WO1987007503A1 - Modificateur de reponse biologique - Google Patents

Modificateur de reponse biologique Download PDF

Info

Publication number
WO1987007503A1
WO1987007503A1 PCT/US1987/001397 US8701397W WO8707503A1 WO 1987007503 A1 WO1987007503 A1 WO 1987007503A1 US 8701397 W US8701397 W US 8701397W WO 8707503 A1 WO8707503 A1 WO 8707503A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
ribosomes
response modifier
membrane
Prior art date
Application number
PCT/US1987/001397
Other languages
English (en)
Inventor
Richard E. Urban
Original Assignee
Cell Technology, Inc.
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 Cell Technology, Inc. filed Critical Cell Technology, Inc.
Priority to KR1019880700142A priority Critical patent/KR880701110A/ko
Priority to AU77864/87A priority patent/AU606075B2/en
Priority to IN432/MAS/87A priority patent/IN165076B/en
Publication of WO1987007503A1 publication Critical patent/WO1987007503A1/fr
Priority to FI880412A priority patent/FI880412A/fi
Priority to NO880517A priority patent/NO880517L/no
Priority to DK066788A priority patent/DK66788A/da

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria

Definitions

  • This invention relates generally to a pharmaceutical product having immunomodulating properties. More particularly, the invention relates to a biologic response modifier (BRM) , defined as an agent that modifies the relationship between a disease and host by modifying the host's biological response to the disease with resultant therapeutic effects.
  • BRM biologic response modifier
  • the BRMs can be divided into two categories: (1) biologic or chemical agents that can stimulate or otherwise alter one or more of the host's resistance mechanisms and (2) purified cellular products that demonstrate direct effects on a particular disease.
  • the first group of BRMs, in which the present invention falls, consists of agents that activate, increase, or otherwise modify host immunologic reactivity and are generally referred to as immunomodulators.
  • a BRM may be used alone, or in combination with other agents, to enhance resistance to or recovery from invasion by pathogens, to modify or induce tolerance to grafts of foreign tissue, to enhance tumor rejection or stabilization and to inhibit tumor recurrences following other forms of therapy, to restore normal helper-suppressor mechanisms, or otherwise promote a normal immune response.
  • immunomodulators/ immunoadjuvants has been developed since the early 1900 's. The vast majority of these agents are of microbial (bacterial/fungal) origin with the most popular ones being derived from the Corynebacteria, Mycobacteria and Nocardia genera (CMN organisms).
  • the various immunoadjuvants are comprised of either intact viable cells, dead cells, cell walls, various cell wall fractions, endotoxin, various types of polysaccharides, or subcellular fractions such as ribonucleic acid or ribosomes.
  • all of these agents have demonstrated immune potentiating/modulating/adjuvant activity in tissue culture, animals, and humans, against infectious and neoplastic disease, they are universally plagued with inconsistencies in production and composition, and have moderate to severe toxicity. Relative to the treatment of neoplasia, none of these agents has thus far been useful in the treatment of established disease. Moreover, these agents typically demonstrate loss of activity with repeated use (anergy), immune deviation, hypersensitivity reactions, development of chronic inflammatory conditions, and/or the development of various other undesirable conditions.
  • ribosomal vaccines prepared from Staphylococcus aureus and Neisseriameningitis do not.
  • ribosomal vaccines prepared from Mycobacterium tuberculosis 3/ and Salmonella typhimurium 4 appear to induce a cell-mediated response, whereas those prepared from Streptococcus pneumoniae and streptococcus pyogenes mediate a humoral response. 5/ Even though the extracted ribosomal fraction is, theoretically, the active agent, considerable controversy exists in many cases as to whether other cell components (RNA, protein, endotoxin, cell wall) present as contaminants are responsible for the observed immune reactivity.
  • Known cell fraction vaccines may sometimes be appropriate for the prophylactic or therapeutic treatment of infectious disease. However, they are, for several reasons, inappropriate for use as nonsensitizing, general immunomodulators for the treatment of neoplastic disease. The presence of cell wall, endotoxin, or poorly degradable components often results in toxicities similar to those obtained with the intact organisms. Moreover, undesired immune deviating and complex immune responses may be elicited since such vaccines are typically derived from organisms which are part of or which readily cross-react (common antigens) with the host's own microflora. Such vaccines also typically contain fractions having physical and/or chemical characteristics which may be suboptimal for general immune potentiating activity. urban, et al. 6 / reported on the ability of polyribosomes (aggregates of ribosomes) obtained from a specific bacterial organism to suppress the development of cutaneous SaD2 fibrosarcomas in DBA/2 mice.
  • Polyribosome fractions were obtained from cell cultures by lysing the cells osmotically or mechanically (depending on the type of bacteria), followed by differential centrifugation. Although the effect on the SaD2 murine tumor was positive, the mechanism of the induced biological response could not be determined from the data. Although toxicity was significantly low, the process described by Urban, et al. resulted in an extreme variation in polysome profile (size distribution) and a very low product yield. Consistency of quality, stability and effectiveness was not attained.
  • Kirsh, et al. 7/ have reported on immune stimulation and modulation via encapsulation of specific antigens or biologic response modifiers in liposomes. Liposomes containing drugs have been utilized for the treatment of metastatic cancer. 8/ However, the treatment of neoplastic disease (cancer) with biologic response modifiers alone or encapsulated in liposomes has thus far been discouraging. Although encapsulated BRM's surpass non-encapsulated BRM's in efficacy, the limitation of therapeutic benefit may be due to immune activation being limited to the macrophage. Tumor cells rarely, if ever, develop resistance to macrophage killing, as compared to natural killer cells and cytotoxic T-cells. The numbers of macrophages in these cases have been too low to effectively mediate, by themselves, the destruction of large tumor burdens.
  • Another object of the invention is to provide an improved biologic response modifier which is capable of manufacture with consistent quality, stability and effectiveness.
  • FIGURE 1 is an electron micrograph depicting the biologic response modifier of the invention at approximately 82,000 magnification
  • FIGURE 2 is a graph illustrating a human natural killer cell assay upon administration of the biologic response modifier of the invention as compared with administration of leukocyte interferon;
  • FIGURE 3 is a graph depicting a standard assay which demonstrates the bacteriocidal properties of the BRM of the invention
  • FIGURE 4 is a comparison of an antibody- dependent cellular cytotoxicity (ADCC) assay upon administration of the biologic response modifier of the invention as compared with administration of leukocyte interferon
  • FIGURE 5 is a graph illustrating the induction of interleukin 1 (IL-1) utilizing the biologic response modifier of the invention
  • FIGURE 6 is a graph illustrating, on natural killer (NK) cell activity, the effect of depleting various populations of human peripheral blood mononuclear cells (This study demonstrates loss of NK cell activity upon removal of NK cells but not upon removal of B-cells or T-cells when utilizing the biologic response modifier of the invention. Removal of monocytes (not shown) also eliminates the effect, suggesting that the NK cell activity is mediated via the monocyte-macrophage population.);
  • FIGURE 7 illustrates the results of in vivo studies of use of the invention on rat prostatic squamous cell carcinoma (R3327A) (rapid growth);
  • FIGURE 8 illustrates the results of in vivo studies of rat prostatic carcinoma (R3327H) (slow growth);
  • FIGURE 9 illustrates the results of in vivo studies of bilateral rat prostatic adenocarcinoma (R3327CF), also showing results in connection with other treatments;
  • FIGURE 10 is a summary of data acquired from the treatment of terminal cancer patients illustrating the effectiveness of the patient's peripheral blood mononuclear cells to kill specific tumor target cells
  • FIGURE 11 is a series of graphs depicting natural killer cell assays comparing, with alpha interferon, the effectiveness of preparations made from the source microorganisms Pseudomonas. E . coli, Enterobacter aerogenes, and E. chloacae; and FIGURE 12 is a series of graphs depicting natural killer cell assays, comparing, with interferon, the effectiveness of preparations made from the source microorganisms Erwinia chcysanthemi and Flavobacterium.
  • the biologic response modifier of the invention comprises natural membrane vesicles and ribosomes in a suspending buffer.
  • the vesicles are comprised of cellular membrane material endogenous to a selected organism.
  • the ribosomes are also endogenous to the selected organism.
  • the biologic response modifier is substantially free of endotoxin, intact cells, cell walls, and cell membrane fragments.
  • the selected organism is one which does not evoke a significant immune deviating response, is non-pathogenic in humans, and is one from which membrane vesicles are capable of being formed from cell membrane material and which vesicles are readily endocytosed by the monocyte-macrophage cell line.
  • the biologic response modifier exhibits a mean diameter in excess of 170 nm on particle size analysis.
  • the invention is capable of directly activating cells of the monocyte-macrophage cell line. Activation of the monocyte-macrophage, or cells of the monocyte-macrophage cell line, results in the induction of monocyte-macrophage mediated bacteriocidal activities (FIGURE 3) and tumor cytotoxicity (Table 3), altered levels of various white blood cells involved in immune function (e.g.
  • Non-toxic means within a level of toxicity which is tolerable by the mammalian host receiving biologic response modifier therapy.
  • Non-immunogenic means evoking a sufficiently low immunogenic response, or no response at all, such that undesired immune deviating, chronic inflammatory and hypersensitivity responses are not elicited, significantly, in the mammalian host.
  • Mean diameter means the mean diameter of MSD Particle Size Distribution Analysis as measured on a BI-90 (Brookhaven Instrument Corp.) particle sizer. This measurement involves an intensity weighting of the size averaging process and is explained more fully in the Operator's Manual for the instrument. Chapter 6, incorporated herein by reference.
  • Substantially non-pathogenic in humans means not or rarely associated with disease in humans of normal health. Since most microorganisms are capable of causing opportunistic infections under the right circumstances, such as in persons whose immune system has been compromised, this definition excludes only those organisms which typically cause non-opportunistic infections. "Substantially free of endotoxin, intact cells, cell walls, and cell membrane fragments” means a low enough level of biologic activity of such fractions to maintain a non-toxic characteristic as defined herein. "Immune deviating response” means an immune response which is directed away from the disease to be treated.
  • the phenomenon of original antigenic sin can cause the immune system to respond in accordance with historical challenges or to respond to common microflora when challenged by an antigen which is similar to those possessed by the historical challenge or the common microflora.
  • polyclonal activation can cause non-specific mis-directed anamnestic responses. Poorly degradable particles can result in distraction of the monocyte-macrophage cell line (i.e. foreign body reaction, chronic hypersensitivity) prohibiting cooperation of that cell line in response to the disease.
  • a "significant" immune deviating response is one which so attenuates the effect of the desired immune response as to be unacceptable for medical purposes.
  • Natural membrane vesicles means membrane vesicles prepared from membranes which are derived from living or dead natural cells.
  • the biologic response modifier of the invention possesses certain distinguishing characteristics.
  • the biologic response modifier of the invention contains two distinct particle classes, namely natural membrane vesicles and ribosomes.
  • the ribosomes may exist as monomers or as larger polymers, but the mean diameter of the ribosome population is less than the mean diameter of the membrane vesicle population. The relative amounts of the two populations appear to affect the efficacy of the product as determined by standard NK cell assays conducted in vitro.
  • the relative populations affect the measured mean diameter of the total population of particles. It is believed that a mean diameter in excess of 170 nm is necessary to achieve the desired efficacy. Below this level, efficacy of product as observed in standard NK cell assays appears to drop significantly. It has also been observed that the size of the vesicles in the vesicle population have an effect on the efficacy of the biologic response modifier of the invention. Thus, in a preferred form of the invention, substantially all of the vesicles exceed 110 nm in diameter and the mean diameter of the vesicle population is at least 180 nm and is preferably about 210 nm.
  • FIGURE 1 is an electron micrograph, enlarged approximately 82,000 times, showing the cross-sectional appearance of the membrane vesicles and also showing free ribosomes of various particle size (i.e. monomers and small polymers).
  • the manufactured vesicles have been measured by two methods: (1) direct measurement of circularized cross-section seen in electron micrographs; and (2) mathematically, using measurement of particle diffusion coefficients obtained from light scatter analysis using a BI90 (Brookhaven Instrument Corp.) particle sizer.
  • the membrane vesicles and the associated ribosomes are derived from (i.e. endogenous to) the gram negative bacterium, serratia marcescens.
  • Serratia marcescens is a well known organism and many strains are available from a number of sources. Sixty strains are available from the American Type Culture Collection, Rockville, Maryland 20852. This organism provides a particularly suitable source for manufacturing the product exhibiting a high level of immunomodulatmg/ immuno-therapeutic activity as compared to other bacterial sources, and is substantially free of toxicity.
  • Serratia 2000 (SM2000) Cell Technology
  • the desired bacterial membrane vesicles and ribosomes may be conveniently and economically isolated from a suitable source of stationary phase or log phase S .
  • marcescens bacterial cells by means of the method of the invention, which is a simple, rapid and reproducible procedure.
  • Reagents are employed which supply the necessary conditions for the maintenance of the integrity and conformation of the specific fractions isolated. Any reagents which might by themselves be toxic (unacceptably tolerated) or in any way influence or otherwise alter an immune response are avoided.
  • the preferred isolation procedure involves cultivating a seed lot of the bacterial cells in a suitable culture medium at a suitable temperature (30-40°C) to a log phase culture (a given phase of growth is related to final product yield and consistency as is the final density of viable cells per unit volume of culture medium processed); rapidly chilling the log phase culture to 0-4°C; harvesting the bacterial cells; washing and suspending the harvested cells to a prescribed density in a suitable buffering system which maintains an environment suitable for the formation and stability of the membrane vesicles and for the stability of the ribosomes; breaking open (lysing) the cells in a suitable cell disrupter or French pressure cell to produce membrane vesicles with a diameter in excess of about 110 nm or 0.11 microns (disruption of the cells occurs in the presence of a suitable detergent for facilitating endotoxin dissociation); clearing the bacterial cell lysates of cellular debris, including intact cells, cell wall fragments, and large ribosomal aggregates and polysomes, layering
  • rapid-chilling of the log phase culture to 0-4°C may be carried out by any suitable means, such as, for example, by employing a dry ice-alcohol mixture, an acetone-ice mixture, an alcohol-ice mixture, or special devices such as cooling coils. All subsequent steps are preferably carried out at 0-4oC.
  • Cell harvesting may be carried out by centrifugation, or cell harvesters/concentrators may be used instead. Clearing of cellular debris and isolation of the specific vesicle fraction may all be carried out by centrifugation.
  • the membrane vesicle and ribosomal fractions may also be isolated via employment of size-exclusion chromatography.
  • Cleared cell lysate is passed through gels such as Sephadex G-25, G-50, G-100, G-200, Sepharose 2B, Sephacryl S-200, Sephacryl-500 Biogel P-30, Sepharose 4B, TSK HW-75F Fractogel (all trademarks) or any other similar gels having a molecular exclusion limit of roughly 5,000 to 40,000,000 daltons.
  • Cleared cell lysate is loaded onto a presaturated column. The membrane vesicles appear in the void volume while other proteins, cell debris and detergent are eluted in larger volumes.
  • TM Sephadex
  • the membrane vesicles appear in the void volume. Other proteins and cell products elute at higher volumes.
  • the product can be repeatedly passed over the column but each passage causes at least a twofold dilution of the sample.
  • the product can be (if necessary) concentrated by ultra-filtration or by centrifugation.
  • TM gel can be autoclaved at 15 lb/in 2 on liquid cycle for 15 minutes. The gel material is allowed to cool to room temperature, is poured into a microcleaned endotoxin-free column and packed at a flow rate of 8-30 ml/hr. The effluent of the column can be checked for endotoxin contamination by the well known LAL assay.
  • a suitable buffering system for isolation of the membrane vesicle and ribosomal fractions of the invention is comprised of 20 mM MgSO 4 , 50 mM NH 4 CI, and 20 mM Tris HCl, pH 7.6; and for final suspension, the above buffer or Tris or phosphate buffered isotonic saline of the same pH may be used.
  • the magnesium sulphate may be replaced by any other suitable source of magnesium ions, such as magnesium acetate.
  • the components of the buffering system and their specific concentrations may be varied as long as the integrity of the membrane vesicle and ribosomal fraction isolated via the described procedure is maintained.
  • Tris-HCl may be replaced by Trizma 7.1, Trizma 7.2 or any other suitable Tris buffering agent adjusted to pH 7.0 to 7.6. Any buffering system/agent that does not alter the integrity of the membrane vesicles or ribosomes and which is acceptably tolerated by tissues and the intact organism at the concentration employed may be utilized. The actual pH of the buffering system must be compatible with the maintenance of the vesicles and ribosomes, and of the tissues into which the material is injected.
  • the cells are lysed in such a way as to cause fracture and shearing of the cell, so as to produce the membrane vesicles.
  • Any suitable technique may be employed which will produce the desired membrane vesicles. It has been found preferable to effect the lysis mechanically, for example, in a cell disrupter or a French pressure cell.
  • the mechanical lysis conducted is operated so as to provide enough shear to produce the membrane vesicles and associated ribosomes.
  • Microfluidizer 110 Model HOT from Biotechnology Development Corporation.
  • Microfluidization is the dynamic interaction of two bacterial fluid streams in precisely defined microchannels resulting in the lysing of the bacteria and in the production of uniformly sized membrane vesicles.
  • the bacterial suspension is pumped through the interaction chamber of the microfluidizer at 10,000 to 14,000 psi 6 to 12 times to ensure lysis of the cells and the proper vesicle size range.
  • Optimal conditions are 11,000 psi and nine passes.
  • a suitable cell concentration for microfluidization (vol. cell: vol. cell + vol. buffer) is 0.16.
  • the operating pressure of the cell should provide a high degree of cellular disruption such that the desired membrane vesicles are formed.
  • a preferred pressure is about 12,000 psi, but pressures in the range of about 10,000 psi to about 35,000 psi are also satisfactory.
  • a satisfactory French pressure cell is model no. J43339, 40,000 psi rating available from S.L.M. Instruments in Champagne, Illinois.
  • An automatic hydraulic press is used to pressurize the cell, preferably at a nominal level of 12,000 psi. The pressure is not allowed to oscillate more than about ⁇ 500 psi. Pressures below 12,000 psi (cell pressure) result in substantially less lysis of the cells and in progressively more of the vesicles being below about
  • the outlet valve is opened to allow an outflow of lysate at a rate of about 20 ml per minute. However, an outflow as low as 1.0 ml/minute is also acceptable (range 1-40 ml/minute). Flow rate must be such as to provide vesicles of the specified size range. Suitable cell concentration range for passage in the French pressure cell is (vol cells: vol cells + vol buffer) 0.16 to 0.32.
  • a detergent should be utilized to dissociate endotoxins and membrane fragments to smaller, more easily separable particles.
  • a particularly suitable nontoxic detergent for use in the cell lysis procedure is sodium deoxycholate.
  • a suitable (deoxycholate) concentration range is 0.15-0.3% final.
  • a non-toxic (well tolerated) detergent should be utilized.
  • the product of the invention is substantially free of cell walls; biologically active endotoxin as indicated by various biological assays and human toxicity studies; and cell membrane fragments. The product is also free of intact cells. In order to accomplish this, two centrifugation steps are preferably employed. The first centrifugation is as follows:
  • Centrifugation times resulting in lysate average S values below 600 result in significant product loss.
  • Average S values significantly above 600 e.g. 900S
  • the higher average S values will provide increased product yield.
  • a usable safe range is about 600-800 S (average).
  • This procedure removes intact viable and dead cells, and all large cell fragments and subcellular components including large polysome aggregates having an average S value greater than 600.
  • the centrifugation procedure is carried out at 0-4oC.
  • the brake is released at 500-1000 rpm and the rotor allowed to come to a complete stop. To prevent swirling of tube contents the brake should not be released below 500 rpm. Release of the brake at rpm's higher than 1000 only prolongs the production process.
  • Other rotors and centrifuges may be utilized (e.g. Sorvall Superspeed and an SS-34 rotor, Beckman Superspeed centrifuges and equivalent or larger capacity rotors), as long as the methodology is equivalent.
  • the lysate is then immediately layered on sucrose gradients in microcleaned, sterile, cold, polycarbonate centrifuge bottles.
  • the diluent used is the buffering system described above.
  • the layered gradients are then subjected to the second centrifugation step.
  • Sample Size 200 ml cleared/filtered lysate.
  • Centrifugation 0-4oC; slow acceleration, 10,000 rpm; 23 hours at speed, brake off at 500 rpm during deceleration.
  • the second centrifugation procedure allows the isolation, via pelleting, of the specifically sized membrane vesicle fraction and the residual ribosome fraction, leaving smaller fractions such as DNA fragments, RNA fragments, protein fragments, cell wall fragments, and membrane fragments, in the upper portions of the discontinuous gradient.
  • the specific centrifugation time in these rotors is such that the final product is not significantly contaminated by unwanted cellular components.
  • the following wash procedure can be used: For example, for the #30 Beckman rotor: a. The pellets (from the #30 Beckman rotor or equivalent rotor, see above) are resuspended with 10 ml of suitable buffer per 12 tubes. The resuspended pellets are transferred to a sterile, non-pyrogenic microclean container. The centrifuge tubes are then rinsed with 2 ml of suitable buffer per 10 tubes. The rinse is then combined with the resuspended pellets in the appropriate container. The solution is mixed by repeatedly pipetting (10 times with a 10 ml syringe) or by swirling for a few seconds. b.
  • the pelleted fraction isolated in accordance with the foregoing described procedure is washed and then suspended in one of the suitable suspending liquids or buffers described above and filter sterilized with a suitable 0.45 or 0.22 micron filter.
  • the suspending liquid may be of any appropriate pharmaceutical or preferably reagent quality devoid of fractions which might contribute to the precipitation, degradation, aggregation or functional inhibition of the suspended fraction.
  • Micrograms Nucleic Acid A 260 /E 260
  • a 0.05 ml sample of the resuspended product concentrate is diluted in the appropriate resuspending buffer so that the A 260 is between 0.4 and 0.5 for standardization purposes.
  • the absorbances at 280 nM, 260 nM, 225 nM and 215 nM are then determined using the suspending buffer as the blank.
  • the product is diluted to a final concentration of 1.0 mg nucleic acid per 0.5 ml buffer.
  • Average product yield using centrifugation is 1.0-1.2 mg per 1.0 ml cleared lysate.
  • the A 260 /A 280 absorbance ratio mean is 1.7626 with a standard deviation of 0.0626.
  • the mean E 260 is 0.0232 with a standard deviatipn of 0.0007.
  • the preparation of the invention is substantially free of biologically active endotoxins and cell wall fractions, clearly reducing toxicity problems.
  • the invention does not induce cutaneous necrosis/ulceration or death in four day old mice as shown below in Table 1.
  • four-day-old C57B1/6 mice were injected with the invention in the nuchal (neck) region and observed for the development of necrosis and/or death.
  • these animals demonstrate normal weight gain, another indicator of the absence of toxic contaminants.
  • Mean weights for 100 ⁇ g group at to and 24 hours post-injection were 1.72 g and 2.94 g respectively.
  • Mean weights for 200 ⁇ g group at t 0 , 24 hours and 6 days post-injection were 1.57 g, 2.16 g and 4.5 g respectively.
  • mice aDeaths per total number of mice treated following i.p. challenge with 0.5 mg histamine diphosphate (Sigma) 90 minutes after administration.
  • Endotoxin 0.5 micrograms administered i.p. results in 50% death following histamine challenge 7 .
  • Mice, rats and guinea pigs typically demonstrate hypothermic responses following the administration of endotoxin or infectious challenge whereas they develop hyperthermia (1-2°C) following the administration of the invention.
  • mice, rats, quinea pigs and humans have not resulted in anergy, pruritis,
  • DIC dissected intravascular coagulation
  • adjuvant arthritis anaphylaxis (hypersensitivity)
  • elevation of hepatic enzymes or necrosis or ulceration of local injection sites, over a dosage range of 5 micrograms to 10 milligrams (administered one to three times weekly in human subjects).
  • the maximum total dose in one week has not exceeded 12 milligrams.
  • Bilateral hemorrhagic necrosis of the kidneys has not occurred when administered intravenously to rabbits.
  • the membrane vesicles in the product of the invention appear to be readily endocytosed by monocytes and macrophages as observed with phase-contrast microscopy. It is known that endocyto ⁇ is results in cell membrane turnover and that membrane cycling in the monocyte-macrophage cell line activates these cells.
  • the cells of the monocyte-macrophage cell line are artificially broken up into several functional categories with each representing a progressively more differentiated cell. These categories (terminology may vary) are: monocyte, normal or resident macrophage, stimulated macrophage, activated non-tumorcidal macrophage, and activated tumorcidal macrophage. The more differentiated the cell, the less refractory it is to various types of stimulation but the more restricted it may potentially become in effector functions. Conversely, in advanced disease and chronic disease states, the monocyte-macrophage cell line becomes progressively more refractory to stimulation as does the immune system in general. Of these stages, only the activated tumorcidal macrophage has been demonstrated to be directly cytotoxic to tumor cells.
  • monocyte-macrophage product endocytosis has been noted in connection with murine cells.
  • Normal monocytes and resident macrophages were harvested from healthy adult C57B1 mice, without any prior manipulative procedures and without heparin, from the peritoneal cavity.
  • the initial adherent cell population consisted of over 90% rounded cells with the remaining having the typical morphology of macrophages.
  • Round cell populations consisted of monocytes and large numbers of cells having the appearance of medium sized lymphocytes. All cells endocytosed product and were therefore members of the monocyte-macrophage cell line.
  • B16 melanoma and kidney epithelial cells did not exhibit phagocytic activity in the presence of the invention or in the presence of endotoxin or BCG cell wall products.
  • a receptor specific for a component of the vesicles and/or ribosomes of the invention exists which would account for the apparent rapid internalization of the invention by the monocytes-resident macrophages.
  • Other explanations involving nonspecific physical and/or chemical interactions are also possible.
  • Table 3 summarizes the results of a monocyte-resident macrophage cytotoxicity study wherein the invention was administered in vitro, using concentrations of 0 to 100 micrograms at 5 microgram intervals to 25 cm 2 flasks containing 10 5 B16 melanoma cells as targets and mouse peritoneal monocytes/resident macrophages as effector cells.
  • the effector to target ratios are indicated in the left-hand column.
  • the cells were fixed in 95% ethanol and stained with Mayer's hematoxylin 96 hours after initiation of the assay.
  • Those results indicated by a plus sign mean that macroscopic growth was readily visible at 80%-100% confluence. A negative sign indicates that no visible macroscopic growth was present with a significantly reduced or absent target cell population confirmed microscopically.
  • Table 4 indicates the WBC and neutrophil levels for a series of terminally ill human patients taken prior to and subsequent to administration of the invention. Dosage levels are indicated. The product was administered once a week for three weeks. It may be seen from the examples in Table 4 that the specified treatment using the invention significantly increases the white blood cell count and neutrophils. It is noted that the alteration of white blood count does not necessarily increase for all dosages nor for all patients. (It does not decrease.) However, certain patients, as shown in Table 4, respond to administration of the invention by significant increase in white blood cell count and neutrophils.
  • the product of the invention therefore does not have a cytostatic effect on bone marrow.
  • Proper immune function requires the coordinate cooperation of several cell types and the sequential production and release of numerous cellular products. It is known that the cells of the monocyte-macrophage cell line are central in this process in that optimal antibody responses (B-cell function), cell mediated immunity (T-cell function), and possibly natural killer (NK) cell activity, do not occur in their absence.
  • B-cell function antibody responses
  • T-cell function cell mediated immunity
  • NK natural killer
  • an agent selected to potentiate/modify immune function should augment any ongoing responses or in the absence of responses, lower the system response threshold thus allowing recognition, activation and selection of the appropriate effector functions.
  • the ideal activating agent should not induce immune deviating, anergic or aberrant hypersensitivity responses directed to itself or cross-reacting entities, or promote or direct the selection of a specific singular effector pathway.
  • the product of the invention fulfills these requirements as none of these phenomena have been observed at significant levels during clinical trials.
  • FIGURE 2 illustrates the ability of the biologic response modifier of the invention to stimulate human natural killer cells in a manner comparable to that of leukocyte interferon.
  • the in vitro assay illustrates the degree of lysis of target cells (measured in terms of the release of radioactive chromium from target cells) by action of human natural killer cells for various dosage levels of the invention and interferon, shown in the respective graphs.
  • the dosage level codes are given below each graph, with the zero dosage level representing the background or leakage level of chromium release from target cells.
  • Control release is the counts per minute (CPM) of radioactivity in the presence of product and effector plus target cells, and control release is counts per minute obtained with the use of effector plus target cells only. Maximum release is obtained by incubating an aliquot of target cells in saponin, a detergent.
  • the graph in FIGURE 3 depicts, on the ordinate, the log number of Listeria monocytogenes in mouse peritoneal cells.
  • the abscissa represents time, in hours.
  • Listeria monocytogenes is a bacterium which causes an acute meningitis in humans with or without associated septicemia.
  • the assay depicted in FIGURE 3 is a standard assay for bacteriocidal activity.
  • the control is proteose peptone which is known to elicit bacteriocidal macrophages but which is not tolerated by humans.
  • the assay is described in Cruprinski, C.J., Henson, P.M., and Campbell, P.A., 1984, J. Leukocyte Biol. 35:193.
  • the lines on the graph indicate the change in the number of bacteria in culture over three hours when they are exposed to cells harvested from the peritoneal cavities of mice at varying times and for several different preconditioning steps as follows:
  • FIGURE 4 The ability of the biologic response modifier of the invention to modulate the activity of antibody dependent cellular cytotoxicity (ADCC) is shown in FIGURE 4. Procedures followed have been described in the literature. 10 / 11 / in this assay, human peripheral blood monocytes are used as the effector cells at the specified effector-target ratios. Target cells were chromium labeled murine YAC cells and the antibody utilized was rabbit anti-mouse cells.
  • ADCC antibody dependent cellular cytotoxicity
  • the killing level at least for relatively high effector to target cell ratios, is higher for the product of the invention than for interferon at product dosage levels of 15 and under micrograms per milliliter, but appeared to be in the background level at 20 micrograms per milliliter (a concentration which results in autophagocytic death of the monocyte population in this assay).
  • FIGURE 5 The ability of the invention to stimulate the release of interleukin 1 (IL-1) is illustrated in FIGURE 5. Assay procedures followed have been described in the literature. 12/ 11/ Peripheral blood mononuclear cells were prepared from blood drawn from human subjects to be tested. The cells were incubated in various concentrations of the product of the invention. After a specified incubation period aliquots of the culture medium were harvested and tested in a blastogenesis assay at various dilutions. Blastogenesis was determined by the counts per minute of radioactive (tritiated) thymidine taken up by 10 6 murine thymocytes following 72 hours incubation in the harvested culture supernatants. In FIGURE 5, the background level is indicated by the solid circles.
  • FIGURE 6 Human peripheral blood mononuclear cells were used for the effector population at the effector-target ratios specified. It may be seen that, without the invention or in the absence of natural killer cells, the background or leakage level is below 20% chromium release. With a non-depleted cell population, administration of the invention results in clear stimulation of natural killer cell cytotoxicity.
  • FIGURE 7 a graph of the results of an in vivo study on rat prostatic squamous cell carcinoma (rapid growth tumors) is illustrated. It may be seen that the administration of the invention induced the regression of large, established, rapidly growing carcinomas in rats. All non-treated tumor bearing controls had expired within a 14 month period whereas all treated animals were alive and 4 out of 6 animals totally regressed their tumors. In conducting these studies, it was discovered that a 1 milligram weekly paralesional injection was effective whereas a 2 milligram weekly injection was not.
  • the specific carcinoma treated was the Dunning tumor subline R3327A, a non-hormonally dependent, moderate-fast growth rate tumor which is metastatic in advanced disease.
  • the tumor host was Copenhagen X Fisher F 1 . This tumor system is a model for the national Prostatic Cancer Group. Implantation was in the left flank. Mean tumor volume at first treatment was 681 cubic millimeters.
  • FIGURE 8 a study of the invention in connection with slow growth rat prostatic carcinoma is illustrated.
  • the tumor hosts were Copenhagen X Fisher F 1 . This represents another tumor model for the National Prostatic Cancer Group. Implant was in the left flank. Treatment consisted of 2 milligram paralesional injections every seven days. The mean tumor volume at initiation of treatment was 1138.8 (> 2 cm 3 ) cubic millimeters.
  • the specific carcinoma was Dunning tumor subline R3327H, which is a well differentiated adenocarcinoma, demonstrating a very slow growth rate.
  • FIGURE 8 it may be noted that the invention induced stabilization/regression of large established slow growing carcinomas in rats. All control, non-treated, tumor bearing animals demonstrated progressive disease, death and weight loss whereas all treated animals showed disease stabilization, progressive slow regressions (indicated by pathology), normal weight and no deaths. In this case, whereas the 2 milligram weekly paralesional injection was effective, a 1.0 milligram weekly paralesional injection was not.
  • the above two tumor systems demonstrate the expected effective dosage variance with different tumors.
  • FIGURE 9 a study of bilateral rat prostatic adenocarcinoma tumors is shown. Comparisons to a control group are made with hosts receiving orchiectomy, orchiectomy plus dosage with the invention, steroid plus dosage with the invention, and dosage with the invention only. Comparisons are with left flank and right flank tumors.
  • FIGURE 9 it may be noted that the invention induced the regression of very large (total greater than 5 cubic centimeters) moderately fast growing adenocarcinomas; Treated animals received weekly 1 milligram paralesional injections at the left flank tumor site only.
  • the data demonstrate the systemic effect of therapy (contralateral untreated lesions regressing) utilizing the invention and lack of inhibition of the biologic response modifier effect by the steroid, dexamethasone. Dexamethasone has not been observed to inhibit the hyperthermic or tumor response in human tumor bearing patients.
  • In vivo human clinical studies have been conducted utilizing the invention pursuant to regulations and procedures for Phase I and Phase II testing promulgated by the Food and Drug Administration of the United States.
  • Phase I studies were for the purpose of determining the toxicity, if any, of the invention. Pursuant to Phase I tests, the patients studied were in an advanced state of disease, were considered terminal, and had failed all previous therapy. These patients received three or six treatments at each of the indicated dosage levels administered every 7 days subcutaneously at a site remote from the site of the tumor. The toxicity trials indicated no significant toxicity problems and that the product is well tolerated in humans.
  • the specific dosage level and treatment interval selected for therapy on a human patient will often vary from patient to patient.
  • the factors which influence dosage level and treatment interval include the patient's overall immune system response characteristics, the particular type and extent of disease, the overall health of the patient, the location of the disease, etc. This is essentially no different from other biologic response modifiers and the most beneficial dosage schedules may be determined in accordance with techniques used in connection with such other biologic response modifiers, and with pharmaceuticals generally.
  • the membrane vesicles and ribosomes used in the invention are readily biodegradable.
  • a particularly noteworthy advantage of the degradation phenomenon is that the particulate populations of the product of the invention stay intact in the biological system only for a short period of time sufficient to activate the immune reaction, and thereafter rapidly degrade. Hence, the development of severe, chronic pathophysiologic reactions, as a result of chronic immune activation, are avoided.
  • Degradation is due to the presence of various enzymes (e.g., RNases proteases, lipases) in various types of cells (e.g. monocytes, macrophages, neutrophiles) and tissue fluids (e.g. blood, lymph). This degradation is more rapid at elevated temperatures (e.g.
  • the product is degraded (in vitro) in less than about two minutes as determined by particle size analysis. The rate of degradation is dependent upon the enzyme concentration and temperature. Once degradation occurs, the efficacy of the composition as an immunomodulating agent is significantly lessened.
  • FIGURE 10 the human in vivo induction of killer cell cytotoxicity against three different tumor targets is depicted.
  • peripheral blood mononuclear cells will kill K562 tumor target cells in vitro to some extent.
  • peripheral blood mononuclear cells typically will not kill the Raji and Colon 38 tumor cell lines.
  • IL-2 interleukin II
  • FIGURE 10 is representative of the in vitro measurement of killer cell cytotoxicity in peripheral blood mononuclear cells obtained from patients 24 hours after treatment with the biologic response modifier of the invention. It may be seen that significant killing results against all three types of tumor targets. The data demonstrate significant in vivo activation of the patient's killer cells. Examination of the effector cell population via specific cell markers shows the presence of activated monocytes, natural killer cells and LAK cells.
  • peripheral blood mononuclear cells used were obtained from whole blood collected from patients 24 hours after injection with the biologic response modifier of the invention. Injections were subcutaneous and varied from one to four milligrams per dose, given 3 times per week. The assays were performed prior to and 24 hours after injection. The level of cytotoxicity obtained with in vivo activated cells compares favorably with the activity of in vitro lymphokine (IL-2) activated killer cells obtained after culturing human peripheral blood mononuclear cells with IL-2.
  • IL-2 in vitro lymphokine
  • Mycobacterium smegmatis, and Propionibacterium acnes were little better than a control group of the mice which received no treatment (tumor appearance and death within 60 days).
  • polysomes obtained from Serratia marcescens, at certain dosages demonstrated absence of tumor development in 60% or more of the animals and suppression in the development of tumors in the remaining animals. Because the biologic response modifier of the invention is obtained from a microorganism that is not a member of the microflora of the patient, is not associated with infectious disease in normal individuals, and because the microorganism's common bacterial antigen does not or is poorly cross-reactive with organisms making up the normal microflora. inappropriate immune responses are avoided.
  • Such responses include for example immune deviation and the phenomenon of original antigenic sin which is commonly observed with currently developed immunopotentiators (e.g. BCG, C. parvum, cell wall fractions).
  • immunopotentiators e.g. BCG, C. parvum, cell wall fractions.
  • polyribosome aggregates prepared from E. coli, M. smegmatis and Streptococcus pneumoniae are very poor sources for biological responses, whereas S. marcescens, which is not part of and does not readily crossreact with host microflora, is an excellent source of the biologic response modifier.
  • microorganisms which are suitable as a source for the membrane vesicles and ribosomes utilized in the invention.
  • the basic characteristics of such microorganisms must be that the microorganism is not a member of the microflora of the patient.
  • the microorganism's common bacterial antigen must not react or at least must be poorly cross-reactive with organisms making up the normal microflora. Thus, the organism should not be one which evokes an immune deviating response.
  • the organism must be one which does not or rarely causes disease in humans.
  • the source microorganism must be one in which the cellular membrane forms vesicles in the appropriate size range.
  • Suitable microorganism sources other than Serratia marcescens are: Erwinia chrysanthemi (Pectobacterium) ATCC 14092; and, to a lesser extent, Enterobacter aerogenes ATCC E13048.
  • FIGURE 11 shows that E. aerogenes demonstrates significantly greater effectiveness than Pseudomonas, E . coli, and E . cloacae, though less than alpha interferon at specific dosage levels.
  • FIGURE 12 compares E. chrysanthemi (effective) with fiavohacterium (not effective) and interferon.
  • Other microorganisms may be used as sources and processed as above to produce ribosomes and vesicles of the size specified. Using the previously described in vitro assays, their effectiveness may be readily assessed to determine whether or not they are useful as a biologic response modifier.
  • the biologic response modifier of the invention is free of viable cells, dead cells, cell wall, and biologically active endotoxin, recognition due to cross-reactivity is minimized, those components that are poorly degradable are eliminated, and those components known to be highly toxic (e.g., endotoxin) or which can result in chronic inflammatory situations e.g. adjuvant arthritis, granulomas, ulcerations, are minimized or eliminated.
  • highly toxic e.g., endotoxin
  • chronic inflammatory situations e.g. adjuvant arthritis, granulomas, ulcerations
  • the advantages of the biologic response modifier of the invention are numerous. By activating cells of the monocyte-macrophage cell line, the cellular cooperation necessary for optimal immune function is promoted. Activation of early differentiated cells of the monocyte-macrophage cell line (monocyte, normal/resident macrophage) promotes multiple effector functions.
  • the composition of the invention has thus far demonstrated induction of multiple types of effector functions dependent upon the condition of the assay or disease host parameters.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Modificateur de réponse biologique (BRM) essentiellement exempt d'endotoxine, de cellules intactes, de parois cellulaires et de fragments de membranes cellulaires, comprenant des vésicules de membrane naturelle et des ribosomes dans une solution tampon de suspension. Les vésicules de membrane comprennent une substance de membrane cellulaire endogène à un microorganisme sélectionné qui ne déclenche pas de réaction immunitaire déviante significative chez les patients et est essentiellement non pathogène pour l'homme. Les ribosomes sont également endogènes au microorganisme sélectionné, qui est du type où les vésicules de membrane peuvent être libérées de la substance de membrane cellulaire. Le BRM, auquel la lignée cellulaire monocyte-macrophage du patient fait aisément subir une endocytose, est produit en cultivant des cellules de souche Serratia marcescens, en recueillant les cellules cultivées, en dissociant l'endotoxine à l'aide d'un détergent approprié, en traitant le concentré cellulaire pour produire des ribosomes et des vésicules de membrane présentant des diamètres non inférieurs à 110 nm, en séparant les ribosomes et les vésicules de membrane et en les remettant en suspension dans une solution tampon appropriée, de sorte que le diamètre moyen des particules soit supérieur à 170 nm.
PCT/US1987/001397 1986-06-09 1987-06-08 Modificateur de reponse biologique WO1987007503A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
KR1019880700142A KR880701110A (ko) 1986-06-09 1987-06-08 생물학적 반응 모디파이어
AU77864/87A AU606075B2 (en) 1986-06-09 1987-06-08 Biologic response modifier
IN432/MAS/87A IN165076B (fr) 1986-06-09 1987-06-10
FI880412A FI880412A (fi) 1986-06-09 1988-01-29 Biologisk respons modifierande aemne.
NO880517A NO880517L (no) 1986-06-09 1988-02-05 Biologisk respons-modifiseringsmiddel.
DK066788A DK66788A (da) 1986-06-09 1988-02-09 Middel til modificering af biologiske reaktioner

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US87213186A 1986-06-09 1986-06-09
US872,131 1986-06-09
USNOTFURNISHED 2006-03-02

Publications (1)

Publication Number Publication Date
WO1987007503A1 true WO1987007503A1 (fr) 1987-12-17

Family

ID=25358910

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1987/001397 WO1987007503A1 (fr) 1986-06-09 1987-06-08 Modificateur de reponse biologique

Country Status (6)

Country Link
JP (1) JPH01500752A (fr)
CN (1) CN87104886A (fr)
DD (1) DD273198A5 (fr)
IL (1) IL82803A (fr)
PT (1) PT85057B (fr)
WO (1) WO1987007503A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989008499A1 (fr) * 1988-03-17 1989-09-21 Nippon Fine Chemical Co., Ltd. Liposome
WO1991004326A1 (fr) * 1989-09-19 1991-04-04 Cell Technology, Inc. Procede d'electrophorese libre pour isoler des ribosomes et des vesicules de membrane naturelle
WO1991012813A1 (fr) * 1990-03-02 1991-09-05 Catherine Anne Mccall Procede du traitement du cancer par l'utilisation d'une combinaison de chimiotherapie et d'un modificateur de la reponse biologique
EP2589391A2 (fr) * 2010-07-01 2013-05-08 Postech Academy-industry Foundation Méthode de diagnostic et de traitement du cancer par des microvésicules cellulaires

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103479597B (zh) * 2012-06-14 2015-02-18 苏州恒宇生物科技有限公司 一种葡萄来源活性成分—纳米级膜性囊泡的制备方法及其用途
CN109576180B (zh) * 2018-12-17 2021-11-26 北京利昂盛生物技术有限公司 一株赤红球菌及其作为免疫佐剂在制备疫苗中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4241046A (en) * 1978-11-30 1980-12-23 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4323561A (en) * 1977-09-06 1982-04-06 Temple University Of The Commonwealth System Of Higher Education Process of enhancing immmunogenic response in mammals by the administration of synthetic glycolipid adjuvants
JPS5849311A (ja) * 1981-09-18 1983-03-23 Eisai Co Ltd 安全なるリポソ−ムの製造法
US4563349A (en) * 1980-07-30 1986-01-07 Takeda Chemical Industries, Ltd. Superoxide dismutase, its immobilized form, and their production and use

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5428813A (en) * 1977-08-09 1979-03-03 Yuuichi Yamamura Solid preparation containing cell membrane extract substance used as suspension when using same
JPS5444601A (en) * 1977-09-12 1979-04-09 Dainippon Pharmaceut Co Ltd Water-soluble peptide glycan decomposition product
JPS607973B2 (ja) * 1977-11-26 1985-02-28 メルシャン株式会社 抗腫瘍性剤
JPS57139019A (en) * 1981-02-20 1982-08-27 Yuichi Yamamura Immunological adjuvant substance, its preparation and carcinostatic agent containing the same
JPS57200315A (en) * 1981-06-04 1982-12-08 Grelan Pharmaceut Co Ltd Antitumor substance
JPS59118712A (ja) * 1982-12-27 1984-07-09 Morinaga Milk Ind Co Ltd 抗腫瘍剤及びその製造法
JPS6153219A (ja) * 1984-08-23 1986-03-17 Tadashi Arai 抗腫瘍剤

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4323561A (en) * 1977-09-06 1982-04-06 Temple University Of The Commonwealth System Of Higher Education Process of enhancing immmunogenic response in mammals by the administration of synthetic glycolipid adjuvants
US4241046A (en) * 1978-11-30 1980-12-23 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4563349A (en) * 1980-07-30 1986-01-07 Takeda Chemical Industries, Ltd. Superoxide dismutase, its immobilized form, and their production and use
JPS5849311A (ja) * 1981-09-18 1983-03-23 Eisai Co Ltd 安全なるリポソ−ムの製造法

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989008499A1 (fr) * 1988-03-17 1989-09-21 Nippon Fine Chemical Co., Ltd. Liposome
WO1991004326A1 (fr) * 1989-09-19 1991-04-04 Cell Technology, Inc. Procede d'electrophorese libre pour isoler des ribosomes et des vesicules de membrane naturelle
WO1991012813A1 (fr) * 1990-03-02 1991-09-05 Catherine Anne Mccall Procede du traitement du cancer par l'utilisation d'une combinaison de chimiotherapie et d'un modificateur de la reponse biologique
EP2589391A2 (fr) * 2010-07-01 2013-05-08 Postech Academy-industry Foundation Méthode de diagnostic et de traitement du cancer par des microvésicules cellulaires
EP2589391A4 (fr) * 2010-07-01 2014-03-05 Postech Acad Ind Found Méthode de diagnostic et de traitement du cancer par des microvésicules cellulaires
US9066971B2 (en) 2010-07-01 2015-06-30 Postech Academy-Industry Foundation Method for treating and diagnosing cancer by using cell-derived microvesicles
KR101752506B1 (ko) * 2010-07-01 2017-06-30 포항공과대학교 산학협력단 세균유래 마이크로베시클을 이용한 암치료 및 암진단 방법

Also Published As

Publication number Publication date
CN87104886A (zh) 1988-04-20
JPH01500752A (ja) 1989-03-16
PT85057B (pt) 1990-03-30
IL82803A0 (en) 1987-12-20
DD273198A5 (de) 1989-11-08
IL82803A (en) 1992-06-21
PT85057A (en) 1987-07-01

Similar Documents

Publication Publication Date Title
Czuprynski et al. Killing of Listeria monocytogenes by inflammatory neutrophils and mononuclear phagocytes from immune and nonimmune mice
Williams et al. The role of the O and K antigens in determining the resistance of Klebsiella aerogenes to serum killing and phagocytosis
Levitz et al. Growth inhibition of Cryptococcus neoformans by cultured human monocytes: role of the capsule, opsonins, the culture surface, and cytokines
JPH08500623A (ja) 新規グルカン調製物
AU691797B2 (en) Immunotherapeutic composition
US4971801A (en) Biologic response modifier
CN105524883B (zh) Capri细胞及其制备方法
Kobayashi et al. Characterization of the pyrogenicity of Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans
JP2006506399A (ja) バクテリアの染色体ｄｎａ破砕物と非毒性リポポリサッカライドを含む免疫強化及び調節組成物
WO1987007503A1 (fr) Modificateur de reponse biologique
Newman et al. Separation of serum bactericidal and opsonizing activities for Haemophilus influenzae type b
JP3995733B2 (ja) 免疫賦活組成物
JP2747293B2 (ja) 細菌感染に対する非特異的防御を刺激するための薬剤
US5093115A (en) Method of preparing activated killer monocytes for treating colorectal cancer
JP2017518373A (ja) スタフィロコッカス・アウレウス感染疾患の予防または治療用組成物
CN114364396B (zh) 铜绿假单胞菌疫苗在抗烧烫伤感染中的应用
GB2077101A (en) Antiallergic composition containing streptococci
Moura et al. Lipids from Mycobacterium leprae cell wall are endowed with an anti‐inflammatory property and inhibit macrophage function in vivo
AU780909B2 (en) Composition and method for inducing apoptosis in prostate cancer cells
Finlay-Jones et al. Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils.
Yamamura Immunological Responses to a Murine Mammary Adenocarcinoma: Cytolysis by Armed Monocyte and by Antibody-Dependent Cellular Cytotoxicity
US6794368B1 (en) Composition and method for inducing apoptosis in prostate cancer cells
Mikkelsen et al. Mechanisms of action of intravesical bacillus Calmette-Guerin for bladder cancer
DE69929781T2 (de) Anwendung von monocyte-abstammende zellen, antigene und antikörper zur optimalen induzierung einer immuntherapeutischen leistungsfähigkeit
US4877616A (en) Process for preparing xerosin II and xerosin III, improved biological response modifiers

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU DK FI JP KR NO SU

WWE Wipo information: entry into national phase

Ref document number: 880412

Country of ref document: FI