CN87104886A - Preparation method of biological response modifier - Google Patents

Abstract

A method for the preparation of biological response modifiers for the treatment of human and animal diseases is described. The products of the method include native membrane vesicles and ribosomes present in the suspension buffer. Membranous vesicles are composed of cell membrane material endogenous to selected microorganisms. Ribosomes are also endogenous to selected microorganisms. The biological response modifier is substantially free of endotoxin, intact cells, cell wall and cell membrane fragments. The selected microorganism does not cause significant immune bias in the patient and is an organism that is substantially non-pathogenic to humans. Membranous vesicles in selected microorganisms were formed from cell membrane material and were readily endocytosed by the patient's monocyte-macrophage cell line.

Description

Preparation method of biological reaction modifying agent

This application is a continuation of U.S. patent application 872, 131 filed on 9.6.1986 and is not in superimposition therewith.

The present invention relates generally to a process for the preparation of a pharmaceutical product having immunomodulatory properties, and in particular to a process for the preparation of a Biological Response Modifier (BRM), defined as: agents that alter the biological response of the host to disease and alter the relationship between disease and host with their resulting therapeutic effect. BRMs can be divided into two categories: (1) biological or chemical agents that stimulate or alter one or more host resistance mechanisms, and (2) purified cell products that have been shown to act directly on specific diseases. The present invention belongs to a first class of BRMs which comprise agents capable of activating, increasing or otherwise altering the immunological reactivity of a host, generally referred to collectively as immunomodulators.

BRMs may be used alone or in combination with other agents to enhance resistance to or to restore from pathogen attack, to alter or induce tolerance to foreign tissue transplants, to enhance tumor rejection or stabilization and to inhibit tumor recurrence after additional treatment, to restore normal helper-suppressor cell mechanisms, or to promote normal immune responses.

Since 1900, a wide variety of immunomodulators/immunoadjuvants have been developed. Most of these agents are of microbial (bacterial/fungal) origin and most commonly are obtained from corynebacteria, mycobacteria and nocardia (CMN organisms). Various immunological adjuvants include intact viable cells, dead bacteria, cell walls, various cell wall fragments, endotoxins, various types of polysaccharides or subcellular fragments such as ribonucleic acids or ribosomes. While all of these formulations have demonstrated immunopotentiating/modulating/adjuvanting activity in tissue culture, in animals and humans against infectious and neoplastic diseases, they are not readily universally applicable and may be severely toxic due to their inconsistent production and composition. These agents are not very useful in treating established diseases relative to treating neoplasias. Furthermore, these agents have also been shown to lose activity (no cellular immunoreactivity), develop immune bias, hypersensitivity reactions, cause chronic inflammatory conditions and/or other various adverse conditions upon repeated use.

These formulations cannot be chemically defined due to their lack of purity and/or complexity, and in an attempt to reduce immune response and toxic complications, many researchers have either extracted various specific parts from these sources, or synthesized a variety of components that have been shown to be immunologically active. Examples of specific moieties that have been investigated include cell wall moiety de-muramyl tripeptides, staphylococcal protein A, various polysaccharides, RNA moieties^（1）And ribosomal moiety^（2-5）。

In the case of the ribosome moiety, the mechanism of action differs depending on the origin of the ribosome. Although most ribosomal vaccines require active adjuvants, ribosomes prepared from Staphylococcus aureus and Diplococcus meningitidis (Neisseria meningitidis) are not required. In addition, the ribosomal vaccines prepared from Mycobacterium tuberculosis (Mycobacterium tuberculosis) and Salmonella typhi (Salmonella typhimurium) induce cell-mediated immune responses, while the ribosomal vaccines prepared from Streptococcus pneumoniae and Streptococcus pyogenes (Streptococcus pyogenes) induce humoral immune responses^（5）. Although it is theoretically possible to assume that the extracted ribosomal moiety is an active agent, there are still many controversies in many cases as to whether other cellular components (RNA, proteins, endotoxins, cell walls) present as contaminants cause the immunoreactivity issues observed.

Known cellular vaccines may sometimes be suitable for the prevention or treatment of infectious diseases. However, for several reasons they are not suitable for use as unsensitised, generally universal immunomodulators for the treatment of malignancies. The presence of cell walls, endotoxins or non-degradable components often causes toxicity similar to that caused by the whole organism. Furthermore, because such vaccines are typically derived from organisms that are themselves part of, or readily cross-reactive with (co-antigens) the host's own microbial system, they can elicit unfavorable immune bias (poor) and complex immune responses. Such vaccines also contain certain moieties that may have less than desirable physical and chemical properties for overall immunopotentiating activity.

Polyribosomes from specific bacterial organisms have been reported by urban et al to have the ability to inhibit the development of SaD2 fibrosarcoma in the skin of DBA/2 mice. The polysome fraction is prepared by varying the osmotic pressure or by mechanical lysis of the cells (depending on the type of bacteria) followed by differential centrifugation. Although positive for the effects of the tumors of the SaD2 mouse, the mechanism of the induced biological response could not be determined based on this data. Although very toxic, the method described by Urban et al results in very variable and low yield profiles of multiple ribosomes and also does not achieve consistency in quality, stability and utility.

Kirsh et al^（1）The immunostimulatory and modulatory effects are reported to be exerted by encapsulating specific antigens or biological response modifiers in liposomes. Liposomes containing drugs have been utilized to treat metastatic cancer^（8）. However, the use of biological response modifiers, either alone or encapsulated within liposomes, to treat malignant diseases (cancer) is largely disappointing. Although encapsulated BRMs are superior in efficacy to unencapsulated BRMs, the therapeutic effect is limited due to limited immune activation of macrophages. Tumor cells develop little, if any, resistance to macrophage killing compared to natural killer cells and cytotoxic T cells. In these cases, the destruction of large tumor dissemination, effectively mediated by macrophages themselves, appears to be too small in number.

It is an object of the present invention to provide a method for preparing improved biological response modifiers.

It is another object of the present invention to provide a method for preparing an improved biological response modifier which exhibits minimal toxicity and immune bias.

It is a further object of the present invention to provide a method for preparing an improved biological response modifier that is stable in quality, stability and utility.

It is still another object of the present invention to provide a method for preparing the aforementioned bioreaction modifier with constant quality and high yield;

other objects of the present invention will become apparent to those skilled in the art from the following description and by reference to the accompanying drawings.

FIG. 1 is an electron micrograph of a bioreaction modifier prepared according to the method of the present invention, the figure being magnified approximately 82,000 times;

FIG. 2 shows the results of an analysis of human natural killer cells after administration of a biological response modifier prepared according to the method of the present invention and administration of leukocyte interferon.

FIG. 3 depicts a standard analytical method demonstrating the bactericidal properties of BRMs prepared in accordance with the method of the present invention;

FIG. 4 is a graph comparing the results of an antibody-dependent cellular cytotoxicity (ADCC) assay following administration of a biological response modifier made according to the methods of the present invention and administration of interferon-leukocyte;

FIG. 5 is a diagram illustrating the induction of Leukopoietin 1 (IL-1) by a biological response modifier made according to the method of the present invention;

FIG. 6 illustrates the effect of various human peripheral blood mononuclear cell populations on Natural Killer (NK) cell activity (this study demonstrates the loss of NK cell activity after removal of NK cells without removal of B cells or T cells when using the biological response modifiers of the present invention.) removal of monocytes (not shown) also eliminates this effect, suggesting that NK cell activity is mediated by monocyte-macrophage populations);

FIG. 7 shows the results of in vivo treatment (rapid growth) of rat prostate squamous cell carcinoma (R3327A) using the method of the present invention;

figure 8 shows the results of an in vivo (slow growth) study of treatment of rat prostate cancer (R3327H);

figure 9 illustrates the results of an in vivo therapeutic study on rat bilateral prostate adenocarcinoma (R3327 CF) showing the results applied in combination with other therapeutic approaches.

FIG. 10 is a summary of data obtained for treatment of patients with advanced cancer illustrating the efficacy of peripheral blood mononuclear cells of the patients to kill specific tumor target cells 24 hours after in vivo administration of a biological response modifier made according to the methods of the invention. This result is comparable to that obtained by in vivo activation of human peripheral blood mononuclear cells with interleukin II.

FIG. 11 shows the results of a series of assays for natural killer cell activity. In which the efficacy of interferon-alpha and preparations derived from microorganisms of the genus Pseudomonas, Enterobacter aerogenes, and Enterobacter aeruginosa (E.chloroacae) were compared, respectively.

FIG. 12 shows the results of a series of natural killer cell activity assays. The efficacy of interferon was compared to formulations obtained from Erwinia chrysanthemi and Flavobacterium.

In general, the biological response modifiers made by the methods of the invention include natural membrane vesicles and ribosomes in a suspension buffer. Vesicles are composed of cell membrane material that is endogenous to a selected organism. Ribosomes are also endogenous to the organism of choice. The biological response modifier is substantially free of endotoxin, intact cells, cell walls and cell membrane fragments. The selected organism cannot cause obvious immune deviation reaction (immune de-viral response), is not pathogenic to human body, and the membrane vesicle in the selected organism can be formed by cell membrane materials and is easily endocytosed by a monocyte/macrophage system. The average diameter of the biological response modifier was greater than 170nm as shown by analysis of particle size.

The biological response modifiers prepared according to the methods of the invention are capable of directly activating cells of the monocyte-macrophage lineage. Activation of cells of the monocyte-macrophage or monocyte-macrophage lineage induces monocyte-macrophage mediated bactericidal activity (fig. 3) and tumor cytotoxicity (table 3), changes in the levels of various white blood cells involved in immune function (e.g., monocytes, neutrophils (table 4)), induces tumor target cell (K562, Raji, Co 38) cytotoxicity (fig. 10) in humans, induces natural killer cell mediated cytotoxicity (fig. 2), induces antibody dependent cellular cytotoxicity (fig. 4), induces interleukin I (IL-I) (fig. 5), and possibly also the release of interleukin ii (IL-ii) (fig. 10).

For purposes of clearly illustrating the invention, the terms used in this specification and the appended claims are defined as follows:

by "non-toxic" is meant that toxicity is limited to levels that are tolerated by a mammal receiving treatment with a biological response modifier.

By "non-immunogenic" is meant a sufficiently low or no immunogenic response in a mammalian host such that no undesirable immune bias, chronic inflammatory and hypersensitivity reactions occur.

"mean diameter" refers to the mean diameter measured by MSD particle size distribution analysis on a BI-90 (Brookhaven Lnstrument Corp.) particle size analyzer. The method includes weighting the intensity of the particle size averaging process, for a more detailed explanation see chapter 6 of the instruction manual for the instrument, which is incorporated by reference.

By "substantially non-pathogenic to the human body" is meant that no or little disease-related changes are caused to a normal, healthy person. Since most microorganisms are capable of causing opportunistic infections under appropriate conditions (e.g., in humans where the immune system has been compromised), this definition excludes organisms that normally cause non-opportunistic infections.

"immune bias response" refers to an immune response directed against a disease that deviates from that being treated. For example, when challenged with an antigen similar to that of a previous challenge or common microbial system, the phenomenon of Original antigen delinquent (Original antigenic sin) can cause the immune system to respond to the previous challenge or to the common microbial system. In addition, polyclonal activation can cause non-specific off-target recall responses (non-specific mis-directed and antisense responses). The poorly degradable particles can cause disturbances in the monocyte-macrophage cell line (i.e., response to foreign objects and chronic hypersensitivity) inhibiting the synergistic effect of the cell line in response to disease. By "significant" immune bias response is meant that the effect of the expected immune response is attenuated to a level that is unacceptable for medical purposes.

"native membrane vesicles" refers to membrane vesicles prepared from the membrane of living or dead native cells.

Although there is insufficient scientific evidence to clarify the reasons for the observed efficacy of the bioresponse modifiers of the invention, it is clear that the bioresponse modifiers of the invention have certain distinct characteristics. To this end, it is to be understood that the biological response modifiers of the invention include two distinct classes of particles, namely natural membrane vesicles and ribosomes. Ribosomes can exist as monomers or as large multimers, but the average diameter of the ribosome population is smaller than the average diameter of the membrane vesicle population. Based on the in vitro NK cell assay results, it appears that the relative amounts of the two populations may affect the efficacy of the product. Of course, the relevant population also affects the average diameter of the total population of particles. It is believed that an average diameter exceeding 160nm is an essential condition for achieving the intended effect. If the mean diameter is below this level, the efficacy of the product (according to NK cell assay) is significantly reduced.

It was also observed that the size of vesicles in the vesicle population also had an effect on the efficacy of the biological response modifier. Thus, preferred forms of the biological response modifiers of the invention are substantially all those having a diameter in excess of 110nm, and the average diameter of the vesicle population is at least 180nm, preferably about 210 nm. Formulations with diameters below the above values have also been observed to be less potent (according to in vitro NK cell analysis). The efficacy of the membrane vesicle only formulation and the ribosome only formulation has also been shown to be below expected levels, suggesting that there may be synergy between the two formulations.

Fig. 1 is an electron micrograph (magnified 82,000 times) showing the cross-section of membrane vesicles and showing ribosomes with various particle sizes (i.e., monomers and small multimers). The vesicles that have been made are measured by two methods: (1) direct measurement of Ring formation Cross section as seen in the Electron micrograph (2) use of BI₉₀A particle size analyzer of the type (Brookhaven Instrument Corp.) was used for light scattering analysis, and the scattering coefficient of the particles was determined by a mathematical calculation.

In a preferred form of the biological response modifier produced by the method of the invention, the membrane vesicles and associated ribosomes are derived from the gram-negative bacterium Serratia marcescens (Serratia marcescens). Serratia marcescens is a well-known microorganism and many strains are available from a number of sources. Sixty strains are available from the American type culture Collection (ATCC, Rockville, Maryland 20852). This organism is particularly suitable for use as a microbial source for the manufacture of products which exhibit a high level of immunomodulatory/immunotherapeutic activity and which are substantially non-toxic compared to other bacterial sources.

The particular serratia marcescens strains actually utilized to obtain the following data were:

serratia 2000 (SM 2000), a pigment-producing and non-pigment-producing variant of an internal strain of cell technology Inc. (Boulder, Colorado, USA);

serratia MSC, a strain of unknown origin obtained from a stock culture of Metropolitan State school (Denver, Colorado, USA);

serratia marcescens ATCC ^＃60, and

serratia marcescens CU, a strain of unknown origin obtained from storage cultures at the university of Colorado (Boulder, Colorado USA).

The desired bacterial membrane vesicles and ribosomes can be conveniently and economically isolated from Serratia marcescens bacterial cells in stationary or log phase by the method of the invention, a simple, rapid and reproducible process. The reagents used must be capable of providing the necessary conditions for maintaining the integrity and morphology of the particular fraction being separated. Any reagent which may itself be toxic (intolerable) or which in any way interferes with or modifies the immune response should be avoided.

Preferred isolation procedures include culturing seed cells of the bacteria in an appropriate medium at an appropriate temperature (30-40 ℃) to a logarithmic growth phase culture (a given growth phase associated with end product yield and consistent with the final density of viable cells per unit volume of medium), rapidly cooling the logarithmic growth phase culture to 0-4 ℃, (harvesting the bacterial cells), washing the harvested cells and suspending them in a suitable buffer system at the predicted cell density to maintain an environment suitable for membrane vesicle formation while favoring stability of membrane vesicles and ribosomes, (disrupting (lysing) the cells in an appropriate cell disruptor French press cell to produce membrane vesicles (cells can be disrupted in the presence of an appropriate detergent to favor endotoxin breakdown) having diameters in excess of about 110nm (or 0.11 micron), clarifying cell debris (including whole cell debris), Cell wall debris, large aggregates of ribosomes and polysomes), layering the clarified vesicle-containing cell lysate, retaining soluble cellular components, on a suitable linear or discontinuous density gradient material that is non-toxic (tolerable) and non-immunogenic, precipitating the applicable vesicle and ribosome fractions while precipitating as few as an unacceptably small fraction as possible, aseptically removing the density gradient material, washing the precipitation fraction, and thereafter resuspending the membrane vesicles and ribosomes in a suitable buffer system with minimal disruption.

In carrying out the above separation, the logarithmic growth phase culture may be rapidly cooled to 0-4 ℃ by any suitable method, for example, a dry ice-ethanol mixture, an acetone-ice mixture, an ethanol-ice mixture, or a special device such as a cooling coil may be used. All subsequent steps are preferably carried out at 0-4 ℃. Cells may be harvested by centrifugation or by using a cell harvester/concentrator. Both clarification of cell debris and separation of specific vesicle fractions can be performed by centrifugation.

Sieving exclusion chromatography (Size-exclusion chromatography-chromatography) can also be used to separate the membrane vesicles from the ribosomal fraction. Cell lysates were clarified by Sephadex G-25, G-50, G-100, G-200, Sepharose 2B, Sepha-crylS-200, Sephacryl-500 Biogel p-30, Sepha-rose 4B, TSK HW-75F Fractogel (all trade names above) or other similar gels having a molecular exclusion limit of about 5,000 to 40,000,000 daltons. The clarified cell lysate was loaded onto a presaturated column. Membrane vesicles appear in the outer water volume, while other proteins, cell debris and detergents are eluted in larger volumes. For example, 1ml of the cleared lysate is applied to a 10ml G-100Sephadex (TM) column and eluted with buffer. Membrane vesicles appear in the outer water volume, with other proteins and cellular products eluting at larger volumes. The product can be run through the column repeatedly, but each run dilutes the sample at least two-fold. If desired, the product may be concentrated by ultrafiltration or centrifugation.

The column and gel can be prepared in a sterile and endotoxin-free condition according to the methods specified by gel manufacturers. For example, a Sephadex (TM) gel may be autoclaved for 15 minutes at 15 psi based on liquid circulation. The gel material was allowed to cool to room temperature, poured into a microscopically clean endotoxin-free column and collected at a flow rate of 8-30 ml/hr. The column effluent can be checked for endotoxin contamination by the known LAL methodAnd (6) dyeing. Suitable buffer systems for isolating membrane vesicles and ribosomal moieties of biological response modifiers prepared according to the methods of the invention comprise 20mM MgSO₄、50mM NH₄Cl and20 mM Tris-HCl, pH7.6, and to prepare the final suspension, the above-mentioned buffer or Tris or phosphate buffered isotonic saline having the same pH can be used. Any other suitable source of magnesium ions, such as magnesium acetate, may be used in place of magnesium sulfate. The composition of the buffer system and its specific concentration can be varied as long as the integrity of the membrane vesicles and the ribosomal fraction isolated by the above steps is maintained. For example, Tris-HCl may be replaced by Trizma7.1, Trizma7.2, or any other suitable Tris buffer adjusted to pH7.0 to 7.6. Any buffer system/buffer that does not alter the integrity of the membrane vesicles or ribosomes and is tolerated by the tissue or intact organism at the concentrations used may be used. The true pH of the buffer system must be suitable for maintaining the vesicles and ribosomes and the tissue into which the material is injected.

The method for lysing cells is required to break the cells and shear the cells to produce membrane vesicles. Any suitable technique for obtaining membrane vesicles may be used. Among them, mechanical methods such as those in a cell disruptor or a French pressure cell are preferred. Sufficient shear force must be provided to generate membrane vesicles and associated ribosomes when mechanical lysis is performed.

One satisfactory method is to use a biotech Development Corp 110T-type microfluidizer. Microfluidization is the dynamic interaction of two bacterial streams in precisely defined microchannels to cause bacterial lysis and the production of uniform-sized membrane vesicles. In the range of 10,000 to 14,000 Psi²) The bacterial suspension was pumped under pressure into the interaction chamber of the microfluidizer and passed through the chamber 6 to 12 times to ensure cell lysis and to obtain membrane vesicles of the appropriate size range. The optimum conditions for this operation were a pressure of 11,000 Psi and nine passes. A suitable cell concentration for microfluidization (cell volume: cell volume + buffer volume) is 0.16.

When French pressure cells are used, the cells are broken down sufficiently to form the desired membrane vesicles. The preferred pressure is about 12,000 Psi, but satisfactory results are also obtained with pressures in the range of 10,000 Psi to 35,000 Psi.

One satisfactory French pressure cell is model J43339 available from s.l.m. instruments (Champagne, Illinois) at a pressure rating of 40,000 Psi. The cells can be crushed using an automatic hydraulic press, with a preferred standard pressure of 12,000 Psi. The pressure fluctuations must not exceed about ± 500 Psi. Pressures below 12,000 Psi (cell pressure) result in greatly reduced cell lysis and progressively more vesicles with diameters below about 110 nm. The outlet valve was opened to allow the lysate to flow out at a flow rate of about 20ml per minute, but the flow rate could be as low as 1.0 ml/min (ranging between 1-40 ml/min). The flow rate used must be such as to provide a range of membrane vesicles of a particular size. A suitable range of cell concentration (cell volume: cell volume + buffer volume) by French pressure cell is 0.16 to 0.32.

The detergent used should dissociate and render the membrane fraction into smaller, more easily separable particles. A particularly suitable non-toxic detergent for use in the cell lysis step is sodium deoxycholate. The final concentration of sodium deoxycholate is 0.15-0.3%. Non-toxic (i.e., fully tolerated) detergents should be used.

The products prepared according to the process of the present invention are substantially free of cell walls, free of biologically active endotoxins and free of membrane debris as shown by various biological assays and human toxicity studies. The product also has no intact cells. To achieve this, two centrifugation steps are preferably used. The first centrifugation was:

(a) micro-clean, sterile, with Backman at-0-1 ℃ during lysis operations^＃20ml of cold bacterial cell lysate was collected in a 30 rotor polycarbonate bottle. The number of such centrifuge bottles to be prepared will depend on the amount of final product to be manufactured. This volume of lysate represents a running,Perpendicular R_minIs 72mm, and effective R_maxIs 95 mm. In a Beckman ultracentrifuge, using normal acceleration and precooled (0-4 ℃) Beckman^＃The tubes were centrifuged at 16,000 rpm at 30 rotors. The centrifugation time is such that it is apparent from R_minAnd R_maxThe average S value between values is 600. If the centrifugation time results in a lysate with an average S value below 600, then a significant loss of product can result. If the average S value is much higher than 600 (e.g., 900S), there may be a risk of contamination of the final product with various toxic cellular components. Higher S values will increase product yield as long as no product contamination occurs. A safety range of about 600-. Using Beckman ^＃30 rotor, polycarbonate bottle and20 ml lysate can be obtained with 3.55X 10⁹A W²t, average 600S, clarified lysate (centrifugation at 16,000 rpm for 20 min +3 min to achieve normal acceleration). The method removes intact viable and dead cells, subcellular components including polysome aggregates with average S values greater than 600 and all large cell debris. The centrifugation is carried out at 0-4 ℃. The brake is released at 500-. To prevent the contents from spinning, the brake is not released below 500 rpm. Releasing the brake at rpm above 1000 only lengthens the production process. Other rotors and centrifuges (e.g., Sorvall Supersped centrifuge and SS-34 rotor, Beck-man Supersped centrifuge and equal or larger capacity rotors) may also be used, as long as the methodology is equivalent.

(b) All steps are carried out at 0-4 ℃. The clarified lysate (total volume 15-16 ml) was carefully aseptically harvested using a cold sterile, pyrogen-free syringe and 18G spinal needle (or equivalent). During harvesting, the tip of the needle is allowed to protrude a few millimeters below the surface of the lysate, taking special care not to touch the side wall of the centrifuge bottle and not to disturb the pellet mass. The harvested lysate is immediately passed through a cold 0.45 micron filter (e.g., Millex-HA, Millipore) and collected in a cold, sterile, pyrogen-free, preferably non-adhesive plastic/glass tube.

Immediately after the first centrifugation step and filtration, the lysate was layered on a sucrose gradient in a microscopically clean, sterile, cold polycarbonate centrifuge flask. The diluent used is the buffer system described above. The stratified gradient was then centrifuged a second time.

The product separation parameters were as follows:

(1) basal product yield (1-1.2 mg product/1.0 ml layered clarified lysate/gradient).

^＃50Beckman rotor:

gradient: in a polycarbonate centrifuge bottle, layering 4-5ml15% (W/W) sucrose on 3.0ml30% (W/W) sucrose-with a distinct interface. Sucrose was sterile and endotoxin negative as determined by quantitative Limulus assay.

Sample size: 1.0ml clarified/filtered lysate.

Centrifuging: slowly accelerating at 0-4 deg.C, and centrifuging at 38,000 rpm for 60 min.

(2) To increase the amount of product separated per centrifugation, the following rotors can be used:

^＃30Beckman rotor:

gradient: in polycarbonate bottles, 11.0ml of 15% (W/W) sucrose (based on the volume of lysate) was layered over 9.0ml of 30% (W/W) sucrose to form a distinct interface. Quantitative Limulus assay detection indicated that the sucrose used was sterile and endotoxin negative.

Sample size: 4-5ml clarified/filtered lysate.

Centrifuging: 0-4 deg.C, slow acceleration, 30,000 rpm, 120 min. The time for high speed centrifugation (+ or-) is adjusted to give maximum yield and minimum contamination, but this depends on the volume of the lysate of the layer. The brake is turned off at 1000rpm (not less than 500 rpm). See manufacturer recommended brake off times for a particular centrifuge/rotor system.

^＃14Beckman rotor:

gradient: 100ml of 25% (W/W) sucrose was added to the polycarbonate bottle. The sucrose used was sterile and was endotoxin negative as determined by the quantitative Limulus method.

Sample size: 100ml clarified/filtered lysate.

Centrifuging: 0-4 deg.C, slowly accelerating, 14,000 rpm, high-speed centrifuging for 12 hr, and cutting off brake at 500rpm during deceleration.

^＃10Beckman rotor:

gradient: 200ml of 25% (W/W) sucrose was filled in a polycarbonate bottle. The sucrose used was sterile and was endotoxin negative as determined by the quantitative Limulus method.

Sample size: 200ml clarified/filtered lysate.

Centrifuging: 0-4 deg.C, slowly accelerating, 10,000 rpm, high-speed centrifuging for 23 hr, and cutting off brake at 500rpm during deceleration.

The second centrifugation process allows the fraction of membrane vesicles of a specific size and the remaining ribosomal fraction to be separated by forming a pellet cake and leaving smaller fractions such as DNA fragments, RNA fragments, protein fragments, cell wall fragments and membrane fragments in the upper part of the discontinuous gradient. The specific centrifugation time in these rotors should be such that the final product is not significantly contaminated with unwanted cellular components. If the centrifugation time cannot be changed, the following washing procedure can be used in order to reduce the amount of the above-mentioned contaminants:

as for^＃30Beckman rotor:

a. resuspend pellet (cake) (from pellet) with 10ml of appropriate buffer (12 tubes each)^＃A 30Beckman rotor or equivalent rotor, see above). The resuspended settled cake is transferred to a sterile, pyrogen-free, microscopically clean container. After thatThe centrifuge tubes (10 tubes each) were rinsed with 2ml of the appropriate buffer and the eluate and resuspended pellet cake were combined in the appropriate container. The solution was mixed by repeated pipetting (10 times with a 10ml syringe) or by vortexing for a few seconds.

b. 5ml of the resuspended pellet cake is taken up in a sterile, pyrogen-free solution containing 15ml of an appropriate buffer^＃30-rotor polycarbonate centrifuge tubes.

c. The tubes were centrifuged for 25 to 40 minutes at normal acceleration and brake initiation.

Other types of centrifuges and rotors may be used, provided that they are methodically equivalent (e.g., a 50.2Ti Beckman rotor, with shorter run times when using the same magnitude of gradient). Instead of a discontinuous gradient, a suitable linear gradient may also be used.

The fraction separated according to the preceding procedure, which is a precipitate cake, is washed, then suspended in the above suitable suspension or buffer solution and sterile filtered through a filter using suitable 0.45 or 0.22 micron pores. The suspension liquid used may be any suitable pharmaceutical or agent of a quality to prevent precipitation, degradation, aggregation or functional inhibition of the suspended fraction.

Product quantification can be performed according to the nucleic acid content according to the following formula:

E260＝0.0373-0.0079（A260/A280）

microgram nucleic acid A260/E260

For standardization purposes, a 0.05ml sample of resuspended product concentrate can be diluted with the appropriate resuspension buffer so that its a260 is between 0.4 and 0.5. The absorbance at 280nm, 260nm, 225nm and 215nm was then determined using suspension buffer as a blank. Once the nucleic acid content has been determined, the product is diluted to a final concentration of 1.0mg nucleic acid per 0.5ml buffer. The protein content of the product can be calculated approximately using the following formula:

microgram protein/ML 144 (a 215-a 225).

The average product yield obtained using centrifugation is 1.0-1.2mg per 1.0mg of clarified lysate. The average of the absorbance ratio of A260/A280 was 1.7626, and the standard deviation was 0.0626. The mean value of E260 was 0.0232 and the standard deviation was 0.0007.

Products with vesicles of this size therefore have a high functional efficacy (see below), for reasons that are not fully understood at present. However, it is believed that the size of the membrane vesicles is specified, i.e. the average diameter is at least 180nm, and it is essential that the minimum diameter is substantially 110nm^〔15〕。

The biological reaction modifier product of the present invention has basically no biologically active endotoxin and cell wall part, and this can lower the toxicity of the product obviously. For example, the bioresponse modifier prepared according to the method of the invention does not cause skin necrosis/ulceration or death of the mice when used on four day mice (see Table 1 below). This study was conducted by injecting the bioreaction modifier of the invention into the neck of C57B1/6 mice on four days and observing the appearance of necrosis and/or death. In addition, observations indicate that these animals all have normal weight gain, which is another indication of non-toxic contamination.

TABLE 1

Skin necrosis test

Necrosis/death-

Animal number biological response modifier dose 18 hours 24 hours 48 hours 96 hours

4 10μg 0/0 0/0 0/0 0/0

7 100μg 0/0 0/0 0/0 0/0

6 200μg 0/0 0/0 0/0 0/0

The average body weights of the 100. mu.g experimental group at 0 hour and 24 hours after injection were 1.72g and 2.94g, respectively. The average body weights of the 200. mu.g group at 0 hour, 24 hours and 6 days after injection were 1.57g, 2.16g and 4.5g, respectively.

Histamine hypersensitivity tests in CFW mice at 8 weeks also demonstrated no toxicity due to endotoxin.

TABLE 2

Histamine hypersensitivity assay

A. Endotoxin dose (. mu.g) administration route D/T^a

Coli (phenol, Sigma) intraperitoneal injection (i.P.) 4/6

0.5

Escherichia coli (TCA, Sigma) i.P. 3/6

0.5

B. Dosage of bioreaction modifier (mug)

100 i.P. 0/6

200 i.P. 0/6

600 i.P. 0/6

2000 i.P. 0/7

a: number of deaths in each group of mice 90 minutes after challenge with 0.5mg histamine diphosphate (Sigma) by intraperitoneal injection. Animals given endotoxin (0.5 μ g, i.P.) resulted in 50% death following challenge with histamine^（1）. Mice, rats and guinea pigs exhibited a low temperature response after endotoxin or infectious challenge, and an increase in body temperature (1-2 ℃) after administration of the bioresponse modifiers of the invention.

Further, mice, rats, guinea pigs and humans (one or more subjects) were injected once or repeatedly (at a dose of 5. mu.g to 10 mg, one to three times per week in human trials) without causing weakness, itching, DIC (disseminated intravascular coagulation), arthritis, anaphylaxis (hypersensitivity), elevated liver enzymes and localized necrosis or ulceration at the injection site. The maximum total dose per week does not exceed 12 mg. When the rabbits were given intravenous injections, no hemorrhagic necrosis of the bilateral kidneys occurred.

The membrane vesicles in the product of the invention appear to be readily endocytosed by monocytes and macrophages when observed under phase contrast microscopy. Endocytosis is known to lead to cell membrane renewal and membrane circulation in the monocyte-macrophage cell line can activate these cells.

Cells of the monocyte-macrophage cell line can be artificially classified into several functional classes, each of which represents a class of cells that gradually differentiate into more complete classes. These classes (the nomenclature may differ) are: monocytes, normal or resting stage macrophages, stimulated macrophages, activated non-tumor-killing macrophages and activated tumor-killing macrophages. The more well differentiated the cell is, the less unresponsive it is to various types of stimuli, and potentially more limited in the performance of effector cell functions. Conversely, in advanced disease and chronic disease states, the monocyte-macrophage cell line, as a general immune system, is increasingly rendered more unresponsive to stimulation. In these stages, only activated tumor-killing macrophages were shown to be directly cytotoxic to tumor cells.

It has been particularly noted that monocyte-macrophage product endocytosis is associated with murine cells. Normal monocytes and resting macrophages were harvested from the abdominal cavity of healthy adult C57B1 mice without any prior procedures and without heparin. The initial adherent cell population contained more than 90% of the round cells, the remainder having a typical macrophage morphology. The circular cell population consists of monocytes and a large number of moderately sized cells with the appearance of lymphocytes. Wherein all cells are endocytosed and are therefore members of the monocyte-macrophage cell line.

Within minutes after endosomal addition of the product of the invention to the purified primary monocyte-resting macrophage (round cell) population, a large number of microvesicles (whose size and number increase with time) begin to appearBelow the cell membrane. Phagocytic activity is directly proportional to the concentration of the added product of the invention. When the concentration of the bioreaction modifier of the invention is added to exceed 50 micrograms/10 per 3ml of medium⁵When cells are used, the extreme vacuole formation that occurs with maintenance of the round cell morphology will lead to autophagic death. Neither phagocytic activity nor cell activation was observed when endotoxin or BCG cell walls were added at different concentrations to the same cell population. Neither the B16 melanoma cells nor the renal epithelial cells exhibit phagocytic activity in the presence of the bioresponse modifiers of the invention, or in the presence of endotoxin or BCG cell wall products. One possibility is that the bioresponse modifier of the invention rapidly enters into monocyte-quiescent macrophages due to the presence of receptors specific for vesicles and/or ribosomal components prepared according to the process of the invention. Other interpretations involving non-specific physical and/or chemical interactions (e.g., relative hydrophobicity) are also possible.

Table 3 below summarizes the results of a monocyte-resting macrophage cytotoxicity study involving in vitro administration of a bioresponse modifier of the invention at concentrations of 0 to 100 micrograms (5 micrograms concentration intervals in each tube) added to a sample containing (10) cells as targets⁵B16 melanoma cells and 25Cm of mouse peritoneal monocytes/resting macrophages as effector cells²In a culture flask. The left column in the table shows the ratio of effector cells to target cells. 96 hours after the start of the assay, cells were fixed in 95% ethanol and stained with Mayer's hematoxylin. The results indicated by the plus sign represent cell growth readily visible under the microscope at 80% -100% confluence, and the minus sign represents no visible growth, i.e., significant reduction or absence of the target cell population as evidenced by microscopic observation.

TABLE 3

Tumor cytotoxicity assay

Visible (confluent) growth at E/T ratio plus different concentrations of product (micrograms) for 96 hours

0 3 5 10 15 20 25 30 35 40 45 50 55 60 65 70-100

2：1 + + - + - + + + + + + - + + + +

1：1 + + - + - + + + + + - + + + +

1：2 + + - + - + + + + + + - + + + +

1：4 + + + + + + + + + + + + + + + +

As can be seen from the above table, the bioresponse modifiers of the invention induce the differentiation of peritoneal monocytes/resting macrophages into a tumor cytotoxic state at very specific concentrations (5, 15 and 50 micrograms). High levels of cytotoxicity are achieved at effector-target cell ratios of 2: 1 or less than 2: 1. The observed tumor cell killing exhibited extreme vacuolization, cell shrinkage, and/or increased phase density, all of which occurred within 8 hours of the addition of an effective concentration of the bioresponse modifier.

Previously published research reports indicate that activated non-tumor-killing macrophages must be in contact with lymphokine for 18 hours before they kill target cells, while monocytes and resting macrophages are rendered unresponsive. The cytotoxic activity induced by the products of the invention in monocytes/resting macrophages is very rapid and unique. The fact that cultures had to be incubated for four days before termination indicates that administration of the bioresponse modifiers of the invention can rapidly and effectively induce effector cell function and inhibit target cells for long periods of time.

The ability of the product of the invention to alter various White Blood Cell (WBC) count levels and neutrophil levels is shown in table 4. Table 4 indicates WBC and neutrophil levels before and after administration of the bioresponse modifier in a series of advanced patients. The doses administered are shown in the table. Administered once a week for 3 weeks. As can be seen from the examples shown in Table 4, the white blood cell count and neutrophil count can be significantly increased by specific treatment with the product of the invention. Note that not all of the various doses, nor all patients showed an increase (but also no decrease) in white blood cell count. However, as shown in Table 4, some patients experienced a significant increase in white blood cell count and neutrophil count following the use of the bioresponse modifiers of the invention.

TABLE 4

Patient numbering bioresponse (modification) WBC × 1000 neutrophils (% WBC)

Dose (mg) dose before administration 24 hours after administration

D59261 1.0 18 27 88 92

D25940 1.0 3.8 6.2 58 70

D78252 1.5 8 10.5 67 82

D45851 1.5 8.6 13.6 77 90

3.0 13.3 22.7 90 92

D80822 2.0 6.7 7.6 59 74

3.0 6.1 9.6 59 75

D82088 4.0 3.2 9.4 70.3 93

6.0 3.5 6.3 56 85

D90607 6.0 8.0 9.6 72 84

Normal value range: WBC7.8 + -3 × 1000

Neutrophil leucocyte 40-70%

Patients with low WBC counts prior to treatment reached the normal range 24 hours after treatment. Administration of the bioresponse modifiers of the invention also resulted in an increase in the count of human peripheral blood mononuclear cells (natural killer cells, cytotoxic T cells and/or monocytes) and did not result in thrombocytopenia (data not shown). Therefore, it can be seen that the product of the present invention has no bone marrow suppression effect.

Proper immune function requires the synergistic action of several cell types and in turn produces and releases large amounts of cellular products. Cells of the monocyte-macrophage cell line are known to play a critical role in this process, and optimal antibody responses (B cell function), cell-mediated immunity (T cell function), and possibly Natural Killer (NK) cell activity do not occur in the absence of these cells. Although it is possible to elucidate various synergistic cellular pathways in tissue culture, it is not possible to identify a large number of pathways and their control mechanisms in patients. Because the mechanisms of action of the various specific and/or non-specific immune effector cells occurring in a patient are essentially unknown, the choice of an agent that enhances/modulates immune function should enhance any ongoing response or reduce the systemic response threshold in the absence of a response, thereby allowing the identification, activation and selection of appropriate effector cell functions. An ideal activator should not induce an immune bias, anergy or abnormal hypersensitivity response to itself or a cross-reactive subject, or contribute to or direct the selection of a specific single effector cellular pathway. Since such phenomena are not significantly manifested during clinical trials, the products of the present invention are considered to meet the above requirements.

FIG. 2 is a graph illustrating the ability of the biological response modifiers of the invention to stimulate natural killer cells in humans in a manner similar to the action of interferon-leukapheres. This in vitro test illustrates the extent of target cell lysis (depending on the amount of radiolabeled Cr released from the target cells) due to the action of human natural killer cells after administration of different dose levels of the product of the invention and interferon, respectively, as shown by the different graphs. Dose levels are given in each graph, with zero dose levels representing background or leakage levels of chromium released from target cells.

The above analysis is based on the literature^{（8）（9）}The method as described in (1). Human K562 cells of a myeloid tumor cell line labeled with radioactive sodium chromate were used as target cells. The effector cells used were human peripheral blood mononuclear cells which were not depleted. The following formula can be used for calculation:

% release x 100 (experimental release-control release)/(maximum release-control release) × 100

The experimental release is the counts per minute (cpm) of radioactivity in the presence of product and effector plus target cells, and the control release is the pulse counts per minute obtained with effector plus target cells alone. Maximum release is the cpm number obtained by incubating aliquots of target cells in saponin, a detergent.

The results of this example demonstrate that although not as high as using interferon for a relatively low effector cell to target cell ratio, human natural killer cell cytotoxicity can be significantly induced in the presence of monocytes, much as using interferon. Statistical analysis of 30 products showed that the products of the invention were comparable or superior to interferon-leukocyte in inducing NK cell cytotoxicity. The NK cell cytotoxicity induced by the product is similar to the cytotoxicity induced by interferon, and also depends on various variable factors such as the yearly order of blood donors, sex, cell storage method, proportion of various types of cells and the like.

FIG. 3 is a graphical representation of the bactericidal properties of bioreaction modifiers prepared according to the method of the present invention. Graph in fig. 3The ordinate of (A) shows the logarithmic value of Listeria monocytogenes (Listeria monocytogenes) in the abdominal cells of mice, and the abscissa shows time (hours). Listeria monocytogenes is a bacterium that causes acute meningitis (with or without associated sepsis) in humans. The assay shown in FIG. 3 is a standard assay for the detection of bactericidal activity. The control is known to induce bactericidal macrophages but is intolerant to humans

Peptone. (for a detailed description of the process see Cruprinski, C.J., Henson, P.M., and Campbell, P.A., 1984, J.Leukoc-yte biol.35: 193).

The curves in the figure indicate the change in the number of bacteria in culture when they are exposed to cells from the abdominal cavity of mice within 3 hours, at different times and in several different predetermined conditional steps described below. These different conditional steps are:

A. no abdominal cells-bacterial growth control;

B. intraperitoneal injection 14 days before harvest

Peptone;

C. injecting the biological reaction modifying agent into the abdominal cavity 14 days before harvesting;

D. injecting the biological reaction modifying agent into the abdominal cavity 7 days before harvesting;

E. injecting the biological reaction modifying agent of the invention into the abdominal cavity 1 day before harvesting, and

F. intraperitoneal injection 1 day before harvest

Peptone.

As can be seen from fig. 3, the upper curve representing the control group indicates that the culture grew stably. When cells were harvested from mice intraperitoneally injected with 0.2 mg of the product of the invention, they showed a dependence on the pre-harvest injection time and were essentially identicalUpward and downward injection

A similar control of peptone (1 day before harvest) showed growth decay with different slopes. These data show that the bactericidal activity induced by the product of the invention is still high 14 days after a single injection. The bioresponse modifier of the invention has a long-lasting effect in inducing macrophages to produce a defined bactericidal activity, indicating that it can be used as a therapeutic agent for bacterial infections.

FIG. 4 shows the ability of a bioresponse modifier prepared according to the method of the invention to modulate antibody-dependent cellular cytotoxicity (ADCC). The method is as described in the literature^{（10，11）}The method as described in (1). In this assay, human peripheral blood mononuclear cells having a specific effector cell-target cell ratio are used as effector cells. The target cells were chromium-labeled murine YAC cells and the antibody used was a rabbit anti-mouse cell antibody.

As can be seen from FIG. 4, at least for relatively high effector cell to target cell ratios, the level of killing was higher than that of interferon at doses of 15 μ g/ml or below the level of the product of the invention used, but was background at a concentration of 20 μ g/ml (the concentration that led to autophagic death of monocytes in this assay).

FIG. 5 is a graphical representation of the ability of the products of the invention to stimulate release of interleukin I (IL-I). The detection is based on literature^{（12，13）}The procedure is carried out. The peripheral blood mononuclear cells are prepared from the blood of the subject and incubated with the product of the invention at various concentrations. After a certain period of incubation, an aliquot of the culture broth was harvested. Lymphocyte transformation experiments were performed at various dilutions. The blast-like transformation test of lymphocytes is to perform 10⁶The mouse thymocytes were incubated for 72 hours in the harvested culture supernatant, after which the radioactivity (counts per minute) of tritiated thymidine uptake by the cells was measured. In FIG. 5, the background level is indicated by a solid circle. It can be seen that, except for the lowest dose (1. mu.g/ml), this can occurSignificant stimulation of IL-1 release. Literature reference^（14）The therapeutic effect of IL-1 release has been described.

FIG. 6 is a graph illustrating that the cytotoxic effect on K562 target cells is due to natural killer cells, not B or T cells. The experiment used human peripheral blood mononuclear cells as the effector cell population at a specific effector cell-target cell ratio. It can be seen that the background or leakage level is below 20% chromium release when no bioreaction modifier prepared according to the method of the invention is added or no natural killer cells are present. For non-depleted cell populations, administration of the product of the invention results in significant stimulation of natural killer cell cytotoxicity. Removal of B or T cells from this cell population with specific monoclonal antibodies did not significantly affect the level of cytotoxicity. However, removal of NK cells with monoclonal antibodies abolished the cytotoxic effect. Removal of monocytes (not shown) with monoclonal antibodies also resulted in loss of NK cytotoxicity, suggesting that NK cells are activated by monocyte-macrophage populations.

The results of an in vivo therapeutic study on rat prostate squamous cell carcinoma (rapidly growing tumor) are shown graphically in figure 7. It can be seen that administration of the product of the invention induces regression of large, established and rapidly growing carcinomas in mice. All untreated control tumor-bearing animals died within 14 months, while all treated animals survived and tumor regression was seen in 4 out of 6 animals. In conducting these studies, it was found that a perilesional injection of 1 mg per week was effective, whereas a weekly injection of 2mg was ineffective. The particular tumor treated was Dunning subline of tumors R3327A, a hormone-independent, moderate-fast growing rat tumor that metastasizes at an advanced stage. The tumor host is Copenh-gen X Fisher F₁. The tumor system is a tumor animal model of the national prostate cancer research group. Is implanted in the left flank. The mean tumor volume at the first treatment was 681 cubic millimeters.

FIG. 8 shows the nodule of slow growing rat prostate cancer treated with the product of the inventionAnd (5) fruit. The tumor host is Copenhagen X Fisher F₁. It is another tumor model for the national prostate cancer research group. Is implanted in the left flank. Treatment included injection of 2mg every seven days at the site paralesioned. Mean tumor volume at the start of treatment was 1138, 8 (> 2 Cm)³) Cubic millimeters. This particular carcinoma is Dunning subline of tumors R3327H, a well differentiated adenocarcinoma and has demonstrated a very slow growth rate.

As can be seen in fig. 8, the bioresponse modifier of the invention induced regression of large established and slowly growing carcinomas in rats. All untreated animals exhibited worsening disease, death or weight loss, while treated animals exhibited stable disease, slow progressive regression (as evidenced by pathological examination), normal body weight and no mortality. In this example, a weekly injection of 2mg to the site of injury is effective, while a weekly injection of 1 mg to the site of injury is ineffective. The two tumor systems demonstrated that the expected effective dose varied from tumor to tumor.

Figure 9 shows the results of treatment of bilateral rat prostate adenocarcinoma. The host receiving orchiectomy, orchiectomy plus the product of the invention, steroid plus the product of the invention were compared to the control group only for the product of the invention. Comparisons were made for left and right flank tumors, respectively.

As can be seen in FIG. 9, the bioresponse modifiers prepared according to the method of the invention induced regression of very large (total volume greater than 5 cubic centimeters), moderately fast growing adenocarcinomas. Treated animals received weekly injections of 1 mg of the product of the invention only at the perilesional site in the tumor area of the left flank. The data indicate that systemic therapeutic effects (regression of contralateral untreated lesions) can be achieved with the product of the invention, and that the effects of the bioresponse modifier are not inhibited by steroids, dexamethasone. Dexamethasone was not observed to inhibit the high fever or tumor response in tumor bearing patients.

Clinical studies in humans have been conducted using the bioresponse modifiers of the invention in accordance with the regulations and procedures of phase I and II trials published by the U.S. food and drug administration. Phase I studies were performed to determine the toxicity, if any, of the bioreaction modifiers of the invention. The subjects in phase I are in advanced disease and are considered patients who have failed all previous treatments. These patients received three to six treatments, injected every 7 days subcutaneously, distant from the tumor site, at the indicated doses. Toxicity tests show that the product of the invention has no obvious toxicity and can be well tolerated by human bodies.

At the same time, a therapeutic effect was obtained in 46% of cases. The results of the phase I and phase II tests are given in the following table.

TABLE 5

Patient response summary

Patient-coded dose (mg) diagnostic response

D44937P stabilization of lung cancer 25,. 50, 1.0

D06277.25 prostate cancer is minor

D32770.25 No. Lung cancer

D01088.50 Breast cancer stabilization

D15546.50 colonic/hepatic anuria

D592611.0, 2.0 lymphoma is minor

D259401.0 germ cell anergy

D782521.5 colonic/hepatic anuria

D458511.5, 3.0, Small adenocarcinoma

5.0, 6.0 rectal/abdominal

D1808492.0, 3.0 Breast cancer

D808222.0, 3.0 lymphoma (LHL) none

D818282.5, 8.0 lymphoma (NHL) moiety

D629842.5, 4.0 lymphoma (HL) stabilization

D820882.5, 4.0, 6.0 malignant glioma

D763394.0, 6.0 pancreatic stabilization

D825684.0 Kaposi's sarcoma stabilization

C033435.0, 8.0 Kidney cell stabilization

D873545.0, 1.5 nodular lymphoma fraction

D906076.0 Lung and adenocarcinoma

D918346.0 melanoma No

A0395850.25 renal cell stabilization

A768642.25,. 5, 1.0, 2.0 Colon/liver stabilization

A6619810.5 Stable for lung cancer

Adenocarcinoma

A0783790.5 minor parotid cancer

A0172310.25 absence of colon cancer

A0550011.0 Lung stabilization

A6665221.0 common Yam rhizome

Large cell

A4039271.0 pulmonary tumor

Squamous cell

A0328961.5 adenocarcinoma, neck part

A7080291.5 bronchioloalveolar stabilization

A0441301.5 Lung cancer stabilization

Squamous cell

A2232112.0 Lung cancer stabilization

D473798.0 prostate hyperplasia

D886525.0 colonic Wu

D935038.0 Colon stabilization

D942458.0 NH lymphoma was minor

D861418.0 colonic Wu

D964648.0 melanoma No

D9425710.0 gallbladder failure

D9773510.0 colonic Wu

E0151410.0 NSC pulmonary stabilization

D9852010.0 colonic Wu

E0140110.0 sarcoma without

D663991.0 mediastinal clump No

61342.0 pulmonary stabilization

33742.0 Lung/prostate stabilization

Unknown primary

42774.0, 6.0 Lung stabilization

50793.0 Gastrodia elata Blume

04214.0 histiocytoma fraction

69186.0 gallbladder free

26888.0 tongue without tongue

87088.0 Colon do not

92228.0 prostate gland diseases

E079420 colonic medicine

E131504.0 Small tissue Lung stabilization

E143564.0 neck none

E157354.0 rectal suppository

D691743.0, 4.0 colonic Wu

E119274.0 NSC Lung Wu

E149834.0 Kaposi's sarcoma-free

D707273.0, 4.0 Lung stabilization

E078762.0, 3.0 Kidney stabilization

E046931.0, 2.0 mammary gland minor

E103113.0 throat lozenge

E092653.0 Kidney-failing

D442960.5 colonic Wu

D818282.0 NH lymphoma

E077271.0, 3.0 mammary gland hyperplasia

E037410.5 colonic Wu

E054872.0 parotid gland without

E030830.5, 1.0 endometrial stabilization

E045451.0 Mixed lymphoma without

E051952.0 thymoma is small

Partial remission is 50% or greater;

the disease condition is relieved by more than 25 percent to less than 50 percent when the disease condition is smaller;

stable ═ no progression of the condition;

no means no reaction.

The data indicate a 48% response rate in patients with advanced disease, failure of previous therapy.

The particular dose and treatment period (course) to be administered to a patient will depend upon the particular circumstances of the patient and factors which affect the dose and treatment period include the general immune response characteristics of the patient, the particular type and extent of the disease, the general health of the patient, the location of the condition, etc. This is essentially the same as other biological response modifiers, and the optimum dosage can be determined according to the relevant techniques for other biological response modifiers and general medicine.

The data in Table 6 below show that the products of the invention may be more effective in treating diseases such as brain tumors (e.g., glioblastomas) than other available treatments. The data in table 6 show that there is a reduction in tumor volume in 3 of 6 brain tumor patients, which is the same as the definitive treatment. This result clearly demonstrates that the bioresponse modifier product of the invention is effective in treating this particular tumor.

TABLE 6

Summary of patient response-brain tumor

Patient-coded dose (mg) diagnostic response

D820882.5, 4.0, 6.0 brains are smaller

E119281.0 brain part

E344151.0, 3.0 brain improvement

E373761.0, 3.0 brain significant function improvement

E452091.0 brain improvement

RT 1.0, 3.0 brain improvement

JB 1.0 brain improvement

DR 1.0, 3.0 brain parts

The membrane vesicles and ribosomes used in the bioreaction modifier product of the invention are readily biodegradable. One particularly noteworthy advantage of the degradation phenomenon is that the population of particles of the product of the invention remains intact in the immune system for only a short time, sufficient to activate the immune system, and then rapidly degrades. Thus avoiding the occurrence of severe, chronic pathophysiological reactions as a result of chronic immune activation. The degradation process is due to the action of various enzymes (e.g., ribonucleases, proteases, lipases) present in various types of cells (e.g., monocytes, macrophages, neutrophils) and interstitial fluids (e.g., blood, lymph). The degradation process is accelerated at elevated temperatures (e.g., body temperature, 37 ℃) and gradually slows as the temperature decreases. The product was degraded in vitro in less than 2 minutes at 37 ℃ and 95% serum concentration as measured by particle size analysis. The rate of degradation depends on the enzyme concentration and temperature. Once degraded, the efficacy of the product of the invention as an immunomodulator is significantly compromised.

Figure 10 shows induction of killer cell cytotoxicity against three different tumor target cells in humans. For most individuals, peripheral blood mononuclear cells can kill K562 tumor target cells to some extent in vitro. Peripheral blood mononuclear cells generally do not kill the Raji and Colon38 tumor cell lines. Various biological response modifiers, including interleukin II (IL-2), are administered in vivo to the patient without altering the level of killing of the latter two target cells. Only after in vitro treatment of peripheral blood mononuclear cells with certain biological response modifiers, including IL-2, can these cells be induced to kill other types of tumor target cells than K562. However, it has been found that the bioresponse modifier products of the invention are capable of altering the ability of peripheral blood mononuclear cells to kill these tumor target cells when used in vivo therapy. This study suggests that induction of LAK cells (cytotoxic T cells) occurs after administration of the product prepared according to the method of the invention.

FIG. 10 shows an in vitro assay for killer cell cytotoxicity in peripheral blood mononuclear cells obtained from patients treated 24 hours after administration of the bioresponse modifier of the invention. It can be seen that the results have significant killing effect on all three tumor target cells. Examination of the effector cell population by specific cell markers shows that activated monocytes, natural killer cells and LAK cells are present therein. Natural killer cells are increased in treated patients (as previously discussed) and the presence of activated natural killer cells is found in this assay, thus demonstrating that endogenous interferon is produced in these patients treated with the products of the invention. Furthermore, the presence of activated cytotoxic "T cells" (LAK cells) demonstrates that treatment with the product of the invention results in the production of endogenous IL-II. Another unique property of the products of the invention is that the in vivo activated killer cell population can be maintained in vitro with very low concentrations (2 UIL-2/ml) of IL-2. In contrast, for example, in vitro maintenance of IL-2-activated killer cells (LAK cells) requires the use of high concentrations of IL-2 (500-1000 u IL-2/ml).

The peripheral blood mononuclear cells used were whole blood obtained from patients 24 hours after injection of the bioresponse modifier product of the invention. Subcutaneous injections were used 3 times a week, 1 to 4 mg each. Detection was performed before injection and 24 hours after injection. The cytotoxicity levels obtained with the cells activated in vivo were compared with the activity of killer cells activated in vitro with lymphokine (IL-2), i.e., obtained after culturing human peripheral blood mononuclear cells together with IL-2.

By comparing the results of the detection of natural killer cells (FIGS. 11 and 12), the advantages of membrane vesicles and ribosomes obtained from organisms such as Serratia marcescens were compared with those from other sources. The desired product is prepared from a given organism using the methods described above. The vesicles produced from these organisms have physical and chemical parameters within the above ranges.

The fact that the source of a given bacterial product may affect the type and/or extent of its biological activity is not new to the art. Different strains of BCG show specific levels of immune activation in animal models and clinically. Ribosomal vaccines from different organism sources have been reported to produce varying degrees of immune system activation, and suggest that polysomes from Serratia are superior to those from other sources^（6）. From the cited data, it is clear that buffer and bacterial polysomes from other species (E.coli, S.pneumoniae, M.bovis [ BCG ], M.cloacae and P.acnes) were administered alone and not much better than untreated control mice (tumor development or death within 60 days). On the other hand, when a polysome derived from Serratia marcescens was administered at a dose, no tumor was developed in 60% or more of the animals, and tumor growth was also inhibited in the remaining animals.

Because the bioresponse modifier product of the invention is derived from a microorganism which is not a member of the patient's microbial system and is not associated with infectious disease in normal individuals, and because the common bacterial antigen of the microorganism does not or rarely cross-react with the organisms which make up the normal microbial system, inappropriate immunological reactions are avoided. Such responses include immune bias and initial antigen delinquent phenomena, which are common to recently developed immunopotentiators (e.g., BCG, c. Perhaps for these reasons, the polysomes produced by e.coli, mycobacterium cloacae and streptococcus pneumoniae are poor inducers of biological responses, while serratia marcescens, which is only partially or poorly cross-reactive with host microbial systems, is an excellent source of bioresponse modifiers.

It should be noted that other microorganisms suitable as sources of membrane vesicles and ribosomes may also be used in the present invention. The essential feature of these microorganisms is that the microorganisms must not be members of the patient's microbial system. Furthermore, the common bacterial antigens of the microorganisms used must not or at most only partially cross-react with the organisms that constitute the normal microbial system. Thus, it should not be immune-biased and must not or rarely cause disease in humans. Finally, the source microorganism must have a cell membrane capable of forming vesicles of suitable size.

Suitable microorganisms other than Serratia marcescens are: erwinia Chrysan-the mi (Pectibacter) ATCC14092 and Enterobacter aerogenes (Enterobacter aerogeenes) ATCCE13048, which is classified to a smaller extent. The preparations obtained from these strains exhibited activities consistent with those of the above-mentioned preparation derived from Serratia marcescens. In particular, as shown in fig. 11, the use of enterobacter aerogenes can be much more effective than pseudomonas, escherichia coli, and e.cloacae, but less effective than the use of a-interferon at a specific dosage level. Similarly, fig. 12 compares e.chrysan-the mi (potent) with Flavobacterium (non-potent) and interferon. Other microorganisms can also be used as a source and processed as described above to produce ribosomes and vesicles of a particular size. Their potency can be readily assessed using the in vitro assays described above to determine whether they can be used as bioresponse modifiers.

Because the bioreaction modifier product of the invention is free of viable cells, dead cells, cell walls and biologically active endotoxins, recognition by cross-reactivity is minimized, components that are not readily degradable are removed, and components known to be highly toxic (e.g., endotoxins) or to cause chronic inflammatory conditions (e.g., arthritis, granulomas, ulcers) are minimized or completely removed. In addition, reducing cell surface components as much as possible eliminates immune response variations and phenotypic variations known to be associated with microbial strains.

The advantages of the bioreaction modifier product of the invention are numerous. By activating cells of the monocyte-macrophage lineage, cellular synergy necessary for optimal immune function is promoted. Activation of the drought-differentiated cells of the monocyte-macrophage lineage (monocytes, normal/quiescent macrophages) can enhance multi-effector cell function. To this end, it is well documented that the bioresponse modifier compositions prepared according to the methods of the invention can induce a wide variety of effector cell functions depending on the conditions tested or the parameters of the diseased host.

In addition to those described herein, various modifications of the product of the invention will be apparent to those skilled in the art from the description herein. Such modifications are intended to fall within the scope of the claims appended hereto.

Claims (5)

1. A method of producing a bioreaction modifier comprising culturing bacterial cells of a strain of Serratia marcescens (Serratia marcocens), harvesting the cultured cells, dissociating endotoxin with a suitable detergent, treating the cell concentrate to produce ribosomes and membrane vesicles having a diameter of no less than 110nm, separating the ribosomes and membrane vesicles from the cellular material remaining in the cell lysate, and resuspending the ribosomes and vesicles in a suitable buffer at a relative concentration such that the average particle diameter exceeds 170nm (based on particle size analysis).

2. The method of claim 1, wherein the lysing is accomplished mechanically.

3. A method of producing a bioreaction modifier comprising culturing bacterial cells of the species Serratia marcescens in a suitable culture medium, cooling said culture to a temperature of 0-4 ℃, harvesting the bacterial cells, washing the harvested cells and suspending them in a non-toxic, well-tolerated buffer system to maintain an environment suitable for cell membrane vesicle formation and integrity, lysing the harvested and suspended cells in the presence of a detergent which effects dissociation of membrane debris and endotoxins, said lysing step producing membrane vesicles having a diameter of at least 110nm, clarifying the bacterial cell lysate comprising cell debris including intact cells, cell walls and membrane debris, layering the clarified cell lysate on a density gradient material which is tolerated by the human body and is non-immunogenic to the human body, centrifuging to isolate membrane vesicles and ribosome fractions substantially free of other minor fractions, the density gradient material is removed aseptically, washed and resuspended in buffer with said membrane vesicles of said size range together with the remaining ribosomes.

4. The method according to claim 3, wherein cell lysis is accomplished mechanically at a pressure in excess of 10,000 Psi.

5. A method for producing a bioresponse modifier which comprises culturing bacterial cells of a strain of microorganisms which are not present in the microbial system of the human being to be treated and which have common bacterial antigens which are not or only rarely cross-reactive with the organisms which form the normal microbial system of the patient to be treated, harvesting the cultured cells, dissociating endotoxin with a suitable detergent, disrupting the cell concentrate sufficiently to produce membrane vesicles having an average diameter of not less than 180nm, separating said membrane vesicles and freeing the ribosomes from the cellular material remaining in the cell lysate, and resuspending said vesicles and ribosomes in a suitable buffer.

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