induced by the effector cell in the target cell mitochondrial electron transport chain (ETC), in particular at Complex I and II. Such lesions result in growth inhibition or, if the damaged cells are not able to conduct adequate levels of glycolysis, death of the target.
  • ETC mitochondrial electron transport chain
  • Kilbourn and co-workers, (1984) J. Immunol., 133:2577, have demonstrated that this cytotoxi mechanism might be accounted for by the secretion of a monokine, termed respiration inhibitory factor (RIF), which appears in large measure to mimic the activated macrophage in the exertion of cytostatic effects on a number of tumor cells.
  • RIF respiration inhibitory factor
  • the candidate mediators include oxygen metabolites (Nat et al. (1979), J. Exp. Med. , 149:100) , arginase (Currie (1978), Nature, 273:758) , thymidine (Stadecker et al. (1977), J. Immunol., 119:1738) , C3a (Ferluga et al. (1978), Clin. Expl. Immunol., _3i : _>12), tumor necrosis factor (Carswell et al. (1975), Proc.
  • cytolytic protease As noted, two groups have previously observed mole cules which appear to depend on protease function for t expression of their cytotoxic function. Adams and coworkers employed the murine BCG-activated peritoneal macrophage as a source of a factor, eventually termed cytolytic protease, which was selectively cytotoxic for tumor cells in vitro (Adams et al. , supra) . This prote had a molecular weight of about 35 kD as determined by molecular sieving, and its lytic activity could be bloc by bovine pancreatic trypsin inhibitor, diisopropyl- fluorophosphate and, notably by serum.
  • MCT had a molecular weight o about 150 kD as determined by molecular sieving, and its cell-lytic activity was susceptible to treatment with
  • novel antitumor compositions which include a novel cytolytic factor in a substantially purified form which may be employed in the treatment of cancer cells.
  • This factor designated Cytolytic Factor by the inventor, in its substantially purified form is shown most generally to b a soluble cytolytic protein having a molecular weight of between about 140 and about 160 kilodaltons upon gel exclusion chromatography.
  • substantially purified relates to the fact that the Factor of the invention may be purified away from other biological factors, which other factors may or may not have similar or interfering anticellular activities.
  • this Cytolytic Factor may be characterized by one or more physical or biological properties which serve to both demonstrate its novelty over previously known biologica factors and further, to demonstrate its usefulness as a important new antitumor agent.
  • the Factor when isola in the manner disclosed herein, the Factor is shown to highly stable upon extended storage, for example, it is stable and retains its biological activity at 4 ⁇ C in physiological buffers for more than 6 months and, typically, much longer.
  • the Cytolytic Factor disclose herein has surprisingly been found to be highly active the presence of serum, as shown by its retention of activity in the presence of 10% fetal calf serum.
  • the Factor is shown be immunologically cross-reactive with polycional anti- serum raised against the well known antitumor factor
  • the biological activity of the Factor is shown to be labile to treatment with trypsin, or upon heating to about 100°C for 10 minutes.
  • Such properties demonstrate, for example, that the Cytolytic Factor includes a proteinaceous component tha is required for its antitumor action.
  • the Factor is shown to require protease activity for ⁇ the expression of its biological action. More particularly, the Factor is sh to be a neutral protease, it being active at a neutral Such activity is further demonstrated by sensitivity, generally a dose-dependent sensitivity, of the factor's biological antitumor activity to protease inhibitors su as TLCK or TAME.
  • Cytolytic Factor is generally active to some extent against a number of tumor cell lines.
  • the Fac is found to be surprisingly efficacious against some tumors and tumor cell lines, such as L-929, SVT-2 A375 human melanoma cells, these cells being generally regar in the art as indicative of activity against human tumo
  • the novel factor of th invention is preferably obtained from macrophages, most preferably _in_ vivo or _in vitro "activated" macrophages, derived from a mammalian origin such as a mouse, rabbit, guinea pig, etc., but preferably of murine origin.
  • activated macrophages refers generally t -macrophages which have been treated with an macrophage activating agent as such regents are generally referred in the art.
  • the Factor is thus most generally isolatable from media or culture supernatant wherein activated n_ vivo and/or jln_ vitro macrophages have been cultured so as to release the Factor therein.
  • media is referred to generally herein as “.conditioned” supernatant in that i has been “conditioned” by virtue of the release of biological factors by the macrophages cultures therein.
  • preparation of the macrophage conditio supernatant includes the steps of harvesting macrophage from a mammal, incubating the macrophages in an incubat medium and separating the macrophages to provide the resultant conditioned supernatant.
  • Any number of tissu culture media or physiologic buffers known in the art c be used as the incubation medium.
  • pre-immunization of the mammal with a macrophage activator will result in the generation of e greater amounts of the factor by the subsequently harvested macrophages.
  • Bacillus Calmette Guerin (“BCG”) is utilized as the activator.
  • the mammal used is a mouse.
  • BCG Bacillus Calmette Guerin
  • the harvested macrophages are incubated in medium to which a triggering agent, for example, bacterial endotoxin, is added.
  • a triggering agent for example, bacterial endotoxin
  • an object of the invention is to provide a method for cytolytically inhibiting the proliferation of tumor cells which includes subjecting tumor cells to an effective dose of a preparation which includes the cytolytic factor in substantially purified form.
  • an effective dose is defined be 1 to 2 ul. conditioned by 1 x 10 BCG activated macrophages per ml. This dose of supernatant causes a 5 cell death of 25 x 10 3 L-929 cells in 18-24 hours.
  • FIGURE 1 Supernatants from LPS-triggered, BCG-activate macrophages cultured in medium supplemented with LMS were concentrated on a YM-10 membrane, and the concentrates subjected to -molecular sieving on Sephacyl S-200. The fractions were assayed for lytic activity on L-929 and EMT-6 targets, both targets were actinomycin D-treated. The resulting lytic activity detected on each target is shown. Molecular weight markers indicated are blue dextran, immunoglobulin G, hemoglobulin, and cytochrome
  • FIGURE 2 Adherent peritoneal exudate macrophages were established in 2 cm 2 wells. The macrophages were expose for 4 hr to LPS, PIC, SMDP, tuftsin, or PMA in the dose range from 3-1000 ng/ml, or A23187 in the range of 0.3 t 10 uM. The resulting lytic activity for each sample was determined by bioassay on L-929 targets. The units/ml o CF in test preparations was normalized with respect to that in supernatants from macrophages releasing CF spontaneously, and expressed as a stimulation index (mean _+ S.E. ) .
  • FIGURE 3 Monolayers of BCG-activated macrophages were est ⁇ a ⁇ b ⁇ — lished at 25 or 100 x 106• cells/78 cm2, and culture in 30 ml of medium supplemented with LMS or LAH after triggering with 100 ng/ml of LPS. One ml samples of supernatant were harvested at intervals up to 24 hr and were assayed for CF on L-929 targets. The resulting lyt activity (mean _+ S.E.) is shown for each sample.
  • FIGURE 4 Macrophage monolayers were established in 2 wells in medium with LMS. Doses of actinomycin D, cycloheximide, monensin, or tunicamycin ranging from 0.
  • FIGURE 5 Cytolytic Factor was pretreated with various levels of TLCK for 1 hr at 37°; after overnight dialysi against DPBS, TLCK-treated and control cultures were assayed on L-929 cells. Similarly, the Factor was coincubated with various levels of TAME or catalase on 929 cells. Lytic activity in treated CF samples was compared to a control Factor preparation, and expressed a percentage (mean _+ S.E.) thereof.
  • FIGURE 6 Purified preparations of Cytolytic Factor an necrosin were incubated with various doses of anti- necrosin antiserum at ambient temperature for 1 hr. Th mixtures were then assayed for lytic activity on L-929 cells; these values were normalized to the nonantibody- treated control value and expressed as a percentage thereof.
  • compositions of the invention are gener ⁇ ally defined as including a factor, termed Cytolytic Factor (CF) r which exhibits very high cytolytic activity against a number of various tumor cell targets and very low, if any, such activity against normal cell targets.
  • CF Cytolytic Factor
  • This cytolytic factor is found to be generally soluble and stable in physiologic buffers, for example, phosphate-buffered saline at neutral pH, for long periods of time (greater than six months) when stored at 4°C. As will be appreciated, this allows for ready formulation of the factor into pharmaceutically acceptable vehicles for administration to patients.
  • the Factor may be characterized in physical terms according to its apparent molecule weight upon gel exclusion chromatography. In general, the factor is fou to exhibit a molecular weight between about 140 and abou 160 kilodaltons, with a peak of activity generally observed at about 150 kilodaltons, when the factor is subjected to chromatography on Sephacryl S-200 (Pharmaci Uppsala, Sweden).
  • the Cytolytic Factor may be characterized according to various biological properties. For example, the factor is found to maintain its cytolyti activity in the presence of serum, thus demonstrating it usefulness for direct administration to patients. Moreover, the factor appears to exert its cytolytic acti by way of a mechanism which involves a "neutral" proteas function. For example, in certain embodiments the Facto is found to be inhibited , in a dose-dependent fashion by protease inhibitors such as TAME (alpha, N-tosyl-L-argin methyl ester) or TLCK (alpha, N-tosyl-L-lysyl- chloromethylketone) .
  • TAME alpha, N-tosyl-L-argin methyl ester
  • TLCK alpha, N-tosyl-L-lysyl- chloromethylketone
  • Cytolytic Factor is immunolo ically cross-reaction with Necrosin, a cytotoxic derived from murine macrophage cell line.
  • Necrosin exists as a holotoxin having two molecular weight forms, 70 and 55 kilodaltons, which are multimers of a 15 kilodolten protein (see, e.g., Kull et al.-, (1984), Proc. Natl. Ac Sci. U.S.A. , j$l_:7932).
  • the molecular or etiological ba for this immunological cross-reactivity is unknown and based on the observation that polyclonal antiseru rais against Necrosin has been found to inhibit the cytolyti activity of the Factor.
  • the Cytolytic Factor i shown to be a protein whose cytolytic activity is dependent upon an intact glycosylation apparatus of the macrophage cells from which it is obtained.
  • the production and release of Cytolytic Factor by macrophages is found to require transcription, transla ⁇ tion, glycosylation and an intact secretory apparatus o the producing cell, as is evident from its inhibition following treatment of the macrophages with actinomycin cycloheximide, tunicamycin and monesin, respectively, during culturing of the cells.
  • the Factor itself appears to lose its biological activity followin treatment with trypsin or heating to 100°C for 10 minut
  • the Cytolytic Factor of the present invention has proved highly active in killing tumor cells of lines which are well accepted in the art as appropriate to demonstrate such activity. Moreover, the Factor is found to exhibi much less activity against normal cells. For example, certain factor preparations were found to be highly act against the murine L-929 or EMT-6 adeno-carcinoma and various human tumor, while much less active against "normal" cell lines such as normal mouse lung fibroblasts or peritoneal exudate cells.
  • Cytolytic Factor as compared to previous tumoricidel or tumoristati factors of biological origin.
  • Cytolytic Factor has no apparent effect on the catalysis of iron- release by tumor cells, and is thus distinguishable from various "iron-releasing" monokines which have been described (Klostergaard et al. (1987), Ly phokine Res, j:14).
  • partially purified preparations of Cytolytic Factor exhibit only a minor ability to inhibit the mitochondrial electron transport system, thus demonstrating that Cytolytic Factor is distinguishable from Respiratory Inhibitory Factor (Kilbourn et al., supra. )
  • Cytolytic Factor preparations may be obtained in accordance with the invention in a substantially purifie form as determined by the relative absence of iron- releasing monokines and Respiratory Inhibitory Factor, such additional products being often present in super- natants which " have been "conditioned” with activated macrophages in the manner of the invention.
  • the first step involv the preparation of a supernatant derived from cultures o activated macrophages.
  • the macrophages to be cultured are generally first primed or "initiated" ij vivo by injecting a mammal (a mouse is preferred, but other, mammals such as guinea pigs, hamsters, rats, rabbits, etc., may be used) with an effective amount of a macrophage activation initiator.
  • BCG is a preferred activator, but other agents known to induce cytotoxic activation of macrophages i ⁇ _ vivo may ⁇ used as well.
  • Immunotone American Biotechnology Co.
  • C. Parvum supernatant derived from mitogen stimulated mur T-cells
  • gamma-interferon gamma-interferon
  • muramyl dipeptide a compound that has been modified by the production of cytotoxic macrophages.
  • the amount administered will vary with the particular initiator or activator used, but in general, effective amount is that amount required to produce cytotoxic macrophages, recognizable by established criteria. More particularly, when BCG is used, a dose approximately 2 x 10 colony forming units is the preferred dose. Intraperitoneal administration is preferred.
  • any regimen of injection sufficient to induce activation of the macrophages in vivo may be used, however, in a preferred embodiment, the mammals are firs injected with the activator 25 days before the macrophag are harvested, then "boosted” with a second dose 4 days before harvest. Furthermore, it should be appreciated that iri vivo activation is not strictly required where t macrophages are sufficiently activated in vitro as described below.
  • the macrophages are harvested from the host mammal by peritoneal lavage.
  • This technique generally involves injecting the peritoneal cavity of the animal with a liquid medium, such as a physiologic buffer, massaging the peritoneal area, and draining the peritone exudate from the animal.
  • the peritoneal exudate contains a mixture of cell types, including the activated macrophages.
  • the cell mixture may be enriched for the activated macrophages by any of a number of .methods known to separate macrophages from contaminating cell types.
  • the macrophages are selected by culturing them in serum free
  • the macrophages may then be recultured in any of a number of suitable culture media.
  • the macrophages should be treated with triggering agent shortly after they are established in
  • the triggering agen is endotoxin derived from E. coli, however it will be appreciated that other agents, such as other bacterial endotoxins, muramyl dipeptide, and phorbol mysistate acetate may be used as triggering agents. ⁇ n fact, wher
  • LAH lactalbumin hydrolysate
  • Production of macrophage conditioned supernatant is generally completed after four to twelve hours of cultur 35 Since macrophages triggered with endotoxin as described above have been shown to produce the Cytolytic Factor almost immediately after triggering, and since the Fact is detectable in cultures incubated up to at least 24 hrs., shorter or longer' incubation times may be used. However, four to twelve hours is considered optimal. A any rate, at the termination of the selected incubation period, the resultant conditioned supernatant is remove from the culture, for example, by centrifugation, siphoning or filtration. It may then be stored at -20° until needed for further use. The cytolytic action of supernatant can be titered on actinomycin D-treated L-9 cells.
  • the advantages of the present invention are realized by identification and separation,of a solu Cytolytic Factor having a relative molecular weight between 140,000 and 160,000 datons from the macrophage conditioned supernatant.
  • the Cytolytic Factor may be identified and separated by any of a number of selectio techniques known to those skilled in the art of separati biological molecules. These techniques include, but are not limited to, selective ultrafiltration, ultracentri- fugation, preparative gel electrophoresis, molecular exclusion chromatography, ion exchange chromatography an the like.
  • a preferred method for separating an identifying the monokine entails clarification of the conditioned supernatant by centrifugation, followed by ultrafiltration and subsequent gel exclusion chroma ⁇ tography. More specifically, the ultrafiltration step involves placing the conditioned supernatant in a stirred cell apparatus preferably pressurized with nitrogen.
  • the apparatus contains a membrane which retains only molecule above a specified molecular weight.
  • membranes ar commercially available and may be used for practicing the invention; however, a YM-10 membrane, which retains molecules with a molecular weight greater than 10,000, is preferred.
  • the conditioned supernatant containing the Factor is concentrated approximately 20 to 100-fold.
  • the concentrated supernatant is chromatographed on a gel filtration column
  • gels suitable for such chromatography include, but are not limited to, agarose gels, Sepharose gels, Sephadex gels, Sephacryl gels, and polyacrylamide gels.
  • a Sephacryl S200 gel filtration column is preferred.
  • the column may be equilibrated with any suitable equilibration solution or physiological buffer; however, Dulbecco's phosphate buffered saline is pre- ferred. Fractions eluting from the column in the same volume with molecular weight standards ranging from 140 t 160 kilodaltons are a particularly preferred source of t Factor.
  • the purified Cytolytic Factor may be formulated into a number of preparations suitable for treatment of tumor patients.
  • the monokine may be formulated into such preparations in neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the monokine)which are formed with inorganic acids such as, for example, hydro chloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, andelic, and the like.
  • Salt formed with the free carboxyl groups may also be derive from inorganic bases such as, for example, sodium, pota sium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like
  • the Factor may be admixed with any vehicle in which it retains function.
  • vehicle in which it retains function.
  • suitable vehicles include aqueous physiological solutions, salin dextrose, glycerol and the like, or combinations thereo
  • any preparation of the monokine to be u in human tumor therapy must be formulated with a nontox excipient.
  • any of a number of pharmaceutica excipients may be used, so long as the factor retains i function when it is administered in the presence of suc excipient.
  • the factor may be administered by any of a number of techniques known to those skilled the art of administering biologic compounds. Such techniques in- elude, but are not limited to, intravenous injection, subcutaneous injection, intraperitoneal injection, and continuous infusion.
  • the factor could be administered in conjunction with ot chemotherapeutic agents known to those skilled in the a
  • BCG-activated macrophages produced according to the protocol described below were used to study the mechanis of activated macrophage induction of Cytolytic Factor release. They were also used to prepare conditioned supernatant from which the Factor was purified.
  • mice Six- to eight-week old male CD-I mice were obtained from the University of Texas, Science Park, Bastrop, TX. Ten- to twelve-week old male CB6F1 mice (Balb/cAnN x C57B1/6N) were provided through the breedin program of the Department of Tumor Biology, The Universi of Texas M.D. Anderson Hospital and Tumor Institute, Houston, TX.
  • Macrophage monolayers were obtained by peri- toneal lavage of mice that had been injected i.p. with 2
  • Mg -free phosphate buffered saline collected bbyy centrifugation, adjusted to the proper density (1-2 x 10 cells/ml) and then allowed to adhere to plastic for 4 h in serum-free culture medium at 37°C. After this time, non-adherent cells were removed by washing with PBS. Remaining cells were judged to be > 90% macrophages by morphological and functional criteria. These cells were then utilized either in cocultures with radiolabeled tu cells to determine cytotoxicity, or for supernatant production.
  • PBS phosphate buffered saline
  • Cytolytic Factor was routinely assayed in vit employing the murine L-929 target (C3H/An; ATCC) in a modification of the microcytotoxicity assay of Fisch an
  • 96-well plate were incubated overnight after' seeding. Fifty microliters of 4 ug/ml of actinomycin-D (Sigma) w added to each well immediately before assay of CF super natants or fractions from biochemical separations.
  • the Cytolytic Factor preparations were titered on drug-treated targets, and incubation carried out over ⁇ night. 'Fifty microliters of a 0.02% neutral red soluti were added to each well; after 1-2 hr, the wells were washed with DPBS, and incorporated stain assessed after treatment with citrate-ethanol (0-.1 M citric acid in 7 ethanol-water) . Quantitation of A ⁇ . Q was achieved using Titertek Multiskan. Units of lytic activity were define as the reciprocal of the dilution of the Cytolytic Facto that caused 50% reduction in dye uptake. In some experiments, cell viability was enumerated by use of the MTT assay Mosmann (1983), J. Immunol. Meth., _6Jj: 55. Uni of lytic activity were defined as the reciprocal of the dilution of Factor that caused 50% reduction in MTT formazan production, measured at 570 nm.
  • BCG-activated macrophage were seeded at 2.5 x 10 /well in 96-well plates (- .33
  • EMT-6 and L-929 targets were labeled as single-cell suspensions with Na ⁇ Cro. (ICN, Irvine, CA) in a 15 ml polypropylene tube (Corning Glass Works,
  • the percent cytotoxicity was calculated by the following expression:
  • Cytolytic Factor was quantitated on L 929 targets as described below.
  • the Cytolytic Factor titer in units/ml for drug-treated macrophages was compared to a non-drug-treated control and expressed as percentage thereof.
  • Cytolytic Factor Protease characteristics of Cytolytic Factor were probed by incubation of Cytolytic Factor, partially purified by conventional molecular sieving on Sephacryl S-200 and HP anion-exchange LC, with TLCK (Sigma)
  • Oxygen metabolite-dependent functions of Cytolytic Factor were probed in two ways: by enzymatic degradatio ' of H.O. produced, and by direct measurement of H-O, or 0 released during lysis. Partially-purified Factor was coincubated with catalase (Sigma) during the lytic assay
  • H 2 0 2 and 0 2 ⁇ were measured by the microeli method of Pick and Mizel (1981) J. Immunol. Meth., _46_:211).
  • the H_0_ reaction mixture was composed of 0.56 M phenol red (Sigma) with 19 units/ml horseradish peroxidase (Sigma) with or without 0.25 ng/ml PMA in Hanks balanced salt solution without phenol red.
  • the 0 2 reaction mixture 1.2 mM ferrictochrome C (Sigma) with or without PMA in Hanks balanced salt solution without phenol red. Absorbance was determined on a microelisa reader (Dynatech, Alexandria, VA) at 600 nm for H 2 0 2 (after alkalanization with 10 ul in NaOH) and at 550 nm for 0 2 The reactions were monitored at 1, 4 and 18 hrs of incubation at 37°C.
  • arginine Sigma was added to the medium of the lytic assay during incubation with CF; the amino acid was dissolved in borate buffer to give a final concentration of 667 ug/ml.
  • the lytic activity of CF against L-929 cells in the presence of DME-F12 with added arginine was compared to the activity of CF in DME-F12 medium alone, and expressed as a percentage thereof.
  • the effect of protease inhibitors in serum on lytic activity was evaluated by assay of Cytolytic Factor on L 929 targets as in the assay described below, except that the serum-free medium of Neuman-Tytell (GIBCO) was used incubate targets instead of DME-F12 with 10% FCS.
  • the lytic activity in the former medium was compared to the activity in the presence of serum and was expressed as a percentage thereof.
  • the heat stability characteristics of Cytolytic Factor were explored by incubation of Cytolytic Factor i screw-capped vials for the appropriate times in water baths at the appropriate temperatures. After treatment, the tubes were brought to ambient temperature and assaye on L-929 targets as described below.
  • the lytic activity in the heated preparations was expressed as a percentage of the control preparation which had been held at 4°C.
  • Cytolytic Facto The dependence of lytic activity of Cytolytic Facto on protein structures were examined by incubation of Cytolytic Factor with trypsin (GIBCO) or control buffer for 15 in at 37°C. The reaction was quenched with 10% FCS, and the samples subjected to bioassay. The lytic activity in the enzyme-treated samples was expressed as percentage of the non-treated control.
  • ETC electron transport chain
  • the EMT-6 murine adenocarcinoma line obtained fr Dr. Gabriel Lopez-Berestein, M.D. Anderson Hospital,
  • L-1210 lymphoblastic leukemia cells were obtained from Dr. Berestein. Normal mouse lung fibrob cultures were established by mincing of aceptically removed lungs of 8 day old mice.
  • Murine macrophages were seeded at 2 10 per 100 mm tissue culture dish. After 4 hr of adherence, the macrophages were washed, recultured in 30 ml of DME/F-12 medium, and triggered for 2-6 hrs with 10 ng/ml of endotoxin (bacterial lipopolysaccharide; phenol extracted E. coli serotype 0128:B12; Sigma Chemical Co., St. Louis, MO). The resultant conditioned supernatant w collected by centrifugation and frozen at -20 ⁇ C until further use.
  • the conditioned supernatant was subjected to various in vitro character izations and molecular weight fractionization.
  • the supernatants were thawed, clarified by centrifugation, and concentrated on YM-10 membrane in a stirred-cell apparatus.
  • the concentrates were subjected to molecular sieving on Sephacryl S-200 (Pharmacia, Uppsala, Sweden) in a 2.5 x cm column (BioRad, Richmond, CA) equilibrated with DPBS (Dulbecco's Phosphate Buffered Saline).
  • FIGURE 1 is a chromatogra which illustrates the elution profile of macrophage conditioned supernatant applied to Sephacryl S200 in the foregoing manner. Each fraction was assayed on both L-929 and EMT-6 targets.
  • FIGURE -1 a single cytolytic species of•approximately 150 kD was detected by bioassay on either type of target cell; the EMT-6 target appeared to be about 20-fold more sensitive to the Factor than th L-929 target, when both were treated with actinomycin D. No species in the 40-50 kD range was detected.
  • D Alternative Triggering Agents for Cytolytic Factor Production
  • tuftsin Calbiochem-Behring, San Diego, CA
  • SMDP stearoyl-mura yl dipeptide
  • EMT-6 and L-929 targets were coincubated with resident or BCG-activated macrophages, with or without endotoxin-triggering, for 16-20 hr.
  • Analysis of isotope release indicated that resident macrophages were incapable of significant lysis (up to 7%) of either EMT-6 or L-929 targets, unless puls with endotoxin; under these conditions, the level of lys of L-929 targets rose to 15-25%.
  • Peritoneal macrophages from BCG-i mune mice were established as- adherent monolayers of either 25 x 1 or 100 x 10 6 cells on 78 cm2 dishes. The cells were triggered with 100 ng/ml of LPS and allowed to release
  • Cytolytic Factor in DME-F12 medium supplemented with LMS (10% v/v) or LAH (0.1%) Release was monitored by removing an aliquot of supernatant at various time point and subjecting it to bioassay on L-929 cells. The activity in lytic units/ml for each sample are shown in
  • Cytolytic Factor was released rapidly after triggering macrophages with LPS, reaching peak levels in 4-8 hr. On a per-cell basis, Cytolytic Factor productio was superior with the lower macrophage density (25 x 1
  • Macrophage monolayers were incubated in LAH- containing medium with actinomycin-D, cycloheximide, monensin, or tunicamycin over a dose range from 0.1 to ugt/ml. After 30 min of preincubation, LPS (100 ng/ml) was added and supernatants were collected after four hr After overnight dialysis against DPBS, each sample was assayed for CF activity on L-929 targets; the lytic activity was normalized with respect to that produced b LPS-triggered macrophages in the absence of drug pretre ment, an expressed as a percentage thereof (Figure 4).
  • actinomyci caused > 50% inhibition of CF production/release; with log 10 higher dose, inhibition was > 95%. Consistent w this dependence on RNA synthesis, cycloheximide pretrea ment of macrophages caused a similar pattern of inhibit of Cytolytic Factor production/release. Active secreto processes appear to be involved in Cytolytic Factor release, as monensin pretreatment of macrophages caused dose-dependent inhibition (about 50% inhibition at 2 ug/ml). Tunicamycin treatment of macrophages before triggering caused a similar pattern of inhibition (50% inhibition at - 2.5 ug/ml) . This suggests that either a properly N-glycosylated Factor is required for secretion or that glycosylation is required or expression of biological activity.
  • Cytolytic Factor was significantly labile when treated with trypsin or upon heating at 100° (Table 3). Arginine addition had no apparent effect on lytic activity. Lytic activity of Cytolytic Factor on L-929 targets was essentially identical whether serum protease inhibitors (e.g., alpha--macroglobulin) were excluded not.
  • serum protease inhibitors e.g., alpha--macroglobulin
  • Cytolytic Factor obtained from BCG-activated macrophages from CD-I mice was assayed for cytolytic/cytostatic effects on these target cell lines _in vitro: L-929, EMT-6, MCA-1, B16F1, L-1210, SVT2, an 3T3.
  • a single Cytolytic Factpr preparation, partially purified by molecular sieving was used for all these cytotoxicity studies summarized in Table 4.
  • the L-929 target was sensitive to the Factor in a 24 hr assay. B cytostatic and cytolytic (nonstaining with neutral red) effects were evident.
  • Pretreatment of the L-929 cells with actinomycin-D caused growth inhibition of both control and Factor-treated targets, and therefore allow evaluation solely of the cytolytic effects of the Facto Drug treatment clearly caused marked sensitization of t L-929 target to Cytolytic Factor (about a 70-fold incre in lytic activity).
  • the EMT-6 target demonstrated marked resistance to the Factor; targets treated with the highest dose emplo were > 99% viable as determined by neutral red staining However, actinomycin-D treatment of these targets rende them even more sensitive to CF (3-4-fold) than the drug treated L-929 cell.
  • the MCA-1 target showed a similar, but less pronounced, ability to be sensitized by drug treatment.
  • the B16F1 melanoma showed significant resistance to the Factpr, as did the L-1210 target, whereas the SVT2 target was sensitive.
  • 3T3 fibroblast line showed some sensitivity a 40 hr assay.
  • Cytolytic Factor treatment of normal peritoneal exudate cells from BCG-immune mice had no effect in 48 hr on the viability of either adherent ( polymorphonuclear leukocytes and macrophages ) or nonadherent (lymphocytes) cells.
  • the purified Cytolytic Factor demonstrates only a minor ability to inhibit ETC-dependent reduction of MTT, but a higher cell-lytic potency.
  • the partially purified RIF is capable of strongly inhibiting MTT-formazan production, but has a low cell-lytic activity.
  • Cytolytic Factor was used t investigate its ability to mediate loss of intracellular iron in lytically resistant L-1210 and EMT-6 targets. No release of 59Fe from these targets above spontaneous release in response to Cytolytic Factor was detectable
  • Necrosin and a rabbit antiserum raised again the electrophqretically homogenous toxin (Kull et al., supra) were obtained from Dr. Frederick Kull, The Wellco Research Laboratories, Research Triangle Park, NC.
  • the antiserum was diluted 1:100 in PBS so that 1 ul would neutralize the lytic activity of 5 units of necrosin. From this stock, serial two-fold dilutions were made fro 1:40 to 1:640. To these diluents were added samples of stocks of either necrosin or Cytolytic Factor purified b molecular sieving. These stocks were adjusted to give a concentration of 1000-5000 units/ml as assayed on actinomycin D-treated EMT-6 cells.
  • the antiserum-toxin mixtures were titered o EMT-6 targets and assayed for lytic activity 18-24 hr later.
  • the percent neutralization of lytic activity of necrosin and Cytolytic Factor by the anti-necrosin antiserum was calculated as follows:
  • Percent neutralization [1- Lytic Activity in the Presence of Antiserum (units/ml) Lytic Activity in the Absence of Antiserum (units/ml)
  • the anti-necrosin antiserum which was shown to cross-react with the Cytolytic Factor produced by BCG- activated macrophages (Figure 6), was used to probe the possible role of the Factor in mediating direct nonspecific tumor cytotoxicity of macrophages in coculture.
  • Table 7 Under the coculture conditions, the antiserum could significantly but not completely, block cytotoxicity expressed by activated macrophages against L-929 targets. The inhibition observed was dependent on the antiserum dose employed. The highest level of inhibition achieved brought the cytotoxic activity of LPS-triggered macrophages close to that observed for non-triggered macrophages.
  • the present invention has been disclosed in terms of examples considered by the inven to be the preferred embodiments for practicing the inv tion. However, they are in no way meant to be the onl modes for practicing this invention.
  • the conditioned supernatant is fractioned by molecular exclusion chromatography
  • other methods of fractioning the supernatant are contemplated.
  • other fractionation methods which employ a molecular sizing or molecular weight fractionation can employed. Such separation might therefore involve gel electrophoresis, ultracentrifugation or any other sepa tion technique based on differences in molecular size weight.
  • macrophages employed herein are murine macrophages, there in no reason why other mammalian macrophages cannot be similarly employe
  • macrophages of the present invention are initiated using BCG and activated using E coli bacterial endotoxin, there is no reason why other initiators such as lymphokines and other activators suc as other bacterial endotoxins, cannot be utilized to ga the advantages of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Compositions et méthodes de traitement relatives à l'utilisation d'un nouveau facteur antitumoral cytolytique stable dérivé d'un macrophage, dénommé Facteur cytolytique (FC). La production et la libération du FC ont exigé la transcription, la translation, la glycosylation du macrophage et un appareil sécrétoire non contaminé dénoté par son inhibition au traitement par l'actinomycine D, le cycloheximide, la tunicamycine et la monensine respectivement, avant et pendant le déclenchement au LPS. Le FC obtenu par la culture de macrophages activés au BCG est rapidement apparu dans le produit surnageant après le déclenchement. En utilisant les cibles L-929 ou EMT-6 traitées à l'actinomycine-D dans un micro-échantillon, le FC sécrété par les macrophages cultivés dans les éléments constitutifs du sérum de faible poids moléculaire a été détecté en tant que l'un des éléments -150 kD dans Séphacryl S-200 et s'est révélé stable à 4°; en revanche, en cultivant les macrophages dans de l'hydrolysat de lactalburmine, le FC s'est révélé instable à cette température. Le FC a révélé un spectre d'activité cytotoxique à l'égard d'un certain nombre de cibles tumorales et normales in vitro. Le FC s'est montré modérément sensible au traitement par TLCK et TAME. Un hétéroantisérum de lapin mis en présence de nécrosine très pure a pu neutraliser extrêmement bien l'activité biologique du FC.
EP88908641A 1987-04-14 1988-04-14 Facteur cytolytique Withdrawn EP0360840A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3811187A 1987-04-14 1987-04-14
US38111 1987-04-14

Publications (1)

Publication Number Publication Date
EP0360840A1 true EP0360840A1 (fr) 1990-04-04

Family

ID=21898150

Family Applications (1)

Application Number Title Priority Date Filing Date
EP88908641A Withdrawn EP0360840A1 (fr) 1987-04-14 1988-04-14 Facteur cytolytique

Country Status (4)

Country Link
EP (1) EP0360840A1 (fr)
JP (1) JPH02502999A (fr)
AU (1) AU1709788A (fr)
WO (1) WO1988007868A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60112718A (ja) * 1983-11-21 1985-06-19 Kyorin Pharmaceut Co Ltd 抗腫瘍作用を示す蛋白性物質及びその製造方法
EP0178050A1 (fr) * 1984-08-13 1986-04-16 The Wellcome Foundation Limited Substance protéique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8807868A1 *

Also Published As

Publication number Publication date
AU1709788A (en) 1988-11-04
JPH02502999A (ja) 1990-09-20
WO1988007868A1 (fr) 1988-10-20

Similar Documents

Publication Publication Date Title
Frevert et al. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation.
Gleich et al. Immunobiology of eosinophils
Gidlund et al. Enhanced NK cell activity in mice injected with interferon and interferon inducers
Roberts Jr et al. Virus-induced interferon production by human macrophages
Barros et al. Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom
EPSTEIN The comparative biology of immune and classical interferons
US5086164A (en) Novel methods and compositions for treatment of angiogenic diseases
JP3159705B2 (ja) 血管形成ペプチド
AU590543B2 (en) Purification of native colony stimulating factor-1
Männel et al. Inhibition of nonspecific tumoricidal activity by activated macrophages with antiserum against a soluble cytotoxic factor
US4844895A (en) Composition containing a peptide fragment of platelet factor four and method for restoring suppressed immune responses
Burnett et al. Pharmacological effects of various venoms on cutaneous capillary leakage
EP0345263B1 (fr) Monokine liberant du fer
EP0360840A1 (fr) Facteur cytolytique
Yodoi et al. Formation of IgE-binding factors by rat T lymphocytes. I. Induction of IgE-binding factors by poly I: C and interferon.
CA2078805C (fr) Preparation de cytokine
CA2072626A1 (fr) Medicament liberant du cd14
JPH0453848B2 (fr)
Klostergaard et al. Tumoricidal effector mechanisms of murine BCG-activated macrophages. I. Parameters of production and initial characterization of a cytolytic factor serologically related to necrosin
US5698519A (en) Polypeptide specifically inhibiting cathepsin L
Rabinowitz et al. Aorta contains extractable immunosuppressant activity
US6306824B1 (en) Uses of lipopolysaccharide binding protein
Klostergaard et al. Effector mechanisms of human monocyte-mediated tumor cytotoxicity in vitro: parameters of induction of cytotoxins from peripheral blood monocytes isolated by counterflow elutriation
Cavaillon et al. Presence of IL 1 activity in the supernatant of human monocytes challenged with purified allergenic constituents
Arseneault et al. Selective inflammatory response induced by intratracheal and intravenous administration of poly-L-arginine in guinea pig lungs

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19891013

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI

17Q First examination report despatched

Effective date: 19900615

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19901025