CN87104886A

CN87104886A - The preparation method of biological respinse modifier

Info

Publication number: CN87104886A
Application number: CN198787104886A
Authority: CN
Inventors: 理查德·W·厄本
Original assignee: Cell Technology Inc
Current assignee: Cell Technology Inc
Priority date: 1986-06-09
Filing date: 1987-06-09
Publication date: 1988-04-20
Also published as: IL82803A; PT85057B; WO1987007503A1; JPH01500752A; PT85057A; DD273198A5; IL82803A0

Abstract

Introduced a kind of preparation method that is used for the treatment of the biological respinse modifier of humans and animals disease.The product of this method comprises natural film vesicle and the ribosome that is present in the buffer suspension liquid.The film vesicle is made of the interior cells of microorganisms membrane material that comes from selection.Ribosome comes from the microorganism of selection in also being.This biological response modifier does not have endotoxin, intact cell, cell wall and cell membrane fragments basically.Microorganism through selecting can't cause tangible immune deviation reaction in the patient body, and is a kind of to the essentially no pathogenic organism of human body.Film vesicle in the microorganism through selecting is to be formed and can be easy to by patient's mononuclear cell-macrophage cell line endocytosis by the cell membrane material.

Description

The application is the further part of 872, No. 131 applications of the United States Patent (USP) submitted on June 9th, 1986, and between the two on the content And do not overlap.

The present invention generally relates to the method that preparation has the medical product of immuno-modulating properties, especially relate to the method for preparing biological respinse modifier (BRM), BRM is defined as: change the host with therapeutical effect that it was produced the biology anti-Ying And of disease is changed the medicament that concerns between disease and the host.BRM can be divided into two classes: (1) can stimulate or change the biology or the chemical agent of one or more host resistance mechanism; (2) be proved to be the cellular products of purifying already that can directly act on special disease.The invention belongs to first kind BRM, it comprises and can activate, increases or change host immune and learn reactive medicament, generally is referred to as immunomodulator.

BRM can use separately, perhaps can be used in combination with other medicament, to improve, perhaps promote normal immunoreaction to being recovered, change in the resistivity of pathogen or the invasion and attack or inducing toleration, improve tumor rejection or surely tailor-madely after treating, suppress tumor recurrence, recover normal accessory cell-inhibition cell mechanism through other with And to external tissue grafts by pathogen.

Since 1900, many kinds of immunomodulator/immunological adjuvants have been developed.In these medicaments great majority be microorganism (antibacterial/fungus) source and prevailing be to be obtained from corynebacterium, mycobacteria and Nocardia (CMN organism).Various immunological adjuvants comprise complete survivaling cell, dead antibacterial, cell wall, various cell wall fragments, endotoxin, various types of polysaccharide or subcellular fraction fragment such as ribonucleic acid or ribosome.Though all these preparations all have been proved to be in tissue culture, in animal and human's body infectiousness and neoplastic disease had immunostimulant/adjusting/auxiliary activity, because they are being produced and are forming inconsistent and difficult generally use and may have serious toxicity.For the treatment tumor formed, these preparations then had not been of great use aspect the established disease of treatment.Moreover, also proved these preparations through use meeting loss of activity (anergy) repeatedly, immune deviation, hypersensitivity reaction occur, cause chronic inflammatory disease infectious condition and/or other multiple unfavorable conditions.

Because the impure and/or complexity of these preparations and can not be from chemically defining it, and in the trial that reduces immunoreation and toxicity complication, many research worker have been extracted all special parts by these sources, have perhaps synthesized the multiple component with immunologic competence that proved.The example of the special part of having studied comprises that cell wall partly takes off wall acyl group tripeptides, SP, various polysaccharide, RNA part ⁽¹⁾With the ribosome part ^(2-5)

With regard to ribosome part, its mechanism of action is according to ribosomal source and difference.Though most of ribosome vaccines need active adjuvant, the ribosome that is made by staphylococcus aureus and meningococcus (Neisseria meningitis) does not then need.In addition, by mycobacterium tuberculosis (Mycobacterium tuberculosis) and salmonella typhi (Salmonella typhimurium) but the immunoreation of the ribosome vaccine inducing cell that makes mediation, the ribosome vaccine that is made by streptococcus pneumoniae and streptococcus pyogenes (Streptococcus pyogenes) then can mediate humoral immune reaction ⁽⁵⁾Though can think that in theory the ribosome that has extracted partly is an active ingredient, but other cell component (RNA, protein, endotoxin, cell wall) that exists as pollutant about whether has caused observed immunoreactivity problem, still has a lot of disputes in many cases.

Known cell vaccine may be suitable for prevention or treatment infectious disease sometimes.Yet owing to some reasons, the immunomodulator non-sensitization, general general that Ta And is not suitable as the treatment malignant tumor uses.Cell wall, endotoxin or the existence that is difficult for the degraded composition usually can cause similar in appearance to by the toxicity due to the whole organism.Moreover, because this class vaccine generally is to be obtained from this as the part of host microorganism system or be easy to produce with host self microflora the organism of cross reaction (common antigen), so can cause no suitable immune deviation (poor) (immune deviating) and complex immunity reacts.This class vaccine also contains some may the not too suitable physics and the part of chemical property for total immune-enhancing activity.

People such as urban once reported the polysome that is obtained from the specific bacteria organism and had the ability that suppresses the development of DBA/2 mouse skin SaD2 fibrosarcoma.Polysome partly is by changing osmotic pressure or with mechanical means (deciding according to bacteria types) dissolved cell, making through differential centrifugation afterwards.Though the effect to the SaD2 mouse tumor is male, still can not be according to the mechanism of the definite biologically of being induced of this data.Though toxicity is very low, the described method of people such as Urban can cause polysome distribution pattern (size distribution) that very Di ， And and also do not reach the concordance of quality, stability and effectiveness of very big variation and productive rate is arranged.

People such as Kirsh ⁽¹⁾Reported by parcel specific antigen or biologically modifier performance immunostimulation and regulating action in liposome.The liposome that comprises medicine has been utilized to treat metastatic carcinoma ⁽⁸⁾Yet use independent or be wrapped in the intravital biologically modifier treatment malignant disease of lipid (cancer) but to make the National People's Congress be disappointed.Though rendeing a service the BRM that is better than not wrapping up through the BRM of parcel, owing to the immune activation to macrophage is limited, so limited therapeutic effect.Compare with natural killer cell and cytotoxic T cell, tumor cell seldom (if any) can produce the resistance that macrophage is killed and wounded.In these cases, mediate destruction to big tumour spread effectively by macrophage itself and seem that just its number very little.

An object of the present invention is to provide a kind of method for preparing the biologically modifier of improvement.

Another object of the present invention provides the method for biologically modifier that a kind of its performance of preparation has the improvement of minimum toxicity and immune deviation.

A further object of the present invention provides the method for the biologically modifier of a kind of all stable on quality, stability and effectiveness, improvement of preparation.

A further object of the invention is to provide a kind of preparation to possess the method for stating the biological respinse modifier before constant-quality and the high yield;

With reference to each accompanying drawing, the one skilled in the art is Jiang Hui Ming Liao other purpose of the present invention according to the Miao Shu And of Xia face.

Fig. 1 is that figure amplifies about 82,000 times according to the electron micrograph of the biological respinse modifier of the present invention's method preparation;

After Fig. 2 is presented at the biologically modifier that the method by the present invention that gives makes and gives LeIF, the result that the natural killer cell of people is performed an analysis.

Fig. 3 describes a kind of standard analytical process, and its proof is by the bactericidal property of the BRM of the present invention's method preparation;

Fig. 4 is comparison at the biologically modifier that the method that gives according to the present invention makes and after giving LeIF, the result that the cytotoxicity of antagonist dependent cell (ADCC) is analyzed;

Fig. 5 biologically modifier that to be the graphic extension utilization make according to the present invention's method is induced leukocyte homoatomic 1(IL-1);

(the research proof is when using biologically modifier of the present invention, remove the NK cell, and do not remove NK cytoactive loss situation behind B cell or the T cell to the influence of NK cell (NK) cytoactive in the various human peripheral blood mononuclear cell of Fig. 6 graphic extension colony.Remove the mononuclear cell (not shown) and also get rid of this effect, prompting NK cytoactive is by mononuclear cell-macrophage colony mediation);

Fig. 7 shows the result with method interior therapeutic rat prostate squamous cell carcinoma of the present invention (R3327A) (ramp);

The result of Fig. 8 display body internal therapy rat prostate cancer (R3327H) (slowly growth) research;

Fig. 9 graphic extension shows the result who combines application with other Therapeutic Method simultaneously to the result of rat bilateral adenocarcinoma of prostate (R3327CF) interior therapeutic research.

Figure 10 is the summary to treatment data that patients with advanced cancer obtains, and graphic extension was complied with behind the biologically modifier that method of the present invention makes 24 hours in vivo, and patient's peripheral blood lymphocytes is killed and wounded the effectiveness of special tumor target cell.This result is with to obtain the result with interleukin II vivo activation human peripheral blood mononuclear cell similar.

Figure 11 shows the result of a series of natural killer cell activity detections.Wherein compare with regard to alpha-interferon and the effectiveness that derives from the preparation of Pseudomonas, escherichia coli clostridium perfringen and green sulfur enterobacteria microorganisms such as (E.chloacae) respectively.

Figure 12 shows the result of a series of natural killer cell activity detections.With interferon with compare by the effectiveness of Erwinia chrysanthemi and Flavobacterium gained preparation.

In general, the biologically modifier that makes according to the present invention's method comprises natural membranes vesicle and the ribosome that is present in the buffer suspension liquid.Vesicle is to be made of the interior cell membrane material of being born in through the organism of selection.Ribosome comes from also being through the organism of selection.This biologically modifier does not have endotoxin, intact cell, cell wall and cell membrane fragments in fact.The organism of this selection can not cause tangible immune deviation reaction (immune de-viating response), to the human body no pathogenicity, and film sex vesicle capsule wherein can become And be easy to by mononuclear cell/macrophage system institute endocytosis by cell membrane material shape.According to the average diameter that the analysis showed that the biologically modifier of granular size greater than 170nm.

According to the direct cell of activated mononuclear cell-macrophage system of the biologically modifier of the present invention's method preparation.The cell of activated mononuclear cell-macrophage or mononuclear cell-macrophage system can be induced mononuclear cell-macrophage-mediated bactericidal activity (Fig. 3) and cytotoxicity (table 3), change and participate in the various leukocyte of immunologic function (as mononuclear cell, the level of neutrophil (table 4), induced tumor target cell (K562 in human body, Raji, Co38) cytotoxicity (Figure 10), the cytotoxicity (Fig. 2) of inducing natural killer cell mediation, induce antibody dependent cellular cytotoxicity (Fig. 4), induce interleukin I(IL-I) (Fig. 5) and may also have interleukin II (IL-II) release (Figure 10).

In order clearly to set forth the present invention, now that used term definition in this description and the claim that awaits the reply is as follows:

" nontoxic " is meant that toxicity is limited within the mammal energy tolerance level of accepting the treatment of biologically modifier.

" non-immunogenicity " is meant and causes the reaction of enough low immunogenicity or not this reaction, the so just immune deviation that can not occur not expecting, chronic inflammation and hypersensitivity reaction fully in the mammalian hosts body.

" average diameter " is meant according to MSD particle size distribution analytic process at BI-90(Brookhaven Lnstrument Corp.) average diameter that records on the grain graininess detector.This method comprises the intensity weighted to the granularity averaging process, and relevant more detailed explanation can be referring to workbook the 6th chapter of this instrument of classifying list of references as.

" is no pathogenicity basically to human body " is meant for the normal healthy people not to be had or seldom causes with the relevant change of disease.Because most of microbe all can cause opportunistic infection (as in the existing vitiator's body of immune system) under proper condition, so this definition just forecloses those organisms that generally cause non-opportunistic infection.

" immune deviation reaction " is meant at those and departs from by the immunoreation of the disease of treatment.For example, when attacking with a kind of antigen similar to the antigen of previously attacking or common microflora is had, original antigen fault (Original antigenic sin) phenomenon can cause immune system to produce and previously attack the reaction that conforms to, and perhaps common microflora is reacted.In addition, the polyclone activation can cause non-specific mistake specific aim anamnesis reaction (non-specific mis-directed anamnestic respo-nses).The granule that is difficult to degraded can cause the disorder of the monocyte-macrophage cell line reaction and the chronic hypersensitivity of foreign body (promptly to), suppresses cell line to the synergism in the disease reaction.The reaction of " significantly " immune deviation is meant and can makes the effect of expection immunoreation be attenuated to unacceptable degree concerning medical purpose.

" natural membranes vesicle " is meant the film vesicle that the film by that live or dead n cell makes.

Still lack enough scientific evidence though understand fully the reason of usefulness of observed the present invention's biologically modifier, it is clear and definite that biologically modifier of the present invention has some totally different feature this point.For this reason, must know that biologically modifier of the present invention comprises the granule that two classes are different, i.e. natural membranes vesicle and ribosome.Ribosome can be used as monomer or big polymer exists, but the average diameter of ribosome colony is littler than the average diameter of film vesicle colony.According to external NK cell detection results, find that as if the relative quantity of two colonies can influence the effectiveness of product.Certainly, Reference Group also has influence on the average diameter of granule total group.It is believed that average diameter surpasses 160nm for the condition of being absolutely necessary that gets a desired effect.Be lower than this level as average diameter, then the effectiveness of product (according to the NK cell detection results) then significantly reduces.

Also observe simultaneously, the size of vesicle is also influential for the effectiveness of biologically modifier in the vesicle colony.Therefore, the preferred form of the present invention's biologically modifier is diameter in fact and surpasses 110nm, and the average diameter of vesicle colony is at least 180nm, is preferably about 210nm.Observed its effectiveness of preparation also relatively poor (according to external NK cell analysis) that diameter is lower than above-mentioned numerical value.Prove that also the effectiveness that only contains the preparation of film vesicle and only contain ribosomal preparation is lower than the level of expection, two kinds of preparations of this prompting exist simultaneously and have synergism.

Fig. 1 is an Electronic Speculum figure (amplifying about 82,000 times), and its transverse section ， And that shows the film vesicle shows the ribosome with various granularities (being monomer and little polymer).Measure Manufactured vesicle by two kinds of methods: (1) directly measures the cross section of the ring formation of seeing in the electron micrograph; (2) use BI ₉₀Type (Brookhaven Instru-ment Corp.) grain graininess instrument carries out light-scattering analysis, the resulting granules scattering coefficient is carried out art calculating try to achieve.

In the preferred form of the biologically modifier that the method according to the present invention makes, film vesicle and relevant ribosome are for being obtained from gram negative bacteria serratia marcescens (Serratia marcescens) person.Serratia marcescens is the microorganism ， And that knows of people and can obtains many bacterial strains by a large amount of sources.Can (ATCC, Rockville Maryland20852) obtain 60 bacterial strains by American type culture collection.With other bacterial origin relatively, this organism is particularly suitable for as making that performance has high-level immunomodulating/immune therapeutic activity and being product microbe-derived that does not have toxicity basically.

Obtaining the specific serratia marcescens bacterial strain that in fact following data utilized is:

Serratieae 2000(SM2000), Cell Technology Inc. (Boulder, Colorado, USA) chromogenesis of inner bacterial strain and not chromatogenous mutation;

Serratieae MSC is by Metropolitan land grant college (Denver, Colorado, the bacterial strain of the unknown origin that storage culture USA) obtains;

Serratia marcescens ATCC ^#60; With

Serratia marcescens CU, the bacterial strain of the unknown origin that obtains by the storage culture of Colorado university (Boulder, Colorado USA).

Can easily and economically be separated to required bacterial membrane vesicle and ribosome by resting stage or exponential phase serratia marcescens bacterial cell by method of the present invention-a kind of simply, rapidly and repeatably method.Used reagent must provide necessary condition for integrity and the form that keeps separated specific part.Should avoid using any itself may deleterious (can not tolerate) or disturb by any way or change immunoreactive reagent.

Preferred separable programming is included in the appropriate culture medium, the seed cell of culture of bacteria becomes exponential phase culture (the given trophophase relevant with the end-product productive rate, And and consistent with the whole density of survivaling cell in the per unit volume culture medium) under proper temperature (30-40 ℃); The exponential phase culture is cooled to 0-4 ℃ rapidly; The results bacterial cell; The Xi Bao And of washing results is suspended in the suitable buffer system with the cell density of prediction, is suitable for the environment that the film vesicle forms the stability that helps film vesicle and ribosome simultaneously to keep; Broken (dissolving) cell in suitable cell breakage device or French pressurized tank (French pressure cell), surpass about 110nm(or 0.11 micron to make diameter) film vesicle (can at smudge cells in the presence of the suitable detergent, to help the endotoxin decomposition); The bacterial cell lysate of clarification cell debris (comprising intact cell, cell wall fragments, big ribosome aggregation and polysome); Make through clarifying and contain cellular lysate that capsule bubble And remains with the soluble cell composition at suitable linearity or discontinuous density gradient material higher slice, this material is nontoxic (can tolerate) And and is non-immunogenicity; Be settled out suitable vesicle and ribosome part and be settled out unacceptable smaller portions simultaneously as few as possible; Remove density gradient material under the sterile working; Flushing precipitation part is suspended in film vesicle and ribosome in the suitable buffer system under the destructive condition of minimum degree is arranged afterwards again.

When carrying out above-mentioned separation, can make the exponential phase culture be cooled to 0-4 ℃ rapidly by any proper method, for example can use dry ice-alcohol mixture, acetone-ice mixture, ethanol-ice mixture or such as special devices such as cooling spiral pipes.All each steps of continuous back are preferably in 0-4 ℃ and finish.Available centrifugal method harvesting, or use cell harvester/concentrator.Clarification cell debris and separate all available centrifuging of specific vesicle part and carry out.

Also can use screening and get rid of chromatography (Size-exclusion chromato-graphy) diffusion barrier vesicle and ribosome part.By Sephadex G-25, G-50, G-100, G-200, Sepharose 2B, Sepha-crylS-200, Sephacryl-500 Biogel p-30, Sepha-rose 4B, the above-mentioned trade name that is of TSK HW-75F Fractogel() or other to have the molecule exclusion limit be about 5,000 to 40,000,000 daltonian similar gel clarification cellular lysate.To be contained in through the cellular lysate of clarificationization on the pre-saturated post.The film vesicle comes across in the void volume, other protein, cell debris and detergent then with larger volume by eluting.For example 1ml is added in And buffer solution elution Shang the 10mlG-100Sephadex(TM) Zhu through clarifying lysate.The film vesicle comes across in the void volume, and other albumen and cellular products be eluting when larger volume.Can make product cross post repeatedly, but each post of crossing all make the diluted sample twice at least.During as needs, available ultrafiltration or centrifuging enriched product.

Can be according to the method for gel manufacturing firm regulation, preparative column and gel under aseptic and no contaminated with endotoxins condition.For example, can be with Sephadex(TM) gel based on liquid circulation with the pressure autoclaving of 15 pounds of/square inch 15 minutes.Make gel rubber material be cooled to room temperature, the no endotoxic Zhu Zhong And of impouring microcosmic cleaning collects it with 8-30ml/ hour flow velocity.Can check whether the effluent of post has contaminated with endotoxins by known LAL method.Be used to separate the film vesicle of the biologically modifier that the method according to the present invention makes and the suitable buffer system of ribosome part and comprise 20mM MgSO ₄, 50mM NH ₄Cl and 20mMTris-HCl, pH7.6; For preparing final suspension, can use the above-mentioned buffer with identical PH or the isotonic saline solution of Tris or phosphate-buffered.Available any other suitable magnesium ion source thing such as magnesium acetate replace magnesium sulfate.As long as can keep the film vesicle that is separated to by above-mentioned steps and ribosome integrity partly, just can change the composition and the specific concentrations thereof of buffer system.Replace Tris-Hcl as available Trizma7.1, Trizma7.2 or any other suitable Tris buffer agent of being tuned to PH7.0 to 7.6.Ren He And does not change the integrity of film vesicle or ribosome and can be organized under used concentration or the buffer system/buffer agent of complete organism tolerance all can use.The true pH of buffer system must be suitable for the tissue keeping vesicle and ribosome and injected this material.

The method of dissolved cell must be able to cause Xi born of the same parents Po Sui And pair cell to produce shear action, to make the film vesicle.Any proper technology that can produce the film vesicle all can use.Wherein better be mechanical means, as in cell breakage device or French pressurized tank, carrying out.When carrying out mechanical molten born of the same parents, must provide enough shearing forces, to produce film vesicle and relevant ribosome.

A kind of gratifying method is to use the 110T type trace fluidisation device 110 of biotechnology development company (Biotechnol-ogy Development Corp.).The Micro Fluid effect is that two antibacterial liquid stream produce the kinetics interaction in the micro-channel that accurately limits, and produces film vesicle of uniform size to cause Xi bacterium Rong Xie And.10,000 to 14,000Psi(pound/inch ²) under the pressure, the interaction cell Nei And that bacterial suspension is pumped into microfluidization device with pump makes it by cell 6 to 12 times, with the film vesicle of guaranteeing that Xi Bao Rong Xie And obtains having suitable magnitude range.The optimal condition of this operation is that pressure is 11, and 000Psi And is by nine times.The suitable cell concentration of Micro Fluid effect (cell volume: be 0.16 cell volume+buffer volume).

When using the French pressurized tank, the operating pressure of groove should be able to make cell fully broken to form the film vesicle of expection.Preferred pressure is about 12,000Psi, but pressure is 10, and 000Psi to 35 also can obtain satisfied effect in the 000Psi scope.

A kind of French pressurized tank of satisfaction is that (Champagne, rated pressure Illinois) is 40, the J43339 type French pressurized tank of 000Psi available from S.L.M.Instruments.Can use automatic water pressure machine crush cell, preferred normal pressure is 12,000Psi.Pressure oscillation must not surpass approximately ± 500Psi.Pressure is lower than 12,000Psi(cell pressure) then cause cytolysis to significantly reduce and have much more gradually vesicle diameters less than 110nm about.Open outlet valve and flow out with the flow velocity of the about 20ml of per minute, but the rate of outflow also can be low to moderate 1.0ml/ minute (scope is between 1-40ml/ divides) to allow lysate.Used flow velocity must be the film vesicle that can provide certain size range.Suitable cell concentration scope (cell volume: be 0.16 to 0.32 cell volume+buffer volume) by the French pressurized tank.

Used detergent should be able to dissociate Nei Du Su And make membrane portions become less, be easier to isolating granule.The non-toxicity detergent of particularly suitable is a NaTDC in the cytolysis step.The suitable final concentration of used NaTDC is 0.15-0.3%.Should use nontoxic (can tolerate fully) detergent.

Product according to method preparation of the present invention does not have cell wall basically, studies show that there is not the biologic activity endotoxin through various biological analysiss and human toxicity, and does not have the film fragment.There is not intact cell in this product yet.For reaching this purpose, preferably use the two times centrifugal step.For the first time centrifugally be:

(a) molten born of the same parents operating period, cleaning, aseptic at-0-1 ℃, microcosmic, Backman arranged ^#Collect 20 milliliters of cold bacterial cell lysates in the polycarbonate bottles of 30 rotors.The amount of the finished product that the number of this centrifuge bottle that should prepare will be made according to desire is decided.This volume of lysate has been represented running, a vertical R _MinBe 72mm, and effective R _MaxBe 95mm.In the Beckman supercentrifuge, use (0-4 ℃) Beckman of normal acceleration and pre-cooling ^#30 rotors are with centrifugal each pipe of 16,000 rpm.Centrifugation time is obvious from R for making _MinAnd R _MaxAverage S value between the value is 600.Cause the average S value of lysate to be lower than 600 as centrifugation time, then can cause a large amount of products to be lost.Be much higher than 600(such as 900S as average S value), the danger that makes finished product polluted by various toxic cell compositions then can be arranged.Only otherwise contamination of products occurs, then higher S value will increase the product yield.Adoptable safety range is about 600-800S(meansigma methods).Use Beckman ^#30 rotors, polycarbonate bottles and 20ml lysate can obtain 3.55 * 10 ⁹Individual W ²600S t, average, clarifying lysate (with 16,000rpm centrifugal 20 minutes+3 minutes to reach the speed of normal acceleration).This method can remove complete survival and dead cell, comprise that average S value is greater than the subcellular component of 600 polysome aggregation and the cell debris that all are big.This centrifugal process carries out at 0-4 ℃.Discharging brake ， And when 500-1000rpm stops rotor gradually.For preventing that managing content turns round and round, when being lower than 500rpm, do not discharge brake.Can only prolong production process as be higher than release brake in 1000 o'clock at rpm.As long as methodology is equal to, also can use other rotor and centrifuge (as the rotor of Sorvall Superspeed centrifuge and SS-34 rotor, Beck-man Superspeed centrifuge and equal or larger capacity).

(b) all under 0-4 ℃, carry out in steps.With cold aseptic, apyrogenic syringe and the clarifying lysates of the aseptic carefully results of 18G spinal injection syringe needle (or its equivalent) (cumulative volume 15-16ml).Harvest time, the top of chien shih syringe needle extend into several millimeters under the molten cytosol liquid level, and the Ce Bi And that SC not touch centrifuge bottle does not want disturbance precipitation agglomerate.(as Millex-HA, Millipore) And collects it in cold, aseptic, apyrogenic, a best non-bonding plastic/glass pipe by 0.45 micron cold filter immediately to make the molten cytosol of gathering in the crops.

Through first centrifugation step with after filtering, immediately with lysate at saccharose gradient upper berth layer microcosmic cleaning, in aseptic, the cold Merlon centrifuge bottle.Used diluent is above-mentioned buffer system.It is centrifugal then stratified gradient to be carried out the second time.

The product separation parameter is as follows:

(1) base product yield (the stratified clarification lysate/gradient of 1-1.2mg product/1.0ml).

^#The 50Beckman rotor:

Gradient: in the Merlon centrifuge bottle, with 4-5ml15%(W/W) sucrose is at 3.0ml30%(W/W) the tangible interface of sucrose upper berth layer-have.Sucrose is aseptic De ， And and is the endotoxin feminine gender through quantitative Limulus assay.

Sample size: 1.0ml is clarifying/filtering lysate.

Centrifugal: 0-4 ℃; Slowly quicken; Speed 38, centrifugal 60 minutes of 000rpm.

(2) in order to improve the amount of each centrifugal product that separates, can use following rotor:

^#The 30Beckman rotor:

Gradient: in polycarbonate bottles, with 11.0ml15%(W/W) sucrose (according to the volume of lysate) is at 9.0ml30%(W/W) sucrose upper berth layer, form tangible interface.Warp quantitatively Limulus analytic process detection shows the aseptic De And of used sucrose Shi and is the endotoxin feminine gender.

Sample size: 4-5ml is clarifying/filtered lysate.

Centrifugal: 0-4 ℃; Slowly quicken; 30,000rpm; 120 minutes.Be transferred to the ultracentrifugal time (+or-) to such an extent that maximum yield and minimum the pollution are arranged, but this to depend on the volume of the lysate of spreading layer.Breakaway braking device when 1000rpm (being not less than 500rpm).Can consult manufacturer and carry the braking break time of recommending with regard to specific centrifuge/rotor-support-foundation system.

^#The 14Beckman rotor:

Gradient: in polycarbonate bottles, add 100ml25%(W/W) sucrose.Used sucrose is aseptic, and detects through quantitative Limulus method and to prove the endotoxin feminine gender.

Sample size: 100ml is clarifying/filtering lysate.

Centrifugal: 0-4 ℃; Slowly quicken; 14,000rpm; High speed centrifugation 12 hours; When 500rpm, cut off brake between deceleration phase.

^#The 10Beckman rotor:

Gradient: 200ml25%(W/W packs in polycarbonate bottles) sucrose.Used sucrose is aseptic, and detects through quantitative Limulus method and to prove the endotoxin feminine gender.

Sample size: 200ml is clarifying/filtering lysate.

Centrifugal: 0-4 ℃; Slowly quicken; 10,000rpm; High speed centrifugation 23 hours; When 500rpm, cut off brake between deceleration phase.

For the second time centrifugal process can make the film vesicle part of specific size and residual ribosome part precipitate cake and separated ， And less part such as dna fragmentation, RNA fragment, protein fragments, cell wall fragments and film fragment are stayed in the top section of discontinuous gradient by forming.Specific centrifugation time in these rotors should be to be unlikely the obvious pollution that makes finished product be subjected to the cell component that do not need.If can not change centrifugation time,, can use following washing procedure for reducing the amount of above-mentioned pollutant:

As for ^#The 30Beckman rotor:

A. (be obtained from the suitable buffer of 10ml (each 12 pipe) the precipitation agglomerate (cake) that suspends again ^#30Beckman rotor or be equal to rotor is seen above-mentioned).Again the precipitation cake that suspends is transferred in the container of aseptic, no thermal source, microcosmic cleaning.With the suitable buffer flushing of 2ml centrifuge tube (each 10 pipe), the precipitation cake with eluate and resuspending is incorporated in the appropriate containers more afterwards.Pressure-vaccum (using 10ml syringe 10 times repeatedly) or several seconds of shaking by swirling are so that the solution mixing repeatedly.

B. that the precipitation cake that 5ml is suspended again sucks is one aseptic, do not have thermal source, contain the suitable buffer of 15ml ^#In the 30 rotor Merlon centrifuge tubes.

C. start with normal acceleration and brake each was managed centrifugal 25 to 40 minutes.

As long as be equal on the methodology, also can use the centrifuge and the rotor (, when using onesize gradient, then available) of other model than the short duration of runs as 50.2Ti Beckman rotor.Also can utilize suitable linear gradient to replace discontinuous gradient.

Washing is according to the part of the isolating one-tenth of aforementioned program precipitation cake, is suspended in above-mentioned suitable suspension liquid or buffer Zhong ， And afterwards with suitable 0.45 or 0.22 micron aperture filter filtration sterilization.Used suspension liquid can be any suitable pharmaceuticals, or its quality should reach the reagent that can prevent by the partly precipitated that suspends, degraded, gathering or function inhibition.

Can be according to nucleic acid content, to carry out product according to following formula quantitative:

E260＝0.0373-0.0079（A260/A280）

Microgram nucleic acid=A260/E260

For the purpose of standardization, available suitable resuspending buffer dilutes the product concentrate sample of 0.05ml resuspending, so that its A260 is between 0.4 and 0.5.It is blank to use buffer suspension liquid to do afterwards, measures the absorptance at 280nm, 260nm, 225nm and 215nm place.Nucleic acid content is once measuring, and being about to product, to be diluted to final concentration be that every 0.5ml buffer contains 1.0mg nucleic acid.Available following formula is calculated the protein content of product approx:

Microgram albumen/ML=144(A215-A225).

Using centrifuging to obtain average product yield is that the clarifying lysate of every 1.0mg contains 1.0-1.2mg.A260/A280 absorptance ratio average is 1.7626, and its standard deviation is 0.0626.The E260 average is 0.0232, and its standard deviation is 0.0007.

Has high functionality effectiveness (stating as follows) so have the product of the vesicle of size like this, the still incomplete at present Ming Liao of its reason.Yet the size that it is believed that the film vesicle is specific, and promptly average diameter is at least 180nm, and minimum diameter is very crucial for the 110nm this point in fact ⁽¹⁵⁾

The present invention's biologically modifier goods do not have biologic activity endotoxin and cell wall part basically, thereby have reduced the toxicity of product significantly.For example, to the biological respinse modifier that the mice of order in four days uses the method according to the present invention to make, can not cause cutaneous necrosis/fester or dead mouse (tabulating 1 as follows).This research is to make the cervical region of C57B1/6 mice inject biological respinse Xiu Bian Ji And of the present invention observe the downright bad and/or dead situation that occurs at four days.In addition, observed result shows that these animals all have normal type to increase, and this promptly shows another indication of avirulence pollutant.

Table 1

The cutaneous necrosis test

-necrosis/death-

Number of animals biological respinse modifier dosage 18 hours 24 hours 48 hours 96 hours

4 10μg 0/0 0/0 0/0 0/0

7 100μg 0/0 0/0 0/0 0/0

6 200μg 0/0 0/0 0/0 0/0

100 μ g experimental grouies are respectively 1.72g and 2.94g in the average weight of back 0 hour of injection and 24 hours.The 200 μ g group average weight when injection back 0 hour, 24 hours and 6 days is respectively 1.57g, 2.16g and 4.5g.

Made in 8 weeks CFW mouse do on one's body histamine hypersensitivity test also proof not owing to endotoxic toxicity.

Table 2

The test of histamine hypersensitivity

A. endotoxin dosage (μ g) route of administration D/T ^a

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

0.5

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

0.5

B. biological respinse modifier dosage (μ g)

100 i.P. 0/6

200 i.P. 0/6

600 i.P. 0/6

2000 i.P. 0/7

A: with 0.5mg histamine bisphosphate (Sigma) after intraperitoneal injection is attacked 90 minutes, each organized the death toll in the mice.(0.5 μ g, animal i.P.) causes 50% death after attacking with histamine to have given endotoxin ⁽¹⁾Mice, rat and Cavia porcellus fervescence (1-2 ℃) then occurs accepting low-temp reaction to occur after endotoxin or infectious the attack after giving the present invention's biological respinse modifier.

Moreover, inject once or repeatedly that (dosage is 5 micrograms to 10 milligram for mice, rat, Cavia porcellus and people's (Yi Shang tested object one by one), human trial is on every Mondays to three times), do not cause unable, pruritus, DIC(disseminated inravascular coagulation), arthritis, anaphylaxis (hypersensitivity), liver enzyme raises and injection site local necrosis or ulcer.Maximum weekly accumulated dose is no more than 12 milligrams.When make the hemorrhagic necrosis that the both sides kidney does not take place intravenous injection Shi ， And to rabbit.

As if observe with phase contrast microscope, the film vesicle in discovery the present invention's the product is easy to by mononuclear cell and macrophage endocytosis.Known endocytosis can cause cell membrane to upgrade, and the circulation of the film in the monocyte-macrophage cell line can activate these cells.

The cell of monocyte-macrophage cell line can be divided into several functional categories artificially, the cell that on behalf of a class, it respectively break up more perfectly gradually.These classes (name can be different) are: mononuclear cell, normal or resting stage macrophage, the macrophage of irriate, activated non-tumor-killing macrophage and activated tumor-killing macrophage.Cell differentiation must be perfect more, and it is not reactive just more little to all kinds stimulation, then may be subjected to more restrictions potentially aspect the performance effector cell function.On the contrary, late under disease and the chronic disease situation, monocyte-macrophage cell line is just bigger not reactive to stimulating performance to have gradually as general immune system.In these stages, have only activatory tumor-killing macrophage to be proved to be tumor cell is had directly Cytotoxic.

Noticed that especially mononuclear cell-macrophage product endocytosis is relevant with the Mus cell.Normal mononuclear cell and immobilized macrophage are not added under the heparin situation and gather in the crops making Cheng Xu And without any in advance behaviour by the intraperitoneal of healthy adult C57B1 mice.Initial adherent cell group comprises the garden shape cell more than 90%, and remaining then has typical macrophage form.Garden shape cell colony is by mononuclear cell and be made up of median size and cell with lymphocyte outward appearance in a large number.The equal endocytosis product of all cells wherein, thereby be the member of monocyte-macrophage cell line.

Outside primary mononuclear cell-static macrophage (garden shape cell) colony's endosome of purification, add in a few minutes after the product of the present invention, a large amount of small vesicle (its size and number are along with the time increases) promptly begin to come across cell membrane below.Activate the phagocytic capacity is directly proportional with a product design of the present invention in addition.When add biological respinse modifier concentration of the present invention surpass every 3ml culture medium 50 micrograms/10 ⁵During cell, will cause autophagy death along with keeping garden shape cellular morphology extreme cavity formation to occur.In same cell colony, just both do not observe activate the phagocytic capacity behind the endotoxin of adding variable concentrations or the BCG cell wall and also do not observed the cell-stimulating phenomenon.In the presence of biological respinse modifier of the present invention, or in the presence of endotoxin or BCG cell wall product, B16 melanoma cells or renal epithelial cell all do not show activate the phagocytic capacity.A kind of probability is, owing to have vesicle and/or the special receptor of ribosome composition by the present invention's method preparation, and biological respinse modifier of the present invention is promptly entered in mononuclear cell-static macrophage.Other explanation that comprises non-specific physics and/or chemical interaction (as relative hydrophobicity) also is possible.

3 results that summed up mononuclear cell-static macrophage Study of cytotoxicity tabulate down, give biological respinse modifier of the present invention comprising external, with concentration be the biological respinse modifier of 0 to 100 microgram (respectively managing concentration 5 micrograms at interval) add to contain as target cell it (10 ⁵Individual) 25Cm of the mouse peritoneal mononuclear cell/static macrophage of B16 melanoma cells and action effect cell ²In the culture bottle.The left side column illustrates the ratio of effector lymphocyte to target cell in the table.Analyze back 96 hours of beginning, place 95% ethanol to decide And MayerShi brazilwood extract dyeing admittedly in cell.Grow by the cell that result's representative of positive sign indication is seen easily at the fashionable microscopically of 80%-100% meeting; Negative sign is then represented does not have visible growth, promptly observes the remarkable reduction of proof targeted cell population or does not have targeted cell population through microscopically.

Table 3

The cytotoxicity test

The E/T ratio adds variable concentrations product (microgram) and cultivates 96 hours visible (junction) growth

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

2：1 + + - + - + + + + + + - + + + +

1：1 + + - + - + + + + + - + + + +

1：2 + + - + - + + + + + + - + + + +

1：4 + + + + + + + + + + + + + + + +

As can be seen from the above table, biological respinse modifier of the present invention can induce abdominal cavity monocyte/static macrophage to be divided into the tumor cytotoxicity sexual state when very special concentration (5,15 and 50 microgram).Be 2: 1 or be lower than at 2: 1 o'clock and reach Cytotoxic high level in effector lymphocyte-target cell ratio.Viewed tumor cytotoxicity effect performance has extreme cavity formation, cellular contraction and/or phase density to increase, and above-mentioned phenomenon all can occur in 8 hours behind the biological respinse modifier that adds valid density.

Before the research report delivered indicate, activatory non-tumor-killing macrophage must contact 18 hours with lymphokines before it kills target cell, simultaneously mononuclear cell and static Ju Shi Xi Bao And Fails To Respond.By product of the present invention inductive cytotoxic activity in mononuclear cell/static macrophage then is very rapid and unique.Before termination, four days these facts of culture incubation must be shown, give biological respinse modifier of the present invention rapidly and effectively the thin born of the same parents' merit of inductive effect can suppress target cell for a long time by And.

Shown in the table 4 that product of the present invention changes the ability of various leukocyte (WBC) count level and neutrophil level.Table 4 has pointed out that a series of end-stage patients are in the WBC and the neutrophil level that give biological respinse modifier front and back.Dosage as shown in Table.Give once continuous 3 weeks weekly.By the example shown in the table 4 as can be seen, use product of the present invention to carry out special treatment and can increase white blood cell count and neutrophil number significantly.Should note the not all various dosage of De Shi And, neither all patients all show white blood cell count increase (but also not reducing).Yet as shown in table 4, some patient the reaction that white blood cell count and neutrophil digital display work increases occurred after using biological respinse modifier of the present invention.

Table 4

Patient number biological respinse (repairing change) WBC * 1000 neutrophils (%WBC)

Before agent dose (mg) administration after the administration before the administration in 24 hours after the administration 24 hours

D59261 1.0 18 27 88 92

D25940 1.0 3.8 6.2 58 70

D78252 1.5 8 10.5 67 82

D45851 1.5 8.6 13.6 77 90

3.0 13.3 22.7 90 92

D80822 2.0 6.7 7.6 59 74

3.0 6.1 9.6 59 75

D82088 4.0 3.2 9.4 70.3 93

6.0 3.5 6.3 56 85

D90607 6.0 8.0 9.6 72 84

Range of normal value: WBC7.8 ± 3 * 1000

Neutrophil 40-70%

There is the patient of low WBC counting to reach normal range in back 24 hours before the treatment in treatment.Give biological respinse modifier of the present invention and also can cause the single nucleus of human peripheral (natural killer cell, cytotoxic T cell and/or mononuclear cell) counting to increase, and can not cause thrombocytopenia (data not shown goes out).Therefore visible product of the present invention does not have inhibitory action to bone marrow.

Suitable immunologic function must have the synergism ， And of several types cell to produce then and discharge a large amount of cellular products.The cell of known monocyte-macrophage cell line plays pivotal role in this process, and the suitableeest antibody response (B cell function), cell-mediated immunity (T cell function) and possible NK cell (NK) cytoactive just can not take place when not having these cells to exist.Though might illustrate the cellular pathways of the various synergism in the tissue culture, but can not determine intravital a large amount of approach of patient and control mechanism thereof.Because esoteric each species specificity of patient and/or nonspecific immunity effector lymphocyte mechanism of action are unknown basically, should be able to promote any ongoing reaction so select to use the medicament of reinforcement/adjusting immunologic function, perhaps when not reacting, reduce the system response threshold, thereby discerned, activate and select suitable effector cell function.Ideal activator should not induced immune deviation, anergy or the unusual hypersensitivity reaction at itself or cross reaction main body, perhaps facilitates or guides selection to specificity single effect cellular pathways.Because during clinic trial, do not show this class phenomenon significantly, so think that product of the present invention can satisfy above-mentioned requirements.

Fig. 2 illustrates biological response modifier of the present invention to stimulate the ability of human body natural killer cell similar in appearance to the model of action of LeIF.This in vitro tests has illustrated behind product of the present invention that gives the various dose level and interferon, because the effect of the natural killer cell of people, cause the dissolved degree of target cell (according to deciding) by the radiolabeled Cr that discharges in the target cell, pictorialization by difference respectively.Dosage level provides in each curve, and the zero-dose level is represented background or from the leakage level of the chromium of target cell release.

Above-mentioned analysis is according to document ^{(8) (9)}Described in method carry out.With the people K562 cell of the marrow sample tumor cell line of radioactivity sodium chromate labelling as target cell.Used effector lymphocyte is unbated human peripheral blood mononuclear cell.Available following formula calculates:

% discharges=(experiment release-matched group discharges)/(maximum release-matched group discharges) * 100

It promptly is the count per minute (cpm) of the radioactivity when product and effector lymphocyte add target cell and exist that experiment discharges, and it is resulting per minute step-by-step counting when only adding target cell with the effector lymphocyte that matched group discharges.Maximum release is that aliquot sample with target cell is incubated resulting cpm number in Saponin (a kind of detergent).

The result of this embodiment proves, though for low relatively effector lymphocyte concerning the target cell ratio, be not so good as to use interferon so high, but then can induce the natural killer cell cytotoxicity of people significantly when having mononuclear cell to exist, this point is and uses interferon much the same.Statistical analysis to 30 parts of products shows, inducing aspect the NK cell cytotoxicity, and product of the present invention is equivalent to or is better than LeIF.The inductive NK cell cytotoxicity of this product is similar in appearance to interferon-induced cytotoxicity ， And and also depend on the various variable factors such as ratio of year the making of blood donor, sex, cell storage method and all types of cells.

Fig. 3 diagram has shown the bactericidal property according to the biological respinse modifier of the present invention's method preparation.The vertical coordinate of curve chart illustrates the logarithm value of bacterium monocytogenes in the mouse peritoneal cell (Listeria monocytogenes) among Fig. 3, the abscissa express time (hour).Bacterium monocytogenes is the antibacterial of a kind of people's of causing acute cerebral meningitis (being with or without relevant septicemia).Analyze shown in Fig. 3 and be a kind of standard detection method that detects bactericidal activity.Tester is that known sterilization macrophage but the human body of inducing can not tolerate again

Peptone.(detailed description of relevant this method is referring to Cruprinski, C.J., Henson, P.M. and Campbell, P.A., 1984, J.Leukoc-yte Biol.35: 193).

The change of its number in 3 hours, in different time and following several different predetermined condition steps, with the cells contacting that derives from mouse peritoneal time of the antibacterial among the figure in the curve indication culture.These different conditionality steps are:

A. there is not abdominal cavity cell-bacterial growth contrast;

B. gather in the crops intraperitoneal injection in preceding 14 days

Peptone;

C. gather in the crops preceding 14 days intraperitoneal injection biological respinse modifiers of the present invention;

D. gather in the crops preceding 7 days intraperitoneal injection biological respinse modifiers of the present invention;

E. gather in the crops preceding 1 day intraperitoneal injection biological respinse modifier of the present invention; And

F. gather in the crops intraperitoneal injection in preceding 1 day

Peptone.

As by among Fig. 3 as can be seen, represent the top curve indication culture of matched group stably to grow.When cell is to gather in the crops in the mice body of 0.2 milligram of the present invention's of intraperitoneal injection product, then demonstrate depend on inject time before the results and basically with injection

The growth decline of the Different Slope that the contrast of peptone (gathering in the crops preceding 1 day) is similar.These data show, the inductive bactericidal activity of the present invention's product behind single injection 14 days still very high.Biological respinse modifier of the present invention is possessed long-acting aspect the clear and definite bactericidal activity, is shown that it can be used as the therapeutic agent of bacterial infection inducing macrophage to produce.

Fig. 4 has shown the ability of regulating antibody dependent cellular cytotoxic activity (ADCC) according to the biological respinse modifier of the present invention's method preparation.Method therefor such as relevant document ^(10,11)Described in.In this analytic process, be so that human peripheral blood mononuclear cell's action effect cell of specific effect cell-target cell ratio to be arranged.Target cell is the Mus YAC cell of chromium labelling, and used antibody is the anti-mouse cell antibody of rabbit.

As seen from Figure 4, at least for higher relatively effector lymphocyte concerning the target cell ratio, in used the present invention's product dosage is 15 μ g/ml or when being lower than this level, the level of its lethal effect is higher than interferon, but is 20 μ g/ml(cause the autophagy death of mononuclear cell in this test concentration in concentration) time be to be background level.

Fig. 5 diagram has shown that the present invention's product stimulates interleukin I(IL-I) ability that discharges.Detection is according to document ^(12,13)Described program is carried out.Used peripheral blood lymphocytes is to make in the blood by the subjects, and the present invention's of cell and variant concentration product together is incubated.Behind the incubation of certain hour, the aliquot of results culture fluid.Do lymphocyte transformation test with various dilution factors.The conversion test of lymphocytic blast cell sample is with 10 ⁶Individual Mus thymocyte cell was added in the culture supernatants of results insulation 72 hours, measured afterwards by the radioactivity of the tritium-labeled thymidine of cell absorption (count per minute).Among Fig. 5, represent background level with solid punctuating.As can be seen, except lowest dose level (1 μ g/ml), the obvious stimulation effect that IL-1 is discharged can take place all.Document ⁽¹⁴⁾Put down in writing the therapeutical effect that IL-1 discharges.

Fig. 6 is graphic extension to the cytotoxic effect of K562 target cell is because natural killer cell, rather than B or T cell are in action.This test is with specific effector lymphocyte-target cell ratio, end user's peripheral blood lymphocytes action effect cell colony.Therefrom as can be seen, when not adding by the biological respinse modifier of the present invention's method preparation or not having natural killer cell, background or leakage level are lower than 20% chromium and discharge.Cell colony for not detracting gives product of the present invention and then causes tangible natural killer cell cytotoxicity stimulation.The B or the T Xi Bao And that remove in this cell colony with monoclonal antibody specific can not influence the cytotoxicity level significantly.But removing the NK cell with monoclonal antibody then makes cytotoxic effect disappear.Remove the mononuclear cell (not shown) with monoclonal antibody and also cause the forfeiture of NK cytotoxicity, this promptly points out the NK cell by mononuclear cell-macrophage group activation.

Diagram has shown the result who rat prostate squamous cell carcinoma (mushroom tumor) is carried out interior therapeutic research among Fig. 7.As can be seen, but give big in the product inducing mouse body of the present invention, the mushroom carcinoma of Xing Cheng And is degenerated.All untreated matched group band tumor animals are all dead in 14 months, and all animals through treatment ， And of all surviving has 4 and sees that tumor regression is arranged in 6 animals.Find that in carrying out these researchs other to inject 1 milligram weekly be effectively in focus, inject weekly 2 milligrams then invalid.By the specific tumor of being treated is Dunning tumor subbreed R3327A, for a kind of do not rely on hormone, the rat tumor of growth of moderate-fast, it shifts late the time.Tumor host is Copenh-agen X Fisher F ₁This tumor system is the animal model for tumour of national carcinoma of prostate research group.Implant in left flank portion.Mean tumour volume is 681 cubic millimeters when treating for the first time.

Fig. 8 shows the result with the rat carcinoma of prostate of the slow growth of product treatment of the present invention.Tumor host is Copenhagen X Fisher F ₁It is another tumor model of national carcinoma of prostate research group.Implant in left flank portion.Treatment comprises per seven days 2 milligrams of infringement other position injections.Tumor average volume is 1138 during the treatment beginning, 8(＞2Cm ³) cubic millimeter.This special carcinoma is Dunning tumor subbreed R3327H, and it has proved that for WD adenocarcinoma ， And the speed of growth is very slow.

As seen from Figure 8, biological respinse modifier of the present invention has induced the carcinoma of established and slow growth big in the rat body to degenerate.All untreated animals show all that sb.'s illness took a turn for the worse, dead or lose weight, the animal of receiving treatment shows then that stable disease, carrying out property slowly disappear (pathologic finding proves already), body weight Zheng Chang And and do not have death.In this example, penetrating 2 milligrams in the damage location sidenote weekly is effectively, weekly in the damage location sidenote penetrate 1 milligram then invalid.The effective dose of above-mentioned two tumor systems proof expection is according to different tumors and different.

Fig. 9 shows the result of treatment bilateral rat prostate adenocarcinoma.Add and add product of the present invention with product of the present invention, steroid and compare accepting orchiectomy, orchiectomy only to give the host and the matched group of the present invention's product.Just left side side of body flank and right side of body flank tumor compare respectively.

As seen from Figure 9, induced the degeneration of the adenocarcinoma of growth fast of very big (cumulative volume is greater than 5 cubic centimetres), moderate by the biological respinse modifier of the present invention's method preparation.Animal through treatment only accepts to inject 1 milligram of product of the present invention weekly at the other position of damage of left side side of body flank tumor region.Data show uses product of the present invention can produce systemic treatment effect (the untreated damage of offside is also degenerated), and the effect of biological respinse modifier is not subjected to the inhibition of steroid, dexamethasone.Do not observe dexamethasone energy inhibition zone tumor people's hyperpyrexia or tumor response.

According to the I phase of U.S. food and Drug Administration's announcement and the regulation and the program of II phase test, use biological respinse modifier of the present invention to carry out the intravital clinical research of people.The I phase research of being done is the toxicity of measuring (if any) the present invention's biological respinse modifier.I phase test to as if be in the And of terminal stage of a disease and think the patient that the treatment before all fails.These patients accept three to six treatments, away from subcutaneous per 7 days of tumor locus by indicated dosage injection once.Toxicity test shows that product of the present invention does not have tangible toxicity and can be tolerated well by human body.

At this simultaneously, there is 46% case to receive therapeutic effect.Provided the result of I phase and the test of II phase in the following tabulation.

Table 5

Patient reaction's brief summary

Patient number dosage (mg) diagnostic reaction

D44937P .25 .50,1.0 pulmonary carcinoma are stablized

D06277 .25 carcinoma of prostate is less

D32770 .25 pulmonary carcinoma does not have

D01088 .50 breast carcinoma is stable

D15546 .50 colon/liver does not have

D59261 1.0,2.0 lymphoma are less

D25940 1.0 sexual celies do not have

D78252 1.5 colons/liver does not have

D45851 1.5,3.0, adenocarcinoma are little

5.0,6.0 rectum/abdominal part

D180849 2.0,3.0 breast carcinoma do not have

D80822 2.0,3.0 lymphoma (LHL) do not have

D81828 2.5,8.0 lymphoma (NHL) parts

D62984 2.5,4.0 lymphoma (HL) are stable

D82088 2.5,4.0,6.0 glioblastomas are little

D76339 4.0,6.0 pancreas are stable

D82568 4.0 Kaposis are stable

C03343 5.0,8.0 nephrocytes are stable

D87354 5.0,1.5 nodositas lymphoma parts

D90607 6.0 lungs, adenocarcinoma does not have

D91834 6.0 melanoma do not have

A039585 0.25 nephrocyte is stable

A768642 .25 .5,1.0,2.0 colons/liver are stablized

A661981 0.5 pulmonary carcinoma is stable

Adenocarcinoma

A078379 0.5 carcinoma of parotid gland is less

A017231 0.25 colon cancer does not have

A055001 1.0 lungs are stable

A666522 1.0 pulmonary carcinoma do not have

Maxicell

A403927 1.0 lung tumors do not have

Squamous cell

A032896 1.5 adenocarcinoma, the cervical region part

A708029 1.5 bronchioloalveolars are stable

A044130 1.5 pulmonary carcinoma are stable

Squamous cell

A223211 2.0 pulmonary carcinoma are stable

D47379 8.0 prostate do not have

D88652 5.0 colons do not have

D93503 8.0 colons are stable

D94245 8.0 NH lymphoma are less

D86141 8.0 colons do not have

D96464 8.0 melanoma do not have

D94257 10.0 gallbladders do not have

D97735 10.0 colons do not have

E01514 10.0 NSC lungs are stable

D98520 10.0 colons do not have

E01401 10.0 sarcomas do not have

D66399 1.0 mediastinum agglomerates do not have

6,134 2.0 lungs are stable

3,374 2.0 lungs/prostate is stable

Unknown constitutional

4,277 4.0,6.0 lungs are stable

5,079 3.0 stomaches do not have

0,421 4.0 histiocytoma part

6,918 6.0 gallbladders do not have

2,688 8.0 tongues do not have

8,708 8.0 colons do not have

9,222 8.0 prostate do not have

E07942 0 colon does not have

E13150 4.0 cell lungs are stable

E14356 4.0 cervical regions do not have

E15735 4.0 rectum do not have

D69174 3.0,4.0 colons do not have

E11927 4.0 NSC lungs do not have

E14983 4.0 Kaposis do not have

D70727 3.0,4.0 lungs are stable

E07876 2.0,3.0 kidneys are stable

E04693 1.0,2.0 mammary gland are less

E10311 3.0 larynxs do not have

E09265 3.0 kidneys do not have

D44296 0.5 colon does not have

D81828 2.0 NH lymphoma do not have

E07727 1.0,3.0 mammary gland do not have

E03741 0.5 colon does not have

E05487 2.0 parotid glands do not have

E03083 0.5,1.0 endometrium are stable

E04545 1.0 CLs do not have

E05195 2.0 thymomas are less

Partly=state of an illness alleviates 50% or more;

Less=state of an illness alleviates more than 25%～below 50%;

Stable=condition of illness gets nowhere;

Do not have=reactionless.

Above-mentioned data sheet where there is light the terminal stage of a disease, before the patient that all fails of treatment in 48% reactive ratio is arranged.

To decide according to patient's concrete condition given dose and treatment cycle (course of treatment) that patient adopted, the factor that influences the used dosage and the course of treatment comprises the specific type of patient's whole immune system response characteristic, disease and degree, patient's general health, position of pathological changes etc.This is identical with other biological response modifier basically, and dose,optimum can be determined according to the relevant technologies that is used for other biological respinse modifier and common medicine.

Shown that by 6 the data of tabulating down product of the present invention is in treatment cerebral tumor diseases such as (as glioblastomas), it may be comparatively effective comparing with other feasible Therapeutic Method.Data in the table 6 shows that have 3 routine gross tumor volumes to reduce, this result is identical with limited treatment in 6 routine cerebral tumor patients.This result clearly illustrates that biological respinse modifier product of the present invention is effective on this specific tumor of treatment.

Table 6

Patient reaction's summary-cerebral tumor

Patient number dosage (mg) diagnostic reaction

D82088 2.5,4.0,6.0 brains are less

E11928 1.0 brain branches

E34415 1.0,3.0 brains improve

The remarkable function of E37376 1.0,3.0 brains is improved

E45209 1.0 brains improve

RT 1.0,3.0 brains improve

JB 1.0 brains improve

DR 1.0,3.0 brain branches

The film vesicle and the ribosome that are used for the present invention's biological respinse modifier product are easy to by biodegradation very much.A noticeable especially advantage of signs of degradation is, the microgranule colony of the present invention's product only is kept perfectly in very short time in immune system, and this time is enough to activating immune system, afterwards just degraded rapidly.Therefore can avoid occurring serious, chronic pathology physiological reaction as the result of chronic immune activation.Degradation process is because the result that existing various enzymes (as ribonuclease, protease, lipase) act in various types of cells (as mononuclear cell, macrophage, neutrophil) and the tissue fluid (as blood, lymph fluid).This degradation process quickens when temperature raises that (as body temperature, 37 ℃ of) ， And reduce with temperature and slow down gradually.Detect with the grain size analysis method, this product when 37 ℃ and 95% serum-concentration, in less than 2 minutes promptly by external degradation.The speed of degraded depends on enzyme concentration and temperature.In case degraded, product of the present invention is promptly significantly impaired as a kind of effectiveness of immunomodulator.

Figure 10 is presented at the killer cell cytotoxicity of inducing anti-three kinds of different tumor target cells in the human body.For most of individualities, peripheral blood lymphocytes all can be killed and wounded K562 tumor target cell to a certain extent in external.But peripheral blood lymphocytes is not is not generally killed and wounded Raji and Colon 38 tumor cell lines.Award various biological respinse modifiers in the body, comprise that interleukin II (IL-2) treatment patient ， And does not change the level of killing and wounding to back two kinds of target cells.Have only with after some biological respinse modifier (comprising IL-2) extracorporeal treatment peripheral blood lymphocytes, just can induce these cell killings K562 its tumor types target cell in addition.Yet, have found that biological respinse modifier product of the present invention can change the ability that peripheral blood lymphocytes is killed and wounded these tumor target cells when being used for interior therapeutic.Inducing action to LAK cell (cytotoxic T cell) behind the product that gives by method preparation of the present invention, has taken place in this research prompting.

Figure 10 has shown that the biological respinse modifier that gives the present invention to hanging oneself treats the Cytotoxic vitro detection of killer cell in back 24 hours patient's the peripheral blood lymphocytes.As can be seen, the result all has tangible lethal effect to all three kinds of tumor target cells.Check that by specific cell sign effector lymphocyte colony shows, wherein has activated mononuclear cell, natural killer cell and LAK cell.Natural killer cell increase in the patient body of treatment (find to have activated natural killer cell as the preceding) And that has discussed and in this detection, thereby proof has produced endogenous interferon at these in the patient bodies after the product treatment of the present invention.In addition, having activated cytotoxicity " T cell " (LAK cell) then proves to treat with product of the present invention and then causes endogenous IL-II to generate.Another unique character of the present invention's product is, activatory killer cell colony can keep in the IL-2 of external use very low concentration (2UIL-2/ml) in the body.In contrast, as the external then essential IL-2(500-1000u IL-2/ml of the activatory killer cell of IL-2 (LAK cell) that keeps) with high concentration.

Employed peripheral blood lymphocytes is to be obtained from behind injection the present invention's biological respinse modifier product 24 hours patient's whole blood.Adopt subcutaneous injection, 3 times weekly, each 1 to 4 milligram.Before injection, detected in back 24 hours with injection.Compare by the activatory killer cell of lymphokines (IL-2) (obtaining after being about to human peripheral blood mononuclear cell and IL-2 and together cultivating) activity with external with activatory cell was obtained in the body cytotoxicity level.

By natural killer cell testing result (Figure 11 and 12) relatively, relatively film vesicle that is obtained by organisms such as serratia marcescens and ribosome are than other source person's advantage.In specified organism, prepare required product with preceding method.By the physics of the prepared vesicle of these organisms and chemical parameters all within above-mentioned scope.

A kind of source of given antibacterial product can influence its bioactive type and/or this fact of degree, has been not new knowledge now for this area.The different strains of BCG shows specific immune activation level clinically in the animal model neutralization.Someone reports the ribosome vaccine that derives from different organisms source can produce in various degree immune system activation effect, and prompting to derive from the polysome of Serratieae more good than other source person ⁽⁶⁾By the data of being quoted as can be known, give buffer and the antibacterial polysome that derives from other strain (escherichia coli, streptococcus pneumoniae, Mycobacterium bovis (BCG), smegma mycobacterium and seat skin ulcer bacterium acidi propionici) separately, how much good And is unlike the control group mice of not receiving treatment (tumor manifests or be dead in 60 days).On the other hand, derive from the polysome of serratia marcescens with doses, then 60% or more animal do not have tumor development on one's body, and also see have tumor propagation to be suppressed all the other animals.

Because biological respinse modifier product of the present invention be obtained from certain microflora that does not belong to patient the member's and with normal individual in the uncorrelated microorganism of infectious disease, again because the common bacillary Kang Yuan And of this microorganism or does not seldom produce cross reaction with the organism that constitutes normal microflora, so can avoid unsuitable immunoreation.This class reaction comprises immune deviation and initial antigen fault phenomenon, and this phenomenon is common in the immunostimulant (as BCG, C.parvum and cell wall part) of exploitation in recent years.Perhaps be exactly because these reasons, the polysaccharide body that is made by escherichia coli, smegma mycobacterium and streptococcus pneumoniae is the very poor source of inducing of biologically, just partly or the serratia marcescens that is difficult for host microorganism system generation cross reaction then be the fabulous source of biological respinse modifier.

It should be noted that the present invention also can use other to be suitable for microorganism as the source of film vesicle and ribosome.The basic feature of these microorganisms is that this microorganism necessarily can not be the member of patient's microflora.Moreover the common bacterial antigens of microorganism used therefor necessarily can not or only partly produce cross reaction with the organism that constitutes normal microflora at the most.Therefore, its should not be can cause immune deviation reaction and also can not or seldom cause human body diseases.At last, this source microorganism also must be that wherein cell membrane can form the suitably vesicle person of size.

Suitable microorganism except that serratia marcescens has: ATCC14092 Erwinia Chrysan-themi(Pectobacterium); And assign to more among a small circle clostridium perfringen (Enterobacter aerogenes) ATCCE13048.The preparation performance that is obtained by these bacterial strains has the above-mentioned corresponding to activity of goods that derives from serratia marcescens.Particularly as shown in figure 11, use clostridium perfringen much better effect can be arranged than pseudomonas, escherichia coli and E.cloacae, but more a little than the alpha-interferon difference of using the given dose level.Similarly, Figure 12 is then resultful with E.chrysan-themi() compare with Flavobacterium (Flavobacterium) (unfruitful) and interferon.Also can be with other microorganism as Lai Yuan And processed as stated above, with ribosome and the vesicle of producing specific size.Use aforesaid analyzed in vitro method, can estimate their usefulness at an easy rate, so that can determine used as the biological respinse modifier.

Because do not have living cells, dead cell, cell wall and biologic activity endotoxin in the biological respinse modifier product of the present invention, thereby make by the identification due to the cross reactivity reduce to minimum, the composition ， And that removed difficult degraded reduces to minimum with the known composition that has high toxicity (as endotoxin) maybe can cause chronic inflammatory state (as arthritis, granuloma, ulcer) or removes fully.In addition, reduce the cell surface composition as much as possible and can get rid of known immunoreation variation and the phenotypic variation relevant with microbial strains.

The advantage of biological respinse modifier product of the present invention is a lot.By the cell of activated mononuclear cell-macrophage system, promoted the necessary cell synergism of the suitableeest immunologic function.The drought period noble cells of activated mononuclear cell-macrophage system (mononuclear cell, normal/phase macrophage with all worries set aside) can be promoted the manifold effect cell function.Just fully prove by the biological respinse modifier compositions of the present invention's method preparation for this reason, can induce polytype effector cell function according to the parameter of the conditioned disjunction diseased host that detects.

Except that these contents as herein described, still can given this descriptionly carry out all changes of repairing to product of the present invention, these will be that those skilled in that art are conspicuous.These are repaiied change and certainly will should fall in the application's the claim scope that awaits the reply.

Claims

1, produce the method for biological respinse modifier, it comprises the bacterial cell of cultivating serratia marcescens (Serratia marcescens) bacterial strain, the results cultured cells, with the suitable detergent endotoxin that dissociates, handle this cell concentration thing and make it to be enough to produce the film vesicle that ribosome and diameter are not less than 110nm, by separating said ribosome and film vesicle in the cell material of staying in the cellular lysate, and said ribosome and vesicle are suspended in the suitable buffer so that its average particulate diameter surpasses 170nm (based on the grain graininess analysis) again with a relative concentration.

2, according to the process of claim 1 wherein that cytolysis finishes with mechanical means.

3, produce the method for biological respinse modifier, it is included in the bacterial cell of cultivating the serratia marcescens strain in the suitable culture medium, said culture is cooled to 0-4 ℃, the results bacterial cell, the Xi Bao And that washing is gathered in the crops is suspended in it nontoxic, can be suitable for to keep one that the cell membrane vesicle forms and the environment of integrity in the buffer system of fine tolerance, influence at certain and to dissolve the cell that Shou Huo And suspends in the presence of film fragment and the dissociated detergent of endotoxin, said dissolving step must can produce the film vesicle that diameter is at least 110nm, clarification comprises intact cell, cell wall and film fragment are at the bacterial cell lysate of interior cell debris, the cellular lysate clarified can be tolerated by human body and to the density gradient material upper berth layer of human body non-immunogenicity, centrifugalize goes out not have basically the film vesicle and the ribosome part of other smaller portions, the aseptic density gradient material of removing is washed and is washed And and be suspended with the said film vesicle of said magnitude range again together with remaining ribosome in buffer.

4, according to the method for claim 3, wherein cytolysis is in surpassing 10, finishing with mechanical means under the pressure of 000Psi.

5, produce the method for biological respinse modifier, it comprise cultivation be not present in by And in treatment people's the microflora and have not with or seldom with constitute the bacterial cell of microbial strains that is played the common bacterial antigens of cross reaction by the organism of treatment patient's normal microflora, the results cultured cells, with the suitable detergent endotoxin that dissociates, destroy this cell concentration thing and make it to be enough to produce the film vesicle that average diameter is not less than 180nm, separate said film Nang Pao And and in cellular lysate, in the remaining cell material ribosome is dissociated out, and in suitable buffer, suspend again said vesicle and ribosome.