EP0300007A1

EP0300007A1 - Partial cationization of protein-containing antigens and their use for immunization and desensitization

Info

Publication number: EP0300007A1
Application number: EP88901145A
Authority: EP
Inventors: Jacob Gabriel Michael
Original assignee: Individual
Current assignee: Individual
Priority date: 1987-01-30
Filing date: 1988-01-25
Publication date: 1989-01-25
Also published as: CA1338078C; IL85256A; KR890700355A; AU1183688A; AU618946B2; WO1988005664A1; IL85256A0; JPH01502024A

Abstract

Procédé de préparation d'une substance antigénique contenant une protéine par cationisation partielle d'une substance contenant naturellement une protéine à un point isoélectrique situé entre environ 6,5 et environ 9,5. La substance partiellement cationisée présente une antigénicité accrue par rapport à la substance contenant naturellement une protéine et est utilisée pour des traitements d'immunisation et de désensibilisation de mammifères, en doses administrées par la voie orale ou parentérale.Process for the preparation of an antigenic substance containing a protein by partial cationization of a substance naturally containing a protein at an isoelectric point situated between approximately 6.5 and approximately 9.5. The partially cationized substance has increased antigenicity compared to the naturally containing protein and is used for mammalian immunization and desensitization treatments in doses administered orally or parenterally.

Description

PARTIAL CATIONIZATION OF PROTEIN-CONTAINING

ANTIGENS AND METHOD OF IMMUNIZATION AND DESENSITIZATION

Cross-Reference To Related Application

This application is a continuation-in-part application Serial No. 07/009,234, filed January 3 1987, in the name of Jacob Gabriel Michael.

Background of the Invention

This invention relates to a method of preparing partially cationized protein-containing substance, su as an antiqen, having increased antigenicity, to t product of this method, and to a method of enhanci immune response to a native antigen in a mammal.

The importance of the use of antigens in the preven tion of infectious disease through immunization and i the treatment of allergies through desensitization i well known.

The basis of immunization is the exposure of th organism to be immunized to dead or weakened infectiou agents (viruses, bacteria, toxins, etc.) or extract thereof which contain a foreign, generally macromolecula substance which is capable of evoking an immune response These substances are generally referred to as antigens Likewise, allerqic reactions can be lessened b desensitization, wherein such a substance is used t suppress the normal allerqic response caused by foreig substances, referred to as allergens. Most antigens and allergens are either wholly o partially composed of orotein.

The action of antiqens is thouqht to be dependent i part on the antioen's affinity for certain binding site on cells of the livinq tissue affected by the antigen Such tissue may be blood, internal organs, skin, eyes etc. Interaction of the antigen to the binding sit stimulates the production of antibodies which in tur defend the organism against infectious agents containin the antigen.

Because antigens are foreign substances, they ca have adverse side affects on the organism sought to b immunized. This is most important in the treatment o humans and livestock. It is therefore desirable to b able to achieve an effective immunogenic response b utilizinq lower levels of the antigen. If antigenicit can be increased, it necessarily follows that a smalle dosage can be administered to achieve a given level o immunization.

One way to increase the antigenicity of a substanc is by using an adjuvant in conjunction with the antigen An adjuvant is a substance which augments the immunogeni response by aiding the antigen's interaction with th living tissue. Examples of adjuvants include alum salt for human use. A problem which militates against the us of adjuvant^'s in _in_ vivo immunization is the toxicity an the side effects these compounds induce. Because o this, adjuvants are generally disfavored for use i humans or in any other organism where toxicity and/o side effects are a concern. The elimination of the nee for adjuvant cooperation is therefore desirable. Some foreign substances do not evoke an antigeni response at all, or do so very poorly, when brought in contact with living tissue. One theory is that suc substances lack the threshold amount of bindin capability or strength to cause such a response. I would be advantageous to be able to convert suc substances from a non-antigenic form to an antigeni form. This would allow vaccines to be produced whic could elicit an immunogenic response to a substance wher this was not previously possible.

Immunization methods are normally carried out b subcutaneous or intramuscular injection of the vaccine Oral intake of an antigen is not feasible in mos instances because it often causes suppression of rathe than increase in the immune response. Exceptions to thi general rule include live attenuated bacterial and vira vaccines. Oral vaccines offer advantages such as lowe expense and ease in administration and packaging Therefore, it would be better to be able to administe vaccines in oral form without loss in immunogenic effect.

The chemical modification of antigens, particularl protein-containing antigens, has been known for some tim in fields of research, such as cell surface labelling see D. Danon, Use of Cationized Ferritin as a Label O Negative Charges On Cell Surfaces, J. Ultrastructur Research, Vol. 38, pp. 500-510 (1972).

Prior methods of antigen cationization have taugh the complete cationization of the antigen molecule. Her "complete cationization" is intended to mean that a give substance has had all its groups amenable to cationiza tion so altered. Completely cationized antigens ar generally unsuitable for _in_ vivo use due to their excessive reactivity.

Other background information is contained in t following references which are hereby incorporated reference:

1. Barnes, J. and M. Venkatachalam, Enhancement Glomerular Immune Complex Deposition by a Circulati Polycation. J. Exp. Med. 160:286 (1984).

2. Olte, T., S.P. Batstord, J.J. Mihatson, Takamija and A. Vogt, Quantitative Studies on in si Immune Complex Glomerulonephritis in the Rat Induced Planted Cationized Antigen. J. Exp. Med. 155:460-4 (1982).

3. Gallo, G. , Caulen, T. Glaser, S.N. Emancipat and M.E. Lamm, Nephritogenicity and Differenti Distribution of Glomerular Immune Complexes Related I munogen Charge. Lab. Invest. 48:460 (1983).

4. Schikwik, J., .B. Van den Berg, L.B.A. van Putte, L.A.B. Joosten & L. van den Bersselaa Cationization or Catalase, Peroxidase, and Superoxi Dismutase: Effect or Improved Intrarticular Retention Experimental Arthritis in Mice. J. Clin. Invest. 76:1 (1985) .

5. Muckerheide, A., A.J. Pesce and J.G. Michae Immunosuppressive Properties of a Peptic Fragment of BS

J. Immunol. 119:1340 (1977).

6. Dosa, S. , A.J. Pesce, D.J. Ford, A. Muckerhei and J.G. Michael, Immunological Properties as Peptic Fragments of Bovine Serum Albumin. Immunol. 38:5 (1979).

7. Muckerheide, A., A.J. Pesce, and J.G. Michae Kinetics of Immunosuppression Induced by Peptic Fragmen of Bovine Serum Albumin. Cell. Immunol. 50:340 (1980) .

8. Muckerheide, A., A.J. Pesce and J.G. Michae Modulation of the IgE Immune Response to BSA by Fragmen of the Antigen. Cell. Immunol. 59:392 (1981).

9. Border, .A. , H.J. Ward, E.S. Hamil and A. Cohen, Induction of Membranous Nephropathy in Rabbits Administration of an Exogenous Cationic Antige J. Clin. Invest. 69:451 (1982).

10. Apple, R. , B. Knauper, A. Pesce and' J. Michael, Shared Determinants of Native and Denatur Bovine Serum Albumin are Recognized by^' Both B- and Cells. Mol. Immunol. 21:901 (1984).

11. Levine, B.B. and N.M. Vaz , Effect Combinations of Inbred Strain Antigen and Antigen Dose Immune Responsiveness and Reagin Production in the Mous Int. Aron. Allergy Appl. Immunol. 39:156 (1970).

12. Ferguson, T. A., T. Peters, Jr., R. Reed, A. Pesce and J.G. Michael, Immunoregulatory Properties Antig —•enic Frag—ments from Bovine Serum Albumin. Cell Immunol. 73:1 (1983).

13. Julius, M.H., E. Simpson and L.A. Herzenberq, Rapid Method for the Isolation or Functiona Thymus-derived Murine Lymphocytes. Eu . J. Immunol 3:645 (1973). 14. Hoare, D.G. and D.E. Kosnland, A Method for th Quantitative Modification and Estimation of Carboxyli Acid Groups in Proteins. J. Biol. Chem. 242:2447 (1967).

15. Daron, D. , L. Goldstein, Y. Markovsky and E Skutelsky, Use of Cationized Ferritin as a Label o Negative Charges on Cell Surfaces. J. Ultrastructur Res. 38:500 (1972).

16. Warren, H.S., F.R. Vogel and L.A. Chedid Current Status of Immunological Adjuvants. Ann. Rev Immunol. 4:369 (1986).

17. Mills, Z.J. and E. Haber, The Effect o Antigenic Specificity of Changes in the Molecula Structure of Ribonuclease. J. Immunol. 91:536 (1963) .

18. Heber-Katz, E. , D. Hansburn and R.H. Schwartz The la-molecule ^"or the Antigen-presenting Cell Plays Critical Role in Immune Responses Gene Regulation o T Cell Activation. J. Mol. Cell. Immunol. 1:3 (1983) .

19. Buus, S. , and 0. Werdelin, Oligopeptid Antigens of the Angiotersin Lineage Compete fo Presentation by Paraformaldehnyde-treated Accessory Cel to T Cells. J. Immunol. 136:459 (1986).

20. Babbit, B.P., P.M. Allen, G. Matsueda, E_*. Habe and E.R. Unanue , Binding of Immunogenic Peptides to l Histocompatibilitv Molecules. Nature (London) 317:35 (1985).

21. Buus, S. , S. Color, C. Smith, J.H. Freed, C Miles and H.M. Grey, Interaction Between a "Processed Ovalbumin Peptide and la Molecules. P.N.A.S. 83:29 ( 1986) .

22. Larey, E.X., E. Margoliasn, F.W. Fitch and S. Pierce, Role of LBT4 and la in the Heteroolitic Respon of T Cells to Cytochrome. J. Immunol. 186:3933 (1986).

23. A.N. Glazer, R.J. DeLange and D.S. Sigma Chemical Modifications of Proteins. Lab. Techniques i Biochemistry and Mol. Biology, Vol. 4, Part I, p. 1-20 North-Holland (Am. Elsevier) (1976).

24. Alexander N. Glazer, The Chemical Modificati of Proteins by Group-Specific and Site Specific Reagents 1-103. In: The Proteins, 3rd ed. Vol. II Acad. Press N.Y. (1976).

Where cited herein, these publications are referre to by their numbers in the above list.

There has heretofore been no recognition in th prior art that cationized antigens can be used in in_ viv treatment and prevention of disease by use as vaccines o desensitization agents. Furthermore, the prior art ha not recognized that partially cationized antigens can b used for this purpose. Indeed, it has been reported tha completely cationized antigenoproteins do not exhibi altered immunological properties (9).

It is an object of the invention to provide partially cationized antigenic protein-containin substance, and a method for the preparation thereof which can be administered orally, and which does no require an adjuvant. SUMMARY OF THE INVENTION

As used herein, "protein-containing substance includes all proteins, as well as substances whos molecular makeup is in some part proteinacious, such a lipoproteins and proteosaccharides. These may b substances which do or do not have antigenic propertie in their native forms. More specific examples of sai substances include, without limitation, bovine seru albumin (BSA) , hen egg albumin (OVA) , bovin gammaglobulin (BGG), ferritin, bacterial endotoxin, vira proteins, diptheria toxin and tetanus toxoid, and kille microorganisms, including bacteria and viruses an protein-containinq products thereof.

As used herein, cationization means the conversio or substitution of functional groups on the protei portion of the protein-containing substance whereby th substance is rendered relatively more cationic. Suc functional groups are normally anionic within physiologic pH range and are converted to or substitute for a cationic or nonionic moiety. An example of such cationization is the reaction whereby anionic side chai carboxyl groups are substituted with polycationi aminoethylamide groups.

As used herein, the unreacted or "native" form o the protein-containing substance is indicated by a prefi "n" and the cationized form is indicated by a prefix "c" For example, bovine serum albumin (BSA) may be expresse as either "nBSA" as the native form, or "cBSA" as th cationized form.

The present invention provides a method of preparin an antigenic protein-containing substance which comprise reacting (1) a protein-containing substance, and (2) reagent capable of cationizing the protein-containin substance; and halting the -reaction between the protein containing substance and the reagent after a tim sufficient to result in partial cationization of th protein-containing substance.

According to the invention there is further provide a partially cationized antigenic protein-containin substance having increased antigenicity as compared t the same native protein-containing substance, th partially cationized substance having an isoelectri point ranging from about 6.5 to about 9.5, determined b isoelectric focusing as hereinafter described.

The invention also provides a method of immunizing mammal by administering to' the mammal an effective amoun of a partially cationized antigenic protein-containin substance having an isoelectric point in the range o about 6.5 to about 9.5 as determined by isoelectri focusing. Administration can be effected orally o parenterally, with or without an adjuvant.

A preferred method of partial cationization involve use of a reagent comprising at least one carbodiimide an at least one amine. Control of the degree of cationiza tion is effected by varying the pH, time parameters an concentration of- the reactants. The reaction may b halted or quenched readily at a desired degree of partia cationization, in a manner known in the art. By way of non-limiting example, BSA reaction with a carbodiimide and amine mixture may be quenched after substitution of about 20 new amino groups in place of anionic carboxyl groups. A fully cationized BSA would contain about 80 new amino groups. In general, an addition or substitution of fro about 20% to about 60% of the theoretical maximu possible number of amino groups will result in a isoelectric point ranging between about 6.5 and 9.5 a determined by isoelectric focusing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The protein-containing substance (PCS) can b cationized by several methods known in the art (15, 9) . The preferred method is the reaction of ethylenediamine l-ethyl-3-( 3-dimethyl amino propyl)-carbodiimide (EDC) with the PCS. This particular reaction involves th activation of the carboxyl groups of a protein wit carbodiimide and the subseguent reaction of the activate carboxyl with a nucleophile of the general type +R- H2 t obtain the primary amine type derivatives. Considerabl versatility can be achieved since both the chemica nature of the modification, i.e. introduction of primary secondary or tertiary amine groups and the degree o modification of the protein carboxyls can be varied b proper choice of reagents, reaction time and pH of th coupling reaction.

The cationization methods suitable for use in th present invention are those which effect cationizatio under mild reaction conditions. As used herein mil reaction conditions are those_, under which the molecula character (e.g. 1°, 2° and 3° structure) of the subjec PCS is not substantially altered so as to adversel affect its antigenic pH immunogenic character. Thes conditions can be most generally described as unde relatively low temperature and low ionic strength as wel as neither very acidic (e.g. below pH 4) nor very basi (e.g. above pH 8) nor presenting a very oxidative o reductive environment.

The pH of the EDC reaction is generally maintaine in the range of from about 4.5 to about 6.8. More rapi substitution of carboxyl groups in the EDC reactio occurs at the lower pH levels within this general range For example, BSA cationized at pH 6.0 for 30 minute migrated during electrophoresis the same distance as BS cationized at pH 4.75 for 15 minutes (i.e., 0.7cm) .

Reaction time is determined by the concentration o the reactants and by the degree of cationization desired The preferred reaction time is in the range of from abou 5 minutes to no more than two hours.

The reaction is maintained within the general rang of from about 4° C to about 37° C and is 'generall maintained at about 25° C.

Each known method of cationization may be halted o quenched according to several methods known in the art The EDC reaction is guenched with a buffer, preferably a acetate buffer, which halts the reaction. Concentratio of the acetate buffer is about 4M.

Cationization can be verified and quantified usin known gel electrophoresis as hereinafter described, an isoelectric focusing techniques known in the art. (14) Ninhydrin reaqent can also be used to determine the number of amino groups substituted in a protein molecule as shown hereinafter.

It is preferred that the protein-containing substance be cationized to an extent whereby it exhibits increased antigenic character such as an increas immunogenic character or an increased allergic respon suppressing character. Such increases may be determin by methods known in the art such as those describ below. Although not a limitation to the scope of t applicability of the invention, for most types of PC this will be at a point where the isoelectric point fal within a range of from about 6.5 to about 9.5.

Also as a nonlimiting guide, most PCS types whi are modified to such an extent so as to increase the antigenic character will generally have from about 20% about 60% of the maximum possible number of those anion groups amenable to modification under "mild" conditio while that PCS is in its native state, so modified. used herein "modified" is intended to mean any type chemical modification which causes cationization of t PCS.

Although most of the PCS types modified according the inventive method fall into the above-describ isoelectric point range, it is within the skill in t art to determine and adjust the degree of parti cationization which increases the antigenic character f all PCS types including those PCS types, in native cationized form, whose isoelectric points may fa outside this range.

The following Examples exhibit the inventive meth as practiced on several antigens. Variations of t parameters and methodology for optimization of the meth for any specific PCS is within the known art. Example 1

Bovine Serum albumin (nBSA), five tim crystallized, was cationized according to the gener procedure described by Border (9).

Five grams of nBSA was dissolved in distilled wat to a volume of 25 ml and admixed with a solution of 67 ethylene diamine in 500 ml distilled water. The pH this solution was adjusted to about 4.75 with 6N HCl. this was added 1.8 grams of 1-ethyl -3-(3 dimethyl ami propyl )-carbodi imide .

The reaction was permitted to react for varyi periods of time with constant stirring, while t temperature was maintained at about 25° C and the pH w held constant. After quenching with 4M_ acetate buff mixture, the reaction was subjected to multiple dialys treatments against distilled water and lyophilized. was then passed through a column of Sephadex G-25 a lyophilized again before use.

EXAMPLE 2

The method of Example 1 with the exception that t pH of the reaction was adjusted to about 6.0 with 6N H and the reaction was permitted to react for about 1 hour.

EXAMPLE 3

The method of Example 1 with the exception that th PCS may be. native bacterial endotoxin instead of nBSA. Example 4

The method of Example 1 with the exception that th PCS used was native tetanus toxoid instead of nBS (purchased from Lederle Laboratories, Pearl River, NY).

Example 5

The method of Example 2 with the exception that th PCS used was native hen egg albumin (OVA) instead o nBSA.

Example 6

The method of Example 1 with the exception that th PCS used was ferritin (purchased from Sigma Chemical Co. St. Louis, MO), samples of which were cationized for minutes, 15 minutes and 30 minutes.

Example 7

The method of Example 1 with the exception that th PCS used was heat killed E.coli bacteria.

Example 8

The method of Example 1 with the exception that th PCS used was bovine gamma globulin (BGG), purchased fro Sigma Chemical Co., St. Louis, MO.

Example 9

The method of Example 1 with the exception tha fluorescein isothiocyanate (FITC), a smal non-immunogenic molecule, was conjugated either to nBSA carrier or to a cBSA carrier (as prepared in Exam 1). These conjugates were used as the PCS. fluorescein isothiocyanate-BSA conjugation method is follows:

FITC is used in 0.5 mg concentration per mq protein. FITC is added to BSA solution and the pH adjusted to about 8.4 with borate buffer. conjugation is allowed to proceed for about 1 hour w continuous mechanical stirring at slow speed.

The suspension is dialyzed in a cold room w frequent changes of saline adjusted to a pH of about with borate buffer. The dialysis reguires several d and is complete when the dialyzate is virtually free yellow-green color under ultraviolet light. conjugate is clarified by centrifugation.

Other hapte -carrier conjugations may require m complex chemical reactions to achieve covalent -bondi between the reacting molecules.

Cationization Assay; Agrose Gel Electrophoresis

Materials:

1) Corning^® Electrophoresis Agarose Universal G Film (1% Agarose, 5% sucrose, 0.035% EDTA, in 0.06 barbital buffer, pH 8.6);

2) Corning^® Universal PHAB Buffer (Sodium barbit 17.7g, barbital 2.6g , sodium chloride l.Og, disodium ED 0.7g , and sucrose octaacetate) Reconstituted wi distilled water to 0.05M buffer with 0.035% EDTA, pH 8.6. 3) Corning® Amido Black 10B Stain

NOTE: Above purchased from Fisher Scientific.

4) Corning^® Cassette Electrophoresis Cell wit Corning Power Supply.

Methods;

1) 1.0 ul of sample at a concentration of 30 mg/ra is loaded onto gel into each well. (For sample of lesse concentration, 1 ul of sample is loaded and allowed t dry between applications until appropriate concentratio is reached) .

2) 95 ul buffer is added to each side of cell.

3) Gel is placed on electrophoresis unit and ru for appropriate time period. (For cBSA - time period i 40 minutes; nBSA is run as a control).

4) Gel is removed from electrophoresic unit an placed in amido black stain for 15 minutes. Gel i destained in 5% acetic acid for 20 seconds.

5) Gel is allowed to dry.

6) Gel is destained again in 5% acetic acid unti good contrast is seen between back and background.

7) Gel is rinsed in 2 separate distilled H2O baths.

8) Gel is again_.allowed to dry. Samples may be subjected to isoelectric focusing determine the isoelectric point (pi) value of t particular cationized product, by a convention procedure described in the prior art.

Results:

Chemical Properties and Electrophoresis

The cationization procedure results in t substitution of anionic side chain carboxyl groups polycationic aminoethylamide groups (14, 15). T products are readily soluble in water and show a sing band on gel electrophoresis in the presence of sodi dodecyl sulfate, with or without a reducing agent such mercaptoethanol . The band migrates more rapidly towa the cathode than the native BSA monomer. Passage throu Sephadex G 200 shows a single peak which appears slight before the nBSA peak.

Electrophoresis on agarose gel (Table 1) shows th the two hour reaction time produces a molecule whi appears to have attained a maximum positive charge in t case of cBSA since preparations which are permitted react for a longer period of time do not exhib increased migration toward the cathode.

The following results were obtained using the abo outlined agarose gel technique: Electrophonesis Results

Distance Migrated Reaction Time Toward Cathode (cm)

cBSA cFerritin cOVA

15 min. 0.7 1.0 3.0 30 min. 1.0 1.3 4.0 60 min. 1.6 1.7 5.0 90 min. 1.9 N/T 5.3 120 min.. 2.1 N/T 5.5 150 min. 2.1 N/T N/T 180 min. 2.1 N/T N/T

N/T = Not Tested

The products were determined to have an isoelectri point greater than 6.5 and up to about 9.5. Th isoelectric point is used as one of the measures t determine the degree to which a given PCS has bee cationized.

Amino Group Determination using Ninhydrin Reagent

The number of amino groups substituted in a protei molecule by cationization can be determined by means of ninhydrin reagent. Ninhydrin reagent, purchased fro Sigma Chemical Co. , was used to prepare a curve wit increasing concentrations of glycine, which served as a standard. An experimental procedure with BSA was as follows :

Ninhydrin reagent was added to a cBSA dilution an to nBSA (control), and the mixtures were heated at 80° for 20 minutes, at which point the color of each mixtur was read on a spectrophotometer at 550. A BSA samp cationized for 15 minutes with ethylene diamine/EDC quenched with an acetate buffer, showed a gain of 20 amino groups over the native BSA control. Fu cationized BSA should contain about 80 new amino grou The isoelectric point of the BSA sample cationized for minutes was 8.0. It is thus possible to determine guan tatively the number of amino group substitutions on molecule of a protein-containing substance.

Such a determination is useful in expressing degree to which a protein-containing substance has b cationized. The degree of cationization may thus expressed as a percentage of those anionic groups am able to substitution under relatively mild conditi (i.e. the reaction conditions of the inventive cationi tion method disclosed herein) while the protein-c taining substance is in its native state, which substituted to effect cationization. This percenta though not all inclusive, is usually in the range of f about 20% to about 60%.

Immunogenicity and Desensitization Studies.

Mice: BDF_]_ and BALB/c mice. 6-8 weeks of age, w purchased from the Jackson Laboratory, Bar Harbor, ME.

Adjuvants: Complete and incomplete Freund's Adjuva ( IFA) and bacterial lipopolysaccharide were purchas from Difco Laboratories, Detroit MI. Aluminum hydroxi gel was prepared in our laboratory according to t method of Levine and Vaz (11) or was in the form commercial Maalox (Rorer Inc., Fort Washington, PA) . Antibody Assays: A quantitative ELISA technique was us as previously described (12). Standard curves were r each time an assay was performed, using known amounts antibody raised against either native or cationized BS The sera were then assayed on antigen-coated ELI plates. The coatings used on plates varied in some tes and these variations are described in the Tables belo where applicable.

T cell proliferation assays: BDF mice were injected the hind footpads and tail base with 100 ug native cationized antigen emulsified in Incomplete Freud' Adjuvant. The inguinal and popliteal lymph nodes we removed 10 days later and the subsequent cell suspensi was passed over a nylon wool column as has been describ (13). The nylon wool non-adherent cells were th resuspended in complete RPMI 1640 medium containing 1 horse serum, ImM n-onessential amino acids, 1 mM sodi pyruvate, 2 mM glutamine, 5 x 10~5 M 2-mereaptoethano 25 mM HEPES and 5 ug/ml gentamycin, and plated in 96 we flat bottom plates (Costar, Cambridge, MA) at 5 x 10 cells/well. Native or cationized antigen were added various concentrations in serum-free complete RPMI 16 to triplicate wells. Serum-free medium served as control. Cells were incubated at a final volume of 2 ul at 37° C with 5% CO2 for 72 hours at which time 1 u ^•^H-thymidine was added to each well. Cells we harvested 20 hours later using a Skatron harvester a radioactivity was determined by liquid scintillati spectrophotometry.

The followinq results were obtained with cBSA cO cFerritin, cE.Coli bacteria, cTetanus Toxoid, cBGG a

FITC-cBSA conjugate, in immunogenicity a desensitization studies comparing their antigenicity that of their native forms.

Table A

Anti BSA Antibodies (ug/ml) Immunization 9d 14d 21d

50 ug n BSA, i.v. injection

5 times 58 28 0

50 ug cBSA, i.v. injection

5 times 393 450 425

cBSA prepared as in Example 1

Tested on BSA plate.

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

Table B

EFFECT OF CATIONIZATION ON IMMUNOGENICITY OF BOVINE SERUM ALBUMIN

Relative

Time of Immunogenicity- Cationization Antigen Specie ug/ l Antibody

None nBSA 350

15 min. cBSA 1220

30 min. cBSA 1450

60 min. cBSA 1135

120 min. cBSA 110

Anti BSA response (ug antbs/ml at 14d)

Cationization was conducted at a pH of 4.75 and at temperature of 25°C.

Table B shows that the length of time of protei cationization controls its immunogenicity. Cationizatio optimizes immune responsiveness. However, if cationiza tion is excessive, immunogenicity is sharply reduced, i not totally eliminated. Administration wa intraperitoneal (i.p.) with alum adjuvant (1 mg/dose).

Table C

EFFECT OF CATIONIZATION ON ANTIGEN REACTIVITY (BOVINE SERDM ALBUMIN)

Time of .. Relative Cationization Antigen Specie Antigen Activity

None nBSA ++ 15 min. cBSA +++ 30 min. CBSA ++++ 60 min. cBSA +++ 120 min. cBSA -

BSA primed T lymphocytes responsiveness to antigen

Table C shows that too extensive cationization o BSA creates a molecule incapable of stimulating BS primed T lymphocytes.

Cationization for a time up to 60 minutes greatl enhances the ability of BSA to stimulate lymphocyt proliferation, but excessive cationization creates non-immunogenic or poorly immunogenic molecule.

Table D

Effect of in vivo pretreatment with soluble nBSA or cBSA on the IgG antibody response to either antigen

PRETREATMENT * ENHANCEMENT ' d -9,8,7 IMMUNIZATION OR SUPPRESSION¹ GROUP (intravenous) (intraperitoneal) IQd 14d 21d

100 ug nBSA 100 ug nBSA -18 -26 -51 in alum B 100 ug nBSA 100 ug nBSA -95 -94 -92 in IFA² C 100 ug nBSA 100 ug cBSA -84 -87 -91 in IFA D 100 ug cBSA 100 ug cBSA 2200 6055 400 in alum E 100 ug cBSA 100 ug cBSA 98 113 28 in IFA F 50 ug cBSA 100 ug cBSA 184 241 42 in IFA

¹ Compared to control groups immunized with nBSA or cBSA and pretreated with physiological saline. Suppression indicated by negative value.

² Incomplete Freund's Adjuvant d = day cBSA was prepared as in Example 1.

Table D shows that cBSA evoked greater immun response following initial pretreatment with cBSA wherea nBSA actually suppressed the production of anti BS antibodies . Table E

Effect of in vivo pretreatment with doses of soluble cBSA on the immune response (IgG) to either antigen

PRETREATMENT % ENHANCEME d -9,8,7 OR SUPPRESS

GROUP (intravenous) IMMUNIZATION lOd 21d

A 25 ug cBSA 100 ug nBSA 259² 413

B 25 ug cBSA 100 ug cBSA 497 274

C 10 ug cBSA 100 ug nBSA 867 2818

D 10 ug cBSA 100 ug cBSA 566 683

E 1 ug cBSA 100 ug nBSA 230 866

F 1 ug cBSA 100 ug cBSA 1321 1018

G 25 ug nBSA 100 ug nBSA -85 -27

^"H 25 ug nBSA 100 ug cBSA -22 NS

I 10 uq nBSA 100 uq nBSA -87 NS

J 10 ug nBSA 100 ug cBSA NS NS

K 1 ug nBSA 100 ug nBSA NS NS

L 1 ug nBSA 100 ug cBSA NS NS Immunizations were given intraperitoneally using 1 alum as adjuvant.

2 Compared to control groups immunized with nBSA or cB and pretreated with physiological saline. Suppression indicated by negative value. NS = not significant (less than 5% change).

Table E shows results of a more detailed study o the immune response to BSA obtained with small quantitie (1-25 mg) of the cationized BSA and the resulting immun response, both in terms of increase in enhancement an the longevity of the increase. Table F

EFFECT OF ORAL ADMINISTRATION OF ANTIGEN ON IgG RESPONSE

Immunization Anti BSA Response - IgG

Feedisrig (100 ug) (ug/ml) (3 x 20 mg) in alum lOd 14d 24d

- BSA 900 812 240

- CBSA 1024 2076 3050

BSA BSA 87 58 57

CBSA BSA 2700 1074 875

BSA CBSA 250 287 675

CBSA cBSA 3700 6300 6300

Tested on BSA plate

Table G

EFFECT OF ORAL ADMINISTRATION OF ANTIGEN ON IgE RESPONSE

Immunization Anti B SA R«ϊsponse - - IgE

Feedi;ng (100 ug) PCA titer (3 x 20 mg) in alum lOd 14d 24d

— BSA 40 82 80

- cBSA 80 160 160

BSA BSA 20 20 40

BSA BSA 20 20 20

BSA cBSA 10 10 10 cBSA CBSA 20 20 20

Tables F and G show the effect of oral adminis tration of the cationized and native form of the antigen Table F records the increase in anti-BSA IgG level resulting from cBSA immunization, while Table G show that cBSA suppresses anti-BSA IgE levels.

Table H

EFFECT OF ORALLY ADMINISTERED cOVA ON IMMUNE RESPONSE TO nOVA and cOVA

Intraperitoneal Immunization wi .th Immune Response

Feeding with 0.1 ug Antigen in Anti OVA at 14d 20 mg Antigen 1 ug Alum (ug/ml)

None nOVA 140

None cOVA 765 nOVA nOVA 35 nOVA cOVA 180 cOVA nOVA 1320 cOVA cOVA 2250

OVA cationized for 1 hour as described in Example 5

Table H shows that feeding with nOVA suppresses immune response to OVA. In contrast, feeding (oral administration) with cOVA enhances greatly the immune response to both nOVA and cOVA.

Table I

IMMUNE RESPONSIVENESS TO cOVA AND nOVA in BOFj MICE

Response Days After Immunization

Antigen Administered (ug/ml Anti-OVA Antibodies) i.p. with 1 mg Alum 9ά 14d 21d

0.1 ug nOVA 80 120 120

1.0 ug nOVA 450 840 660

10 ug nOVA 1250 1420 1300

0.1 ug cOVA 425 976 770

1.0 ug cOVA 1300 2010 1660

10 ug cOVA 1500 2350 3200

OVA cationized as described in Example 5.

Table I shows cOVA is far more immunogenic than nO most notably at lower doses of antigenic challenge.

Table J

EFFECT OF INTRAPERITONEALLY ADMINISTERED CATIONIZED FERRITIN IN BDFi MICE

Percent enhancement c Ferritin over n Ferritin (control)

Bled 5 min. 15 min. 30 min at days cF cF cF

9 169 97 217

15 134 101 490

21 122 123 241

35 195 206 457

Cationized Ferritin, cF, was produced as described in Example 6, and administered i.p. to BDF_]_ mice. Antibody levels were determined from time-interval bleeds measured by ELISA on plates coated with native ferritin.

Table J shows a substantial increase in immunogenicity of partially cationized ferritin.

Table K

EFFECT OF I.p. ADMINISTERED CATIONIZED E COli BACTERIA IN BDF MICE

Percent enhancement over controls

No. of bacteria injected 10 days 20 days 30 days

1 x 10⁶ 160 210 180

5 x 10⁶ 220 450 650

1 x 10⁷ 260 500 460

5 x 10⁷ 320 800 650

Heat killed E^ coli bacteria were cationized as Example 7 and washed 3 times with saline and inject i.p. into mice, and the mice were later bled. Antibo concentration was determined by ELISA in which plast plates were coated with untreated bacteria. Antiba terial antibody titers in mice immunized with untreat bacteria served as controls and were compared wi antibody titers from mice immunized with partial cationized bacteria.

Table K shows a substantial increase i immunogenicity of partially cationized bacteria. Table L

EFFECT OF I.p. ADMINISTERED CATIONIZED TETANUS TOXOID IN BDFi MICE

Anti-tetanus antibody Percent enhancement cTT over nTT (control)

Immunization i.p. with

1 mg alum 14 days 28 days 35 days

1 ug cTT 220 300- 320

10 ug cTT 325 560 720

100 ug cTT 280 450 660

Tetanus Toxoid was cationized as described in Example 4.

Animals were bled at various time intervals and antibo levels were determined by ELISA on native TT.

Table L shows that partial cationization increases the immunogenicity of tetanus toxoid.

Table M

EFFECT OF I.p. ADMINISTERED CATIONIZED BGG IN BDFi MICE

Anti- -BGG antibodies ug/ml

Immunization i.p. with 1 mg alum 9 da ys 14 days 21 days

nBGG

1 ug 55 80 60

10 ug 110 400 800

50 ug 350 850 1000

100 ug 320 1200 1200

cBGG

1 ug 110 180 180

10 ug 450 1200 1600

50 ug 1200 2500 2500

100 uq 850 2100 2600

Bovine Gamma Globulin (BGG) was cationized as describe in Example 8.

Animals were bled at various time intervals and antibod levels were measured by ELISA. Column purifi.ed anti-BG antibodies were used as standards. Each group comprise 5 mice, and the entire experiment was repeated twice.

Table M demonstrates that partial cationizatio greatly increases the immunogenicity of Bovine Gamm Globulin. Table N

EFFECT OF FITC CONJUGATED TO cBSA IN BDFi MICE

percent enhancement Bled FITC-cBSA over at days FITC-nBSA (control)

10 240 15 280 22 330 30 180 37 160

FITC-BSA conjugates were prepared as described i Example 9.

Immune response was determined by measuring antibodies produced against FITC-nBSA or FITC-cBSA in mice on ELIS plates coated with FITC-KLH conjugate.

Table N shows that a small non-immunogenic molecule, a hapten such as FITC, becomes highly immunogenic whe conjugated to a partially cationized protein (cBSA) .

The above data establish that the method immunizing a mammal or suppressing allergic response in mammal in accordance with the invention includ administering an effective amount of a partial cationized antigenic protein having an isoelectric poi ranging from about 6.5 to about 9.5, the administrati being either oral or parenteral, and with or without adjuvant. A method of enhancing immune response to native antigen in a mammal comprises administeri parenterally to a mammal an effective amount of th antigen which has been partially cationized such that i isoelectric point ranges from about 6.5 to about 9.5, a thereafter administering parenterally to the mammal amount of that native antigen sufficient to evoke immune response.

Although the invention is in no way limited by a theory as to why beneficial results, are -achieved, it postulated that cationized proteins may be mo immunogenic for the following reasons:

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

2. Alterations in the antigen processing b the APC.

3. More efficient recognition by T helpe cells .

4. Greater affinity for self recognitio antigens ( la) .

Greater affinity for T cell receptors 6. Activation of a new type of T helper cell with more efficient recognition of the antigen.

In light of the teachings, examples and result presented above, it is within the skill in the art t make modifications in the inventive methods withou departing from the invention's spirit.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing an antigenic protein containing substance which comprises:

(a) reacting:

(1) a protein-containing substance, and

(2) a reagent capable of cationizing said protein-containing substance; and

(b) halting said reaction between sai substance and said reagent after a time sufficien to result in partial cationization of sai protein-containing substance.

2. The method of claim 1 wherein sai protein-containing substance is an antigen.

3. The method of claim 1, wherein sai protein-containing substance is selected from the grou consisting of bovine serum albumin, hen egg albumin bovine gamma globulin, ferritin, bacterial endotoxin viral proteins, tetanus toxoid, and killed microorganism and protein-containing products thereof.

4. The method of claim 3, wherein sai protein-containing substance is bovine serum albumin.

5. The method of claim 3, wherein sai protein-containing substance is bovine gamma globulin.

6. The method of claim 3, wherein said protein-containing substance is ferritin.

7. The method of claim 3, wherein sai protein-containing substance is E^ coli bacteria.

8. The method of claim 3, wherein sai protein-containing substance is tetanus toxoid.

9. The method of claim 1, wherein said cationizin reagent is a mixture of at least one carbodiimide and a least one amine.

10. The method of claim 9, wherein said at leas one carbodiimide is l-ethyl-3-( 3-dimethyl amin propyl)-carbodiimide.

^■ 12. The method of claim 1 wherein said protein containing substance is cationized to an extent whereb said protein-containing substance exhibits increase antigenic character.

13. The method of claim 1, wherein said protein containing substance is cationized such that it isoelectric point is less than 9.5.

14. The method of claim 1, wherein said protein containing substance is cationized such that it isoelectric point is in the range of from about 6.5 t about 9.5.

15. The method of claim 1, wherein said reaction i quenched by acetate buffer.

16. The method of claim 1, wherein said protei containing substance is cationized such that from abo 20% to about 60% of the maximum possible number of tho anionic groups amenable to modification under mi conditions while said protein-containing substance is its native state, are modified to effect cationization said substance.

17. A method of preparing an antigen havi increased antigenicity which comprises:

(a) reacting:

(1) an antigen and

(2) ethylene diamine/carbodiimide, and

(b) halting said reaction between sa antigen and said ethylene diamine/carbodiimide aft sufficient time to allow partial cationization said antigen.

18. The method of claim 17, wherein said antigen selected from the group consisting of bovine ser albumin, hen egg albumin, bovine gamma globuli ferritin, bacterial endotoxin, viral proteins, tetan toxoid, and killed microorqanisms and protein-containi products thereof.

19. The method of claim 17 wherein said protei containing substance is cationized to an extent where said protein-containing substance exhibits increas antigenic character.

20. A method of immunizing a mammal comprisi administering to said mammal an effective amount of partially cationized antigenic protein containi substance which has been cationized to an extent where said protein-containing substance exhibits increas antigenic character.

21. The method of claim 20 wherein said protei containing substance has an isoelectric point within t range of from about 6.5 to about 9.5.

22. The method of claim 20, wherein said partial cationized protein containing substance is administer orally.

23. The method of claim 20, wherein said partial cationized protein containing substance is administer parenterally.

24. The method of claim 22, wherein said partial cationized protein containing substance is administer without an adjuvant.

25. The method of claim 20, wherein said partial cationized protein containing substance is administer with an adjuvant.

26. A method of enhancing immune response to native antigen in a mammal, which comprises administeri parenterally to said mammal an effective amount of partially cationized antigen which has been cationized an extent whereby its immunogenic character is increase and thereafter administering parenterally to said mamm an amount of a native antigen sufficient to evoke immune response, said partially cationized antigen being prepared from the same native antigen administer thereafter.

27. The method of claim 26 wherein said partial cationized antigen has an isoelectric point in the ran of from about 6.5 to about 9.5 as determined isoelectric focusing.

28. The method of claim 26, wherein said nati antigen and said partially cationized antigen are chos from the group consisting of bovine serum albumin, h egg albumin, bovine gamma globulin, ferritin, bacteri endotoxin, viral proteins, tetanus toxoid, and kill microorganisms and protein-containing products thereof.

29. A method of suppressing allergic response in mammal susceptible to a specific allergen, whic comprises administering orally to said mammal a effective amount of a partially cationized allergen whic has been cationized to an extent whereby its allergi response suppressing character is increased, prior t exposure to said specific allergen, said partiall cationized allergen being prepared from said specifi allergen.

30. The method of claim 29 wherein said partiall cationized allergen has an isoelectric point in the rang of from about 6.5 to about 9.5, as determined b isoelectric focusing.

31. The method of claim 29, wherein said allerge is chosen from the group consisting of bovine seru albumin, hen egg albumin, bovine gamma globulin ferritin, bacterial endotoxin, viral proteins, tetanu toxoid, and killed microorganisms and protein-containing products thereof.

32. The method of claim 1, wherein said protein containing substance is conjugated with a hapten.

33. A partially cationized antigenic protein containing substance having increased -antigenicity a compared to the same native protein-containing substance.

34. The partially cationized substance of claim 33 wherein said partially cationized substance has a isoelectric point ranging from about 6.5 to about 9.5 a determined by isoelectric focusing.

35. The partially cationized substance of claim 33 wherein said native protein-containing substance is a antigen.

36. The partially cationized substance of claim 33 wherein said native protein containing substance i chosen from the group consisting of bovine serum albumin hen egg albumin, bovine gamma globulin, ferritin bacterial endotoxin, viral proteins, tetanus toxoid, an killed microorganisms and protein-containing product thereof.

37. The partially cationized substance of claim 33 wherein from about 20% to about 60% of the maximu possible number of those anionic groups amenable t modification under mild conditions while said protein containing substance is in its native state, are modifie to effect cationization of said substance.

38. The partially cationized substance of claim 33 wherein said protein-containing substance is a conjugate.