CA1338078C - Partial cationization of protein-containing antigens and method of immunization and desensitization - Google Patents

Abstract

A method for preparing an antigenic protein-containing substance by partially cationizing a native protein-containing substance to an isoelectric point ranging from about 6.5 to about 9.5. The partially cationized substance exhibits increased antigenicity as compared to the native protein-containing substance and is useful in mammalian immunization and desensitization treatments by oral or parenteral administration.

Description

PARTIAL CATIONIZATION O~ PROTEIN-CONTAINING
ANTIGENS AND METHO-I O' -MMUNIZATION AND
DESENS_TI~.A'ION
Thls lnventlon relates to a method of preparlng a partlally catlonlzed proteln-contalnlng substance, such as an antlgen, havlng lncreased antlgenlclty, to the product of thls method, and to a method of enhanclng lmmune response to a natlve antlgen ln a mammal.
The lmportance of the use of antlgens ln the preventlon of lnfectlous dlsease through lmmunlzatlon and ln the treatment of allergles through desensltlzatlon ls well known.
The basls of lmmunlzatlon ls the exposure of the organlsm to be lmmunlzed to dead or weakened lnfectlous agents (vlruses, bacterla, toxlns, etc.) or extracts thereof whlch contain a forelgn, generally macromolecular substance whlch is capable of evoking an lmmune response. These substances are generally referred to as antlgens. Llkewlse, allerglc reactlons can be lessened by desensltlzatlon, whereln such a substance ls used to suppress the normal allerglc response caused by forelgn substances, referred to as allergens.

-1 Most antiqens and allergens are either wholly or partially composed of protein.

The action of antiqens is thouqht to be dependent in part on the antiqen's affinity for certain binding sites 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 bindinq site stimulates the production of antibodies which in turn defend the orqanism aqainst infectious agents containing the antiqen.

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

One way to increase the antiqenicity of a substance is by using an adjuvant in conjunction with the antigen.
An adjuvant is a substance which augments the immunoqenic response by aiding the antigen's interaction with the livinq tissue. Examples of adjuvants include alum salts for human use. A problem which militates against the use of adjuvants in in vivo immunization is the toxicity and the side effects these compounds induce. Because of this, adjuvants are generally disfavored for use in humans or in any other organism where toxicity and/or side effects are a concern. The elimination of the need for adjuvant cooperation is therefore desirable.

-1 Some foreiqn substances do not evoke an antigenic response at all, or do so very poorly, when brought into contact with living tissue. One theory is that such substances lack the threshold amount of binding capability or strength to cause such a response. It would be advantageous to be able to convert such substances from a non-antigenic form to an antigenic form. This would allow vaccines to be produced which could elicit an immunogenic response to a substance where this was not previously possible.

Immunization methods are normally carried out by subcutaneous or intramuscular injection of the vaccine.
Oral intake of an antiqen is not feasible in most instances because it often causes suppression of rather than increase in the immune response. Exceptions to this general rule include live attenuated bacterial and viral vaccines. Oral vaccines offer advantages such as lower expense and ease in administration and packaging.
Therefore, it would be better to be able to administer vaccines in oral form without loss in immunogenic effect.

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

Prior methods of antigen cationization have taught the complete cationization of the antigen molecule. Here "complete cationization" is intended to mean that a qiven substance has had all its groups amenable to cationiza-tion so altered. Completely cationized antigens are qenerally unsuitable for in vivo use due to their excessive reactlvity.
Nakamura et al. Jpn. J. Nephrol, 28(1), 37-44 (1986) disclose chemical catlonlzatlon of bovlne serum albumln.
Muckerhelde et al., Fed. Proc., 45(3), abstract No. 55 (1986) dlsclose that catlonlzed bovlne serum albumin lnduces an antlbody response.
Other background lnformatlon ls contalned in the following references:
1. Barnes, J. and M. Venkatachalam, Enhancement of Glomerular Immune Complex DePosltlon by a Clrculatlnq PolYcatlon. J. Exp. Med. 160:286 (1984).

2. Olte, T., S.P. Batstord, J.J. Mlhatson, H.
Takaml~a and A. Vogt, Quantltatlve Studies on in situ Immune Complex GlomerulonePhrltis ln the Rat Induced bY Planted Catlonlzed Antigen. J. Exp. Med. 155:460-474 (1982).

3. Gallo, G., Caulen, T. Glaser, S.N. Emanclpator and M.E. Lamm, NePhrltoqenicity and Differentlal Distrlbutlon of Glomerular Immune ComPlexes Related to Immunoqen Charqe. Lab.
Invest. 48:460 (1983).

4. Schlkwlk, J., W.B. Van den Berg, L.B.A. van de Putte, L.A.B. Joosten & L. van den Bersselaar, Catlonlzatlon or Catalase, Peroxldase, and Superoxlde Dlsmutase: Effect or Improved Intrartlcular Retentlon on ExPerlmental Arthrltls ln Mlce. J. Clln. Invest. 76:195 (1985).

5. Muckerhelde, A., A.J. Pesce and J.G. Michael, ImmunosuPpresslve ProPertles of a Peptlc Fraqment of BSA. J.
Immunol. 119:1340 (1977).

6. Dosa, S., A.J. Pesce, D.J. Ford, A. Muckerheide and J.G. Mlchael, Immunoloqlcal Propertles as PePtlc 1 Fra~ments of Bovine Serum Albumin. Immunol. 38:509 (1979).

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

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

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

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

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

12. Ferguson, T. A., T. Peters, Jr., R. Reed, A.J.
Pesce and J.G. Michael, Immunoregulatory Properties of Antigenic Fragments from Bovine Serum Albumin. Cell.
Immunol. 73:1 (1983).

13. Julius, M.H., E. Simpson and L.A. Herzenberg, A
Rapid Method for the Isolation or Functional Thymus-derived Murine Lymphocytes. Eur.- J. Immunol.
3:645 (1973).

_ 1 338078 14. Hoare, D.G. and D.E. Kosnland, A Method for the Quantitative Modification and Estimation of Carboxylic 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 or Negative Charges on Cell Surfaces. J. Ultrastructure 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, 2.J. and E. Haber, The Effect on Antigenic Specificity of Chanqes in the Molecular Structure of Ribonuclease. J. Immunol. 91:536 (1963).

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

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

20. Babbit, B.P., P.M. Allen, G. Matsueda, E. Haber and E.R. Unanue, Binding of Immunogenic Peptides to Ia Histocompatibility ~olecules. Nature (London) 317:359 (1985).

21. Buus, S., S. Color, C. Smith, J.H. Freed, C.
Miles and H.M. Grey, Interaction Between a "Processed"

1 Ovalbumin Pe~tide and Ia Molecules. P.N.A.S. 83:2968 (1986).

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

23. A.N. Glazer, R.J. DeLange and D.S. Sigman, Chemical Modifications of Proteins. Lab. Techniques in Biochemistry and Mol. Biology, Vol. 4, Part I, p. 1-205.
North-Holland (Am. Elsevier) (1976).

24. Alexander N. Glazer, The Chemical Modification 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 referred to by their numbers in the above list.
There has heretofore been no recoqnition in the prior art that cationized antigens can be used in in vivo treatment and prevention of disease by use as vaccines or desensitization agents. Furthermore, the prior art has not recognized that partially cationized antigens can be used for this purpose. Indeed, it has been reported that completely cationized antigenoproteins do not exhibit altered immunological properties (9).

It is an object of the invention to provide a partially cationized antigenic protein-containing substance, and a method for the preparation thereof, which can be administered orally, and which does not require an adjuvant.

SUMMARY OF THE INVENTION
As used hereln, "proteln-contalnlng substance"
lncludes all proteins, as well as substances whose molecular makeup ls ln some part protelnaclous, such as llpoprotelns and proteosaccharides. These may be substances which do or do not have antlgenlc propertles ln their natlve forms. More speclflc examples of sald substances lnclude, wlthout llmltatlon, bovlne serum albumln (BSA), hen egg albumln ~OVA), bovlne gammaglobulln (BGG), ferrltln, bacterlal endotoxln, vlral protelns, dlptherla toxln and tetanus toxold, and kllled mlcroorganlsms, lncludlng bacterla and vlruses and proteln-contalnlng products thereof.
As used hereln, catlonlzatlon means the converslon or substltutlon of functlonal groups on the proteln portlon of the proteln-contalnlng substance whereby the substance ls rendered relatlvely more catlonlc. Such functlonal groups are normally anlonlc wlthln a physlologlc pH range and are converted to or substltuted for a catlonlc or nonlonlc molety. An example of such a cationizatlon ls the reactlon whereby anlonlc slde chaln carboxyl groups are substltuted wlth polycatlonlc amlnoethylamlde groups.
As used hereln, the unreacted or "natlve" form of the proteln-contalnlng substance ls lndlcated by a preflx "n" and the catlonlzed form ls lndlcated by a preflx "c". For example, bovlne serum albumln ~BSA) may be expressed as elther "nBSA" as the natlve form, or "cBSA" as the catlonlzed form.
Accordlng to one aspect of the present lnventlon there ls provlded a proteln-contalnlng substance, for use as a vacclne or a densensltlzation agent ln the treatment or preventlon of dlsease, said substance belng partlally catlonlzed through the attachment of at least one agent selected from prlmary amlnes, secondary amlnes and tertlary amlnes, whereln a) sald partlally catlonlzed proteln-contalnlng substance has an isoelectrlc polnt, as measured by lsoelectrlc focuslng ln a polyacrylamlde gel, less than 9.5; and b) the lmmunologlcal response of an anlmal to the partlally catlonlzed proteln-contalnlng substance ls greater than to the unmodlfled, natlve proteln-contalnlng substance.
Accordlng to a further aspect of the present lnvention there ls provlded the use of a partlally catlonlzed antlgen ln the manufacture of a preparatlon for enhanclng the lmmune response of an anlmal to an unmodlfled, proteln-contalnlng antlgen, sald antlgen belng partlally catlonlzed through use of an agent selected from the prlmary amlnes, secondary amlnes and tertlary amlnes so that a) the lsoelectrlc polnt of the modlfled antlgen ls less than 9.5; and b) the partlally catlonized antlgen induces ln sald anlmal a stronger lmmunologlcal response than the unmodlfled antlgen.
Accordlng to the lnventlon there ls provlded a partlally catlonlzed antlgenlc proteln-contalnlng substance havlng lncreased antlgenlclty as compared to the same natlve proteln-contalnlng substance, the partlally catlonlzed substance havlng an lsoelectrlc polnt less than 9.5 and preferably from 6.5 to 9.5, determlned by lsoelectrlc focuslng as herelnafter descrlbed.

~ "

9a 1 3 3 8 0 7 8 62804-989 The inventlon also provides such a partlally cationlzed protein-containlng substance for immunlzlng a mammal.
Adminlstratlon can be effected orally or parenterally, wlth or wlthout an ad~uvant.
A preferred method of partlal catlonlzatlon lnvolves use of a reagent comprlslng at least one carbodllmlde and at least one amlne. Control of the degree of catlonlzation ls effected by varylng the pH, tlme parameters and concentratlon of the reactants. The reactlon may be halted or quenched readlly at a deslred degree of partlal catlonlzatlon, ln a manner known ln the art. By way of non-llmltlng example, BSA
reactlon wlth a carbodllmlde and amlne mlxture may be quenched after substltutlon of about 20 new amlno groups ln place of anlonlc carboxyl groups. A fully catlonlzed BSA would contaln about 80 new amlno groups.

C

In general, an addition or substitution of ~rom about 20% to about 60% of the theoretical maximum possible number of amino groups will result in an isoelectric point ranging between about 6.5 and 9.5 as determined by isoelectric focusing.

DETAILED DESCRIPTION OF TRE PREFERRED EMBODI~ENTS

The protein-containing substance (PCS) can be 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 the activation of the carboxyl groups of a protein with carbodiimide and the subsequent reaction of the activated carboxyl with a nucleophile of the general type +R-NH2 to obtain the primary amine type derivatives. Considerable versatility can be achieved since both the chemical nature of the modification, i.e. introduction of primary, secondary or tertiary amine groups and the degree of modification of the protein carboxyls can be varied by proper choice of reagents, reaction time and pH of the couplinq reaction.
The cationization methods suitable for use in the present invention are those which effect cationization under mild reaction conditions. As used herein mild reaction conditions are those under which the molecular character (e.g. 1, 2 and 3 structure) of the subject PCS is not substantially altered so as to adversely affect its antigenic pH immunogenic character. These conditions can be most generally described as under relatively low temperature and low ionic strength as well as neither very acidic (e.g. below pH 4) nor very basic 1 (e.g. above pH 8) nor presenting a very oxidative or reductive environment.

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

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

The reaction is maintained within the general range of from about 4 C to about 37 C and is generally maintained at about 25 C.
Each known method of cationization may be halted or quenched according to several methods known in the art.
The EDC reaction is quenched with a buffer, preferably an acetate buffer, which halts the reaction. Concentration of the acetate buffer is about 4M.

Cationization can be verified and quantified usinq known gel electrophoresis as hereinafter described, and isoelectric focusinq 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 12 l 338078 1 increased antiqenic character such as an increased immunogenic character or an increased allergic response suppressinq character. Such increases may be determined by methods known in the art such as those described below. Although not a limitation to the scope of the applicability of the invention, for most types of PCS, this will be at a point where the isoelectric point falls within a range of from about 6.5 to about 9.5.

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

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

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

1 E~ample 1 Bovine Serum albumin (nBSA), five times crystallized, was cationized according to the general procedure described by Border (9).

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

The reaction was permitted to react for varying periods of time with constant stirring, while the temperature was maintained at about 25 C and the pH was held constant. After guenching with 4M acetate buffer mixture, the reaction was subjected to multiple dialysis treatments against distilled water and lyophilized. It was then passed through a column of Sephadex G-25 and lyophilized again before use.

EXA~PLE 2 The method of Example 1 with the exception that the pH of the reaction was adjusted to about 6.0 with 6N HCl and the reaction was permitted to react for about 1 hour.

EXA~PLE 3 The method of Example 1 with the exception that the PCS may be native bacterial endotoxin instead of nBSA.

t 1 Example 4 The method of Example 1 with the exception that the PCS used was native tetanus toxoid instead of nBSA
(purchased from Lederle Laboratories, Pearl River, NY).

E~aople 5 10 The method of Example 2 with the exception that the PCS used was native hen egg albumin (OVA) instead of nBSA.

Exa ple 6 The method of Example 1 with the exception that the PCS used was ferritin (purchased from Sigma Chemical Co., St. Louis, MO), samples of which were cationized for 5 minutes, 15 minutes and 30 minutes.
E~ample 7 The method of Example 1 with the exception that the PCS used was heat killed E.coli bacteria.
Ex~ple 8 The method of Example 1 with the exception that the PCS used was bovine gamma globulin (BGG), purchased from Sigma Chemical Co., St. Louis, MO .

E~a~ple 9 The method of Example 1 with the exception that ~luorescein isothiocyanate (FITC), a small 1 non-immunogenic molecule, was conjugated either to an nBSA carrier or to a cBSA carrier (as prepared in Example 1). These conjugates were used as the PCS. The fluorescein isothiocyanate-BSA conjugation method is as follows:

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

The suspension is dialyzed in a cold room with frequent changes of saline adjusted to a pH of about 7.8 with borate buffer. The dialysis requires several days and is complete when the dialyzate is virtually free of yellow-green color under ultraviolet light. The conjugate is clarified by centrifugation.

20Other hapten-carrier conjugations may require more complex chemical reactions to achieve covalent bonding between the reacting molecules.

Cationization Assay: A~o~ Gel Electrophoresis ~aterial~:

1) Corning~ Electrophoresis Agarose Universal Gel Film (1% Agarose, 5% sucrose, 0.035% EDTA, in 0.065M
30barbital buffer, pH 8.6):

2) Corning~ Universal PHAB Buffer (Sodium barbital 17.7g, barbital 2.6g, sodium chloride 1.0g, disodium EDTA
0.79, and sucrose octaacetate) Reconstituted with 35distilled water to 0.05M buffer with 0.035% EDTA, pH 8.6.

16 l 338078 62804-989 3) Cornlng ~ Amido Black 10B Staln NOTE: Above purchased from Flsher Sclentlflc.
4) Cornlng ~ Cassette Electrophoresls Cell wlth Cornlng Power Supply.
METHODS:
1) 1.0 ~l of sample at a concentration of 30 mg/ml ls loaded onto gel lnto each well. (For sample of lesser concentratlon, 1 ~l of sample ls loaded and allowed to dry between appllcatlons untll approprlate concentratlon ls reached).
2) 95 ~l buffer is added to each side of cell.
3) Gel ls placed on electrophoresls unlt and run for approprlate tlme perlod. (For cBSA - tlme perlod ls 40 mlnutes;
nBSA ls run as a control).
4) Gel ls removed from electrophoreslc unlt and placed ln amido black staln for 15 mlnutes. Gel ls destalned ln 5% acetlc acld for 20 seconds.
5) Gel ls allowed to dry.
6) Gel ls destalned agaln ln 5% acetlc acld untll good contrast ls seen between back and background.
7) Gel ls rlnsed ln 2 separate dlstllled H2O baths.
8) Gel ls agaln allowed to dry.

G

--Samples may be subjected to isoelectric focusing to determine the isoelectric point (pI) value of the particular cationized product, by a conventional procedure described in the prior art.

Result~:

Che~ical Properties and Electrophore~is The cationization procedure results in the substitution of anionic side chain carboxyl groups by polycationic aminoethylamide groups (14, 15). The products are readily soluble in water and show a single band on gel electrophoresis in the presence of sodium dodecyl sulfate, with or without a reducing agent such as mercaptoethanol. The band migrates more rapidly toward the cathode than the native BSA monomer. Passage through Sephadex G 200 shows a single peak which appears slightly before the nBSA peak.

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

The following results were obtained using the above outlined agarose gel technique:

--1 Electrophonesis Results Distance ~igrated Reaction Ti~e To~ard 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 isoelectric point greater than 6.5 and up to abou-t 9.5. The isoelectric point is used as one of the measures to determine the degree to which a given PCS has been cationized.

A~ino Group ~etermination U~ing Ninhydrin Reagent The number of amino groups substituted in a protein molecule by cationization can be determined by means of ninhydrin reagent. Ninhydrin reagent, purchased from Sigma Chemical Co., was used to prepare a curve with 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 and to n8SA (control), and the mixtures were heated at 80C
for 20 minutes, at which point the color of each mixture 1 was read on a spectrophotometer at 550. A BSA sample, cationized for 15 minutes with ethylene diamine/EDC and quenched with an acetate buffer, showed a gain of 20 new amino groups over the native BSA control. Fully cationized BSA should contain about 80 new amino groups.
The isoelectric point of the BSA sample cationized for 15 minutes was 8Ø It is thus possible to determine quanti-tatively the number of amino group substitutions on a molecule of a protein-containing substance.
Such a determination is useful in expressing the degree to which a protein-containing substance has been cationized. The degree of cationization may thus be expressed as a percentage of those anionic groups amen-able to substitution under relatively mild conditions(i.e. the reaction conditions of the inventive cationiza-tion method disclosed herein) while the protein-con-taining substance is in its native state, which are substituted to effect cationization. This percentage, though not all inclusive, is usually in the range of from about 20% to about 60~.

I~munogenicity and Desensitization Studies,.

Mice: BDFl and BALB/c mice. 6-8 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, ME.

Adjuvants: Complete and incomplete Freund's Adjuvant (IFA) and bacterial lipopolysaccharide were purchased from Difco Laboratories, Detroit MI. Aluminum hydroxide gel was prepared in our laboratory according to the method of Levine and Vaz (11) or was in the form of - ~~ commercial Maalox (Rorer Inc., Fort Washington, PA).
. 1~, ~ TrQde ~ r k l 3 3 8 0 7 8 62804-989 AntibodY AssaYs: A quantltatlve ELISA technlque was used as prevlously descrlbed (12). Standard curves were run each time an assay was performed, uslng known amounts of antibody ralsed agalnst elther natlve or catlonlzed BSA. The sera were then assayed on antlgen-coated ELISA plates. The coatlngs used on plates varled in some tests and these variations are described in the Tables below, where applicable.
T cell Prollferatlon assays: BDFl mlce were ln~ected ln the hind footpads and tail base with 100 ~g native or cationlzed antlgen emulsifled in Incomplete Freud's Adjuvant.
The inguinal and popllteal lymph nodes were removed 10 days later and the subsequent cell suspenslon was passed over a nylon wool column as has been descrlbed (13). The nylon wool non-adherent cells were then resuspended in complete RPMI 1640 medium contalnlng 10% horse serum, lmM nonessentlal amlno aclds, 1 mM sodlum pyruvate, 2 mM glutamlne, 5 x 10 5 M 2-mercaptoethanol, 25 mM HEPES and 5 ~g/ml gentamycln, and plated in 96 well flat bottom plates (Costar, Cambridge, MA) at 5 x 10 5 cells/well. Natlve or catlonlzed antlgen were added at varlous concentratlons ln serum-free complete RPMI 1640 to trlpllcate wells. Serum-free medlum served as a control. Cells were lncubated at a flnal volume of 200 ~1 at 37C wlth 5% CO2 for 72 hours at which time 1 ~Ci 3H-thymidlne was added to each well. Cells were harvested 20 hours later uslng a Skatron harvester and radloactlvlty was determined by llquid scintlllatlon spectrophotometry.
The followlng results were obtained wlth cBSA cOVA
cFerrltln, cE.Coll bacterla, cTetanus Toxold, cBGG and FITC-~`

21 l 3 3 8 0 7 8 62804-989 cBSA con~ugate, ln immunogenlcity and desensltizatlon studies comparing their antigenlcity to that of thelr natlve forms.
Table A
Antl BSA Antlbodles (yg/ml) Immunlzatlon 9d 14d 21d 50 yg n BSA, l.v. ln~ectlon 5 tlmes 58 28 0 10 50 yg cBSA, l.v. in~ection 5 times 393 450 425 cBSA prepared as in Example 1.
Tested on BSA plate.
Table A shows that cBSA is immunogenlc when ln~ected lnto mice.
Table B
EFFECT OF CATIONIZATION ON IMMUNOGENICITY
OF BOVINE SERUM ALBUMIN
Relative Time of Immunogenicity-Cationization Antigen sPecie uq/ml AntibodY
None nBSA 350 15 min. cBSA 1220 30 min. cBSA 1450 60 min. cBSA 1135 120 min. cBSA 110 Antl BSA response yg antbs/ml at 14d) Catlonlzatlon was conducted at a pH of 4.75 and at a temperature of 25C.
Table B shows that the length of tlme of protein catlonlzatlon controls lts lmmunogenlclty. Cationlzation ~, optlmizes immune responsiveness. However, if cationlzatlon ls excesslve, lmmunogenlclty ls sharply reduced, lf not totally ellmlnated. Admlnlstratlon was lntraperltoneal (i.p.) with alum ad~uvant (1 mg/dose).

.

1 Table C

~r~ OF CATIONIZATION ON ANTIGEN REACllvllr (Bu~ SER~M ALBUMIN) Time of Relative Cationization Antigen SpecieAntigen Activity None naSA ++
15 min. cBSA +++
10 30 min. cBSA ++++
60 min. cBSA +++
120 min. cBSA

BSA primed T lymphocytes responsiveness to antigen Table C shows that too extensive cationization of BSA creates a molecule incapable of stimulating BSA
primed T lymphocytes.

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

24 l 3 3 8 0 7 8 62804-989 Table D
Effect of ln vlvo pretreatment wlth soluble nBSA or cBSA
on the IgG antibody respbnse to elther antlgen % Enhancement PRETREATMENT OR
d -9,8,7 IMMUNIZATION SUPPRESSIONl GrouP (lntravenous~ (lntraperltoneal) 10d 14d 21d A 100 ~g nBSA 100 ~g nBSA ln alum -18 -26 -51 B 100 ~g nBSA 100 ~g nBSA ln IFA2 _95 -94 -92 C 100 ~g nBSA 100 ~g cBSA ln IFA -84 -87 -91 D 100 ~g cBSA 100 ~g cBSA ln alum 2200 6055 400 E 100 ~g cBSA 100 yg cBSA ln IFA 98 113 28 F 50 ~g cBSA 100 ~g cBSA ln IFA 184 241 42 1 Compared to control groups immunlzed with nBSA or cBSA and pretreated wlth physlologlcal sallne. Suppresslon lndlcated by negatlve value.
Incomplete Freund's Ad~uvant d = day cBSA was prepared as ln Example 1.
Table D shows that cBSA evoked greater lmmune response followlng lnltlal pretreatment wlth cBSA whereas nBSA actually suppressed the productlon of antl BSA antlbodles.

25 l 3 3 8 0 7 ~ 62804-989 Table E
Effect of ln vlvo pretreatment wlth doses of soluble cBSA
on the immune response (IgG) to elther antlgen PRETREATMENT % ENHANCEMENT
d -9,8,7 OR
SUPPRESSION
GrouP (intravenous) IMMUNIZATION 10d 21d A 25 ~g cBSA 100 ~g nBSA 2592 413 B 25 ~g cBSA 100 yg cBSA 497 274 C 10 ~g cBSA 100 ~g nBSA 867 2818 D 10 ~g cBSA 100 yg cBSA 566 683 E 1 ~g cBSA 100 ~g nBSA 230 866 F 1 ~g cBSA 100 ~g cBSA 1321 1018 G 25 ~g nBSA 100 ~g nBSA -85 -27 H 25 ~g nBSA 100 ~g cBSA -22 NS3 I 10 ~g nBSA 100 ~g nBSA -87 NS3 J 10 ~g nBSA 100 ~g cBSA NS NS
K 1 ~g nBSA 100 ~g nBSA NS NS
L 1 ~g nBSA 100 ~g cBSA NS NS

1 Immunizations were given intraperltoneally using l mg alum as ad~uvant.

Compared to control groups immunized wlth nBSA or cBSA and pretreated wlth physiological saline. Suppresslon lndlcted by negatlve value.
3 NS = not slgnlficant (less than 5% change).
Table E shows results of a more detalled study of the lmmune response to BSA obtained with small quantities (1-25 mg) of the cationized BSA and the resulting immune response, both ln terms of lncrease in enhancement and the longevity of the increase.

~'.

26 ~ ~38078 62804-989 Table F
EFFECT OF ORAL ADMINISTRATION OF ANTIGEN
ON IgG RESPONSE
Immunlzatlon Antl BSA Response - IgG
Feedlng (100 ~g) (~g/ml) ~3 x 20 mq) ln alum 10d 14d 24d - cBSA 1024 2076 3050 cBSA BSA 2700 1074 875 BSA cBSA 250 287 675 cBSA cBSA 3700 6300 6300 Tested on BSA plate ~`~

27 1 3 3 8 0 7 8 62804-98~
Table G

EFFECT OF ORAL ADMINISTRATION OF ANTIGEN
ON IgE RESPONSE

Immunlzatlon Antl BSA Response - IgE
Feedlng (100 ~g) PCA tlter (3 x 20 mq) ln alum 10d 14d 24d - cBSA 80 160 160 BSA cBSA 10 10 10 20cBSA cBSA 20 20 20 Tables F and G show the effect of oral admlnistratlon of the catlonlzed and natlve form of the antlgen. Table F
records the lncrease ln antl-BSA IgG levels resultlng from cBSA
lmmunlzatlon, whlle Table G shows that cBSA suppresses antl-BSA
IgE levels.

~r Table H
EFFECT OF ORALLY ADMINI~L~ cOVA
ON IMMUNE RESPONSE TO nOVA AND cOVA
Intraperitoneal Immunization withImmune Response Feedlng with 0.1 ~g Antigen inAnti OVA at 14d 20 mq Antiqen 1 ~q Alum (~q/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 descrlbed in Example 5.
Table H shows that feedlng wlth nOVA suppresses lmmune response to OVA. In contrast, feedlng (oral admlnlstratlon) wlth cOVA enhances greatly the lmmune response to both nOVA and cOVA.

~j~

29 . l 3 3 8 0 7 8 62804-989 Table I
IMMUNE RESPONSIVENESS TO cOVA
AND nOVA ln BDFl MICE
Response Days After Immunization Antlgen Admlnlstered (~g/ml Antl-OVA Antlbodles) i.P. with 1 mq Alum 9d 14d 21d 0.1 yg nOVA 80 120 120 1.0 ~g nOVA 450 840 660 10 ~g nOVA 1250 1420 1300 0.1 ~g cOVA 425 976 770 1.0 ~g cOVA 1300 2010 1660 10 ~g cOVA 1500 2350 3200 OVA cationlzed as described in Example 5.
Table I shows cOVA is far more immunogenic than nOVA
most notably at lower doses of antigenic challenge.

~, 1 ` Table J

OF INTRAPERITONEALLY ADMINISTERED
CATIONIZED FERRITIN IN BDFl MICE

Percent enhancement c Ferritin over n Ferritin (control) Bled 5 min. 15 min. 30 min.
at days cF cF cF

Cationized Ferritin, cF, was produced as described in Example 6, and administered i.p. to BDFl 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 1~

rrrt~ OF I.p. AD~IINISTERED
CATIONIZED E coli BACTERIA IN BDFl I~ICE

Percent enhance~ent over controls No. of bacter~a 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 in Example 7 and washed 3 times with saline and injected 20 i.p. into mice, and the mice were later bled. Antibody concentration was determined by ELISA in which plastic plates were coated with untreated bacteria. Antibac-terial antibody titers in mice immunized with untreated bacteria served as controls and were compared with 25 antibody titers from mice immunized with partially cationized bacteria.

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

1 Table L

EFFECT OF I.p. AD~INISTERED
CATIONIZED TETANUS TOXOID IN BDFl ~ICE

Anti-tetanus antibody Percent enhancement cTT
over nTT (control) I _ unization i.p. ~ith 1 mg alua 14 days 28 days 35 days 1 ug cTT 220 300 320 1510 ug cTT 325 560 720 100 uq cTT 280 450 660 Tetanus Toxoid was cationized as described in Example 4.

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

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

33 l 3 3 8 0 7 8 62804-989 Table M

EFFECT OF I.p. ADMINI~
CATIONIZED BGG IN BDFl MICE

Antl-BGG antibodles ~g/ml Immunlzatlon l.p. wlth 1 mg alum 9 daYs 14 daYs 21 daYs nBGG
1 ~g 55 80 60 10 ~g 110 400 800 50 ~g 350 850 1000 100 ~g 320 1200 1200 cBGG
1 ~g 110 180 180 10 ~g 450 1200 1600 50 ~g 1200 2500 2500 100 ~g 850 2100 2600 Bovlne Gamma Globulln (BGG) was catlonlzed as descrlbed ln Example 8.
Anlmals were bled at varlous tlme lntervals and antlbody levels were measured by ELISA. Column purlfled antl-BGG antlbodies were used as standards. Each group comprlsed 5 mlce, and the entlre experlment was repeated twlce.
Table M demonstrates that partlal cationizatlon greatly lncreases the lmmunogenlclty of Bovlne Gamma Globulln.

~ 34 1 338078 1 Table N

OF FITC CONJUGATED TO cBSA
IN BDFl ~ICE

percent enhancement Bled FITC-cBSA over at days FITC-nBSA (control) FITC-BSA conjugates were prepared as described in Example 9.

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

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

-1 The above data establish that the method of immunizing a mammal or suppressing allergic response in a mammal in accordance with the invention includes administering an effective amount of a partially cationized antigenic protein having an isoelectric point ranging from about 6.5 to about 9.5, the administration being either oral or parenteral, and with or without an adjuvant. A method of enhancing immune response to a native antigen in a mammal comprises administering parenterally to a mammal an effective amount of that antigen which has been partially cationized such that its isoelectric point ranges from about 6.5 to about 9.5, and thereafter administering parenterally to the mammal an amount of that native antigen sufficient to evoke an immune response.

Although the invention is in no way limited by any theory as to why beneficial results are achieved, it is postulated that cationized proteins may be more immuno~enic for the following reasons:

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

2. Alterations in the antigen processing by the APC.

3. More efficient recognition by T helper cells.
4. Greater affinity for self recognition antigens (Ia).

5. Greater affinity for T cell receptors.

` 1 338078 6. Actlvatlon of a new type of T helper cells wlth more efficient recognition of the antigen.
The above examples are for illustrative purposes only.

. ~

Claims (9)

1. A protein-containing substance, for use as a vaccine or a densensitization agent in the treatment or prevention of disease, said substance being partially cationized through the attachment of at least one agent selected from primary amines, secondary amines and tertiary amines, wherein a) said partially cationized protein-containing substance has an isoelectric point, as measured by isoelectric focusing in a polyacrylamide gel, less than 9.5; and b) the immunological response of an animal to the partially cationized protein-containing substance is greater than to the unmodified, native protein-containing substance.

2. The partially cationized protein-containing substance according to claim 1, having an isoelectric point in the range from 6.5 to 9.5.

3. The partially cationized protein-containing substance according to claim 1 or claim 2 wherein the unmodified protein-containing substance is selected from bovine serum albumin, hen egg albumin, bovine gamma globulin, ferritin, bacterial endotoxin, viral proteins, tetanus toxoid, diphtheria toxin, and killed microorganisms and protein-containing products thereof.

4. The partially cationized protein-containing substance according to claim 1 or 2, being conjugated with a hapten, and wherein the immunological response of an animal to the hapten, conjugated to the partially cationized protein-containing substance, is greater than to the hapten, conjugated to the unmodified native protein-containing substance.

5. The use of a partially cationized antigen in the manufacture of a preparation for enhancing the immune response of an animal to an unmodified, protein-containing antigen, said antigen being partially cationized through use of an agent selected from the primary amines, secondary amines and tertiary amines so that a) the isoelectric point of the modified antigen is less than 9 5; and b) the partially cationized antigen induces in said animal a stronger immunological response than the unmodified antigen.

6. The use of a partially cationized antigen according to claim 5, wherein the partially cationized antigen has an isoelectric point in the range 6.5 to 9.5.

7. The use of a partially cationized antigen according to claim 5 or claim 6, wherein the unmodified antigen is selected from bovine serum albumin, hen egg albumin, bovine gamma globulin, ferritin, bacterial endotoxin, viral proteins, tetanus toxoid, diphtheria toxin, and killed microorganisms and protein-containing products thereof.

8. The use of a partially cationized antigen according to claim 5 or 6, wherein the partially cationized antigen is conjugated with a hapten, and wherein the level of antibodies produced by the animal to the conjugated hapten/partially cationized antigen is greater than to the hapten conjugated with the unmodified antigen.

9. The use of a partially cationized antigen according to claim 5 or 6 wherein the cationization is effected by coupling the agent to the antigen through an amide bond.