JPH01502024A - Partial cationization of protein-containing antigens and methods of immunization and desensitization - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Partial cation of protein-containing antigens Methods of immunization and desensitization Description of related applications This 8th petition is written by Jacob Gabriel Michael. U.S. Patent Application No. 07 filed on January 30, 1987 in the name of No. 1009.234, a continuation-in-part application.

Background of the invention The present invention provides partially cationized protein-containing substances with increased antigenicity. , for example, methods for producing antigens, products of this method, and mammalian ) relates to a method of enhancing an immune response to an antigen.

Antigens are used to prevent infectious diseases through immunosensitization and to treat allergies through desensitization. The importance of its use is well known.

The basics of immune sensitization are killed or weakened infectious substances (viruses, bacteria, toxins, etc.) , or contain foreign, usually macromolecular, substances capable of producing an immune response. It consists in exposing the organ to be immunized to its extract containing the immunization. these substances is commonly referred to as an antigen. Similarly, allergic reactions are reduced by desensitization In this case, this type of substance is a foreign substance called an allergen. Therefore, it is used to suppress the normal allergic response that occurs.

Many antigens and allergens are composed entirely or partially of proteins. Ru.

The action of an antigen is based on specific binding sites on the cells of the living tissue affected by the antigen. It is believed that this depends in part on the antigen's affinity for the antigen. As such an organization Examples include blood, internal organs, skin, and eyes. Interaction with antigen binding site The drug stimulates the production of antibodies, thereby making the organ more susceptible to antigen-containing infectious agents. be protected.

Since antigens are foreign substances, they may have harmful side effects on the organs to which they are immunized. Sometimes it has a purpose. This is of paramount importance in human and veterinary treatment.

Therefore, an effective immune response can be achieved by utilizing low levels of antigen. It is desirable to be able to do so. If antigenicity can be increased, it is possible to reduce the dose. It becomes necessarily possible to achieve a constant level of immunization.

One way to increase the antigenicity of a substance is to use an adjuvant along with the antigen. is to use. Adjuvants help antigens interact with living tissues. A substance that increases the immune response by An example of Ajiji I Kunto is For use with salt, there is Myonokun salt. Adjuvant in vivo Problems that adversely affect their use in immunization (in vivo) are the The toxicity and side effects caused by For this reason, adjuvants can be toxic and/or It is not recommended for use in humans or other organisms if side effects or side effects are a problem. stomach. Therefore, it is desirable to avoid the need for adjuvants. .

Some foreign substances may produce no or no antigenic response when they come into contact with living tissue. There are also very few. One theory is that this kind of material could produce such a response. There is a lack of a threshold amount of binding possibility, or strength. like this It would be advantageous if a substance could be converted from a non-antigenic form to an antigenic form. This is it This means that vaccines can elicit immune responses to substances that were previously impossible. Chin can be manufactured.

Immunization methods are usually carried out by subcutaneous or intramuscular injection of the vaccine. anti Oral ingestion of raw materials often produces suppression rather than augmentation of the immune response and is therefore not recommended in many cases. I don't like it. Exceptions to this general rule include attenuated bacteria or viruses. There is a live vaccine. Therefore, the vaccine can be administered orally without losing its immunogenic effect. It is preferable if it can be administered.

Chemical denaturation of antigens, especially protein-containing antigens, is used in this field of research, e.g. Surface labeling has been known for some time.

Danon (D, Danon)'s cationized fluorophores as labels for negative charges on the cell surface. Use of erritin.

f Cationized Ferritin asa Label of N aggressive Charges on Ce1l 5 surfaces), Ja Null on Ultrastructure Research (J, Ultrastru ture Research), Volume 38, Pages 500-510 (1972) Please refer to

Conventional antigen cationization methods teach complete cationization of antigen molecules. child Here, "complete cationization" means that all cationizable groups of a given substance are cationized. This indicates that it is turned on. Fully cationized antigen is Due to its reactivity, it is usually unsuitable for use in vivo. Ru.

Additional background information is contained in the following references, which are incorporated herein by reference. I will list it as a reference.

1. Barnes et al. (J. Barnes and M. Venkatachalam) , promotion of the deposition of spherical immune complexes by circulating polycations (i:nhancem entof Glomecular Immune Complex Depo position by a Circulating Poycation), Journaon of Experimental Medicine (J, Exp.

Med, ), 160: 286 (1984).

2. Orte et al. son, H., Takamija and A. Vogt), for transplanted cationized antigens. Quantitative study of local immune complex system globulonephritis in rats induced by Quant itative 5tudies onin 5itu Immune Com plex Glomerulonephritis in the Rat In duced by Planted Cationized Antigen) , Journal Fist-On, Experimental Medicine (J, Exp, Med, ), 155:460-474 (1982).

3. Gallo et al. (G, Gallo, CaulenST, Glaser, S, N, Em ancipator and M.

):, Lamm), glomerular immune complexes associated with nephritis and immunogen charge. Differential distribution of bodies (Nephrif.

Genicity and Differential Distribution n of Glomerular Immune Complexes Re1 ated to Immunogen Charge), Labo Invest (L ab, Invest, )%48:460 (1983).

4. Schifflik et al. (J, Schikwik SW, B, Vanden Berg, L, B, A, vande Putte, L, A, B, Joosten andori , vanden Bersselaar), catalase, peroxidase and and superoxide dismutase cationization: effects on experimental arthritis in mice. Improved intra-articular retention effect (Cationization of Cata) lase, Peroxidase Sand 5uperoxide Dismu tase:Effect, of Improved Intrarticula r　Retention　on　Experimental　Arthriti s In Mice), Journal on Clinical Investigation J. Cln. Invest, 76:195 (1985).

5. Mutzkerheide et al.

A, J, Pe5ce and J, G, Michael), Immunology of digestible fragments of BSA. Immuno 5uppressive Properties f　aPeptic　Fragment　of BSA), journal competition on Immunology (J. Immunol.), 119:1340 (1977).

6. Dosa et al.

J., Ford, A., Muckerheide and J.

G., Michael), Immunological properties of bovine serum albumin as a digestible fragment. (ImmunologicalProperties as Peptic F ragments of Bovine Serum Albumin), im Immunol, 38:509 (1979).

7. A. Muckerheide et al.

A, J, Pe5ce and J, G, Michael) of bovine serum albumin. Kinetics of Immunosuppression Induced by Digestible Fragments mmunosu pressure Induced by Peptic F ragments of BovineSerum Albumine), cell Vascular Immunology (Ce 11. Immuno le), 50: 340 (1980).

8. Mutzkerheide et al.

A, J, Pe5ce and J, G, Michae l), B by fragments of the antigen. Modulation of the IgE immune response to SA gE Immune Re5ponse to BSA by Fragnen ts of the Antigen), Cell Fist Immunology (Ce 11 I mmuno 1. ) 59:392 (1981).

9. Border et al. (W.A., Border, H.J., Ward, E.S., Hami 1 and A, H, Cohen), in rabbits by administration of exogenous cationic antigens. Induction of membranous nephropathy ropathy in Rabbitsby Administration of an Exogenous Cationic Antigen), Clinical Investigation (J, Cl1n, Inve. St.), 69:451 (1982).

10, Apple et al. (R, App 1 e, B, Knaupers A, Pe5ce , J.G., Michael), Common Determination of Native and Denatured Bovine Serum Albumin The group is recognized by both B cells and T cells (Shared Determinant). nants of Native and Denatured Bovine SerumAlbumin are Recognized by both B-andT-Cel1g), Molecular Immunology (Mo1.Imm uno 1. ), 21:901 (1984).

11, Levine et al. (B, B, Levine and N, M.

V az), trafficking for immune responsiveness and reagin production in mice Effect of combination of system antigen and antigen dose tions.

f InbredStrain Antigen and Antigen D ose on ImmuneResponsiveness and Reag inProduction in the Mouse) India Aron Are Lugi, Abul Imzul (Int.

AronSAl 1 e rgy, App 1. Immn.

1, ), 39:156 (1970).

12. Ferguson et al.

Peters, J. r., R. Reed, A. J. Pe5ce and J. G. Micha e l), immunoregulatory properties of antigenic fragments from bovine serum albumin ), Cell Immunology (Ce 11cl Immuno 1.), 73: 1 (1983).

13, Julius et al. (M, H, Julius, E, Simp S On% and A, Herzenberg) Rapid analysis of functional mouse lymphocytes from leg line ARapid Method for the l5olation of Functional Thymus-derived Murine Lymphocytes), European Journal of Conflict on Immunology (Eur, J. Immunol.), 3:645 (197B).

14. Hoare et al. (D.G. and E. Kosnland), Tang Quantitative denaturation and estimation method of carboxylic acid groups in protein (A Method for the Quantitative Modification and E stimulation of Carboxylic Aid Groups in Pr.

teins), Journal on Biological Chemistry ( J. Biol. Chem.), 242:2447 (1967).

15, Daron et al. (D, DaronSL, Go1dsteinsY, Markovsky, and E., 5kutelsky), a marker of negative charge on the cell surface. Use of Cationized Ferritin Ferritin as a Label of Negatire Charg e on Ce1l 5 surfaces), Journal on Ultrastra Future fist search (J, Ultrastructure Res,), 38: 500 (1972).

16, Warren et al.

gel, and A, Ched id), Current status of immunological adjuvants ( Current 5 status of Immunological Adjuv ants), Annual Review on Immunology (Ann.

Rev, Immuno 1. ), 4: 369 (1986).

17. Mills et al. (Z, J, Mills and E, Haber), Ribonuku The Effect of Changes in the Molecular Structure of Rease on Antigen Specificity on Antigenic 5 specificity of Changes in the Mo1ecular 5structure of Ribonu clease), J.

Immuo 1.91: 536 (196B).

18, Hebelukatz et al.

D, Hansburn and R, H, Schwartz), Ia molecules or antigens Presenting cells play an essential role in immune response gene regulation of T cell activation (The la-molecule or the Antigen-presentin g Ce1l Plays a Cr1tical Role in Immu ne Re5ponses Gene Regulation.

f T, Ce1l Activation) J, Mo1. Ce1l, Immuno 1. ), 1: 3 (1983).

19. Buss, S. and Werdelin, O., Angiotensi A type of oligopeptide antigen is transferred to accessory cells treated with paraformaldehyde. (Oligopeptide of the An giotersin Lineage Compete for Rresen tation by Paraformaldehnyde-treated Accessory Ce1l t.

T Ce1ls), Journal on Immunology (J, Immunol,) , 136:459 (1986).

20, Babbitt et al. (B, P, Babb it, p, M, A11en%G, Ma Ia histocompatibility Binding of immunogenic peptides to molecules enic Peptides to Ia) Nature (London) Don), 317: 359 (1985).

21, Booth et al. (S, Buss S, Co1or, C.

Smlth, J. H., Freed, C. Miles and H. M. Gray); Interaction between “processed” ovalbumin peptides and Ia molecules ionBetween a “Pressed’Ovalbumin Pep tide and Ia Mo1ecules), P, N, A, S, 83:29 68 (1986).

22, Rarey et al. (E, X, Rarey, E, Marg.

1iasn, F., W., Fitch and S., Pierce), T cell LBT4 and Ia in heteroolitic responses to cytochromes Role of LBT4 and Ia in the Heter oolitic Re5ponseof T, Ce1ls to Cytoch Rome), Journal on Immunology (J, Immunol.

) 186: 393B (1986).

23, Glazer et al. (A, N, Glazer, R, J.

DeLange and p.S., Sigman), chemical denaturation of proteins, biogenesis. Experimental techniques in chemistry and molecular biology (Chemical Modificat ionof Proteins, Lab, Techniques in Bio Chemistry and Mol.

Bio 1 o g y), Volume 4, Part 1, pp. 1-205, KaHolland ( North-Holland%Am, Else (1976) 24, Alexander N, Glaz e”), chemical denaturation of proteins with group-specific and site-specific reactants (T he Chemical M.

dification of Proteins by Group-5peci fic and 5ite-specific Reagents), 1-10 3. The Proteins, 3rd edition Volume ■, Academic Press Acad, Press, New York (N, Y,) (1976).

When cited herein, publications are referred to by number from the list above. .

So far, the prior art has not been able to use cationized antigens as vaccines or desensitizers. By using it for ±n vivo treatment and disease prevention. It is not recognized that it can be used. Furthermore, in the prior art, partial It is not recognized that cationized antigens can be used for this purpose. actual, It has been reported that fully cationized antigenic proteins do not exhibit altered immunological properties. (9)

The object of the present invention is to provide a partially positive drug which can be administered orally and does not require adjuvants. The object of the present invention is to provide a substance containing ionized antigen protein and a method for producing the same. .

Summary of the invention As used herein, "protein-containing material" refers to any protein , as well as substances whose molecular composition is partly proteinaceous, such as lipoproteins and and proteosatucharides. These substances are antigenic in their native form. It may be a substance with or without a substance. Specific examples of the above substances include: Bovine serum albumin (BSA), chicken egg albumin (OVA), bovine goo Globulin (BGG), ferritin, bacterial endotoxin, viral tan proteins, diphtheria toxoid and tetanus toxoid, and killed microorganisms, e.g. Bacteria and viruses and their protein-containing products are It is not limited to.

As used herein, "cationization" refers to cationization of protein-containing substances. Conversion or substitution of functional groups in the protein moiety makes the substance relatively more cationic. It means that. This type of functional group is usually negative within the physiological pH range. Ionic and can be converted or substituted into cationic or nonionic moieties. Ru. An example of this type of cationization is to convert anionic side chain carboxyl groups into polycations. There is a reaction that involves substitution with an ionic aminoethylamide group.

As used herein, proteins in their unreacted, i.e., "native" form Contained substances are indicated by prefixing rnJ, and cationic forms are indicated by prefixing rcJ. .

For example, bovine serum albumin (BSA) is present in its native form, rnBSAJ or can be represented as the cationized form, rcBSAJ.

In producing an antigen protein-containing substance, the present invention provides (1) protein-containing substances; substance and (2) a reactant capable of cationizing the protein-containing substance. react, and the reaction between the protein-containing substance and the reactant is caused by the above-mentioned protein. Stopping after sufficient time has elapsed for partial cationization of the contained substances to occur. Provides a method consisting of:

The invention further provides that the present invention has increased antigenicity compared to the same native protein-containing material. and the isoelectric point measured by isoelectric focusing electrophoresis described below is in the range of about 6.5 to about 9.5. A partially cationized antigen protein-containing substance is provided.

The present invention also provides for measuring immunosensitization in mammals by isoelectric focusing, with an isoelectric point of about 6 ．． A partially cationized antigen protein-containing substance ranging from 5 to about 9.5. A method is provided by administering an effective amount to a mammal.

Administration can be done orally or parenterally with or without adjuvants. I can do that.

Preferred partial cationization methods include at least one carbodiimide and at least Both use reactants containing one type of amine. The degree of cationization is determined by pH, controlled by varying the time parameters, and the concentrations of reactants.

Reactions are easily stopped with the desired degree of partial cationization by methods known in the art. Can be stopped or quenched. For example, BSA and carbodiimi Reaction with the compound/amine mixture converts the anionic orboxyl group into approximately 20 new amines. Quenching can be performed after substitution with a mino group, but is not limited to this. stomach. Fully cationized BSA would contain 80 new amino groups.

Generally, about 20 to about 60% of the maximum number of amino groups theoretically possible are added or substituted. Then, the isoelectric point measured by isoelectric focusing is in the range of about 6.5 to 9.5. Become.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Protein-containing substances (PC8) can be prepared by several methods known in the art (15,9) can be cationized by A preferred method is ethylenediamine/1- Ethyl-3-(3-dimethylaminopropyl)-carposiimide (EDC) and P This is a reaction with O5. This particular reaction converts the carboxyl group of the protein into Activation with bodiimide and then the activated carboxyl of the general type 10R-NH2 A primary amine type derivative is obtained by reacting with a nuclear reagent.

The chemistry of modification, i.e. the introduction of primary, secondary or tertiary amino groups, also The degree of denaturation of protein carboxyl groups also depends on reagents, reaction time, and coupling. The pH of the reaction can be changed by appropriate selection, so A wide variety of uses can be obtained.

Cationization methods suitable for use in the present invention include mild reaction conditions for cationization. It is something to do. As used herein, mild reaction conditions refer to Substantial changes in the molecular properties of PO2 (e.g. -order, secondary and tertiary structure) conditions that do not adversely affect its immunogenic properties at antigenic pH. Ru. These conditions are most commonly relatively low temperatures and low ionic strengths, and extremely Whether it is acidic (e.g. pH 4 or less) or extremely basic (e.g. pH 8 or more) It can be stated that conditions do not present a highly oxidizing or reducing environment. Wear.

The pH of the EDC reaction is typically kept in the range of about 4.5 to about 6.8. EDC reaction Substitution of the carboxyl group with is recommended at lower pH levels within this general range. even faster. For example, BSA cationized for 30 minutes at pH 6.0 is During the run, the same distance (i.e. 0.7 (7)).

The reaction time is determined by the concentration of reactants and the degree of cationization desired.

Suitable reaction times range from about 5 minutes to 2 hours or less.

The reaction is maintained at a general range of about 4°C to 37°C, typically 25°C.

Each of the known cationization methods can be stopped or stopped by several methods known in the art. You can enqueue it. The EDC reaction is quenched with a buffer, preferably an acetate buffer. Confirmation and quantification of anti-cationization can be performed using known gel electrophoresis described below or those skilled in the art. This can be done by isoelectric focusing techniques known in the art (14). ninhydrin The reagent is also used to measure the number of substituted amino groups in a protein molecule, as shown below. It can be used to

Protein-containing substances have increased antigenicity, e.g. increased immunogenicity or increased It is preferable to cationize to the extent that it exhibits the ability to suppress allergic responses. This way This increase can be measured by methods known in the art, such as those described below. Ru. For many types of PO2, this results in isoelectric points ranging from about 6.5 to about 9.5 However, the scope of applicability of the present invention is not limited thereby.

For many types of PO2 that have been altered to the extent that their antigenicity has increased, the PO2 Maximum possible number of anionic groups that can be modified under "mild" conditions while in the denatured state About 20% to about 60% of the . As used herein, "denatured" refers to chemical modification of any kind.

Many types of PO5 modified by the method of the present invention fall within the above-mentioned isoelectric point range. contains types of PO5 whose isoelectric points are outside this range in unmodified or cationized form. For all types of PO2 including Determination and adjustment are within the skill of those skilled in the art.

The following examples demonstrate the methods of the invention performed with several antigens. any specific Varying parameters and methods to optimize the method for PC5 is within the known technology.

Example 1 Bovine serum albumin (nBSA) crystallized five times was added as described in the border (9). It was cationized using a standard method.

Dilute 5 g of nBSA in distilled water to a volume of 25 ml, add 67 ml of ethylene diluted A solution of the amine in 500 ml of distilled water was mixed. Adjust the pH of this solution to 6N H Adjusted to approximately 4.75 with CI+. To this was added 1.8 g of 1-ethyl-3-(3-dimethyl thylaminopropyl)-carbodiimide was added.

With constant stirring, the reaction was allowed to proceed for various times, while the temperature was maintained at approximately 25°C. After quenching with a 4M acetate buffer mixture kept at a constant pH, Dialysis treatment against distilled water was performed multiple times and freeze-drying was performed. Next, add this to Sephadex It was passed through a column of G-25 and lyophilized again before use.

Example 2 Except that the pH of the reaction was adjusted to about 6.0 with 6N HCI) and the reaction was run for about 1 hour. , the method of Example 1 was carried out.

Example 3 Same as Example 1 except that PO2 is non-denatured bacterial endotoxin instead of nBSA. Implement the method.

Example 4 The PO2 used was not nBSA but undenatured tetanus toxoid (Ledery Laboratories). Lederle Laboratories, the No. 1 company in New York, NY Except for the example (purchased from Pearl River, NY) Method 1 was implemented.

Example 5 The Pcs used was non-denatured chicken egg albumin (OVA) instead of nBSA. Except for the above, the Pcs obtained by actually using the method of Example 2 were treated with ferritin (Sigma Chemical Co., Ltd.). (Sigma Chemical Co.) St. Louis, Missouri, USA (S) (Purchased from t, Lou i s, Mo) and the sample was heated for 5 minutes, 15 minutes and The method of Example 1 was carried out, except that cationization was carried out for 30 minutes.

Example 7 The method of Example 1 was carried out, except that the Pcs used was heat-killed E. coli. Ta.

Example 8 The Pcs used were bovine-7-globulin (BGG) (Sigma Chemical Co., USA). The method of Example 1 was carried out except that the following steps were taken (St. Louis, Missouri).

Example 9 Fluorescein inthiocyanate (FITC), a small non-immunogenic molecule, was conjugated to either the BSA carrier or the CBSA carrier (prepared in Example 1). The method of Example 1 was carried out except that Using these complexes as Pcs Ta. The method for combining fluorescein inthiocyanate and BSA is as follows. .

FITC is used at a concentration of 0.5 mg per rag of protein. FITC Add to the BSA solution and adjust the pH to about 8.4 with borate buffer. Mechanically agitating the bond Let stand for about 1 hour while stirring at low speed.

The suspension was kept in a cold room, and the pH was adjusted to approximately 7.81 with borate buffer, and the saline solution was frequently added. Dialyze while exchanging. Dialysis takes several days, and the dialysate turns yellow-green under ultraviolet light. It is complete when it disappears. The complex is cleared by centrifugation.

Other hapten-carrier linkages require additional steps to achieve covalent bonding between reactive molecules. Complex chemical reactions are required.

1) Corning Electrophoresis Agarose Universal Gel Filler lum (1% agarose, 5% sucrose, 0.035% EDTA) M's barbital buffer, pH 8,6), 2) Corning Uni Versal PHAB buffer (harbital sodium 17.7 g, barbiturate 2 ．． 6g.

Sodium chloride 1.0g5 EDTA disodium 0.7g1 and sinicrose octaacetate) with 0.035% EDTA, reconstituted with distilled water, 05M buffer, pH 8.6.

3) Corning ■Amido Black IOB stain.

Note: This was purchased from Fisher Scientific.

4) Corning Cassette Electrophoresis Cell with Corning Power Supply .

Method: 1) Load 160 gg of sample at a concentration of 30 μ/ml onto the gel in each well.

(For samples with lower concentrations, add 1 gg sample until the appropriate concentration is reached.) The sample is loaded and allowed to dry between applications. ) 2) Add 95 μp of buffer to each side of the cell.

3) Place the gel on an electrophoresis unit and run it for an appropriate period of time.

For cBSA, the period was 40 minutes, and nBSA was run as a control).

4) Remove the gel from the electrophoresis unit and place it in Amido Black stain for 15 minutes. . Destain the gel in 5% acetic acid for 20 seconds.

5) Dry the gel.

6) Re-wash the gel in 5% acetic acid to obtain a good contrast between background. The cells were destained until they were removed.

7) The gel was washed in two separate distilled H20 baths.

8) Dry the gel again.

The sample was subjected to isoelectric focusing by conventional methods described in the prior art. It is also possible to measure the isoelectric point (pI) value of a particular cationized product.

As a result of the cationization process, the anionic side chain carboxyl groups become polycationic amino Substituted with an ethylamide group (14.15). The product dissolves easily in water and reduces dodecyl sulfate with or without agents such as mercaptoethanol. In the presence of sodium chloride, gel electrophoresis shows a single band. This band is It moves toward the anode more quickly than the unmodified B5A11j. Sephadex G2 00, a single peak appeared slightly before the DBSA peak. Indicates peak.

From electrophoresis on agarose gel (Table 1), a reaction time of 2 hours revealed that cBS It can be seen that molecules that appear to have achieved maximum positive charge are produced in A. this is that preparations reacted for longer times will migrate farther toward the anion. This is because it shows no movement.

By using the agarose gel method described above, the following results were obtained.

Electrophoresis results Reaction time Distance traveled toward the cathode ((1)) CBSA7z litine cOVA ]5 minutes 0.7 1.0 3.0 30 minutes 1.0 1.3 4.0 60 minutes 1.6 1.7 5.0 90 minutes 1.9 N/T 5.3 120 minutes 2. I N/7 5.5 150 minutes 2. I N/T N/T 180 minutes 2. I N/T N/T N/T- (not tested) When the product was measured, the isoelectric point was 6.5 or more and about 9.5 or less. isoelectric point It was used as one of the scales to measure the degree of cationization of a given Pcs.

The number of amino groups substituted in a protein molecule by cationization is It can be measured using a reagent. Ninhi purchased from Sigma Chemical Company Curve with increasing concentrations of glycine, which serves as a standard using Dolin's reagent. was created. The procedure for the experiment using BSA was as follows.

Ninhydrin reagent was added to cBsA dilution and nBSA (control) and the mixture was Heat at 0°C for 20 minutes, at which point the color of each mixture was read on a spectrophotometer at 550. I took it. Cation with ethylenediamine/EDC for 15 minutes and clean with acetate buffer. The quenched BSA sample added 20 new amino acids compared to the native BSA control. group was increasing. Fully cationized BSA contains approximately 80 novel amino acids. It should contain a group. Isoelectric point of BSA sample cationized for 15 minutes was 8.0. Therefore, the placement of amino groups on molecules of protein-containing substances It is possible to quantitatively measure the conversion number.

Such measurements are useful in expressing the degree of cationization of protein-containing substances. be. Therefore, the degree of cationization determines whether a protein-containing substance is in its native state. under relatively mild conditions (i.e., the cations of the invention disclosed herein) Among the anionic groups that can be modified under the reaction conditions of can be expressed as the rate of substitution. This rate is typically 20% to 60% (within the scope of, but not limited to) Bar Harbor, Maine, U.S.A. Bar Harb.

r, ME) Jaquelin/Laboratory (Jacks.

It was purchased from N Laboratory.

Adjuvants: complete and incomplete 70-indoadzinibant (IFA), and Fungal lipopolysaccharide was produced in Detroit, Michigan, USA. Difco Laboratories (IT, MI) purchased from ries). Aluminum hydroxide gel was prepared by Levin et al. and Vaz) (11) or commercially available markers. Maalox (Rorer InC1), USA pencil Uses Fort Washington, Vanier (Fort Washington, PA) did.

Antibody Assay: Quantitative EL ISA method as previously described (12 )used. using known amounts of antibodies against native or cationized BSA. A standard curve was drawn for each test. Next, EL coated with serum and antigen I Assayed on SA plate. Depending on the test, the coating on the plate This variation is shown in the table below where applicable.

T cell proliferation assay: Incomplete flocculation was performed on the hindlimb hopping and tail base of BDPI mice. Inject 100 μg of native or cationized antigen emulsified in indoor adjuvant. I shot it.

Inguinal and axillary lymph nodes were removed after 10 days, and the subsequent cell suspension was placed on a nylon It was passed through a wool column as previously described (13). Next is Nairo Non-adsorbable cells were added to wool with 10% horse serum, 1 mM non-essential amino acids, 1 mM Sodium Pyruvate, 2mM Glutamine, 5X10-'M2-Mercapto Ethanol, 25mM HEPES, and 5μg/mfl gentamicin Resuspend in complete RPM 11640 medium containing Costar, Cambridge, Mass. idge.

MA) at 5 x 10' cells/well. Undenatured or Cationized antigen was added to serum-free complete RPM 11640 and wells were set up in triplicate.

Serum-free medium served as a control. Cells were grown in a final volume of 200 μm at 137 °C and 5% C. Incubate with O2 for 72 hours, at which point each well receives 1 μCi of 3H-thymidine. Added gin.

Cells were harvested after 20 hours with a Skatron harvester and placed in a liquid system. Radioactivity was measured by anti-silane spectrophotometry.

cBsA, cOVASc7e! J Chin, C Escherichia coli, C tetanus toxoid, c B GGs and FITC-cBSA complex to reduce their antigenicity to their native state. The following results were obtained for immunogenicity and desensitization studies comparing morphology.

Table A Anti-BSA antibody Cu g/mfl) Immunization 9th, 14th, 21st 50ug nBSA, 58 28 0 intravenous injection (5 times) 50ug cBSA, 393 450 425 intravenous injection (5 times) cBSA was prepared in the same manner as in Example 1.

BSAS-Plate Test.

Table A shows that cBSA is immunogenic when injected into mice.

Table B Effect of cationization on the immunogenicity of bovine serum albumin Time of cationization Type of source Relative immunogenicity (μg/antibody mal) None nBSA 350 15 minutes cBSA 1220 30 minutes cBsA 1450 60 minutes cBSA 1135 120 minutes cBsA 110 Anti-BSA response (μg/ml of antibody after 14 days) cationization at pH 4,7 It was carried out at temperatures of 5 and 25°C.

Table B shows that the length of time a protein is cationized controls its immunogenicity. I understand. Cationation optimizes immune responsiveness. However, cation Excessive oxidation will rapidly reduce immunogenicity unless it is completely removed. The administration is Administered intraperitoneally (i, p,) with alum azinibant (1 mg/dose). It was.

Table C Effect of cationization on antigen reactivity (bovine serum albumin) Cationization time Type of antigen Relative antigenic activity nBSA ++ 15 minutes cBSA +++ 30 minutes cBSA +++++ 60 minutes CBSA +++ 120 minutes cBsA - Responsiveness of BSA-sensitized T lymphocytes to antigens Table C shows that the cationization of BSA is excessive. Excess may result in the molecule being unable to stimulate BSA-sensitized T lymphocytes. Recognize.

Cationization for less than 60 minutes significantly increases the ability of BSA to stimulate lymphocyte proliferation. However, excessive cationization produces non-immunogenic or less immunogenic molecules.

Table D Anal (in vivo) pretreatment with soluble nBSA or cBsA is effective against each antigen. Effect on IgG antibody response Glue Pretreatment Day - 9, 8, 7 Immunization Enhancement or Suppression Rate 1) (%) A 100μgnBsA 100μgnBSA -18-26-51 (alum (in progress) B 100μgnBsA 100μgnBsA -95-94-92 (IFA” During) CI00μgnBsA 100μgcBSA -84-87-91 (in IFA) D I00μg eBsA 100μg eBsA 2200 8055 40 0 (medium alum) E100μg cBSA 100μg cBsA 98 113 2g (in IFA ) F 50ug cBsA 100μg cBsA 184 241 42 (IF A middle) 1) Compared to a control group immunized with nBSA or cBSA and pretreated with physiological saline compared. Suppression is indicated by negative values.

2) Incomplete Freund's adjuvant cBsA was prepared in the same manner as in Example 1.

From the table, nBSA virtually suppresses the production of anti-BSA antibodies, whereas CBSA is shown to generate an even higher immune response after initial pretreatment with cBsA. .

Effect that pretreatment has on the immune response (IgG) to each antigen Glue Pretreatment Day - 9, 8, 7 Immunization Enhancement or Suppression Rate 1゜(%) - Bu (intravenous) 10th and 21st A 25u g cBsA 100. cz g nBSA 2592413B 25ug cBsA 100μgcBsA 497 274C10ug cBS A 100μg nBSA 867 2818D lOμg cBSA 100 μg cBsA 56B 683E lu g cBsA 100μg nBS A 230 g86F lug cBsA 100μg cBsA 1321 1018G 25ug nBSA lon μg nBSA -85-27H25 u g nBSA 100. cz　g　cBsA　-22NS　”1　10ug nBSA 100u g nBSA-87NS”J 10ug nBSA 100μg cBsA NS NSK 1μg nBsA 100μg nB SA　NS　NSL　101μg　nBSA　100μg　cBsA　NS　N 51) Immunization was done by intraperitoneal injection using 1 mg alum as an adjuvant. It was done by giving.

2) Compared to a control group immunized with nBSA or cBSA and pretreated with physiological saline Comparison. Suppression is indicated by a negative value.

3) NS: Not significant (change rate less than 5%).

Table E shows BSA obtained using small amounts (1-25 mg) of cationized BSA. the immune response obtained, both in terms of increased rate of enhancement and duration of increase. We present the results of a more detailed study on epidemic response.

Table F Effect of oral administration of antigen on IgG response Oral administration Anti-BSA response in alum Answer - Immunization with 1 gG (μg 1 country 1) (3x20+ag) (100μg) 10th 14th 24th - BSA 90 0 812 240 cBsA 1024207 [i 3050BSA BSA 87 58 57 cBsA BSA 27001074 875BSA cBsA 250 2ε 7675cBsA cBsA 37006300 [Tested on 1300BSA board] Table G Effect of oral administration of antigen on IgE response Oral administration Anti-BSA response in alum Answer - Immunization with 1gE (PCA titer) (3x20Img) (100u g’) lod 14d 24d-BSA 40 82 116 - eBsA 80 160 160 BSA BSA 20 20 40 BSA BSA 20 20 2G BSA cBsA 10 10 10 cBsA cBsA 20 20 20 Tables F and G show the cationized form and Figure 3 shows the effect of oral administration of the antigen in its native and native forms. Table F shows immunization with cBsA. has been shown to increase the levels of anti-BSA IgG, while Table G shows that has shown that CBSA suppresses the levels of anti-BSAIgE.

Table H C0vA administered orally stimulates the immune response to n0VA and cOV A1: impact on 1.0μg alum immune response, No resistance after 14 days with 0.1 μg of antigen in 20 mg of antigen n0VA 140 None cOVA 765 nOV＾　n0VA　35 nOVA cOVA IPfO eOVA n0VA 1320 - 憇■-------Normal and 1111111-μ''--OVA is explained in Example 5. Ionization was performed at 1 hour intervals as instructed.

Table H shows that administration of n0VA suppresses the immune response to OVA. Karu. In contrast, when administering (oral administration) b5J I:, C0VA, n0V The immune response to both A and cOVA (7) is significantly enhanced.

Table 1 BDF, immune response to cOVA and nOVA in mice 111 g of alum intraperitoneally with the response mortar after immunization (μg/anti-OVA Antibody ml 9 days 14 days 21 days Administered antigen □ 0.1ug n0VA 80 120 1201, Oug n0VA 450 B2O66010u g n0VA 1250 1420 13000.1 ug eOVA 425 97G 7701, Ou g cOVA 1B00 2010　1660　1660　Yu　　　　　　235G　3μ1　　　　　235G　3μ1　- OVA was conducted Cationization was performed as described in Example 5.

From Table 1, cQVA has a higher nO It can be seen that it is much more immunogenic than VA.

Table J Effects of intraperitoneally administered cationized ferritin on BDF in mice n ferritin (control) Is　134　101　490 Cationized ferritin (cF) was produced as in Example 6, and BDF was inoculated into mice. Administered intracavitally. Antibody levels are determined from bleeding after various periods of time; Blood was measured by ELISA on plates coated with native ferritin. did.

Table J shows that the immunogenicity of partially cationized ferritin is substantially increased. Recognize.

Table K Effects of intraperitoneally administered cationized E. coli in BDFI mice lXl06160 210 180 5X106220 450 650 IXIO' 260 500 480 5XlO' 320 800 850 Heat-killed E. coli was cationized as in Example 7, and cationized with saline three times. Washed, mice were injected intraperitoneally, and then the mice were bled. The antibody concentration is It was measured by ELISA using a plastic plate coated with native bacteria.

Antibacterial antibody titers from mice immunized with native bacteria served as controls; were compared with antibody titer fractions from mice immunized with partially cationized bacteria.

Table K shows that the immunogenicity of partially cationized bacteria is substantially increased. .

Table Alum of BDlag of cationized tetanus toxoid administered intraperitoneally cT T nTT (control) 18g eTT 220 800 32010μ g cTT 325 580 720100μgcTT 280 450 66 Tetanus toxoid was cationized as in Example 4.

Animals were bled after various times and antibody levels were measured on native TT. Measured by EL I SA.

The table shows that partial cationization increases the immunogenicity of tetanus toxoid. I understand.

Table M Effects of intraperitoneally administered cationized BGG on BDPI mice 1 μg 55 80 60 1Oμg 110 400 800 50μg 350 850 1000 100μg g20 1200 1200 discharge average 1 μg 110 180 180 10μg 450 1200 160050μg 1200 2500 250 0100μg 850 2100 2800 Bovine-γ-globulin (BGG) Cationization was performed as described in Example 8.

Animals were bled after various times and antibody levels were determined by ELISA. Measured by. Column purified anti-BGG antibody was used as a standard. Each group The loop consisted of 5 mice and the whole experiment was repeated twice.

From Table M, partial cationization significantly increases the immunogenicity of bovine γ-globulin. It can be seen that it increases.

Table N Effects of FITC complexed with cBSA on BDF1 mice Progress of bleeding FITC-cBsA to FITC-nBA (control) Reinforcement rate (%) against day The FITC-BSA conjugate was prepared as described in Example 9.

The immune response was induced in mice by FITC-nBSA or FITC-cBS A: Antibodies raised against were coated with FITC-KLH [conjugate] Determined by measuring on ELISA plates.

From Table N, it can be seen that haptens, which are small non-immunogenic molecules, such as FITC, are partially Can be highly immunogenic when complexed with cationized protein (cBSA) Wow.

From the above data, it is clear that the method of immunizing mammals according to the present invention or The allergy response suppression method is based on partial positive It includes the step of administering an effective amount of ionized antigen protein, and the administration can be done orally or parenterally. It is clear that the adjuvant may or may not be used. Breastfeeding A method for enhancing an immune response to a native antigen in an animal includes an isoelectric point of about 6.5 to about An effective amount of the partially cationized antigen is administered parenterally to a mammal in the range of 9.5 administering and subsequently feeding a sufficient amount of native antigen to generate an immune response. It consists of the step of parenterally administering to animals.

The present invention is not limited to any theory as to why the beneficial results are achieved. However, cationized proteins are more immunogenic for the following reasons: It is presumed to be of primary origin.

1. Increased affinity for antigen presenting cells (APC).

2. Changes in antigen processing by APC.

3. More efficient recognition by helper T cells.

4. Increased affinity for self-recognized antigen (Ia).

Increased affinity for 5.7 cell receptors.

6. Activation of a new type of helper T cell that recognizes antigens more efficiently.

With reference to the above teachings, examples and results, there are no deviations from the concept of the present invention. It is within the skill of the art to modify the method of the invention without further modification.

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Claims (1)

[Claims] 1. (a) (1) protein-containing substance and (2) the above protein-containing substance in cationic form. react with a reactant that can be converted into (b) the reaction between the substance and the reactant is carried out on a portion of the protein-containing substance; stop after sufficient time has elapsed for target cationization to occur. A method for producing an antigen protein-containing substance comprising: 2. 2. The method according to claim 1, wherein said protein-containing substance is an antigen. 3. The protein-containing substance may be bovine serum albumin, chicken egg albumin, Bovine-7-globulin, ferritin, bacterial endotoxin, viral protein Tetanus toxoid and killed microorganisms, as well as their protein-containing raw materials. 2. The method of claim 1, wherein the method is selected from the group consisting of: 4. 4. The method of claim 3, wherein said protein-containing substance is bovine serum albumin. 5. The method according to claim 3, wherein the protein-containing substance is bovine-7-globulin. . 6. 4. The method according to claim 3, wherein the protein-containing substance is ferritin. 7. 4. The method according to claim 3, wherein the protein-containing substance is E. coli. 8. 4. The method of claim 3, wherein said protein-containing substance is tetanus toxoid. 9. The cationized reactant is at least one carbodiimide and at least one carbodiimide. 2. A method according to claim 1, wherein the method is a mixture with one amine. 10. At least one of the above carbodiimides is 1-ethyl-3-(3-dimethyl 10. The method according to claim 9, wherein the compound is carbodiimide. 11. Claim 9, wherein said at least one amine is ethylenediamine. Method. 12. to the extent that said protein-containing substance exhibits increased antigenicity; 2. The method according to claim 1, wherein the substance containing a substance is cationized. 13. Contains protein until the isoelectric point of the above-mentioned substance-containing substance is less than 9.5. 2. The method of claim 1, wherein the substance is cationized. 14. The isoelectric point of the protein-containing substance is in the range of about 6.5 to about 9.5. 2. The method according to claim 1, wherein the protein-containing substance is cationized. 15. 2. The method of claim 1, wherein said reaction is quenched with an acetate buffer. 16. While the above protein-containing material is in its native state, it can be modified under mild conditions. The above product is obtained by modifying about 20% to about 60% of the maximum possible number of anionic groups. Claim 1, wherein the protein-containing substance is cationized to achieve high quality cationization. Method described. 17. (a) (1) Antigen and (2) React with ethylenediamine/carbodiimide, then (b) The reaction between the antigen and the ethylenediamine/carbodiimide as described above Stopping after sufficient time has elapsed for partial cationization of the antigen to occur. A method for producing an antigen with increased antigenicity comprising: 18. The above antigen is bovine serum albumin, chicken egg albumin, bovine Robulin, ferritin, bacterial endotoxin, viral protein, tetanus Consisting of toxoids and killed microorganisms and their protein-containing products 18. The method of claim 17, wherein the method is selected from the group. 19. To the extent that the protein-containing substance exhibits increased antigenicity, 18. The method according to claim 17, wherein the substance containing a substance is cationized. 20. Partially cationized to the extent that it exhibits increased antigenicity in mammals. A mammal comprising administering an effective amount of a cationized antigenic protein-containing substance. How to immunize. 21. A claim in which the protein-containing substance has an isoelectric point in the range of about 6.5 to about 9.5. The method according to item 20. 22. Claim 2, wherein the partially cationized protein-containing substance is orally administered. The method described in 0. 23. A claim that the above partially cationized protein-containing substance is administered parenterally. 20. The method described in 20. 24. The above partially cationized protein-containing substances are administered without adjuvant. 23. The method of claim 22, wherein the method provides: 25. Administer the above partially cationized protein-containing substance with an adjuvant 21. The method according to claim 20. 26. Efficacy of partially cationized antigens that have been cationized to the extent that immunogenicity is increased amount is administered parenterally to a mammal, and then an amount sufficient to generate an immune response is administered to the mammal. comprising administering a native antigen to said mammal, said partially cationized antigen; to a mammal that is manufactured from the same native antigen that is subsequently administered. A method of enhancing immune responsiveness to native antigens in patients. 27. The isoelectric point of the above partially cationized antigen was determined by isoelectric focusing to about 6. 27. The method of claim 26, wherein the nucleation rate is in the range of 5 to about 9.5. 28. The above native antigen and the above partially cationized antigen are incorporated into bovine serum albumin. chicken egg albumin, bovine 7-globulin, ferritin, bacterial endo Toxins, viral proteins, tetanus toxoid and killed microorganisms, and 27. The method of claim 26, wherein the protein-containing product is selected from the group consisting of: . 29. Allergic response suppressive properties in mammals prior to exposure to specific allergens. Oral administration of an effective dose of a partially cationized allergen that has been cationized to an extent that increases administration of the partially cationized allergens mentioned above to the specific allergens mentioned above. in mammals susceptible to certain allergens that are manufactured from allergens. How to suppress allergic responses. 30. The isoelectric points of the above partially cationized allergens were measured by isoelectric focusing. 30. The method of claim 29, wherein the molecular weight is in the range of about 6.5 to about 9.5. 31. The above allergens are bovine serum albumin, chicken egg albumin, bovine 7-Globulin, ferritin, bacterial endotoxin, viral protein, Tetanus toxoid and killed microorganisms and their protein-containing products. 30. The method according to claim 29, wherein the method is selected from the group consisting of: 32. The method according to claim 1, wherein the protein-containing substance is complexed with a hapten. . 33. Partially with increased antigenicity compared to the same native protein-containing material Cationized antigenic protein-containing substance. 34. The isoelectric point of the partially cationized substance described above is approximately 6.0 as measured by isoelectric focusing. 34. The partially cationized material of claim 33, which ranges from 5 to about 9.5. 35. 34. The partial positive according to claim 33, wherein said native protein-containing substance is an antigen. Ionized substances. 36. The undenatured protein-containing substances listed above are bovine serum albumin, chicken egg protein, etc. rubumin, bovine-7-globulin, ferritin, bacterial endotoxin, virus infectious proteins, tetanus toxoid and killed microorganisms, and their proteins. 34. The partially cationized substance of claim 33, wherein the partially cationized substance is selected from the group consisting of . 37. While the above protein-containing material is in its native state, it can be modified under mild conditions. The above materials are modified by modifying about 20 to about 60% of the maximum possible number of anionic groups that can be used. 34. The partially cationized substance according to claim 33, which cationizes. 38. 34. The partially cationic compound according to claim 33, wherein the protein-containing substance is a complex. substances.