DD273198A5 - METHOD FOR PRODUCING A MODIFYING AGENT - Google Patents

Abstract

The invention relates to a process for the preparation of a biological reaction modifier for the treatment of a patient consisting of natural membrane bladders and ribosomes in a suspension buffer solution. According to the invention, these membrane bladders and ribosomes are endogenous to a selected microorganism which does not cause a significant immunodeficiency reaction in the patient and is substantially nonpathogenic in humans, the selected microorganism is also one in which membrane bladders can be formed from cell membrane material which blasts lightly from the cell membrane material Monocytic macrophage cell line of the patient are endocytosed, the biological reaction modifier is substantially free of endotoxin, intact cells, cell walls and cell membrane fragments, the biological reaction modifier further having an average diameter greater than 170 nm after grain size analysis.

Description

For this 9 pages drawings

Field of application of the invention

The invention relates to a biological reaction modifier for treating a patient consisting of natural membrane vesicles and ribosomes in a suspension buffer solution.

The invention generally relates to a pharmaceutical product having immunomodulatory properties. It more particularly relates to a biologic response modifier (BRM), defined as an agent that modifies the relationship between a disease and the host by conferring the host's biological response to the disease modified therapeutic effects.

Characteristic of the known state of the art

The BRM can be divided into two categories:

1. biological or chemical agents capable of stimulating or otherwise altering one or more host resistance mechanisms, and 2. purified cell products that exhibit direct effects on a particular disease. The first group of BRMs covered by the invention consists of agents that activate, enhance or otherwise modify the host's immunological responsiveness, and are generally referred to as immunomodulators.

A BRM may be used alone or in conjunction with other agents to increase resistance to or recovery from infestation by pathogens, to modify or induce the tolerance of foreign tissue transplantation, to promote tumor rejection or stabilization, and to inhibit tumor recurrence following other forms therapy, to restore normal helper-suppressor mechanisms, or to otherwise promote a normal immune response.

Since the beginning of this century, a wide variety of immunomodulators / immunoadjuvants have been developed. The vast majority of these agents are of microbial (bacterial / fungal) origin, the most popular being derived from the genera Corynebacteria, Mycobacteria and Nocardia (CMN organisms). The various immunoadjuvants consist of either intact viable cells, dead cells, cell walls, various cell wall fragments, endotoxin, various types of polysaccharides, or subcellular fractions such as ribonucleic acid or ribosomes. Although all of these agents have demonstrated immunopotentiating modulating reconstitution activity in tissue culture, animals and humans against infectious and tumorous diseases, they generally suffer from contradictions in production and composition and exhibit moderate to severe toxicity. As far as the treatment of tumors is concerned, none of these agents has hitherto been useful in the treatment of a detected disease. In addition, these agents typically exhibit loss of efficacy upon repeated application (anergy), immune deviation, hypersensitivity reactions, development of chronic inflammatory conditions, and / or the development of various other undesirable conditions.

Due to the inability to chemically define these agents because of their contamination and / or complexity, and in an attempt to reduce the variability of immune responses and toxic complications, a number of researchers have either extracted different specific fractions of these sources, or several components that have one show immunological activity synthesized. Examples of specific fractions investigated are the cell wall fraction muramyl tripeptide, staphylococcal protein A, various polysaccharides, RNA fraction ¹¹ and the ribosomal fraction. ² " ⁵¹

With regard to the ribosomal fraction, the mechanism of action differs depending on the ribosome source. Although the majority of ribosomal vaccines require an adjuvant for efficacy, ribosomal vaccines made from Staphylococcus aureus and Neisseria meningitis are not. ²¹ In addition, ribosomal vaccines prepared from Mycobacterium tuberculosis ³ 'and Salmonella typhimurium ⁴¹ appear to cause a cell-mediated reaction, while ribosomal vaccines prepared from Streptococcus pneumoniae and Streptococcus pyogenes mediate a humoral response. ⁸¹ Although the extracted ribosomal fraction is theoretically the active substance, in many cases there is considerable controversy as to whether other cell components (RNA, protein, endotoxin, cell wall) present as contaminants are responsible for the observed immune responsiveness.

Known cell fraction vaccines may sometimes be useful for the prophylactic or therapeutic treatment of infectious diseases. However, they are unsuitable for use as non-sensitizing general immunomodulators for the treatment of tumorous diseases for various reasons. The presence of cell wall, endotoxin, or poorly degradable components often results in toxicities similar to those achieved with the intact organisms. In addition, undesirable immune-aberrant responses and complex immune responses may be produced because such vaccines are typically derived from organisms that are part of, or readily responsive to, the host's own microflora (common antigens). Such vaccines characteristically also contain fractions having physical and / or chemical characteristics which may be suboptimal for general immunopotentiating activity.

Urban et al. ⁶ 'reported the ability of polyribosomes (aggregates of ribosomes) derived from a specific bacterial organism to suppress the development of cutaneous SaD2 fibrosarcomas in DBA / 2 mice. Polyribosome fractions were recovered from cell cultures by osmotic or mechanical dissolution (depending on the type of bacteria) followed by differential centrifugation. Although the effect on the SaD2 murine tumor was positive, the mechanism of the evoked biological response could not be determined from the data. Although the toxicity was very low, Urban et al. described process to an extreme change of Polysomenprofils (size distribution) and a very low product yield. No consistency of quality, stability and effectiveness was achieved.

Kirsh et al. ^{Figures 7} 'have reported immune stimulation and modulation by the inclusion of specific antigens or modifiers of the biological response in liposomes. Liposomes containing drugs are used for the treatment of metastatic cancer. ⁸¹

However, the treatment of tumor diseases (cancer) with modifiers of biological response alone or included in liposomes has been daunting to date. Although included BRMs outperform unencluded BRM in efficacy, the limitation of therapeutic benefit could be attributed to the fact that immune activation is limited to the macrophages. Tumor cells develop rarely, if at all. Resistance to macrophage killing in comparison to natural killer cells and cytotoxic T cells. The number of macrophages in these cases is too small for them to effectively mediate even the destruction of large tumor loads.

Object of the invention

It is an object of the invention to provide an improved biological reaction modifier.

It is a further object of the invention to provide an improved biological reaction modifier having minimal toxicity and immune deviation.

Another object of the invention is to provide an improved biological reaction modifier which can be prepared with consistent quality, stability and efficacy.

Explanation of the essence of the invention

It is an object of the present invention to find suitable membrane vesicles and ribosomes from a selected microorganism and a method of preparing the biological reaction modifying agent having the desired properties, which provides consistent quality and high yield.

The biological reaction modifier of the present invention very generally includes natural membrane vesicles and ribosomes in a suspension buffer solution. The vesicles consist of cell membrane material that is endogenous to a selected organism. The ribosomes are also endogenous to the selected organism. The biological reaction modifier is substantially free of endotoxin, intact cells, cell walls and cell membrane fragments. The selected organism is an organism that does not elicit a significant immunodeficient reaction, which is non-pathogenic in humans and whose membrane vesicles can be formed from cell membrane matter and whose vesicles are readily endocytosed by the monocyte / macrophage cell line. The biological reaction modifier has an average diameter greater than 170 nm in particle size analysis.

The invention can directly activate cells of the monocyte / macrophage cell line. Activation of the monocyte-macrophages or cells of the monocyte-macrophage cell line results in the production of monocyte-macrophage mediated bactericidal activities (Figure 3) and tumor cytotoxicity (Table 3), altered levels of various white blood cells involved in immune function (e.g. B. Nonocytes, neutrophilia (Table 4), for in vivo production of tumor target cytotoxicity (K562, Raji, Co38) in humans (Figure 10), elicitation of natural killer cell-mediated cytotoxicity (Figure 2), elicitation of antibody-dependent cell cytotoxicity (FIG. 4) and the release of interleukin I (IL-1) (FIG. 5) and possibly of interleukin II (IL-II) (FIG. 10).

For the purposes of accuracy, the following terms used in this specification and the appended claims are defined:

"Non-toxic" means within a level of toxicity that is tolerable to the mammalian host that includes therapy with the biological response modifier.

By "non-immunogenic" is meant inducing a sufficiently low immunogenic response, or no reaction at all, that the undesirable immune-aberrant, chronic inflammatory and hypersensitivity reactions in the mammalian host are not materially evident.

"Mean Diameter" means the median diameter of the MSD Grain Distribution Analysis measured on a BI-90 Grain Screen Classifier (Brookhaven Instrument Corp.) This measurement includes an intensity weighting of the size averaging process and is explained in detail in the Equipment Operation Manual, Chapter 6 ,

"Substantially non-pathogenic in humans" does not or rarely means having disease in normal human health

be connected. Since most microorganisms can cause opportunistic infections in the right circumstances, eg. For example, in individuals whose immune system is compromised, this definition excludes only those organisms that normally cause non-opportunistic infections.

Substantially free of endotoxin, intact cells, cell walls and cell membrane fragments, "a sufficiently low level of biological activity of such fractions means to maintain a non-toxic trait defined herein.

"Immunodeficient reaction" means an immune response directed away from the disease being treated, for example, the appearance of the original antigenic sin can make the immune system respond to historical challenges or respond to the common microflora, when challenged by an antigen similar to those dominated by the historical challenge or the common microflora, polyconal activation may also induce nonspecific misdirected anamnestic reactions., Badly degradable particles may result in distraction of the monocyte-macrophage cell line ( ie, foreign body reaction, chronic hypersensitivity), which prohibits the involvement of this cell line in response to the disease A "significant" immune-aberrant response is one which attenuates the effect of the desired immune response is not acceptable for medical purposes.

"Natural membrane vesicles" means membrane vesicles made from membranes derived from living or dead natural cells.

Although scientific evidence is not available to clarify the reasons for the observed efficacy of the biological reaction modifier of the present invention, it will be appreciated that the biological reaction modifier of the present invention has certain distinguishing characteristics. It is thus clear that the biological reaction modifier according to the invention contains two different classes of particles, natural membrane vesicles and ribosomes. The ribosomes may exist as monomers or as larger polymers, but the mean diameter of the ribosome population is smaller than the mean diameter of the membrane vesicle population. The relative amounts of the two populations appear to influence the efficacy of the product as determined by in vitro standard NK cell samples. Of course, the relative populations also affect the measured mean diameter of the total population of particles. It is believed that a mean diameter greater than 170nm is required to achieve the desired effectiveness. Below this value, the efficacy of the product observed in standard NK cell samples appears to be significantly reduced.

It has also been observed that the size of the bubbles in the vesicle population has an effect on the efficacy of the biological response modifier of the present invention. Thus, in a preferred embodiment of the invention, substantially all bubbles are larger in diameter than 110 nm and the mean diameter of the bubble population is at least 180 nm, preferably about 21 μl. It has been observed that preparations with diameters below these values have a lower than desired activity in in vitro NK cells. It is also clear that preparations containing only membrane vesicles and preparations containing only ribosomes fall below the desired efficacy values, suggesting a possible synergism in the presence of the two populations.

The invention further encompasses a method for producing a biological reaction modifier characterized by growing bacterial cells of the strain Serratia marcescens, harvesting the cultured cells, dissolving endotoxin with a suitable detergent, subjecting the cell concentrate to a treatment which is sufficient to produce ribosomes and to produce membrane vesicles having a diameter of not less than 110 nm, separating the ribosomes and membrane vesicles from the remaining cell material in the cell lysate, and reanalyzing the ribosomes and vesicles in a suitable buffer solution at appropriate concentrations, such as that the mean particle diameter after the particle size analysis is more than 170 nm. The cell dissolution is mechanical.

In particular, the method of the invention is characterized by growing bacterial cells of the species Serratia marcescens in a suitable culture medium, cooling the culture to 0-4 ⁰ C, harvesting the bacterial cells, washing and flooding the harvested cells in a non-toxic, well-tolerated buffer system maintaining a suitable environment for the formation and integrity of the cell membrane vesicles, dissolving the harvested and suspended cells in the presence of a detergent to induce membrane fragment and endotoxin dissociation, producing at least 110 nm diameter membrane vesicles in this dissolution step, cleaning the bacterial cell lysate of cell debris including intact cells, cell walls and membrane fragments, layering the purified cell lysate on density gradient material that is well tolerated by humans and is non-immunogenic, pelletizing the membrane vesicle and ribosome fractions substantially without pelletizing other smaller fractions, aseptically removing the density gradient material, and rinsing and rinsing the membrane vesicles of said region together with the remaining ribosomes in the buffer solution.

Cell dissolution occurs mechanically at a pressure of over 10,000 psi (703.1 kp / cm ² ).

In a further embodiment of the method according to the invention, the preparation of the modifying agent is carried out by cultivating bacterial cells of a strain of a microorganism which is not present in the microflora of the patient to be treated and which has a common bacterial antigen in common with the normal microflora Cropping the cultured cells, dissolving endotoxin with a suitable detergent, subjecting the cell concentrate to cleavage sufficient to produce membrane vesicles having an average diameter of not less than 180 nm, which does not or only poorly cross-react with the patient-forming organisms Separating the membrane vesicles and free ribosomes from the remaining cell material in the cell lysate and rinsing the vesicles and ribosomes in a suitable buffer solution.

embodiment

The invention is explained in more detail below with reference to some examples. In the attached drawing show:

Fig. 1: an electron micrograph showing the modifier of the biological reaction of the invention in an approximately

82,000 times magnification represents; Fig. 2 is a graphical representation of a human killer cell sample upon administration of the bioconversion modifier of the present invention as compared to administration of

leukocyte; Fig. 3: a graphical representation of a standard sample showing the bactericidal properties of the invention

Modifier demonstrates; Fig. 4: a comparison of an antibody-dependent cell cytotoxicity sample (ADCC) when administering the inventive

Modifier of the biological response in contrast to the administration of leukocyte interferon; Fig. 5: a graphic representation of the induction of interleukin 1 (IL-D using the inventive

Modifier of the biological reaction; Fig. 6 is a graph showing the effectiveness of natural killer cell (NK) the effect of depletion of various populations of human peripheral blood mononuclear cells (This study demonstrates, but does not demonstrate, the loss of potency of the NK cell upon removal of NK cells Removal of B-lines or T-cells when applying the biological response modifier of the invention.

The removal of monocytes (not shown) also eliminates this effect, suggesting that the

NK cell activity is mediated via the monocyte-macrophage population.); Fig. 7 shows the results of in vivo studies of the application of the invention in rat prostate squamous cell carcinomas (R3337A) (rapid growth);

Fig. 8: the results of in vivo studies of prostate carcinomas in rats (R 3327 H) (slow growth); Fig. 9: the results of in vivo studies of bilateral prostatic adenocarcinomas (R3327 CF) in rats

illustrates and also results related to other treatments; Fig. 10: A summary of data obtained from the treatment of terminal cancer patients demonstrating the efficacy of the patient's peripheral blood mononuclear cells to kill specific tumor target cells 24 hours after in vivo administration of the invention. The results are comparable to the in vitro

Activation of human peripheral blood mononuclear cells with interleukin II; Fig. 11: a series of graphs of natural killer cell samples showing the efficacy of preparations prepared from the source microorganisms Pseudomonas, E. coli, Enterobacter aerogenes and E. chloacae

Compare alpha interferon; Fig. 12: a series of graphical representations of natural killer cell samples comparing the efficacy of preparations prepared from the source microorganisms Erwin la chrysanthemi and Flavobacterium with interferon.

Figure 1 is an electron micrograph of about 82,000 magnifications showing cross-sectional appearance of the membrane vesicles as well as free ribosomes of different particle size (i.e., monomers and small polymers). The bubbles produced have been measured by two methods:

1. direct measurement of the circularized cross-section seen on the electron micrographs, and

2. mathematically by measuring the particle diffusion coefficients obtained from light scattering analysis using a B190 Grain Screen Classifier (Brookhaven Instrument Corp.).

In a preferred embodiment of the invention, the membrane vesicles and the associated ribosomes are derived from the Gram-negative bacterium Serratia marcescens. Serratia marcescens is a well-known organism, and many strains are available from a number of sources. Of the American Type Culture Collection, Rockville, Maryland 20852, sixty strains are available. This organism provides a particularly suitable source for producing the product with a high level of immunomodulatory / immunotherapeutic activity compared to other bacterial sources and is substantially free of toxicity.

The specific strains of Serratia marcescens actually used to develop the data set forth below were: Serratia 2000 (SM 2000) Cell Technology (Boulder, Colo., USA) strain of the Company, pigmented and non-pigmented variants;

Serratia MSC of unknown origin from stock culture at Mteropolithan State College (Denver, Colorado USA); Serratia marcescens ATCC 60; and

Serratia marcescens CU of unknown origin from stock culture at the University of Colorado (Boulder, Colorado, USA). The desired bacterial membrane vesicles and ribosomes can conveniently and economically be isolated from a suitable source of S. marcescens stationary phase or log phase bacterial cells by the method of the invention, which is a simple, rapid and reproducible method. Reagents are used which provide the necessary conditions for maintaining the integrity and conformation of the isolated specific fractions. Any reagents that may be inherently toxic (unacceptably tolerated) or otherwise affect or otherwise alter an immune response are avoided.

The preferred isolation method involves growing a bacterial cell seed portion in a suitable culture medium at a suitable temperature (30-40 ° C) to a log phase culture (a given growth phase is related to end product yield and consistency as well as the final density of viable cells per volume of processed culture medium); rapid quenching of the log phase culture to 0-4 ° C; harvesting the bacterial cells; washing and flooding the harvested cells to a prescribed density in a suitable buffer system that maintains an environment suitable for the formation and stability of the membrane vesicles and for the stability of the ribosomes; disrupting (disrupting, rupturing) the cells in a suitable cell disruptor or French pressure cell to produce membrane vesicles greater than about 110nm or 0.11 microns in diameter (rupture of the cells)

Cells take place in the presence of a suitable detergent for easier endotoxin dissolution); purifying the bacterial cell lysates of cell debris including intact cells, cell wall fragments and large ribosomal clusters and polysomes; placing the purified cellular lysate containing the vesicles and residual soluble cell constituents on a suitable linear or discontinuous density gradient material that is non-toxic (allowable tolerance) and non-immunogenic; pelletizing the appropriate vesicle and ribosome fractions while minimizing pelleting of unacceptably smaller fractions; the aseptic removal of the density gradient material; rinsing the pelleted fraction and then uniformly rinsing the membrane vesicles and ribosomes with minimal disruption in a suitable buffer system.

In carrying out the isolation process described above, the rapid quenching of the log phase culture to 0-4 ° C can be accomplished by any suitable means, for example, by the application of a dry ice-alcohol mixture, an acetone-ice mixture, an alcoholic acid. Ice mix or special devices like cooling coils. All subsequent steps are preferably carried out at 0-4 ° C. Cell harvesting may be done by centrifugation, but cell harvesting devices / thickeners may be used instead. Purification of cell debris and isolation of the specific vesicle fraction can all be achieved by centrifugation. The membrane vesicle and ribosome fractions can also be isolated by using grain size exclusion chromatography. Purified cell lysate is purified by gels such as Sephadex G-2S, 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 other similar gels with a molecular exclusion limit of about 5,000 to 40,000,000 Daltons passed. Purified cell lysate is placed in a presaturated column. The membrane vesicles appear in the volume of the interstices, while other proteins, cell debris and detergent are dissolved out in larger volumes. For example, 1 ml of purified lysate is loaded into a 10 ml G-100 Sephadex (TM) column and dissolved out with buffer solution. The membrane vesicles appear in the volume of the interstices. Other proteins and cell products are liberated at higher volumes. The product can be passed repeatedly through the column, but each run will cause at least a two fold dilution of the sample. The product can be thickened (if necessary) by ultrafiltration or by centrifugation. Columns and gels can be prepared in a sterile and endotoxin-free manner as indicated by the gel manufacturers. For example, Sephadex Gel (TM) can be autoclaved at 15 pounds per square inch in the liquid circuit for 15 minutes. The gel material is cooled to room temperature, poured into a micro-cleaned endotoxin-free column and filled at a flow rate of 8-30 ml / h. The column effluent can be tested for endotoxin contamination using the well-known LAL assay.

A suitable buffer system for isolating the membrane vesicle and ribosome fractions of the invention consists of 20 mM MgSO ₄ , 50 mM NH ₄ Cl and 20 mM Tris HCl, pH 7.6; and for the final suspension, the above buffer solution or Tris or phosphate buffered isotonic saline solution having the same pH can be used. The magnesium sulfate can be replaced by any other suitable source of magnesium ions, such as magnesium acetate. The components of the buffer system and their specific concentrations can be changed as long as the integrity of the membrane vesicle and ribosome fraction isolated by the described procedure is maintained. Tris-HCl can be replaced by Trizma 7.1, Trizma 7.2, or any other suitable Tris buffering agent adjusted to a pH of 7.0 to 7.6. Any buffer system / buffer that does not alter the integrity of the membrane vesicles or ribosomes and that are tolerably tolerated by the tissues and the intact organism at the concentration used can be used. The actual pH of the buffer system must be consistent with the maintenance of the bubbles and ribosomes and the tissues into which the material is injected.

The cells are dissolved so as to cause breakage and shearing of the cell to produce the membrane vesicles. Any suitable procedure that produces the desired membrane bubbles may be used. It has been found that a mechanical design of the resolution (lysis), z. In a cell splitter or a French pressure cell, is preferable. The mechanical dissolution (lysis) is carried out in order to achieve sufficient shear to produce the membrane vesicles and associated ribosomes.

A satisfactory dissolution method is the use of a microfluidizer (Microfluidizer 110, Model 110T from Biotechnology Development Corporation). Microfluidization is the dynamic interaction of two bacterial fluid streams in well-defined microchannels, resulting in the dissolution of the bacteria and the production of membrane bubbles of uniform size. The bacterial suspension is pumped through the microfluidizer interaction chamber at 10,000 to 14,000 psi (709 to 984 kp / cm ² ), 6 to 12 times, to ensure the dissolution of the cells and the correct size range of the bubbles. Optimal conditions are 11000 psi (773 kp / cm ² ) and nine passes. A suitable cell concentration for microfluidization (vol. Cell: vol. Cell + vol. Buffer solution) is 0.16. Where a French pressure cell is used, the operating pressure of the cell should ensure a high degree of cell cleavage so that the desired membrane vesicles are formed. A preferred pressure is about 12000 psi (844 kp / cm ² ), pressures in the range of about 10,000 psi to about 35,000 psi (709 to 2460 kp / cm ² ), but are also satisfactory. A satisfactory French pressure cell is the Model No. J 43339,40000 psi (2812 kp / cm ² ) rated operation available from SLM Instruments of Champagne, Illinois. An automatic hydraulic press is used to pressurize the cell, preferably to a nominal level of 12000 psi (844 kp / cm ² ). The pressure must not fluctuate more than about ± 500 psi. Pressures below 12,000 psi (844kp / cm ² ) (cell pressure) result in significantly lower cell lysis and increasingly more vesicles less than about 110nm. The outlet valve is opened to ensure lysate effluent in an amount of about 20 ml per minute. However, a drain of only 1.0 ml / minute is also acceptable (range 1-40 ml / minute). The flow rate must be such that bubbles of the specified size range are provided. The suitable cell concentration range for the run in the French pressure cell (vol. Cells: vol. Cells + vol. Buffer solution) is 0.16 to 0.32.

A detergent should be used to break down endotoxins and membrane fragments into smaller, more easily separable particles. A particularly suitable detergent for use in the cell dissolution process is sodium deoxycholate. A suitable (deoxycholate) final concentration range is 0.15-0.34. A non-toxic (well-tolerated) detergent should be used.

The product of the present invention is substantially free of cell walls, biologically active endotoxin, as determined by various biological and human toxicity studies, and free of cell membrane fragments. The product is also free of intact cells. To achieve this, preferably two centrifugation steps are used. The first centrifugation is as follows:

a) Twenty milliliters of cold bacterial lysates are collected during the dissolution process in a micro-cleaned, sterile polycarbonate flask of a Beckman # 30 rotor at a temperature of 0-1 ° C. The number of such centrifuge bottles thus prepared is determined by the amount of final product to be produced . This Lysatvolumen represents a running, vertical _Rmin. of 72mm and a useful R _max. of 95 mm. The tubes are in a pre-cooled (0-4 ⁰ C) Beckman # 30 rotor at 16000 U / min, using a normal The centrifugation time is so high that the average S value, calculated between R _min and Rmax, is 600. Centrifugation times, which lead to average S values of the lysate below 600, result in one significant product loss, average S-values substantially above 600 lbs, eg, 900 s), there is a risk that the prod Finally, it is contaminated with various toxic cell components. As long as there is no product contamination, the higher averages of S will result in increased product yield. An applicable safe range is around 600 to 800S (on average). Using a Beckman # 30 rotor, polycarbonate bottles and a 20.0 mL lysate volume, a purified lysate is recovered at an average of 600 S with W * t = 3.55 χ 10 ⁹ (20 min run at 16000 rpm + 3 min to reach the speed at normal acceleration). This procedure removes intact viable and dead cells and all large cell fragments and subcellular components including large polysomal aggregates with an average S-value greater than 600. The centrifuging process is carried out at 0-4 ⁰ C. The brake is at 500-1000rpm. released, and the rotor can be completely stopped. To avoid swirling around the tube contents, the brake should not be released below 500 rpm. Releasing the brake at rotation rates above 1000 only prolongs the production process. Other rotors and centrifuges may be used (eg Sorvall superspeed and SS-34 rotor, Beckman superspeed centrifuges and equivalent or larger rotors) as long as the method is equivalent.

b) All procedures are carried out at 0-4 ⁰ C. The purified lysate is carefully and aseptically harvested with a cold sterile, non-pyrogenic syringe and 18G spinal needle (or equivalent) (15-16 ml total volume). The needle tip is held a few millimeters below the surface of the lysate and care must be taken that the sides of the centrifuge bottle are not touched and the pellet is not disturbed. The harvested lysate is immediately passed through a cold 0.45 micron filter (eg, Millex HA, Millipore) and collected in one or more cold, sterile, non-pyrogenic, preferably non-binding, plastic / glass tubes.

After the first centrifugation step and filtration, the lysate is then immediately layered on sucrose gradients in micro-cleaned, sterile, cold polycarbonate centrifuge bottles. The diluent used is the buffer system described above. The coated gradients are then subjected to the second centrifuging step.

The production parameters are the following:

(1) Basic product yield (1-1.2mg of product / 1.0ml of purified layered lysate / gradient).

# 50 Beckman rotor:

Gradient: 4-5ml 15% (mass in mass concentration) sucrose layered to 3.0ml 30% (mass in mass concentration) sucrose in polycarbonate bottles - sharp interface.

The sucrose is sterile and endotoxin negative after a quantitative Limulus determination.

Sample size: 1.0ml purified / filtered lysate

Centrifugation: 0-4 ° C; slow acceleration; 38000U / min .; 60 minutes at speed.

(2) To increase the amount of product isolated by centrifuging, the following rotors can be used: # 30 Beckman rotor:

Gradient: 11.0 ml 15% (mass in mass concentration) sucrose (depending on the layered lysate volume) layered to 9.0 ml 30% (mass in mass concentration) sucrose in polycarbonate bottle sharp interface. The sucrose is sterile and endotoxin negative after a quantitative Limulus determination. Sample size: 4-5ml purified / filtered lysate

Centrifugation: 0-4 ⁰ C; slow acceleration; 30000U / min .; 120 minutes at speed. The time at speed (+ or -) is set to maximize yield and minimize contamination. This depends on the volume of the layered lysate. Braking takes place at 1000 rpm. (Not below 500 rpm) Refer to the manufacturer's instructions for braking recommendations for specific centrifuges / rotor combinations.

# 14 Beckman rotor:

Gradient: 100ml 25% (mass in mass concentration) sucrose buffer in polycarbonate bottles. The sucrose is sterile and endotoxin negative after a quantitative Limulus determination.

Sample size: 100ml purified / filtered lysate.

Centrifugation: 0-4 ⁰ C, slow acceleration; 14000U / min .; 12 hours at speed; Braking at 500rpm

during the slowdown.

# 10 Beckman Rotor:

Gradient: 200ml 25% (mass in mass concentration) sucrose buffer in polycarbonate bottles. The sucrose is sterile and endotoxin negative after a quantitative Limulus determination.

Sample size: 200ml purified / filtered lysate.

Centrifugation: 0-4 ⁰ C; slow acceleration; ЮОООи / Міп .; 23 hours at speed; Braking at 500rpm

during the slowdown.

The second centrifugation procedure allows pelletization to isolate the membrane vesicle fraction of specific size and residual ribosomal 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 of these rotors is such that the final product is not essential

contaminated by unwanted cellular components. If the centrifuge times can not be changed to reduce the amount of the above impurities, the following washing procedure can be used: For example, for the # 30 Beckman rotor:

a. The pellets (from the # 30 Beckmann rotor or equivalent rotor, see above) are rinsed with 10 ml of a suitable buffer solution to each of 12 tubes. The rewashed pellets are placed in a sterile, non-pyrogenic, micro-clean container. The centrifuge tubes are then rinsed with 2 ml of a suitable buffer solution for every 10 tubes. The rinse solution is then mixed with the rewashed pellets in the appropriate container. The solution is mixed by repeated pipetting (ten times with a 10 ml syringe) or by pivoting for a few seconds.

b. 5 ml of the rewashed pellets are pipetted into a sterile, nonpyrogenic # 30 rotor polycarbonate centrifuge tube containing 15 ml of appropriate buffer solution.

с The tubes are centrifuged for 25 to 40 minutes with normal acceleration and deceleration.

Other types of centrifuges and rotors may be used as long as the method is equivalent

(For example, the 50.2 Ti Beckman rotor has a shorter run time when using gradients of the same size). Instead of the discontinuous gradient, suitable linear gradients can also be used.

The pelleted fraction isolated by the method described above is washed and then floated in one of the appropriate suspending liquids or buffer solutions described above and filter sterilized with a suitable 0.45 or 0.22 micron filter.

The suspending fluid may be of any suitable pharmaceutical or preferably reagent grade quality and without fractions which could contribute to the precipitation, degradation, aggregation or functional inhibition of the suspended fraction.

The product quantization is based on the nucleic acid content determined according to the following formulas:

E ₂₆₀ = 0.0373 -

Microgram nucleic acid = A _2M / E ₂₆ o

A 0.05 ml sample of the reconstituted product concentrate is diluted in the appropriate resuspending buffer solution such that A _{280 is} between 0.4 and 0.5 for standardization purposes. Using the suspending buffer solution as a blank, the absorbance at 28OnM, 26OnM, 225nM and 215nM is then determined. When the nucleic acid content is determined, the product is diluted to a final concentration of 1.0 mg nucleic acid per 0.5 ml buffer solution. The protein content of the product can be approximated using the following formula:

Microgram protein / ML = 144 (A ₂₁₅ - A ₂₂₅ ).

The average product yield using centrifugation is 1.0-1.2 mg per 1.0 ml of purified lysate. The average of the extinction ratio A ₂₆₀ ZA ₂₈₀ is 1.7626 with a standard deviation of 0.0626. The mean E ₂₆₀ is 0.0232 with a standard deviation of 0.0007.

The reason for the high functional efficacy (described below) of product with bubbles of this size is not fully understood at present.

Nevertheless, it is believed that the specified size of the membrane vesicles, i. a mean diameter of at least 180 nm, and essentially a minimum size of about 110 nm are significant. The preparation according to the invention is essentially free of biologically active endotoxins and cell wall fractions, thereby clearly reducing toxicity problems. For example, the invention causes no cutaneous necrosis / ulceration or death in four day old mice, as shown in Table 1 below. In this study, four-day-old mice (C57B1 / 6) injected the invention in the nuchal (neck) area and were observed for the development of necrosis and / or death. In addition, these animals showed normal weight gain to be another sign of absence of toxic contaminants.

Table 1 Dermon Croquet Test

Invention no. v. animals dose 18h 24 hours NecrosisZTod 48 h 96 h 4 7 6 10 цд ЮОцд 200 цд 0/0 0/0 0/0 0/0 0/0 0/0 OZO OZO OZO OZO OZO OZO

The mean weight of the group 100K at to and 24 hours after the injection was 1.72 g and 2.94 g, respectively. The mean weight of the 200 μς group at to, 24 hours, and 6 days after injection was 1.57 g, 2.16 g, and 4.5 g, respectively. A study of histamine hypersensitivity in 8 week old CFW mice also revealed the lack of toxicity due to endotoxin.

Table 2 histamine hypersensitivity test A.

B.

endotoxin path D / T dosage (Micrograms) E. coli (phenol, Sigma) i.p. 4.6 0.5 E. coli (TCA, Sigma) i.p. 3.6 0.5 invention dosage (Micrograms) i.p. 0/6 100 i.p. 0/6 200 i.p. 0/6 600 i.p. 0/7 2000

'Deaths on the total number of treated mice after one i.p. Assault with 0.5 mg histamine diphosphate (Sigma) 90 minutes after administration. Endotoxin (0.5 micrograms) with i.p. Administration results in 50% death from histamine attack. Mice, rats and guinea pigs typically exhibit hypothermic reactions after administration of endotoxin or infectious attack while developing hyperthermia (1-2 ° C) after administration of the invention.

In addition, single or repeated injections in mice, rats, guinea pigs and humans (over 100 subjects) do not result in anergy, pruritis, DIC (disseminated intravascular coagulation), adjuvant arthritis, anaphylaxis (hypersensitivity), liver enzymes elevation or necrosis or ulceration of local injection sites a dose range of 5 micrograms to 10 milligrams (two to three times a week to individuals). The maximum total dose in a week did not exceed 12 milligrams. When given intravenously to rabbits, bilateral haemorrhagic necrosis of the kidneys has not occurred.

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 endocytosis 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 split into various functional categories, each of which represents an increasingly differentiated cell. These categories (terminology may be different) are: monocytes, normal or resisectant macrophages, stimulated macrophages, activated non-tumoral macrophages, activated tumor-promoting macrophages. The more differentiated the cell, the less persistent it is over the different types of stimulation, but the more limited it may possibly be in effector functions. In contrast, in advanced and chronic disease states, the monocyte-macrophage cell line becomes progressively more resistant to stimulation, as is generally the case with the immune system. Of these stages, only the activated tumoricidal macrophages have been shown to be directly cytotoxic to tumor cells. More specifically, monocyte-macrophage product endocytosis has been observed in association with murine cells. Normal monocytes and resident macrophages were removed from the abdominal cavity in healthy adult C57B1 mice without previous manipulative procedures and without heparin. The initial adherent cell population consisted of more than 90% rounded cells with the remaining cells having the typical macrophage morphology. Round cell populations consisted of monocytes and large numbers of cells that had the appearance of medium sized lymphocytes. All cells endocytosed product and were thus members of the monocyte-macrophage cell line. Within minutes after the in vitro delivery of the product of the invention to a purified fresh population of monocytes and resident macrophages (rounded cell), countless minute blisters that increased in size and number over time began to appear directly under the cell membranes. The phagocytic activity changed in direct proportion to the concentration of the supplied product according to the invention. Autophagocytic death with previous extreme vacuolization while maintaining a rounded cell morphology occurred when the concentration of the product of the present invention exceeded 50 micrograms per 3 ml medium / 10 ⁵ cells. No phagocytic activity or cell activation was observed after endotoxin or BCG cell walls were delivered at different concentrations to identical cell populations. B16 melanoma and kidney epithelial cells did not show phagocytic activity in the presence of the product of the invention or in the presence of endotoxin or BCG cell wall products. It is possible that a receptor specific for a component of the blisters and / or ribosomes of the invention exists which would explain the apparent rapid intemalization of the product of the invention by the monocyte-resident macrophages. Other explanations that include nonspecific physical and / or chemical interactions (such as relative hydrophobicity) are also possible.

Table 3 below summarizes the results of a monocyte-resident macrophage cytotoxicity assay using the product of the invention at concentrations of 0 to 100 micrograms at 5 microgram intervals in 25 cm ² flasks, the 10 ⁶ B16 melanoma cells as target cells, and peritoneal monocytes / resident mouse macrophages as effector cells were administered in vitro. The ratio of effector to target cells is given in the left column. The cells were fixed in 95% ethanol and stained with Mayer's hematoxylin 96 hours after the start of the experiment. The plus-sign results indicate that macroscopic growth was readily apparent at 80% -100% confluence. A negative sign indicates that there was no visible macroscopic growth, whereby a significantly reduced or missing target cell population was confirmed microscopically.

Table 3 Tumor cytotoxicity experiment

E / Z ratio

Visible (confluent) growth at 96 hours

at product concentration (micrograms)

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

It can be seen from the above table that very specific concentrations (5.15 and 50 micrograms) of the product of the present invention cause the differentiation of peritoneal monocytes / resident macrophages to a tumor cytotoxic condition. High levels of cytotoxicity are achieved at effector-to-target ratios of 2: 1 or less. The observed tumor cytocidal effects were extreme vacuolization, cell contraction and / or increase in phase density; they were observed within only eight hours after the delivery of the effective concentrations to the product of the invention.

Previously published studies indicate that activated non-tumor macrophages must be exposed to lymphokines for 18 hours before they kill target cells, with monocytes and resident macrophages unresponsive. The cytotoxic activity produced by the product of the invention in monocytes / resident macrophages is very rapid and unique. The fact that the cultures were incubated for four days prior to termination demonstrates the rapid and efficient induction of effector cell function and long-term target cell monitoring as a result of administration of the product of the present invention. The ability of the product of the present invention to alter levels of various white blood cell counts (WBC) and neutrophils is shown in Table 4. Table 4 contains the white blood cell count and neutrophil counts for a number of terminally ill patients removed before and after administration of the product of the invention. The dosage heights are indicated. The product was administered once a week for three weeks. From the examples in Table 4 it can be seen that the indicated treatment with the product according to the invention substantially increases the number of white blood cells and neutrophilia. It should be noted that the change in the number of white blood cells is not necessarily an increase in all doses or in all patients. (However, it is not a decrease.) However, certain patients respond to the administration of the product of the invention with a substantial increase in the number of white blood cells and neutrophilia, as shown in Table 4.

Table 4

product WBC x 1000 24 hours later neutrophils 24 hours later dose before 27 (% WBC) 92 Patient no. (Mg) 18 6.2 before 70 D59261 1.0 3.8 10.5 88 82 D25940 1.0 8th 13.6 58 90 D78252 1.5 8.6 22.7 67 92 D45851 1.5 13.3 7.6 77 74 3.0 6.7 9.6 90 75 D80822 2.0 6.1 9.4 59 93 3.0 3.2 6.3 59 85 D82088 4.0 3.5 9.6 70.3 84 6.0 8.0 56 D90607 6.0 72

normal ranges: WBC 7.8 ± 3 x 1000 neutrophils 40-70%

Patients with low pre-therapy WBC levels reach normal within 24 hours of therapy. The administration of the product according to the invention also leads to an increase in the number of peripheral blood mononuclear cells (natural killer cells, cytotoxic T cells and / or monocytes) in humans and does not cause thrombocytopenia (data not shown). The product according to the invention thus has no cytostatic effect on the bone marrow.

Proper immune function requires the coordinated interaction of different cell types and the regular generation and release of countless cellular products. It is known that the cells of the monocyte-macrophage cell line play a central role insofar as optimal antibody responses (B-cell function), cell-mediated immunity (T-cell function) and possibly the activity of the natural killer cell (NK cell) in their absence do not occur. Although various cooperative cellular pathways can be elucidated in tissue culture, it is not possible to determine the multiplicity of pathways and the effective control mechanisms in humans as a patient. Since the various specific and / or non-specific immune effector mechanisms occurring in humans are essentially unknown, an agent selected to potentiate / modify the immune function should augment any reactions occurring or, in the absence of responses, reduce the system response threshold and thus the detection, activation and selection of the corresponding Enable effector functions. The ideal activating agent should elicit non-immunogenic, anergic or aberrant hypersensitivity reactions directed to itself or cross-reacting moieties or to promote or direct the selection of a specific, unique effector trajectory. The product of the invention meets these requirements, as none of these phenomena have been observed during clinical tests with significant values.

Figure 2 illustrates the ability of the biological response modifier of this invention to stimulate natural killer cells in humans in a manner comparable to that of leukocyte interferon. The in vitro experiment illustrates the degree of target cell resolution (measured in terms of release of radioactive chromium from the target cells) by the action of the human natural killer cells at various dose levels of the product of the present invention and interferon as shown in the corresponding graphs , The dose value codes are shown below each graph, with the zero dose being the background or zero rate for chromium release from the target cells.

The experiments were carried out according to the experiments described in the literature / 8,9 /. Human K562 cells from the myeloid tumor line were labeled with radioactive sodium chromate and serve as the target cells. The effector cells used were non-depleted human peripheral blood mononuclear cells. The killing is calculated by the following formula:

Experimental Release - Control Release

% Release =: - ^ z - x 100

° maximum release - control release

Experimental release is the count of radioactivity per minute (CPM) in the presence of the product and effector plus target cells, and the control release is the count per minute (CPM), which is determined only by the effector plus target cells. Maximum release is obtained by incubating an aliquot of target cells in saponin, a detergent. The results in this example indicate that, although not as high as interferon at relatively low effector to target cell ratios, it nevertheless shows significant levels of NK cell cytotoxicity in humans in the presence of monocytes at a significant level comparable to that of interferon comes. Statistical analysis of more than 30 product lots shows that the product of the invention is the same or better in producing NK cell cytotoxicity than leukocyte interferon. The NK cell cytotoxicity produced by the product of the invention is similar to interferon-induced cytotoxicity and is dependent on a number of variables, including blood donor age and gender, method of cell storage, and the ratio of cell types present.

Figure 3 shows the bactericidal properties of the biological reaction modifier of the invention. The graph in Fig. 3 shows on the ordinate the logarithmic number of Listeria monocytogenes in peritoneal cells in the mouse. The abscissa represents the time in hours. Listeria monocytogenes is a bacterium that causes acute meningitis in humans with or without associated septicemia. The experiment shown in Figure 3 is a standard experiment for bactericidal activity. The basis of comparison is proteosepeptone, which is known to produce bactericidal macrophages but which is not tolerated by humans. The experiment is described in: Cruprinski, C.J., Henson, P.M., and Campbell, P.A., 1984, J. Leukocyte Bioi. 35: 193rd

The lines in the graph show the change in the number of bacteria in the culture over three hours when exposed to cells taken from the abdominal cavity of mice at different times and for several different pretreatment steps as follows:

A. no peritoneal cells - reference basis of bacterial growth;

B. intraperitoneal injection of proteose peptone 14 days before collection;

C. intraperitoneal injection of the product of the invention 14 days prior to collection;

D. intraperitoneal injection of the product of the invention 7 days prior to collection;

E. intraperitoneal injection of the product of the invention 1 day before collection;

F. Intraperitoneal injection of proteosepeptone 1 day prior to collection.

As can be seen in Figure 3, the upper reference lines represent that the cultures have grown steadily. In cases where the cells were harvested from the mice after 0.2 milligram of the product of the invention had been injected intraperitoneally, there were several decreases in growth differential with time of injection prior to collection, which corresponded substantially to the proteose-peptone control sample at 1 day , These data show that the bactericidal, energizing efficacy of the product remains high even 14 days after a single injection. The prolonged action of the biological reaction modifier of the present invention in the stimulation of macrophages with marked bactericidal activity thus demonstrates its usefulness as a therapeutic agent for bacterial infections.

The ability of the biological reaction modifier of the invention to modulate the activity of antibody-dependent cell cytotoxicity (ADCC) is shown in FIG. Applicable methods are described in the literature. In this experiment, human peripheral blood monocytes are used as the effector cells at the fixed effector-target ratios. The target cells were chromium labeled YAC Murine cells and the antibody used was rabbit anti-mouse cells.

From Figure 4 it can be seen that the level of killing, at least at relatively high effector-target cell ratios, is higher for the product of this invention than for interferon at product dosage levels of 15 micrograms per milliliter and below, but at 20 micrograms per milliliter (one Concentration that leads to autophagocytic death of the monocyte population in this experiment) seemed to be at the zero rate level. The ability of the product of the invention to stimulate the release of interleukin 1 (IL-I) is illustrated in FIG. The experimental procedures used are described in the literature. 13 / Peripheral blood mononuclear cells were prepared from blood taken from the subjects. The cells were incubated in different concentrations of the product according to the invention. After a specified incubation period aliquots of the culture medium were "harvested" and tested at various dilutions in a Blastogeneseversuch. The blastogenesis was determined by counts of radioactive (tritiated) thymidine per minute, from 10 ^е Murinthymozyten after 72 hours incubation in the supernatant fluids of The height of the zero rate is indicated by the solid circles in Figure 5. It can be seen that with the exception of the lowest dose of

1 microgram per milliliter of significant stimulation of IL-1 release occurred. The therapeutic benefits of IL-1 release are documented in the literature.

Figure β illustrates that the cytotoxic effect against K562 targets is due to natural killer cells and is not affected by B or T cells. Human peripheral blood mononuclear cells were used for the effector population at the indicated effector-target ratios. It can be seen that without the product according to the invention or in the absence of the natural killer cells, the zero rate or the blank value is below 20% chromium release. In a non-depleted cell population, administration of the product of the present invention results in a clear stimulation of natural killer cell cytotoxicity. The removal of B or T cells from the population with specific monoclonal antibodies does not significantly affect the degree of cytotoxicity. The removal of NK cells with monoclonal antibodies has been shown to eliminate the cytotoxic effect. Removal of monocytes with monoclonal antibodies (not shown) also results in a loss of NK cytotoxicity, suggesting that the NK cell is activated by the monocyte-macrophage population.

Figure 7 is a graphical representation of the results of an in vivo study of prostate cell carcinoma (fast growing tumors) in rats. It appears that the administration of the product according to the invention has caused the regression of large, proven, rapidly growing carcinomas in rats. All non-treated tumor bearing control animals died within a 14-month period while all treated animals were alive and 4 out of 6 animals had completely receded their tumor. In carrying out these studies, it was found that a weekly paralesional injection of 1 milligram was effective, while a weekly injection of 2 milligrams was not effective. The specific carcinoma treated was the Dunning tumor, subline R3327A, a non-hormone dependent, moderately fast-growing tumor that is metastatic in the advanced stage of the disease. The tumor host was Copenhagen X Fisher F ₁ . This tumor system is a model for the National Prostate Cancer Group. An implantation took place in the left switch. The mean tumor volume was 681 cubic millimeters at the first treatment. FIG. 8 shows an examination of the product according to the invention in connection with slow-growing prostate carcinoma in rats. The tumor hosts were Copenhagen X Fisher F ₁ . This is another tumor model for the National Prostate Cancer Group. The implantation took place in the left switch. The treatment consisted of paralesional injections of 2 milligrams every seven days. The mean tumor volume at the beginning of the treatment was 1138.8 cubic millimeters (> 2 cm ³ ). The specific carcinoma was the Dunning tumor, subline R3327H, a well-differentiated adenocarcinoma with a very slow growth rate.

From Figure 8 it can be seen that the product according to the invention has caused the stabilization / regression of large, proven, slowly growing carcinomas in rats. All untreated comparative tumor bearing animals showed progressive disease, death and weight loss, while all treated animals had disease stabilization, increasing slow regression (indicated by pathology), normal weight and no deaths. In this case, a weekly paralesion injection of 2 milligrams was effective, while a 1.0 milligram weekly paralesion injection was ineffective. Both of the above tumor systems show the expected variance in the effective dose for different tumors.

FIG. 9 shows an examination of bilateral prostate adenocarcinoma tumors in rats. Comparisons are made with a control panel in which hosts received orchiectomy, orchiectomy plus administration of the product of the invention, steroid plus administration of the product of the invention, and administration of the inventive product alone. The comparisons are made with tumors of the left switch and the right switch. From Figure 9 it can be seen that the product according to the invention has caused the regression of very large (in total greater than 5 cubic centimeters), moderately fast-growing adenocarcinomas. The treated animals received weekly paralesional injections of 1 milligram alone on the left vertex tumor stool. The data demonstrate the systemic effect of the therapy (recovering contralateral untreated lesions) using the product of this invention and the lack of inhibition of the action of the biological response modifier by the steroid, dexamethasone. Dexamethasone has not been shown to inhibit the hyperthermic or tumor response in tumor-challenged patients.

Human-scale in vivo human in vivo studies using the product of the present invention were conducted in accordance with the Phase I and Phase II testing and testing procedures promoted by the United States Department of Food and Drugs. Phase I studies were conducted to determine possible toxicity of the product of the invention. According to the Phase I tests, the patients studied were in an advanced disease state, considered to be at the terminal stage, and any previous therapy had failed. These patients received three or six treatments at each of the indicated dosage levels administered subcutaneously every 7 days at a site remote from the tumor. The toxicity tests showed that there were no significant toxicity problems and that the product is well tolerated by humans.

At the same time, 46% of the cases showed a therapeutic effect. The results of Phase I are shown in the following table:

Table 5: Summary of patient responses

Patient no. Dose (mg) diagnosis reaction D44937P 0,25,0,50,1,0 Lung Ca stable D06277 0.25 Prostate Ca low D32770 0.25 Lung Ca none D01088 0.50 Breast Ca stable D15546 0.50 Colon / Liver none D59261 1,0,2,0 Lymphoma (HL) low D25940 1.0 germ cell none

Continuation of Table 5

Patient no. Dose (mg) diagnosis reaction D78252 1.5 Colon / Liver none D45851 1,53,0, adenocarcinoma low 5,0,6,0 Rectum / abd D180849 2,0,3,0 Breast Ca none D80822 2,0,3,0 Lymphoma (LHL) none d81828 2,5,8,0 Lymphoma (NHL) pertiell D62984 2,5,4,0 Lymphomy (HL) stable D82088 2,5,4,0,6,0 glioblastoma low D76339 4,0,6,0 pancreas stable D82568 4.0 Kaposi Sarcoma stable C03343 5,0,8,0 renal cell stable D87354 5,0,1,5 Nodular lymphoma partial D90607 6.0 Adenocarcinoma lung none D91834 6.0 melanoma none A039585 0.25 renal cell stable A768642 0,25,0,5,1,0,2,0 Colon / Liver stable A661981 0.5 Lung Ca, stable a de no A078379 0.5 Parotid-Ca low AOI7231 0.25 Colon Ca none A055001 1.0 lungs stable A666522 1.0 Lung Ca none big cell A403927 1.0 Lung Ca none scaly A032896 1.5 Adenocarcinoma, neck partial A708029 1.5 Bronchioalveolar stable A044130 1.5 Lung Ca stable scaly A223211 2.0 Lung Ca stable D47379 8.0 prostate none D88652 5.0 colon none d93503 8.0 colon stable D94245 8.0 NH lymphoma low D86141 8.0 colon none d96464 8.0 melanoma none D94257 10.0 gallbladder none d97735 10.0 colon none E01514 10.0 NSC lung stable D98520 10.0 colon none E01401 10.0 Sarcoma none D66399 1.0 mediastinal mass none 6134 2.0 lung stable 3374 2.0 Lung / prostate stable unk. primary 4277 4,0,6,0 lung stable 5079 3.0 stomach none 0421 4.0 histiocytoma partial 6918 6.0 gallbladder none 2688 8.0 tongue none 8708 8.0 colon none 9222 8.0 prostate none E07942 2.0 colon none E13150 4.0 lung stable small cell E14356 4.0 neck none E15735 4.0 rectal none D69174 3,0,4,0 colon none E11927 4.0 NSC lung none E14983 4.0 Kaposi Sarcoma none D70727 3,0,4,0 lung stable E07876 2,0,3,0 renal stable E04693 1,0,2,0 chest low E10311 3.0 larynx none

Continuation of Table 5

Patient no.

Dose (mg)

diagnosis

reaction

E09265 3.0 D44296 0.5 d81828 2.0 E07727 1,0,3,0 E03741 0.5 E05487 2.0 E03083 0,5,1,0 E04545 1.0 E05195 2.0

renal none colon none NH Lymphoma none chest none colon none parotid none endometrium stable mixed lymphoma none thymoma low

partial - 50% or greater decline in the disease

low = more than 25%, but less than 50% decrease in the disease

stable = disease not progressive

no = no reaction

The above data show a 46% rate of progression in advanced disease, in end stage patients, in whom all have failed the previous therapy.

The specific dosage level and treatment interval chosen for therapy in a human often varies from patient to patient. The factors that influence dose level and treatment interval include the overall reaction characteristics of the patient's immune system, the type and extent of the disease, the overall health of the patient, the site of the disease, and so on. This is essentially no different from other biological reaction modifiers, and the most advantageous dosing schedules can be determined according to the methods used in conjunction with other biological reaction modifiers and with pharmaceuticals in general.

The fact that efficacy in certain diseases can be very significant compared to other available treatments is evidenced by the data presented in Table 6 below, which relates to the treatment of brain tumor, e.g. Glioblastoma, refer. The data in Table 6 show that in 6 cases of brain tumor in three cases, even with limited treatment, there was a reduction in tumor size. This clearly indicates that the bioconversion modifier of the invention exhibits efficacy with respect to this particular tumor type.

Table 6: Summary of patient reactions - brain tumor

Patient no.

Dose (mg)

diagnosis

reaction

D82088 E11928 E34415 E37376

RT DR

2,5,4,0,6,0 1,0

1.0.3.0 1.03.0

1.0.3.0 1.0.3.0

Brain brain brain brain

Brain brain

low

partial

progress

significant

functional

improvement

progress

partial

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 remain intact in the biological system for only a short time, sufficient to activate the immune response, and then rapidly degrade. Thus, the development of severe, chronic pathophysiological reactions as a result of chronic immune activation is avoided. Degradation is due to the presence of various enzymes (e.g., RNases, proteases, lipases) in various cell types (e.g., monocytes, macrophages, neutrophils) and tissue fluids (e.g., blood, lymph). This degradation is faster at elevated temperatures (eg, body temperature, 37 ° C) and becomes progressively slower as the temperature decreases. At 37 ° C and 95% serum concentration, the product is degraded (in vitro) in less than about 2 minutes, as determined by particle size analysis. The rate of degradation depends on the enzyme concentration and temperature. Once degraded, the effectiveness of the composition as an immunomodulatory agent is significantly reduced.

Figure 10 shows the induction of killer cell cytotoxicity in humans in vivo against three different tumor targets. In most individuals, the peripheral blood mononuclear K562 tumor target cells are killed to some degree in vitro. However, peripheral blood mononuclear cells do not kill the Raji and Colon 38 tumor cell lines. Treatment of patients by in vivo administration of various biological reaction modifiers including interleukin II (IL-2) has not perceptibly altered the degree of killing with respect to the latter two target cells. Only the in vitro treatment of peripheral blood mononuclear cells with specific modification agents in the biological reaction, including IL-2, causes these cells to kill other types of tumor target cells than K562. However, it has been found that the biological reaction modifier of the present invention can alter the ability of peripheral blood mononuclear cells to kill such tumor target cells after in vivo treatment. This indicates that induction of LAK cells (cytotoxic T cells) occurs after administration of the product of the present invention.

Figure 10 is representative of the in vitro measurement of killer cell cytotoxicity in peripheral blood mononuclear cells recovered from patients 24 hours after treatment with the bioconversion modifier of the present invention. It can be seen that all three types of tumor targets undergo substantial killing. The data demonstrate significant in vivo activation of the patient's killer cells. Examination of the effector cell population via specific cell-labeling agents shows the presence of activated monocytes, natural killer cells and LAK cells. The increases in natural killer cells observed in treated patients (previously discussed) and the presence of activated natural killer cells in this experiment demonstrate the endogenous production of interferon in these patients after treatment with the product of the present invention. In addition, the presence of activated cytotoxic "Z cells" (LAK cells) indicates that treatment with the product of the present invention results in endogenous IL-II production Another unique property of the product of the invention is that it activates in vivo Killer cell populations can be maintained in vitro at very low concentrations of IL-2 (2U IL-2 / ml), in contrast to the high levels of IL-2 (500-1000 U IL-2 / ml) required for maintenance IL-2 activated killer cells (LAK cells) are required in vitro.

The peripheral blood mononuclear cells used were recovered from whole blood taken from the patient's biological response 24 hours after injection with the modifier of the present invention. The injections were subcutaneous and ranged from 1 to 4 milligrams per dose and were given three times a week. The experiments were carried out before and 24 hours after the injection. The level of cytotoxicity achieved with in vivo activated cells is consistent with the activity of in vitro lymphokine (IL-2) activated killer cells obtained after culturing human peripheral blood mononuclear cells with IL-2.

The superiority of membrane vesicles and ribosomes derived from organisms of the claimed type such as Serratia marcescens, as compared to other sources, is indicated by the values obtained from comparative experiments with natural killer cells (Figures 11 and 12). The product was prepared according to the method described above from the specified organisms. The bubbles produced from each of these organisms were within the above-identified physical and chemical parameters.

The fact that the source of a given bacterial product affects the type and / or level of biological activity is not new to this area. Different strains of BCG show specific levels of immune activation in animal models as well as clinically. Ribosome vaccines (previously discussed) cause differential activation of the immune system depending on the source organism, and the superiority of polyribosomes obtained from Serratia compared to other sources has been suggested. In the cited publication, the administration of buffer solution alone and bacterial polysomes was indicated , which were derived from other bacterial species (Eschericia coil, Streptococcus pneumoniae, Mycobacterium bovis [BCG], Mycobacterium smegmatis and Propionibacterium acnes), slightly better than a control group of mice receiving no treatment at all (tumor incidence and death within 60 days). On the other hand, at certain doses, polysomes obtained from Serratia marcescens showed the absence of tumor development in 60% or more of the animals and suppression of tumor development in the remaining animals. Since the biological reaction modifier of the present invention is derived from a microorganism which is not part of the microflora of the patient, is not related to the infectious disease in normal individuals, and the common bacterial antigen of the microorganism does not cross react weakly or weakly with organisms which normal microflora, inappropriate immune reactions are avoided. Such reactions include, for example, immune deviation and the appearance of the so-called original antigenic violation commonly observed in currently-developed immunopotentiators (e.g., BCG, C. parym, cell wall fractions). For these reasons, polyribosome aggregates produced from E. coil, M. smegmatis, and Streptococcus pneumoniae are likely to be very poor sources of biological responses, whereas S. marcescens, which is not part of the host's microflora and does not readily cross-react with it, is an excellent source for this Modifier of the biological reaction is. It should be noted that there are other microorganisms which are useful as a source of the membrane vesicles and ribosomes used in the invention. The basic feature of such microorganisms must be that the microorganism is not part of the microflora of the patient. In addition, the common bacterial antigen of the microorganism must not react with the organisms that form the normal microflora, or at least cross-react poorly. The organism should therefore not be one which causes an immune-deviating reaction. The organism must be one which does not or rarely causes a disease in man. And finally, the source microorganism must be one in which the cell membrane forms vesicles of the appropriate size range. Examples of suitable microorganism sources other than Serratia marcescens are: Erwinia chrysanthemi (Pectobacterium) ATCC 14092 and to a lesser extent Enterobacter aerogenes ATCC E13048. Preparations from the above strains show efficacy of the type described above in connection with preparations from S. marcescens. More precisely. Figure 11 shows that E. aerogenes show significantly higher potency than Pseudomonas, E. coli and E. cloacae, albeit with low potency as alpha interferon at specific dosage levels. Similarly, Figure 12 compares E. chrysanthemi (active) with Flavobacterium (not effective) and interferon. Other microorganisms may also be used as sources and processed as above to produce ribosomes and bubbles of the specified size. Using the in vitro experiments described earlier, their effectiveness can be readily assessed to determine whether or not they are suitable as a biological response modifier.

Since the biological reaction modifier of the present invention is free of viable cells, dead cells, cell wall and biologically active endotoxin, recognition due to cross-reactivity is as low as possible, those components which are poorly degradable are eliminated and are the components known to be are highly toxic (eg endotoxin) or that can lead to chronic inflammatory situations, eg. As adjuvant arthritis, granulomas, ulcerations, reduced or excluded. In addition, by minimizing cell surface components, changes in the immune responses that are known to be related to the strain of the organism are eliminated, as well as eliminating phenotypic changes.

The advantages of the biological reaction modifier according to the invention are numerous. By activating cells of the monocyte-macrophage cell line, cell co-operation required for optimal immune function is promoted. Activation of early differentiated cells of the monocyte-macrophage cell line (monocytes, normal / resident macrophages) promotes multiple effector functions. The composition of the product of the invention has so far demonstrated the elicitation of multiple types of effector functions that depend on the experimental conditions or the parameters of the host of the disease.

Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the description of this specification. These modifications are included within the scope of the appended claims.

footnotes

1 Millman, et al., Proc. Soc. Exp. Biol. Mad. 147: 765-768, 1974, Infect. Immun., 14: 929-933, 1976.

2 Thomas, D.W., and Weiss, E., Infect. Immun., 6: 355-363, 1972.

3 Klun, D.L., and Youmans, G.P., RES J. Recticuloendo Thel. Soc., 13: 263-274, 1973.

4 Smith, R.A., and Bigley, N.J., Infec. Immun., 6: 384-389, 1972.

5 Swendsen, C.L., and Johnson, W., Infec. Immun., 14: 345-354, 1976

 β Cancer Research, 40: 1501-1505, 1980.

7 Bergman, R.K., et al. Infection end Immunity, 18: 352-355, 1977.

8 West, W.H., Cannon, G.V., Kay, H.D., Bonnard, G.D., and Herberman, R.B. (1977) Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells. (Natural Cytotoxic Responsiveness of Human Lymphocytes to a Myeloid Cell Line: Characterization of Effector Cells.) J. Immunology 118: 355.

9 of Landazuri, M.O., Silva, Α, Alvarez, J., and Herberman, RB (1979), Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations in that natural cytotoxicity and antibody-dependent cell cytotoxicity in humans are mediated by the same effector cell populations), J. Immunology 123: 252.

10 Fuson, E.W., Whitten, H.O., Ayers, R.D., and Lamon, E.W., (1978), Antibody-dependent cell-mediated cytotoxicity by human lymphocytes. I. Comparison of IgM and IgG-induced cytotoxicity. (Antibody-Dependent Cell-Mediated Cytotoxicity by Human Lymphocytes I. Comparison of IgM- and IgG-Mediated Cytotoxicity.) J. Immunology 120: 1726.

11 Kay, H. 0., Bonnard, W.H., West, W.H., and Heberman, R.B., (1977), A functional comparison of human Fc-receptor-bearing lymphocytes active in natural cytotoxicity and antibody-dependent cellular cytotoxicity. (A functional comparison of human Fc receptor-bearing lymphocytes that are effective in natural cytotoxicity and antibody-dependent cell cytotoxicity.) J. Immunology 123: 252.

12 Conlon, P.J. (1983) A rapid biologic assay for the detection of interleukin 1. (A rapid bioassay for the detection of interleukin 1) J. Immunology 131: 1280.

13 Wood, DD, Bayne, EK, Goldring, MB, Gowen, M., Hamerman, 0., Humes, JL, Ihrie, EJ, Lipsky, PE, and Staruch, MJ, (1985), The four biochemically distrinct species of human Interleukin 1 allae similar biologic activities. (The four biochemically distinct types of human interleukin 1 all exhibit similar biological activities.) J. Immunology 134: 895.

14 Dinarello, CA, 1984, New England J. of Med., 311: 1413-1418.

15 Kreuter, J., and Haenzel, I., 1978, Infec. and immune. 19: 667-675.

Claims (2)

A process for producing a biological reaction modifier characterized by cultivating bacterial cells of a strain of a microorganism which is not present in the microflora of the patient to be treated and which has a common bacterial antigen in common with the normal microflora of the microorganism to be treated Patients are not or poorly cross-reacting patient-forming organisms, harvesting the cultured cells, dissolving endotoxin with a suitable detergent, subjecting the cell concentrate to cleavage sufficient to produce membrane vesicles having a mean diameter of not less than 180 nm Separating the membrane vesicles and free ribosomes from the remaining cell material and ribosomes in a suitable buffer solution. A method according to claim 1, characterized in that the cell dissolution occurs mechanically at a pressure of over 10,000 psi.