PT85057B - PROCESS OF PREPARATION OF A BIOLOGICAL RESPONSE MODIFIER - Google Patents

Abstract

A biologic response modifier (BRM) substantially free of endotoxin, intact cells, cell walls, and cell membrane fragments, which comprises natural membrane vesicles and ribosomes in a suspending buffer. Membrane vesicles are comprised of cellular membrane material endogenous to a selected microorganism which does not evoke a significant immune-deviating response in patients and is substantially non-pathogenic in humans. The ribosomes are also endogenous to the selected microorganism, which is one in which membrane vesicles can be formed from cell membrane material. The BRM, readily endocytosed by the patient's monocyte-macrophage cell line, is produced by cultivating cells of strain Serratia marcescens, harvesting cultivated cells, preferably dissociating endotoxin with appropriate detergent, treating cellular concentrate to produce ribosomes and to produce membrane vesicles with diameters < 110 nm, separating the ribosomes and membrane vesicles, and resuspending them in appropriate buffer such that means particle diameter is > 170 nm.

Description

The present invention relates to a process for the preparation of a biological response modifier for the treatment of humans and animals, which comprises culturing bacteria cells from a strain of microorganisms that are not present in the microflora of the patient to be treated and which have a common bacterial antigen that does not cross-react or that reacts poorly with organisms that constitute the normal microflora of the patient to be treated, harvest the cultured cells, dissociate the endotoxin with a suitable detergent, subject the cell concentrate to sufficient disruption to produce membrane vesicles with an average diameter of not less than 180 nm, separate said free membrane vesicles and ribosomes from the cell material remaining in the cell lysate and resuspend said vesicles and rysomas in a suitable buffer.

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-2 Descriptive MEMORY

The present invention relates in general to the preparation of a pharmaceutical product with immunomodulatory properties. More particularly, the invention relates to the preparation of a biological response modifier (MRB), defined as an agent that modifies the relationship between a disease and a host by modifying the biological response of the host to the disease, thereby resulting in therapeutic effects. MRBs can be divided into two categories: (1) biological or chemical agents that can stimulate or otherwise alter one or more of the host's resistance mechanisms and (2) purified cellular products that demonstrate direct effects on a particular disease. The first group of MRB, included in this invention, consists of agents that activate, increase or otherwise hinder the host's immune reactivity and are generally referred to as immunomodulators.

MRB alone, or in combination with other agents, can be used to increase resistance to pathogens or recovery from invasion of pathogens, modify or induce tolerance to foreign tissue grafts, to increase tumor rejection or stabilization and to inhibit the recurrence of tumors after other forms of therapy, to restore normal mechanisms that assist suppression, or that otherwise promote a normal immune response.

A wide variety of immunomodulators / immunoadjuvants has been developed since about 1900. The vast majority of these agents are of microbial origin (bacteria / fungi) and the most popular are derived from the genera Corynebacteria, Mycobacteria and Nocardia (CMN organisms) ). The various immunoadjuvants are comprised of intact viable cells, dead cells, cell walls, various cell wall fractions, endotoxin, various types of polysaccharides, or subcellular fractions such as ribonucleic acid or ribosomes. Although all of these agents have demonstrated immune enhancing / modulating / adjuvant activity in animal and human tissue culture, against infectious diseases

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-3e neoplastics, they are attributed, universally inconsistencies in production and composition and have moderate to severe toxicity. Regarding the treatment of neoplasms, none of the des. These agents have so far been useful in the treatment of established disease. In addition, these agents typically demonstrate loss of activity with repeated use (anergy), immune deviation, hypersensitivity reactions, development of chronic inflammatory conditions, and / or development of several other undesirable conditions.

Due to the inability to chemically define these

I agents given their impurity and / or complexity, and in an attempt to reduce the variability of immune responses and toxic complications, certain researchers have extracted several specific fractions from these sources or synthesized various components demonstrating immunological activity. Examples of specific fractions that have been investigated include muramyl tripeptide from a cell wall fraction, staphylococcal protein A, various polysaccharides, RNA fraction (1) and ribosomal fraction (2-5).

In relation to the ribosomal fraction, the mechanism of action differs according to the source of ribosomes. Although most ribosomal vaccines need an adjuvant to be active, ribosomal vaccines prepared from Staphylococçus aureus and Neisseria meninqitis do not need it (2). In addition, ribosomal vaccines prepared from Mycobacterium tuberculosis (3) and Salmonella typhimurium (4) appear to induce a mediated cellular response, whereas those prepared from Streptococcus pneumoniae and Streptococcus pyoqenes mediate a humoral response (5). Even if the extracted ribosomal fraction is, theoretically, the active agent, there is considerable controversy in many cases about whether other cellular components (RNA, protein, endotoxin, cell wall) present as contaminants, are responsible for the observed immune reactivity.

Vaccines of known cell fractions may

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-4Sometimes suitable for prophylactic or therapeutic treatment of infectious diseases. However, they are, for various reasons, unsuitable for use as general non-sensitizing immunomodulators for the treatment of neoplastic disease. The presence of cell walls, endotoxin, or poorly degradable components often results in toxicities similar to those obtained with intact organisms. In addition, unwanted complex immune-deviant and immune responses can be eliminated since these vaccines are typically derived from organisms that are part of or that easily cross-react (common antigens) with the host's own microflora. Such vaccines also typically contain fractions with physical and / or chemical characteristics that may be good for general immune enhancing activity.

Urban et al (6) reported the ability of poly-ribosomes (aggregates of ribosomes), obtained from a specific bacterial organism, to suppress the development of cutaneous SaD2 fibrosarcomas in DBA / 2 rats. The poly-ribosome fractions were obtained from cell cultures by osmotic or mechanical lysis of the cells (depending on the type of bacteria), followed by differential centrifugation. Although □ effect on SaD2 murine tumor was positive, the mechanism of the induced biological response could not be determined from the data. Although the toxicity was significantly low, the process described by Urban, et al., Resulted in an extreme variation in the polysome profile (size distribution) and in a very low product yield. There was no consistency in quality, stability and effectiveness.

Kirsh, et al. (7) reported immune stimulation and modulation through the encapsulation of specific antigens or biological response modifiers in liposomes. Liposomes containing drugs were used to treat metastatic cancer (8). However, the treatment of neoplastic disease (cancer) with biological response modifiers isolated or encapsulated in liposomes has so far been a desire.

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-5 courageous, although □ encapsulated MRB is superior in efficacy to non-encapsulated MRB, the limitation of the therapeutic benefit may be due to the fact that immune activation is limited to the macrophage. Tumor cells rarely, if ever, develop resistance to death by macrophages when compared to natural Iciller cells and cytotoxic T-cells. The number of macrophages in these cases was. too low to effectively mediate, on their own, the destruction of large masses of tumors.

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

It is another object of the invention to present an improved biological response modifier that exhibits minimal toxicity and immune deviation.

Another objective of the invention is to present an improved biological response modifier that is capable of manufacturing with consistent quality, stability and effectiveness.

Yet another object of the invention is to provide a process for preparing the preceding biological response modifier that has consistent quality and high yield.

Other objects of the invention will become apparent to those skilled in the art from the following description when related to the accompanying drawings, in which:

Figure 1 is an electron micrograph showing a biological response modifier of the invention with a magnification of approximately 82000x;

Figure 2 is a graph illustrating a test to the natural human killer cell, by comparison after administration of the biological response modifier of the invention and after administration of leukocyte interferon;

Figure 3 is a graph showing a standard assay that demonstrates the bactericidal properties of the MRB of the invention;

Figure 4 is a comparison between the cytotoxicity assay

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-6 antibody dependent cell toxicity (ADCC) after administration of the biological response modifier of the invention and after administration of leukocyte interferon;

Figure 5 is a graph illustrating the induction of interleukin 1 (IL-1) using the biological response modifier of the invention;

Figure 6 is a graph illustrating, in natural killer cell (NK) activity, the depletion effect of various populations of human peripheral blood mononuclear cells. (This study demonstrates loss of NK cell activity after removal of NK cells but not after removal of B-cells or T-cells when using the biological response modifier of the invention. Removing monocytes (not shown) also eliminates the effect, suggesting that the activity of NK cells is mediated by the monocyte-macrophage population);

Figure 7 illustrates the results of studies of the in vivo use of the invention in rat prostate squamous cell carcinoma (R3327A) (rapid growth);

Figure 8 illustrates the results of in vivo studies of rat prostatic carcinoma (R3327H) (slow growth);

Figure 9 illustrates the results of in vivo studies of bilateral rat prostatic adenocarcinoma (R3327CF) also showing results in relation to other treatments;

Figure 10 is a summary of the data obtained from the treatment of terminal cancer patients illustrating the efficacy of the patient's peripheral blood mononuclear cells to kill targeted tumor cells 24 hours after in vivo administration of the product of the invention. The results are comparable with the in vitro activation of human peripheral blood mononuclear cells with interleukin II;

Figure 11 is a series of graphs showing natural killer cell assays comparing, with alpha interferon, the efficacy of preparations obtained from microorganisms

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-7 Pseudomonas, E .. coli, Enterobacter aeroqenes and £ _. chloacae; and Figure 12 is a series of graphs showing natural killer cell assays, comparing, with interferon, the efficacy of preparations obtained from microorganisms Eruiinia chrysanthemi and Flavobacterium.

Generally, the biological response modifier of the invention comprises vesicles of natural membranes and ribosomes in a suspension buffer. Vesicles consist of cell membrane material endogenous to a selected organism. Ribosomes are also endogenous to the selected organism. The biological response modifier is essentially free of endotoxin, intact cells, cell walls and fragments of cell membranes. The selected organism is one that does not prevent a significant immune-deviant response and is non-pathogenic in Hcmem, and is one in which the membrane particles are likely to be formed from cell membrane material and in which the vesicles are easily endocytosed by the monocyte / macrophage cell line. The biological response modifier has an average diameter greater than 170 nm in the particle size analysis.

The invention is capable of directly activating cells of the monocyte-macrophage cell line. Activation of the monocyte-macrophage, or cells of the monocyte-macrophage cell line results in the induction of monocyte-macrophage-mediated bactericidal activities (Figure 3) and in the induction of tumor cytotoxicity (Table 3), altered levels of various white blood cells involved in immune function (eg monocytes, neutrophils (Table 4)) and in vivo induction of target tumor cytotoxicity in humans (K562, Raji, Co38), (Figure 10), induction of natural killer cell-mediated cytotoxicity ( Figure 2), induction of antibody-dependent cellular cytotoxicity (Figure 4), and induction of interleukin I (IL-1) release (Figure 5) and possibly interleukin II (IL-II) (Figure 10).

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-8 For better accuracy, the following terms used in this specification and appended claims are defined:

'* Non-toxic' ’means being at a level of toxicity that is tolerable by the host mammal receiving biological response modifier therapy.

Non-immunogenic means eliciting a sufficiently low immunogenic response or no response at all, such that it does not elicit significant chronic inflammatory responses and unwanted immuno-deviant immensity in the mammalian host.

Mean diameter means the mean diameter measured by Particle Size Distribution Analysis, MSD, on a BI-90 particle size determination device (Brookhaven Instrument Corp.). This measurement involves weighting the intensity of the process of determining average sizes and is explained in more detail in the instrument's Operation Manual, chapter 6, incorporated here as a reference.

Substantially non-pathogenic in man '.' Means rarely or never associated with diseases in humans of normal health. Since most microorganisms are likely to cause opportunistic infection in the right circumstances, such as in people where the immune system is compromised, this definition excludes only those organisms that typically cause non-opportunistic infections.

Essentially free of endotoxin, intact cells, cell walls and fragments of cell membranes signifies a biological activity and sufficiently low level of these fractions to maintain a non-toxic characteristic as defined herein.

Immuno-deviant response means an immune response that is directed out of the disease to be treated. For example, the phenomena of the original antigenic attack may compel the immune system to respond according to historical challenges ”or to respond to ordinary microflora when chal-

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-9lenged by an antigen that is similar to those possessed by the historical challenge or by the common microflora. In addition, polyclonal activation can cause poorly directed and non-specific anamnesic responses. Weakly degradable particles can result in the diversion of the monocyte-macrophage cell line (i.e., reaction to foreign bodies, chronic hypersensitivity) preventing the action of that cell line in response to disease. A Significant immuno-deviant response is one that so attenuates the effect of the desired immune response that it is unacceptable for medical purposes.

Natural membrane vesicles means membrane vesicles prepared from membranes that are derived from natural living or dead cells.

Although there is no scientific evidence to clarify the reasons for the observed effectiveness of the biological response modifier of the invention, it is obvious that the biological response modifier of the invention has certain specific characteristics. Thus, it is obvious that the biological response modifier of the invention contains two distinct particle classes, namely vesicles of natural membranes and ribosomes. Ribosomes can exist as monemers or as larger polymers, but the average diameter of the ribosome population is less than the average diameter of the membrane vesicle population. The relative quantities of the two populations appear to affect the product's effectiveness as determined by standard NK cell assays conducted in vitro. In addition, of course, relative populations affect the measured average diameter of the total particle population. It is believed that it is necessary. An average diameter greater than 170 nm is required to achieve the desired efficiency. Below this value, the product's effectiveness, measured in standard NK cell assays, decreases significantly.

It was also observed that the size of the vesicles in the vesicle population has an effect on the effectiveness of the biological response modifier of the invention. Thus, in a preferred form of the invention, essentially all vesicles exceed 110 nm in diameter and the average diameter of the vesicle population

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-10las is at least 180 nm and is preferably about 210 nm. It was found that preparations with diameters below these values have less than desirable efficacy in z tests

in vitro NK cells. It is also obvious that preparations containing only membrane vesicles and preparations containing only ribosomes have lower than desired levels of effectiveness, suggesting a possible synergism in the presence of the two populations.

Figure 1 is an electron micrograph enlarged approximately 82000x, showing the cross sectional aspect of the membrane vesicles and also showing free ribosomes with various particle sizes (i.e., monomers and small polymers). The vesicles produced were measured by two processes: (1) direct measurement of the circularized cross-section observed in electron micrographs; and (2) mathematically, using measurement of particle diffusion coefficients obtained by light scattering analysis using a BI90 particle analyzer (Brookhaven Instrument Corp.).

In a preferred form of the invention, the membrane vesicles and the associated ribosomes are derived from (i.e., dogenous) chemical-negative bacteria, Serratia marcescens. Serratia marcescens is a well-known organism and many strains are available from various sources. Sixty strains are available at the American Type Culture Collection, Rockville, Maryland 20852. This organism gives rise to a particularly suitable source for the production of the product with a high level of immuno-modulating / immuno-therapeutic activity when compared to other bacterial sources. , and is virtually free of toxicity.

The specific strains of Serratia marcescens currently used in the development of the data presented below were:

Serratia 2000 (SM2000) Cell Technology (Boulder, Colo, rado, USA), variants of pigmented and non-pigmented strains within;

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-11SERP MSC of unknown origin obtained from a culture stored at Metropolitan State College (Denver, Colorado, USA);

Serratia marcescens ATCC No. 60; and

Serratia marcescens CU of unknown culture origin stored at the University of Colorado (Boulder, Colorado, USA).

The vesicles of bacterial membranes and desired ribosomes can be conveniently and economically isolated from a suitable source of S. bacterial cells. marcescens, in stationary or log phase by means of the process of the invention, which is a simple, fast and reproducible procedure. Reagents are used that provide the necessary conditions for maintaining the integrity and conformation of the specific isolated fractions. Any reagents that may themselves be toxic (tolerated unacceptably) or that influence in any way or otherwise alter an immune response are avoided.

The preferred isolation procedure involves cultivating a bacterial cell sowing lot in a suitable culture medium at an appropriate temperature (302C-40SC) to obtain a culture in the log phase (a given growth phase is related to the yield and consistency of the final product as well as the final density of viable cells per unit volume of the processed culture medium); rapidly cool the culture in the log phase to 0-42C; collect bacterial cells; washing and suspending the harvested cells to a specified density in a suitable buffer system that maintains a suitable environment for the formation and stability of the membrane vesicles and for the stability of the ribosomes; rupture by opening (lysis) the cells in a suitable cell disruption device or in a French pressure cell to produce membrane particles with a diameter greater than about 110 nm or 0.11 micron (cell disruption occurs in the presence of a suitable detergent to facilitate dissociation

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-12 endotoxin action); remove bacterial lysates from cell debris, including intact cells, fragments of cell walls and large aggregates of ribosomes and polysomes, deposit the clean cell lysate containing the vesicles and the remaining soluble cell constituents, on a suitable density gradient material linear or discontinuous, which is non-toxic (acceptable tolerance); and not immunogenic; aggregate the appropriate vesicle and ribosome fractions while minimizing the aggregation of less unacceptable fractions; aseptically remove the density gradient material; wash the aggregate fraction and then resuspend the membrane vesicles and ribosomes evenly with minimal disruption, in a suitable buffer system.

By conducting the isolation procedure described above, the culture in the log phase can be rapidly cooled to 0-4 ° C by any suitable means, such as using a dry ice-alcohol mixture, a mixture of acetone- ice, an alcohol-ice mixture, or special devices such as cooling coils. All subsequent steps are preferably carried out at 0-42C. Cell collection can be performed by centrifugation, or cell collection / concentration devices can be used instead. The cleaning of cellular debris and the isolation of the fraction of specific vesicles can all be carried out by centrifugation.

The ribosome and membrane vesicle fractions can also be isolated using size exclusion chromatography. The clarified cell lysate is passed through gels such as Sephadex G-25, G-50, G-100, G-200, Sepharose 2B, Sephacryl S-200, Sephacryl-500 Biogel P-30, Sepharose 4B, TSK HW-75F Fractogel (all trademarks) or any other similar gels with a molecular exclusion limit of about 5000 to 40,000,000 daltons. The clarified cell lysate is loaded onto a pre-saturated column. The membrane vesicles appear, 100 in the free volume while the other proteins, residues

4 ’

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-13 cell phones and detergents are eluted in larger volumes. For example, 1 ml of clarified lysate is loaded onto a 10 ml column of Sephadex G-100 (TM) and eluted with buffer. The ve. membrane particles appear in the free volume. Other proteins. and cellular products elute to larger volumes. The product can be passed through the column repeatedly but each passage causes at least a double dilution of the sample. The product can be (if necessary) concentrated by ultrafiltration or by centrifugation.

The columns and gels can be prepared in sterile and endotoxin-free ways as specified by the gel manufacturers. For example, you can treat the Sephadex (TM) gel in an autoclave at 103 kPa in a liquid cycle for 15 minutes. The gel material is allowed to cool to room temperature, poured onto an endotoxin-free micro-clean column and packaged at a flow rate of 8-30 ml / hour. Endotoxin contamination of the column effluent can be verified by the well-known LAL assay.

A suitable buffer system for isolating the membrane and ribosome vesicle fractions of the invention consists of 20 mM MgSO4, 50 mM NH4Cl and 20 mM Tris HCl, pH 7.6; and for the final suspension, this same buffer or isotonic saline solution buffered with Tris or phosphate can be used. The magnesium sulfate can be replaced by any other suitable magnesium ion source, such as magnesium acetate. The components of the buffer system and their specific concentrations can vary as long as the integrity of the ribosome fraction and isolated membrane vesicle is maintained through the procedure described. You can replace it. replace Tris-HCl with Trizma 7.1, Trizma 7.2 or any other. another suitable Tris buffer agent adjusted to pH 7.0 to 7.6. Any buffering agent / system may be used which does not alter the integrity of the membrane vesicles or ribosomes and which is acceptably tolerated by the tissues and intact organism, at the concentration used. The actual pH of the breadboard system must be compatible with the maintenance of vesicles and ri66 328

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-14bosomes and the tissues in which the material is injected.

The cells are lysed in order to cause the cell to fracture and cut, in order to produce the membrane vesicles. Any suitable technique that produces the desired membrane vesicles can be used. It was found that it was preferable to carry out the lysis mechanically, for example, in a cell disruption device or French pressure cell. The conducted mechanical lysis is operated in order to give a sufficient cut to produce the associated membrane vesicles and ribosomes.

A satisfactory lysis procedure is to use a Micro fluidizer 110, Model 110T from Biotechnolo gy Development Corporation. Microfluidification is the dynamic interaction of the two streams of bacterial fluids in precisely defined microchannels resulting in the lysis of bacteria and the production of uniformly sized membrane vesicles. The bacterial suspension is transferred by pa pump. for the microfluidizer interaction chamber at a pressure between 69 MPa and 97 MPa, 6 to 12 times for. insure cell lysis and the proper shape of vesicle sizes. The optimum conditions are 76 MPa and nine passes. A cell concentration suitable for microfluidification (cell volume; (cell volume + buffer volume)) is 0.16.

When using a French pressure cell, the operating pressure of the cell must cause a high degree of cell disruption in order to form the appropriate membrane vesicles. A preferred pressure is about 83 MPa, but pressures in the range of 69 to about 241 MPa are also satisfactory.

A satisfactory French pressure cell is model n3. 343339, with a capacity of up to 276 MPa, sold by S.L.

M. Instruments in Champagne, Illinois. An automatic hydraulic press is used to pressurize the cell, preferably at a nominal value of 83 MPa. The pressure is not allowed to oscillate more than about +. 3.5 MPa. Pressures below 83 MPa (cell pressure) result in substantial lysis | 66 328

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-15% lower than cells and progressively more vesicles appearing below about 110 nm. The outlet valve is opened to allow the lysate to escape at a flow rate of about 20 ml per minute. However, an output flow rate as low as 1.0 ml / minute is also acceptable (range 1 to 40 ml / minute). The flow rate must produce vesicles of the specified range of dimensions. The cell concentration range suitable for passage in the French pressure cell is 0.16 to 0.32 (cell volume: (Cell volume + buffer volume)).

A detergent should be used to dissociate endotoxins and membrane fragments into smaller, more easily separable particles. A non-toxic detergent particularly suitable for use in the cell lysis procedure is sodium deoxycholate. A suitable final concentration range (of deoxycholate) is 0.15 to 0.3%. A non-toxic (well-tolerated) detergent should be used.

product of the invention is essentially free of cell walls; biologically active endotoxin as revealed by various biological assays and human toxicity studies; and fragments of cell membranes. The product is also free of intact cells. To achieve this, two centrifugation steps are preferably used. The first centrifugation is done as follows:

(a) 20 ml of cold bacterial cell lysate are collected during the lytic process in a polycarbonate bottle with Beckman N9 30, 0-1SC, micro-clean, sterilized. The number of such prepared centrifuge bottles is determined by the amount of final product to be produced. This volume of lysate represents a vertical ^R _m j_ _n , in operation of 72 mm and a useful R _max of 95 mm. The tubes are centrifuged in a pre-cooled (0-42C) Beckman N2 30 rotor at 16000 rpm, using normal acceleration, in a Beckman ultracentrifuge. The centrifugation time should give an average value of S clarified between R. and R _x of 600. The maximum centrifugation values that give mean values of S of

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-16 flats less than 600 result in significant product losses. S values significantly above 600 (eg 900S) can lead to contamination of the final product with various toxic cellular components. As long as there is no contamination of the product, the higher S values will give a higher product yield. A safe and useful range is around 600-800S (average). Using a Beckman N2 30 rotor, polycarbonate bottles and a volume of lysate of 20.0 ml, a clarified lysate with 600S is obtained on average with W t of 3.55 x 10 (20 minutes operation at | 16,000 rpm + 3 minutes to reach speed with normal acceleration). This procedure removes viable cells, dead cells and cells and all large cell fragments and subcellular components including large polysome aggregates with an average S value greater than 600. The centrifugation procedure is performed at 0-4Â ° C. The brake is released at 500-1000 rpm and the rotor is allowed to stop. To avoid swirling the contents of the tube, the brake must not be released below 500 rpm. Releasing the brake at rpm values above 1000 only prolongs the production process. Other rotors and centrifuges (eg Sorvall Superspeed and an SS-34 rotor, Beckman Superspeed centrifuges and rotors of equivalent or greater capacity) may be used, provided the methodology is equivalent.

(b) All procedures are carried out at 0-4 ° C. The clarified lysate is collected carefully and aseptically (15-16 ml of total volume), with a cold, sterile, non-pyrogenic syringe and an 18G spinal needle (or equivalent). The tip of the needle is kept a few millimeters below the surface of the lysate during collection and care must be taken to avoid touching the side walls of the tube. centrifugation screen and not to disturb the aggregate. The collected liquid is passed immediately through a cold filter of 0.45 ytun (for example Millex-HA, Millipore) and collected in cold, sterile, non-pyrogenic tube (s), plastic / glass, preferably non-binding (s).

After the first centrifugation step and after filtering

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-17tion, the lysate is immediately deposited in carose gradients in polycarbonate, micro-clean, sterilized and cold centrifugation bottles. The diluent used is the buffer system described above. The stratified gradients are then subjected to the second centrifugation step.

The isolation parameters of the product are as follows:

(1) basic product yield (1-1.2 mg of product / 1.0 ml of deposited clarified lysate / gradient).

Beckman NQ 50 rotor:

gradient: 4-5 ml of sucrose at 15% by weight, afterwards.

over 3.0 ml of 30% by weight sucrose in polycarbonate bottles - sharp interface. Sucrose is sterile and without endotoxin as revealed by a quantitative Limulus assay.

Sample volume: 1.0 ml of clarified / filtered lysate.

Centrifugation: 0-42C, slow acceleration; 38000 rpm for 60 minutes at speed.

(2) to increase the amount of product isolated by centrifugation, the following rotors can be used:

Beckman N9 30 rotor:

gradient: 11.0 ml of sucrose at 15% by weight (depending on the volume of lysate deposited) deposited on 9.0 ml of sucrose at 30% by weight in polycarbonate bottles - sharp terface. Sucrose is sterile and without endotoxin as determined by a quantitative Limulus assay.

Sample volume: 4-5 ml of clarified / filtered lysate.

Centrifugation: 0-42C; slow acceleration; 30,000 rpm;

120 minutes at speed. The speed time (approximately) is adjusted to maximize throughput and minimize contamination. This depends on the volume of lysate deposited. The unlock point occurs at 1000 rpm. (Do not

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File 46667 —18— greater than 500 rpm). See manufacturers' instructions for unlocking recommendations for specific centrifuge / rotor combinations.

Beckman Ne 14 rotor:

gradient: 100 ml of 25% by weight sucrose layer in polycarbonate bottles. Sucrose is sterile and without endotoxin as determined by a quantitative Limulus assay.

Sample volume: 150 ml of clarified / filtered lysate.

Centrifugation: 0-42C, slow acceleration, 14000 rpm, hours at speed, unlocking at 500 rpm during deceleration.

Beckman NQ 10 rotor:

gradient: 200 ml of a 25% by weight sucrose layer in polycarbonate bottles. Sucrose is sterile and without endotoxin as determined by a quantitative Limulus assay.

Sample volume: 200 ml of clarified / filtered lysate.

Centrifugation: 0-4 ° C, slow acceleration, 10,000 rpm;

hours in speed, unlocking at 500 rpm during deceleration.

second centrifugation procedure allows isolation, through aggregation, of the white mejn vesicle fraction of specified size and of the residual ribosome fraction, leaving smaller fractions, such as DNA fragments, RNA fragments, protein fragments, fragments of pa . networks of cells and fragments of membranes in the supe regions. discontinuous gradient. The specific centrifugation time in these rotors is such that the final product is not significantly contaminated by undesirable cellular components. If the centrifugation times cannot be changed to reduce the amount of the contaminants mentioned above, the following washing procedure can be used :

ç

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-19For example, for the Beckman N9 30 rotor:

The. The aggregates (from the Beckman Rotor N9 30 or equivalent rotor, see above) are resuspended with 10 ml of suitable buffer for each 12 tubes. The resuspended aggregates are transferred to a sterile, non-pyrogenic micro-clean container. The centrifuge tubes are then washed with 2 ml of suitable buffer for every 10 tubes. The washing is then combined with the resuspended aggregates in the appropriate container. Mix the solution repeatedly using a pipette (10 times with a 10 ml syringe) or stir for a few seconds.

B. transfer, with the aid of a pipette, 5 ml of the resuspended aggregates to a sterile, non-pyrogenic polycarbonate centrifuge tube, with an NB 30 rotor, containing 15 ml of suitable buffer.

ç. tubes are centrifuged for 25 to 40 minutes, with normal acceleration and braking.

Other types of centrifuges and rotors can be used provided the methodology is equivalent (for example, the Beckman 50.2 Ti rotor leads to a shorter operating time using the same size gradients). Suitable linear gradients can be used instead of the discontinuous gradient.

The isolated aggregate fraction is washed according to the procedure described above and then suspended in one of the appropriate suspension liquids or buffers described above and sterilized through a suitable 0.45 or 0.22 æm filter. The suspension liquid can be any pharmaceutically suitable or preferably reagent quality product without fractions that may contribute to precipitation, degradation, aggregation or functional inhibition of the suspended fraction.

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-2 0 The product is quantified based on the nucleic acid content which is determined by the following formulas:

E ₂₆₀ = 0.0373 -D, 0079 (« ₂₆₀ / A ₂₈₀ )

Nucleic Acid micrograms = ^Α 26θ / ^Α

280

A 0.05 ml sample of the resuspended product concentrate is diluted in the appropriate resuspension buffer so that the A _26Q value is between 0.4 and 0.5 for standardization purposes. The absorbances at 280 nm, 260 nm, 225 nm and 215 nm are then determined using the suspension buffer as a control solution. After the nucleic acid content has been determined, the product is diluted to a final concentration of 1.0 mg of nucleic acid per 0.5 ml of buffer. Approximately the protein content can be calculated using the following formula:

micrograms of Protein / ML = 144 (A ₂₁₅ -A ₂₂₅ ).

average product yield using centrifugation is 1.0 to 1.2 mg per 1.0 ml of clarified lysate, the average absorbance ratio Α, ^ θ standard of 0.0626. The average value of E / OO & Oe ^ »^ 232 with a standard deviation of 0.0007.

The reason for the high functional efficacy (described below) of a product with vesicles of this size is not yet fully understood. However, it is believed to be import / A θ óe 1 »7626 with a deviation pa.

have the size of the membrane vesicles as specified, notably an average diameter of at least 180 nm, and essentially a minimum size of about 110 nm (15).

The composition of the invention is essentially free of biologically active e dotoxins and cell wall fractions, clearly reducing toxicity problems. For example, the product of the invention does not induce skin necrosis / ulceration or death in four-day-old rats as shown below in Table 1. In this study, four-day-old C57B1 / 6 mice were injected with the composition of the invention in the nape of the neck (neck) and were observed for verifying the development of necrosis and / or death. In addition

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-21, these animals showed normal weight gain, another indicator of the absence of toxic contaminants

TABLE 1

DERMONECROTIC TEST

----------- Necrosis / Death --------- Invention

N8 of Animals Dosage 18 hr. 24 hr. 48 hr. 96 hr 4 10 / g 0/0 0/0 0/0 0/0 6 100 o / o o / o 0/0 o / o 200 piQ 0/0 0/0 0/0 0/0

The average weights for the 100 yU-g group at tg and 24 hours after injection were 1.72 g and 2.94 g respectively. The average weights for the 200 µg cat _{Q group} , 24 hours and 6 days after injection were 1.57 g, 2.16 g and 4.5 g respectively.

Histamine hypersensitivity assay in CFW rats at 8 weeks of age also showed an absence of toxicity due to endotoxin.

TABLE 2) HISTAMINE HYPERSENSITIVITY TEST

A. DOSAGE.DE

ENDOTOXIN (micrograms) VIA D / T ^a E. coli (PHENOL , SIGMA) 0.5 1 * p * 4/6 E. coli (TCA, SIGMA) 0.5 i.p. 3/6 DOSAGE OF INVENTION (micrograms) 100 1 · p · 0/6 200 i.p. 0/6 600 i.p. 0/6 2000 i.p. 0/7

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File N2 46667 ³ Deaths by total number of rats treated following a challenge ip with 0.5 mg of histamine diphosphate (Sigma) 90 minutes after administration. Endotoxin (0.5 micrograms) administered ip results in 50% of deaths after chalenge with histamine (7). Rats, rats and guinea pigs typically demonstrate hypothermic responses after administration of endotoxin or infectious challenge while developing hyperthermia (1-22C) after administration of the composition of the invention.

In addition, single or repeated injections in rats, rats, guinea pigs and humans (more than 100 patients) did not result in anergy, itching, DIC (disseminated intravascular coagulation), adjuvant arthritis, anaphylaxis (hypersensitivity), elevation of liver enzymes, or necrosis or ulceration of the injection sites, for a dosage range of 5 yvg to 10 mg (administered one to three times a week in humans). The maximum total dose in one week did not exceed 12 mg. No bilateral hemorrhagic kidney necrosis occurred when rabbits were administered intravenously.

The membrane vesicles in the product of the invention are easily endocyted by monocytes and macrophages as seen by phase contrast microscopy. It is known that endocytosis results in alteration of the cell membrane and that the circularization of the membrane in the monocyte-macrophage cell line activates these cells.

The cells of the monocyte-macrophage cell line are artificially broken down into several functional categories, each representing a progressively more differentiated cell. These categories (terminology may vary) are: monocyte, normal or resident macrophage, stimulated macrophage, activated non-tumoricidal macrophage and activated tumoricidal macrophage. The more differentiated the cell, the less refractory it is to various types of stimulation but the more restricted it can become in effector functions. On the contrary, in states of advanced disease and chronic disease, the monocyte-macrophage cell line becomes progressively more reactive.

| 66 328

File N ° 46667 —2 3— fracture to stimulation as with the immune system in general. Out of these states, the activated tumoricidal macrophage was only shown to be directly cytotoxic against the tumor cells.

More specifically, production endocytosis was observed. monocyte-macrophage in relation to murine cells. Normal monocytes and resident macrophages from healthy C57B1 adult rats were collected from the peritoneal cavity, without any previous manipulative procedures and without heparin. The initial population of adherent cells consisted of more than 90% of round cells with the rest having the typical macrophage morphology. The round cell populations consisted of monocytes and a large number of cells with the apa. of medium-sized lymphocytes. All cells endocyte product and were therefore members of the monocyte-macrophage cell line.

A few minutes after the in vitro addition of the product of the invention to a purified virgin population of resident monocyte-macrophage (rounded cell), mere small vesicles began to appear, which increased in size and number over time, just below the membranes. of the cells. The action phagocytic activity varied in direct proportion to the concentration of the added composition of the invention, auto-phagocytic death occurred preceded by extreme vacuolization with maintenance of a round cell morphology, when the concentration of the composition of the invention exceeded 50 by 3 ml of medium / 10 ^ cells. Neither phagocytic activity nor cell activation was observed following the addition of endotoxin or BCG cell walls at various concentrations to identical populations of cells. Epithelial cells of the kidney and melanoma B16 did not show phagocytic activity in the presence of the composition of the invention or in the presence of endotoxin or BCG cell walls. There is a possibility that there is a specific receptor for a component of the vesicles and / or ribosomes of the invention that may be responsible for the apparent rapid internalization of the composition of the invention by resident monocyte-macrophages. They are also possible.

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-24 possible other explanations involving non-specific physical and / or chemical interactions (such as hydrophobicity).

Table 3 below, summarizes the results of a monocyte-macrophage cytotoxicity study in which the composition of the invention was administered in vitro, using concentrations from 0 to 100 µg in 5 µg intervals,

5 to 25 cm flasks containing 10 B16 melanoma cells as targets and peritoneal resident monocyte macrophages of the rat as effector cells. Effector to target ratios are indicated in the left column. The cells were fixed in 95% ethanol and stained with Mayer's hematoxylin 96 hours after the start of the assay. The results indicated with a + sign mean that macroscopic growth with 80-100% confluence was easily visible. A - sign indicates that macroscopic growth was not visible with an absent or significantly reduced target cell population confirmed microscopically.

TABLE 3

TUMOR CYTOXICITY TEST

VISIBLE GROWTH REASON (CONFLUENT) AT 96 H0

and the RAS THE PRODUCT CONCENTRATION (MICROGRAMS) 0 3 5 10 15 20 25 30 35 40 45 50 55 60 65 70-100 2: 1 + 4- - + + 4- 4 · 4- + 4 · 4- + + 1: 1 + 4- - + + + 4 * 4 * 4 ' 4 ’ + 4 · + + 1: 2 + + - + - + d * 4 · 4 4 * 4- - 4 · 4 · + + 1: 4 + + + + + 4- 4- + + 4- 4- 4 · 4 · + +

From the previous table, it can be seen that very significant specific co-concentrations of the composition of the inversion (5, 15 and 50 yx, g) directly induce the differentiation of peritoneal resident monocyte-macrophages into a cytotoxic tumor state. High levels of cytotoxicity are achieved for effector - target ratios of 2: 1 or less. The cytocidal effects of the tumor observed were of extreme vacolization, cell contraction and / or increase in phase density, and were observed only 8 hours after the addition of the cojn.

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-25position of the invention in effective concentrations.

Previously published studies showed that activated non-tumoricidal macrophages required 18 hours of exposure to lymphokines before killing target cells with non-responding resident monocytes and macrophages. The cytotoxic activity induced in resident monocytes / macrophages by the product of the invention is very rapid and unique. The fact that the cultures were incubated for four days before termination demonstrates the rapid and effective induction of the effector cell function and long-term control of the target cell as a result of administering the composition of the invention.

The ability of the product of the invention to change white blood cell count (WBC) levels and neutrophil levels is shown in Table 4. Table 4 indicates WBC and neutrophil levels for a series of terminally ill human patients. observed before and after administration of the composition of the invention. Dosage levels are indicated. The product was administered once a week for three weeks. It can be seen from the examples in Table 4 that the specific treatment with the composition of the invention significantly increases the number of white blood cells and neutrophils. It is noted that the change in the number of white blood cells does not necessarily increase for all dosages or for all patients. (It doesn't decrease). However, some patients, as shown in Table 4, respond to the administration of the composition of the invention with a significant increase in the number of white blood cells and neutrophils.

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-26TABLE 4

Dosage of Invention WBC x 1000 Neutrophils (% WBC) Patient N2. (mg) Before After 24h. Before After 24h 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 70.5 93 6.0 3.5 6.3 56 85 D90607 6.0 8.0 9.6 72 84 Normal ranges: WBC 7.8 + 3 X 1000

Neutrophils 40-70%

Patients with a low number of WBCs before therapy enter the normal range 24 hours after therapy.

Administration of the composition of the invention also results in increased mononuclear count of human peripheral blood (natural killer cells, cytotoxic T-cells and / or monocytes) and does not result in thrombocytopenia (data not shown). The product of the invention thus does not have a cytostatic effect on the spinal cord.

Adequate immune function requires the coordinated cooperation of various types of cells and the sequential production and release of numerous cellular products. Monocyte-macrophage cell line cells are known to be critical in this process since optimal antibody responses (B-cell function), cell-mediated immunity (T-cell function), and possibly killer cell activity (NK) do not occur in their absence. Although it is pos. It is not possible to elucidate several cooperative cell pathways in tissue culture, it is not possible to determine the multiplicity of pathways and operative control mechanisms in the human patient. Since, in the patient, the various effector mechanisms

) 66 328

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-27 specific and / or non-specific immune that occur are essentially unknown, an agent chosen to enhance / modify immune function should increase any responses. in progress or in the absence of responses, decrease the level of response in the system thus allowing the recognition, activation and selection of the appropriate effector functions. The ideal activating agent should not induce anergic or aberrant, immuno-deviant hypersensitivity responses directed at themselves or cross-reactive entities, or promote or direct the selection of a specific single effector pathway. The product of the invention fulfills these requirements since none of these phenomena have been observed to a significant degree during clinical trials.

Figure 2 illustrates the capacity of the res modifier. biological proposal of the invention to stimulate human natural killer cells in a manner comparable to that of leukocyte interferon. The in vitro assay illustrates the degree of lysis of target cells (measured in terms of the release of radioactive chromium from target cells) by the action of natural human killer cells for various dosage levels of the product of the invention and of interferon, shown in respective graphics. The dosage level codes are given below each graph, with the zero dosage level representing the background or escape level of chromium release from the target cells.

The tests were conducted according to those described in the literature (8, 9). Human K562 cells from the myeloid tumor line were labeled with radioactive sodium chromate which served as target cells. The effector cells used were non-depleted peripheral human blood mononuclear cells. Mortality is calculated according to the following formula:

Experimental release- (less) Release control% release = --------------------- x 100

Maximum release - (less)

Release of control

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-28The experimental release is the count per minute (CPM) of radioactivity in the presence of product and effector cells plus target cells, and the control release is the count per minute obtained using only effector cells plus target cells. Maximum release is achieved by incubating an aliquot of the target cell in saponin, a detergent.

The results in this example indicate that although the cytotoxicity induction of human natural killer cells in the presence of monocytes is not as high as with interferon, for relatively lower ratios of cell effect. target cell / cell, it definitely occurs at a significant level comparable to that of interferon. Statistical analysis of more than 30 product batches demonstrates that the product of the invention is equivalent or superior to leukocyte interferon in inducing cytotoxicity of NK cells. The NK cell cytotoxicity of the product is parallel to the interferon-induced cytotoxicity and depends on certain variables including the blood donor's age and sex, cell storage process and proportion of cell types present.

Referring now to Figure 3, the bactericidal properties of the biological response modifier of the invention are illustrated. The graph in Figure 3 shows, in ordinates, the Listeria monocytogenes logo in rat peritoneal cells. In abscissa, time is represented in hours. Listeria monocytogenes is a bacterium that causes acute meningitis in humans with or without associated septicemia. The assay shown in Figure 3 is a standard assay for determining bactericidal activity. The control is peptone proteose which is known to produce bactericidal macrophages but which is not tolerated by man. The test is described in Cruprinski, C.

Henson, P.M., and Campbell, P.A., 1984, J. Leukocyte Biol. 35: 193.

The lines in the graph indicate the change in the number of bacteria in culture for three hours when they are exposed to cells harvested from the rat's peritoneal cavities at various heights and for several different preconditioning steps

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-29tes as follows:

A. without control of bacterial growth, of peritoneal cells;

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

C. intraperitoneal injection of the product of the invention 14 days before harvest;

D. intraperitoneal injection of the product of the invention 7 days before harvest;

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

F. intraperitoneal injection of proteose peptone 1 day before harvest.

As can be seen in Figure 3, the upper lines, representing the controls, indicate that the cultures have grown constantly. The situations in which the cells were removed from the rats after 0.2 mg of the product of the invention were injected intraperitoneally show that growth slows with several slopes depending on the injection time before harvest, essentially coinciding with the control of peptone proteose in the 1 day situation. These data show that the bactericidal inducing activity of the product remains high even 14 days after a single injection. Thus, the prolonged effect of the biological response modifier of the invention on inducing macrophages with pronounced bactericidal activity demonstrates its usefulness as a therapeutic agent for bacterial infections.

Figure 4 shows the ability of the biological response modifier of the invention to modulate the cellular cytotoxicity activity dependent on ADCC antibodies). The procedures followed have been described in the literature (10, 11). In this assay, peripheral human blood monocytes are used as effector cells in the specified target effector ratios. The target cells were murine YAC cells labeled with chromium and the antibodies used were antigen cells

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-30 rabbit mouse.

It can be seen in Figure 4 that the level of mortality, at least for relatively high ratios between effector cells and target cells, is higher with the product of the invention than with interferon for product dosage levels of 15 µg or less per milliliter, but appeared to be at the bottom level at 20 µg / ml (a concentration that results in auto-phagocytic death of the monocyte population in this trial).

The ability of the product of the invention to stimulate the release of interleukin 1 (IL-1) is illustrated in Figure 5. The test procedures followed are described in the literature (12, 13). Peripheral blood mononuclear cells were prepared from blood taken from human subjects to be tested. The cells were incubated with various concentrations of the product of the invention. After a specific incubation period, aliquots of the culture medium were collected and tested in a blastogenesis assay at various dilutions. Blastogenesis was determined by counting per minute of radioactive (tritiated) thymidine absorbed by ¹⁰⁶ murine thymocytes after 72 hours of incubation in the supernatants of the harvested culture. In Figure 5, the bottom level is indicated by solid circles. It can be seen that, with the exception of the minimum dose of 1 / Ag / ml, significant stimulation of IL-1 release occurred. The therapeutic benefits of IL-1 release are documented in the literature (14).

It is illustrated in Figure 6 that the cytotoxic effect against K562 targets is due to natural killer cells and is not affected by B or T cells. Peripheral human blood non-nuclear mg cells were used for the effector population for reasons of effector / specified target. It can be seen that, without the product of the invention or in the absence of natural killer cells, the background or leakage level is less than the release of 20% chromium. With an unreduced cell population, administration of the product of the envy results in an evident stimulation of the cytotoxicity of

) 66 328

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-31 natural killer cells. The removal of B or T cells from the population with specific monoclonal antibodies does not significantly affect the level of cytotoxicity. It is found that the removal of NK cells with monoclonal antibodies eliminates the cytotoxic effect. Removal of monocytes with monoclonal antibodies (not shown) also results in pej? cytotoxicity of NK cells suggesting that the NK cell is activated by the monocyte-macrophage population.

Referring now to Figure 7, a graph of the results of an in vivo study on rat prostate scaly cell carcinoma (fast growing tumors) is presented. It can be seen that administration of the product of the invention induces regression of large, stable, fast-growing carcinomas in rats. All controls with untreated tumors died over a period of 14 months while all treated animals survived and 4 out of 6 animals had a complete regression of their tumors. In conducting these studies it was found that a weekly injection of 1 milligram was effective while a weekly injection of 2 milligrams was not. The specific carcinoma treated was Dunning suhlirihageni R3327A tumor, a non-hormone-dependent tumor, with moderate to rapid growth rate that is metastatic in advanced disease states. The tumor host was Copenhagen X Fisher F1. This tumor system is a model of the National Prostatic Cancer Group. The implantation was carried out on the left flank. The average tumor volume in the first treatment was 681 mm.

Referring now to Figure 8, a state of the product of the invention is illustrated with respect to rat slow-growing prostatic carcinoma. The tumor hosts were Copenhagen X Fisher F ^. This system represents another tumor model of the National Prostatic Cancer Group. The implantation was carried out on the left flank. The treatment consisted of 2 mg paralesional injections every seven days. The average tumor volume at the start of treatment was 1138.8 mm (<2 cm). The specific carcinoma was Dunning tumor R3327H, which is an adenocarcinone well

328

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-32 differentiated, demonstrating a very fast growth rate. so slow.

In Figure 8, it can be noted that the product of the invention induced the stabilization / regression of large slow-growing carcinomas in established rats. All control animals, untreated, with tumors, showed progressive disease, death and weight loss while all treated animals showed stabilization of the disease, slow progressive regressions (indicated by the pathology), normal weight and no deaths. In this case, while the 2 mg weekly paralesional injection was effective, a 1.0 mg weekly parallel injection was not. The two tumor systems mentioned above demonstrated the effective dose variance expected with different tumors.

Referring now to Figure 9, a study of adenocarcinoma tumors, bilateral prostate of the rat is presented. Comparisons are made with a control group with hosts undergoing orchiectomy, orchiectomy plus dosage with the product of the invention, steroids plus dosage with the product of the invention and only dosage with the product of the invention. Comparisons are made with tumors on the left and right flank.

In Figure 9, it can be seen that the product of the invention induced regression of moderately fast, very large growth adenocarcinomas (total volume greater than 5 cm). Treated animals received weekly paralesional injections of 1 mg in the left flank tumor only. The data demonstrate the systemic effect of therapy (regression of untreated contralateral lesions) using the product of the invention and the loss of inhibition of the effect of the res modifier. biological use by the steroid, dexamethasone. There was no inhibition of the hyperthermic or tumoral response by dexa metasone in human patients with tumors.

In vivo human clinical studies have been conducted using the product of the invention according to regulations and procedures for Phase I and Phase II tests promulgated by) 66 328

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United States Food and Drug Administration. Phase I studies were carried out to determine the toxicity, if any, of the product of the invention. According to Phase I trials, the patients studied were in a state of advanced disease, which was considered terminal, and in which all previous therapy had failed. These patients received three to six treatments at each of the indicated dosage levels, administered every 7 days subcutaneously at a site away from the tumor. Toxicity tests did not indicate any significant toxicity problems and revealed that the product was well tolerated by man.

At the same time, however, a therapeutic effect was noted

46% of those submitted Patient N9 cases. The results obtained in Phase I are answer in the following table. TABLE RESPONSE SUMMARY 5 PATIENTS Dosage (mg) Diagnosis D44937P 0.25, 0.50, 1.0 Pulmonary CA stable D06277 0.25 Prostatic CA smaller D32770 0.25 Pulmonary CA none D01088 0.50 Chest CA stable D15546 0.50 Colon / Liver none D59261 1.0, 2.0 Lymphoma (HL) smaller D25940 1.0 Germ cell none D78252 1.5 Colon / Liver none D45851 1.5, 3.0, Adeno CA smaller. 5.0, 6.0 rectum / abd D180849 2.0, 3.0 Chest CA none D80822 2.0, 3.0 Lymphoma (LHL) none D81828 2.5, 8.0 Lymphoma (NHL) partial D62984 2.5, 4.0 Lymphoma (HL) stable D82088 2.5, 4.0, 6.0 Glioblastoma smaller D76339 4.0, 6.0 Pancreatic stable D82568 4.0 Kaposi's sarcoma stable

) 66 328

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C03343 5.0, 8.0 Renal Cell stable D87354 5.0, 1.5 Nodular lymphoma partial D90607 6.0 Adeno CA 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 Pulmonary CA, adeno stable AC78379 0.5 Parotid CA smaller A017231 0.25 Colon CA none A055001 1.0 Pulmonary stable A666522 1.0 Pulmonary CA big cell none A403927 1.0 Pulmonary CA scaly none A032896 1.5 Adeno CA, neck partial A708029 1.5 Bronchialveolar stable A044130 1.5 Pulmonary CA scaly stable A223211 2.0 Pulmonary CA stable D47379 8.0 prostate none D88652 5.0 colon none 093503 8.0 colon stable D94245 8.0 NH lymphoma smaller 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 / pros tata

unknown stable primary

4277

4.0, 6.0 stable lung

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5079 3.0 stomach none 0421 4.0 histiocytoma partial 6918 6.0 gallbladder none 2688 8.0 language none 8708 8.0 colon none 9222 8.0 prostate none E07942 2.0 colon none E13150 4.0 small cell of the lung stable 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's sarcoma none D70727 3.0, 4.0 lung stable E07876 2.0, 3.0 renal stable E04693 1.0, 2.0 chest smaller E10311 3.0 larynx none E09265 -5.0 renal none D44296 0.5 colon none D81828 2.0 NH lymphoma none E07727 1.0, 3.0 chest none E03741 0.5 colon none E05487 2.0 parotid none E03083 0.5 ,. 1.0 endometrium stable E04545 1.0 Mixed lymphoma none E05195 2.0 thymoma smaller partial = 50% reduction or higher of the disease minor = upper reduction 25% but less than 50% of disease stable = disease without progress none = no answer

The data presented above reveal a response rate. of 46 $ in patients with advanced terminal disease, in which all previous therapy failed.

specific dosage level and the chosen treatment interval for therapy in a human patient will vary

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-36 often from patient to patient. The factors that influence the dosage level and treatment interval include characteristics, particular type and extent of the disease, general health of the patient, location of the disease, etc. This is not essentially different from other biological response modifiers and treatment regimens. more beneficial dosages can be determined according to techniques used in relation to these other biological response modifiers and with other pharmaceutical products in general.

The fact that the efficacy can be highly significant in certain diseases when compared to other available treatments is evidenced by the data presented in Table 6, below, in relation to the treatment of the brain tumor, for example glioblastoma. The data in Table 6 indicate that for six cases of brain tumors, three showed a decrease in tumor size even with limited treatment. Es. The fact clearly indicates that the biological response modifier of the invention demonstrates an efficacy with respect to this particular type of tumor.

TABLE 6

SUMMARY OF PATIENT RESPONSES - BRAIN TUMORS

Patient Ne Dose (mg) Diagnosis answer D82088 2.5, 4.0, 6.0 Brain smaller E11928 1.0 Brain partial E34415 1.0, 3.0 Brain progression E37376 1.0, 3.0 Brain significant functional improvement RT 1.0, 3.0 Brain progression DR 1.0, 3.0 Brain partial The vesicles membrane and ribosomes used in prodjj of the invention are easily biodegradable. An advantage

particularly notable of the degradation phenomenon are the particulate populations of the product of the invention that remain

| 66 328

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-37 intact in the biological system only for a period of time sufficient to activate the immune reaction, and then degrade rapidly. Thus, the development of serious chronic pathophysiological reactions is avoided as a result of chronic immune activation * Degradation is due to the presence of several enzymes (eg RNases, proteases, lipases) in various types of cells (eg monocytes , macrophages, neutrophils) and tissue fluids (eg blood, lymph). This degradation is more rapid at elevated temperatures (eg body temperature, 372C) and tor | it becomes progressively slower as the temperature decreases. At 37 ° C and serum concentration of 95%, the product is graded (in vitro) in less than about two minutes as determined by particle size analysis. The rate of degradation is dependent on the concentration of enzymes and temperature. When degradation occurs, the effectiveness of the composition as an immunomodulating agent is significantly decreased.

Referring now to Figure 10, in vivo induction of killer cell cytotoxicity against three different target tumors is shown in humans. For most individuals, peripheral blood mononuclear cells. will kill K562 tumor target cells in vitro to some extent. However, peripheral blood mononuclear cells will not kill the Raji and Colon 38 tumor cell lines. Treatment of patients by in vivo administration of various biological response modifiers, including interleukin II (IL-2) has not been shown to alter the level of mortality in relation to the last two target cells. Only in the in vitro treatment of peripheral blood mononuclear cells with certain biological response modifiers, including IL-2, does it induce these cells to kill other types of tumor target cells other than K562. However, it has been found that the biological response modifier of the invention is capable of altering the ability of peripheral blood mononuclear cells to kill such target tumors following in vivo treatment. This fact suggests that the induction of

) 66 328

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-38 LAK cell (cytotoxic T cell) occurs after administration of the product of the invention.

Figure 10 represents the measurement of killer cell cytotoxicity in peripheral blood mononuclear cells obtained from patients 24 hours after treatment with the biological response modifier of the invention. It can be seen that significant mortality results against the three types of target tumors. The data demonstrate significant in vivo activation of the patient's killer cells. Examination of the effector cell population using specific cell markers reveals the presence of monocytes, kil cells. read natural and activated LAK cells. The increases in natural killer cells observed in treated patients (previously discussed) and the presence of activated natural killer cells in this assay demonstrate the endogenous production of interferon in these patients after treatment with the product of the invention. In addition, the presence of activated cytotoxic T cells (LAK cells) demonstrates that treatment with the product. All of the invention results in the production of endogenous IL-II. Another unique feature of the product of the invention is that in vitro populations of killer cells activated in vivo can be maintained with very low concentrations (2U IL-2 / ml) of IL-2. This contrasts with the high concentrations of IL-2 (500-1000 U IL-2 / ml) necessary to maintain IL-2 activated killer cells (LAK cells) in vitro.

The peripheral blood mononuclear cells used were obtained from whole blood collected from patients 24 hours after infection with the invention's biological response modifier. The injections were subcutaneous and ranged from one to four milligrams per dose, given 3 times per week. Tests were performed before and 24 hours after injection. The level of cytotoxicity obtained with cells activated in vivo compares favorably with the activity of lymphokine-activated killer cells in vitro (IL-2) obtained after culturing human blood mononuclear cells in the peripheral with IL-2.

) 66 328

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-39The superiority of membrane vesicles and ribosomes derived from organisms of the claimed type, such as Serratia marcescens, compared to other sources, is suggested by those obtained from comparative tests with natural killer cells ”(Figures 11 and 12). The product was prepared by the above described process from the indicated organisms. The vesicles prepared from each of these organisms were within the physical and chemical parameters established above.

The fact that the source of a given bacterial product influences the type and / or degree of biological activity is not new in this field. Different strains of BCG show specific levels of immune activation in animal models and 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 has been suggested when compared to other sources (6). In the cited publication, the administration of isolated buffer and bacterial polysomes derived from other bacterial species (Escherichia coli, Streptococcus pneumoniae, Mycobacterium bovis (BCG), Mycobacterium smegmatis, and Propionibacterium acnes) were slightly superior to a control group of rats that did not receive treatment (appearance of tumor and death in 60 days). On the other hand, ospolysomes obtained from Serratia marcescens, in certain dosages, demonstrated the absence of tumors in 60 / or more of the animals and suppression of tumor development in the other animals.

Since the biological response modifier of the invertion is obtained from a microorganism that is not a member of the patient's microflora, it is not associated with infectious diseases in normal individuals, and given that the bacterial antigen common to microorganisms is not or is not weakly as a result of cross-reactions with organisms that are part of the normal microflora, inadequate immune responses are avoided. These responses include, for example, immuno-deviation and the

328

File N9 46667 —4 Οπό of the original antigenic offense that is commonly observed with currently developed immuno-enhancers (eg BCG, Ç. Parvum, cell wall fractions). Probably due to these reasons, polyribosome aggregates prepared from E. coli, M. smegmatis and Streptococcus pneumoniae are very weak sources for responses. biological tests, while S_. marcescens, which is not a part of and does not cross-react with the host's microflora, is an excellent source of the biological response modifier.

It should be noted that there are other microorganisms that are suitable as a source of membrane vesicles and ribs. sums used in the invention. The basic characteristic of this microorganism must be that the microorganism is not a member of the patient's microflora. In addition, the microorganism's common bacterial antigen must not react or at least have weak cross-reactions with organisms that are part of the normal microflora. Thus, the organism must not elicit an immune-deviating response. The organism must be one that does not cause or rarely causes disease in humans. Finally, the source microorganism must be one in which the cell membrane forms vesicles in the appropriate size range.

Examples of sources of microorganisms other than Serratia marcescens are: Erwinia chrysanthemi (Pectobacterium) ATCC 14092; and, to a lesser extent, Enterobacter aeroggnes ATCC E13048. The preparations of the strains mentioned above show activity of the type described above in connection with preparations of S. marcescens. More particularly, Figure 11 shows that E / aerogens demonstrate significantly higher efficiency than Pseudomonas, E .. coli and E ,. cloacae, although lower than that of interferon alpha for specific dosage levels. Similarly, Figure 12 compares E_. chrysanthemi (effective) with Flavobacterium (not effective) and interferon. Other microorganisms can be used as sources and processed as described above to produce ribosomes and vesicles of the specified size. Using the in vitro assays described above, their effectiveness can be easily assessed | 66 32Θ

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-41cia to determine whether or not they are useful as biological response modifiers.

Since the biological response modifier of the invention is free of viable cells, dead cells, cell walls and biologically active endotoxin, recognition due to cross-reactivity is minimized by eliminating components that are poorly degradable and components that are poorly degradable. they are known to be highly toxic (for example endotoxin) or to result in chronic inflammatory conditions, for example adjuvant arthritis, granulomas, ulcerations, are minimized or eliminated. In addition, by minimizing cell surface components, variations in immune responses that are known to be associated with the organism strain and phenotypic variations are eliminated.

The advantages of the biological response modifier of the invention are numerous. Activating cells from the monocyte-macrophage cell line promotes the necessary cellular cooperation for optimal immune function. Activation of newly differentiated cells from the monocyte-macrophage cell line (monocyte, normal / resident macrophage) promotes multiple effector functions. The composition of the invention has so far demonstrated induction of multiple types of effector functions depending on the condition of the assay or parameters of the disease host.

Various modifications of the invention will become obvious to the skilled person, in addition to those presented and described here from the teachings of the specification. It should be noted that these modifications fall within the scope of the appended claims.

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-42NOTES

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

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

3) Klun, D.L. and Youmans, G.P., RES 3. Recticuloendo Thel. Soc., 13: 263-274, 1873.

4) Smith, R.A., and Bigley, N.3., Infec. Immun., 6: 384-389, 1972.

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

6) Cancer Research, 40: 1501-1505, 1980.

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

8) West, W.H., Cannon, G.B., Kay, H.D., Bonnard, G.D. and Herberman, R.B. (1977) Natural cytotoxic reactiuity of human lymphocytes against a myeloid cell line: characterization of effector cells. 3. Immunolaqy 118: 355.

9) deLandazuri, M.O., Silva, A., Alvarez, 3. and Herberman, R.B. (1979) Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations. 3. Immunoloqy 123: 252.

10) Fuson, E.W., VJhitten, H.D., Ayers, R.B. and Lamon, E.W. (1978) Antibody-dependent cell-mediated cytotoxicity by human lymphocytes. I. comparison of Igm- and IgG-induced cytotoxicity. 3. Immunoloqy 120: 1726.

11) Kay, H.D., 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 anti body-dependent cellular cytotoxicity. 3, Immunoloqy 123: 252.

12) Conlon, P.3. (1983) A rapid Biologic assay for the detection.

) 66 328

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13) Wood, D.D., Bayne, E.K., Goldring, M.B., Gowen, M., Hamerman, D., Humes, 3.L., Ihrie, E.3., Lipsky, P.E. and Staruch, M.3. (1985) The four biochemically distinct species of human Interleukin 1 all exhibit similar biologic activities. 3. Immunolooy 134: 895.

14) Dinarello, C.A., 1984, Neui Enqland 3. of Med., 311-1413-1418.

15) Kreuter, 3. and Haenzel, I., 1978, Infec. and Immun. 19:: 667-675.

Claims (14)

1 - Process of preparing an answer modifier. biological stain, characterized by the cultivation of spleen cells, serratia marcescens strains, harvesting cultured cells, dissociating the endotoxin with a suitable detergent, subjecting the cell concentrate to a treatment sufficient to produce ribosomes and to produce membrane vesicles with diameters of not less than 110 nm, separating said ribosomes and membrane vesicles from the remaining cellular material in the cell lysate, and resuspending said ribosomes and vesicles in a suitable buffer, in relative concentrations such that the diameter particle size is greater than 170 nm in the determination of particle size. Process according to claim 1, characterized in that the lysis of the cells is carried out mechanically. 3 - Process of preparation of a biological response modifier, characterized by culturing cells of bacteria of the species Serratia marcescens in a suitable culture medium, cooling that culture to 0-490, harvesting the bacterial cells, washing and suspending cells harvested in a non-toxic and well-tolerated buffer system that man-) 66 328 File Mo. 46667 -44 has a suitable environment for the formation and integrity of membrane cell vehicles, lysing harvested and suspended cells in the presence of a detergent to effect the dissociation of the membrane fragment and the endotoxin, said lysis step being such which produces membrane vesicles with diameters of at least 110 nm, cleaning bacterial cell lysates from cell debris including intact cells, cell walls, and membrane fragments, depositing the clean cell lysate on a density gradient material well tolerated and non-immunogenic in humans, aggregate the membrane vesicle and ribosome fractions without essentially adding other smaller fractions, aseptically remove the density gradient material, and wash and resuspend the said membrane vesicles of said range together with the ribs. remaining sums in the buffer. Process according to claim 3, characterized in that the lysis of the cells is carried out mechanically at a pressure greater than 69 MPa. Process according to the preceding claims, characterized in that a biological response modifier is prepared comprising membrane vesicles and ribosomes suspended in a buffer, said membrane vesicles consisting of cell membrane material endogenous to the Serratia marcescens organism, the said ones being ribosomes also endogenous to the organism Serratia marcescens, the said membrane vesicles having an average diameter of at least 180 nm, the biological response modifier also exhibiting an average particle diameter of natural membrane vesX membranes and ribosomes greater than 170 nm, being said essential biological response modifier, free of endotoxin, intact cells, cell walls and cell membrane fragments. 6 - Process according to claim 5, characterized in that the biological response modifier demonstrates 66 328 File N2 46667 -45a detectable activation of the monocyte-macrophage cell line. Process according to claim 5, characterized in that essentially all said vesicles have a diameter greater than 110 nm. 8 - Process of preparation of a biological response modifier, characterized by the cultivation of bacteria cells from a microorganism strain that is not present in the microflora of the patient to be treated and that has an antigen, in the common bacterial that does not cross-react or that reacts little with organisms that constitute the normal microflora of the patient to be treated, harvested cultured cells, dissociate the endotoxin with a suitable detergent, subject the cell concentrate to sufficient disruption to produce membrane vesicles with a mean diameter not less at 180 nm, said membrane vesicles and free bossomas are separated from the remaining cell material in the cell lysate, and the resuspended vesicles and ribosomes are resuspended in a suitable buffer. Process according to claim 8, characterized in that a biological response modifier is prepared to treat a patient, comprising natural membrane vesicles and ribosomes suspended in a buffer, said membrane vesicles and ribosomes being endogenous to a microorganism. selected that does not produce a significant immune response deviation in the patient and that is essentially non-pathogenic in humans, the said microorganism being also the one in which the membrane vesicles are likely to form from the cell membrane material, said vesicles being easily endocytosed by the patient's monocyte-macrophage cell line, said biological response modifier being essentially free of endotoxin, intact cells, cell walls and cell membrane fragments, with the said biological response modifier having a higher average diameter 66 328 File N5 46667 -46 to 170 nm by particle size analysis. Process according to claim 9, characterized in that the biological response modifier has two populations of main particles having one of these populations. smaller particles made up of ribosomes. but and the other population being made up of natural membrane vesicles with a mean diameter greater than 180 nm. Process according to claim 10, characterized in that each of said populations is represented by essentially different peaks in a size distribution curve determined by particle size analysis. Process according to claim 9, characterized in that essentially all said natural membrane vesicles have a diameter greater than 110 nm. Process according to claim 9, characterized in that the biological response modifier demonstrates detectable activation of the patient's monocyte-macrophage cell line. Process according to claim 9, characterized in that said predetermined microorganism is selected from the group consisting of Serratia marcescens, Erminia chrysanthemi and Enterobacter aerogenes.