CA1099258A

CA1099258A - Antigen-protein complex for blocking allergic reactions

Info

Publication number: CA1099258A
Application number: CA288,312A
Authority: CA
Inventors: Arthur Malley
Original assignee: Research Corp
Current assignee: Research Corp
Priority date: 1976-10-12
Filing date: 1977-10-07
Publication date: 1981-04-14
Also published as: SE8205313D0; FR2382460B1; SE8205313L; FI58501C; GB1552056A; AU510639B2; FR2382458B1; FR2382458A1; NO773465L; ES463124A1; US4140679A; LU78273A1; SE7711422L; FI58501B; US4158705A; NO147860C; CH636524A5; NO147860B; DE2744983A1; NL7711026A

Abstract

ANTIGEN-PROTEIN COMPLEX FOR
BLOCKING ALLERGIC REACTIONS

ABSTRACT OF THE DISCLOSURE

A multivalent complex suitable for blocking allergic reactions comprising (1) at least one Timothy antigen D frag-ment having the antigenic determinant structure:

wherein: G represents glucose, and T represents threonine or a peptide linked to said structure through a threonine molecule;
covalently bonded through at least one glucose moiety to a peptide or protein, (2) a conjugate having the structure:

or (3) a conjugate comprising at least one conjugate of the structure:

covalently bonded to a protein or peptide by a peptide linkage through the glutathione moiety.
A method of forming complex (1) by activating a glucose moiety of the Timothy antigen D fragment and reacting the thus activated intermediate with a peptide or protein.
A method of treating allergies comprising in-jecting one of the above complexes into an individual to inhibit antigen-induced histamine release.
An injectable composition for blocking allergic reactions containing the above complex.

Description

BACKGROUND ~)F TIiE INVENTION
_ _ _ _ .
Methods of treating allergies and allergic reactions have heretofore embodied the administration -to an individual of an antigen which initiates the formation of antibodies which compete with the antibodies initiated by the allergen thereby dampening or reducing the allergic reaction.
- The u~ciual prior a-rt approach in the treatment of allergic patients involves the preparation of a cxude extract of the aller~y-producing substance in an aqueous buffered solution. The patients are then treated with first minute, then increasing doses of the crude extract over a long ~eriod of tims~ Frequently, three or more years of treatment are necessary before siynificant lasting improvment is observed. As the treatment progresses, the amount of crude extract that a patient receives is gradually increased.
The extent of increased dosage and rate depends upon the patient's individual sensitivity and the degree of reaction to previous dosages.
The in~ection of whole crude allergen extract over long periods of time induce the production of blocking or competing antibodies. It is theorized that the blocking antibodies compete with allergic antibody for the antigen and provides protection to the allergic patient.
These are numerous and obvious disadvantages associated with this expensive and time-consuming process bm~

~9~
of desensitizing the allergic patien-t. very often the administration of the crude extract results in a severe allergic reaction if the dosages have not been carefull.y monitored and administered. In addition, the process of desensitization is a long and time-consuming and very often e~pensive processO In addition, the production of competing antibodies in the allergic patient Yery often result in side e~fècta which may be more damaging to the pàtiënt thàn the allergic reaction itself.
More importantly, the method of desensiti~ation ~eretofore employed in the prior art has not been altogether successful. In many instances, the time-consuming desensitization procedure does not result in an ade~uate degree of protect.ion to the allergic patient w~o still continues to suffer with the allergy when exposed to antigen.
It is an object of the present invention to provide a complex suitable for administration to an allergic patient which neutraliæes existing anitbodies and completely blocks`the formation of antibodies, either competing or specific to the aller~enic antigen.
It is a further object of the invention to provide a met~od for the production of complexes that ~ill block the allergic reaction.
It is still a further object of the invention to provide a ~ethod for the treatment of an aller~ic individual to block the allergic reaction.

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2~3 It is still a further object of the present invention to provide an injectable composition for administration to an allergic patient for blocking the allergic reaction.

SUMMA~Y OF THE INVE21TION
The invention comprises a multivalent complex suitable for blocking allergic reactions comprising (1) at least one Timothy antigen D fragment having the an~igenic / determinant structùre:

OH

HO ~ ~ ~ H

¦l O~G-G-O-T
OH O

wherein: G represents glucose, and T represents threonine or a peptide linked to said structure through a -threonine molecule;
covalently bonded through at least one glucose moiety to a peptide or protein, (2~ a conjugate having the structure:

~ ; OH

\ OH 5 - ~Jlutathione 0~1, 0 bm:

~' ~9g~
or (3) a conjugate comprising at leas~ onc conjuyate of the st.ructureO

10 ,~ // ~ .OEI

~ H ~ S - glutathione - . ~ 1~ - . . .
OH

covalently bonded to a protein ox peptide by a peptide linkage through the glutathione moiet~O
The invention also includes a method for preparing complex (1) by activating a glucose moiety of ~he Timothy ;~
anitgen D fragment and thereafter reacting the thus formed intermediate with a peptide or protein.
The invention further includes a method of treating allergies comprising injecting into an individual an amount of one of the above complexes sufficient to inhibit antigen-induced histamine release~
The invention further includes an injectable composition suitable for administration to allergic patients for blocking allergic reactions comprising a medium suitable for inj~ction containing the above-identified complex.

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DET~ILED DESCRIPTION OF T IE IN~7F,NTION
Various allergenic antigen fragments of Timothy pollen have been recently isolated and characterized. See Malley et al, Imunoc emistry, 1975, Volume 12, payes 551-SS4.
A ma~or multi-yalent allerg n contained in Timothy pollen is that known in the prior art as ~tigen ~ The monovalent antigenic determinant strUcture of antigen B which is commonly `; ` ~ ermèd. ~:nelgen D is: . .- . . . . . - .. - -1 / `~ ~\;=~ 0~1 ~ ~ O-G-G-O-T
OH O

wherein: G represents glucose, and T represents threonine or a peptide linked to said structure through a threonine molecule.
Fragments of antigen D~ identified in the prior art as antigen D~, antigen D2 and antigen D~ differ in the size of the peptide "tail" linked to the glucose moiety of the antigenic determinant structure through the threonine molecule. [See J. Immunology 99, 825~ 1967; Immunochemistry, 1, 237, 1964, 12, 551, 1975; WHO Symposium on ~ller~en Standardization ed. by F. T. Perkins! GeneYa~ 1974; Develop.
biol. standard 29, 29~ 1975]. Antigen Dl has a molecular bm: i ~9~325~ ~

weight of about 5~000O An~igen D2 has a molecular weight of about 2,500. Antigen D3 is ~he fragment wherein the molecule bonded to the glucose moiety is threanine alone. In some ca~es serine, ins~ead of threonine 9 is attached to the glucose moiety due to a misreading of *he code responsible for the addition of ~`
specific amino acids during protein synthesis.
The querci~in fractions of the antigen structural formula is linked to the glucose moieties Yi a an ether linkage through OH groups on the respective molecules.
The threonine linkage to the glucose moiety is also an etller linkage through an OH group on the cellobiose frac-tion and the OH group of threonine. It is presumed, based on stoichiometric considerations, that the activation occurs primarily at the glucose molecule adjacent to the quercitin molecule. However, it is to be understood that the complexes of the invention are formed through a glucose ac~ivation mechanism notwithstanding the precise locus of activation l~ithin the cellobiose fraction. It is not pre-cisely knol~n which OH group in cellobiose fraction comprisesthe linkage siteO
As is apparent from the above structure, the mole-cule is open at vario-ls sites to conjugation ~ith a ~ide variety of reactants. ~lany multivalent conjugates can be prepared ~ihich ~ould be expected to initiatc allergic re-actions by virtue of their structure. Indeed, it has been ~0~$2~;~

found that the attachment of proteins or peptides to the molecule at si~es other than ~he glucose or carbo-hydrate moie~ies results in cQnjugates which initiate allergic reactionsO
Unexpectedly 9 it has been found that the linkage o a peptide or a protein to the antigen D fragment through the glucose moiety results in a complex which does not initiate allergic reactions 9 but rather, completely neu-t~alizes allergic antibody and inhibits or blocks the ormation of allergic antibodies.
Thus, these complexes or conjugates may be administered to an allergic patient for the complete inhibition of the allergic reaction without the need for the long and time-consuming and expensive desensitiza-` 15 tion process described above. Since administration of ~he complex inhibits the formation o-f allergic antibodies altogether, the allergic reaction itself is inhibited as well as any side effects produced by the formation of com-peting antibodies produced according to the conventional desensitization method.
Similar results have been shown to be produced by a conjugate having the s~ruc~ure:

0~1 ~ tathione OH O
' ' or a conjugate comprising at least one quercitin-glutathione conjugate covalently bonded to a protein or peptide by a peptide linkage through the glutathione moiety. Administration of these complexes or con-jugates also results in a complete inhibition of the formation of allergic antibodies, either specific to the antigen or competing antibodies and a complete blockage of the allergic reaction.
The Timothy antigen D fragment may be covalently bonded through at least one glucose moiety to any suitable peptide or protein. It is to be understood that any pep-tide or protein suitable for administration to an allergic patient may be covalently bonded to the antigen fragment~
Preferred among the peptides or proteins are human serum albumin> a d-amino acid copolymer of lysine and glutamic acid ~d-GL)~ human IgG as well as other homologous and similar proteins or d-amino acid copolymers.
As noted above, it is essential that the peptide or protein be linked to the glucose moiety of the antigen D fragment. Bonding of the protein or peptide to any other portion o~ the molecule will result in a complex which initiates rather than inhibits the allergic reaction. Co-valent bonding may be achieved by activatillg the antigen D
fragment ~ith a cyanogen halide. The method is described in detail by A~en et al, Nature~ 214, 1302-1304 ~1967) and Cuatrecasas et al, Proc. Natl Acad. Sci., U.S., 61, G36-643 2~;~

~19~8)o The cyanogen halide reacts with the hydroxyl . group o~ the glucose groups to form cyana~e es~ers. The reaction occurs mos~ rapidly at a pH between 10 and 12.
The cyanate may react further with additional hydroxyl groups to ~orm an imidocarbonate intermediate. The cyclic imidocarbonate formed as a major product :following the reaction of the cyanogen halide with the glucose mo;ety - is highly reactive with amines to form N-imidocarbonates, isoureas or N-carbamates. The exact nature of the linkage bet~een the protein and the cyanogen halide actiYated glu-cose moiety is unknown. Notl~ithstanding the exact mechanism, however, the intermediate formed by the reaction of a cyano-gen halide with the antigen D fragment is highly reactive with peptides and proteins to form a complex wherein the peptide or protein is covalently linked through the glucose moiety to the antigen D fragment.
The celIobiose raction of the antigen D frag-ment may also be activate~ for linkage to the protein or peptide by the method described in Inununol. 20, 1061, (1971):

--].1 ~0~$2S~

- O ~ oxidation ~ \
~ \
O - ~ NaIO4 '~ - IH H
OH ~

CHzOH

protein~ -reduction, e.g.
R-NH2_ o _ H C NaBH~
HO I OH
R

~

R

Where the carboxyl groups of the antigen D
fragment were activated by Woodward~s reagent K leaving the quercitin-glucose-glucose moiety unaffected, the resulting conjugate with human serum albumin, for example, induced S antigen-induced histamine release reactions as sho~n by direct skin tests. Thus~ it is apparent that the allergic reaction inhibition capabilities of the complcx depend upon - ~ conjugation of the peptide or protein with the antigen D-fragment through the ~lucose moiety.

-~2-The quercitin-glutathione and quercltin-glutathione-protein conjugates described above are also potent allergic reaction blocking agents.
The quexci-tin-glutathione conjugate is formed by an oxidation-addition reaction utilizing A~O or other suitable oxidizing agent:

OH
o~ ~ ~ OH

~ \ ~ OH

OH O ~ glutamyl-cysteinyl-glycine A~O~
(glutathione) 0~1 ~ 0~1 OH O glutamyl-cysteine-glycine The resulting con~ugate may then be conjugated with a peptide or protein by activation of the carhoxyl ~roups of the glutathione moiety with for example Woodward s reagent carbodiimides etc~ followed by conjuga-tion with bm:

.. ,;, .

~O~Z~B

~he protein or peptide moleculeO The quercitin-gluta~hione is linked to the protein via a peptide bond through either the glutamic acid or glycine portion of the glutathione moiety.
The peptide or protein conjugated ~o the quercitin-glutathione conjuga~e may be any peptide or pro-tein sui~able for administration to an allergic patient.
Preferred among the proteins and pep~ides are human serum albumin9 a d-amino acid copolymer of lysine and glutamic l0` acid (d-GL), human IgG or other homologous proteins or d-amino acid copolymers.
Obviously, the antigen D fragment and quercitin-- glu~athione molecule may be bonded to the protein or pep-tide molecule at a variety of sites on the latter. Generally, it is preferred to bind from about 2.0 to about 30 moles~
preferably 5 to 20 moles, of the antigen D fragment or quercitîn-glutathione compound to 1 mole of protein or peptide. The molar ratio may be varied by varying the molar ratio of reactants in the method of preparation.
The antigen-protein complexes described above may be incorporated in any suitable medium such as buffered saline for administration to an allergic patient.
Conventional additives, adjuvants~ etc., may be included in the composition for injection; however, it is ~5 to be understood that they are not required.

.

Generally~ the compositions are injected sub-cutaneously9 intra-muscularly or intra-venously.
Generally9 dosages in th~ range of from abou~
- 0.01 to about 0.25 mg9 preferably 0.025 to 0.1 mg, per kg body weight of the complex in ~he composition injected is su~ficient to provide an acceptable degree of allergic reaction blocking capability.
It is to be understood that the parameters of the dosage range are governed by the duration of the c.om^
plex in the circulation, the antibody titer of the patient undergoing therapy, the immune status of the patient and the duration of the suppression of the IgE response.
The complexes and methods of the present invention are useful in the treatment o grass-sensitive patients to reduce allergy symptoms. The complexes suppress the forma-tion of allergic antibodies against grass pollen antigens and further act to neutralize existing antibodies in the patient's system. It is to be understood that the invention is useful in blocking not only Timothy allergic reactions but also rye grass an~ other grass pollens -that cross-react ti-e-, June grass9 etc.)~
The follol~ing e~amples illustrate methods of preparation o the comple~es of the in~ention.
EXA~IPLE 1 25- Preparation o~ Antigen D-protein Complex 90 my of antigen Dl fragment ( mol w-t.~ 5000) were placed in an ice ba-th and the pI-I adjusted to 10.5 with NaOH solution. The mixture was provided wi-th a motorized stirrer and 2 g CNBr was added. The pH was main-tained at 10.5 ~25 by the addition of NaOH over the next 10 m,inutes.
15 ml of 0.1 M NaHCO3 buffer (pH 9) containing 0.15 M NaCl was added. Then, 45 mg of human serum albumin (HSA) was , added and the mixture stirred at 4C for 24 hours. The, mixture was concentrated under negative pressure to yield a product having an antigen Dl protein ratio of abou-t 5:1.

Unreacted antigen Dl was removed by dialysis.
Complexes with antigen D2 and D3 fragments and other protein or peptide carriers may be prepared in identical fashion.
'EXAMPLE 2 Preparation of Quercitin-Glutathione 1.5 g of AgCl was mixed with 10 ml of 4~ NaOH
for 20 minutes. The AgO precipitate was filtered on a Buchner funnel.

0.2 g of glutathione was dissolved in 4 ml of 0.25 N NaOH and placed in an ice bath. 0.2 g ~uercitin was dissolved in 8 ml of 0.25 N NaOH. 1.2 g of the freshly prepared AgO was added to the latter and the mïxture stirred 15-20 minutes on an ice bath.
The glutathione solution was added and -the mixture stirred for 1 hour in an ice bath. Excess AgO was removed by filtering. The pH of the resulting solution was adjus-ted to 1-2.5 with HCl and the solution s-t,ixred for 30 minutes.

~,~ bm:, ~'.~.

The solution was filtered to remove unbound que~citin and the pH readjus~ed t~ 7 wi~h NaOH. Pree glutathione was determined by the DNTB me~hod (Anal. Biochem.
48, 1557, 1972).

Preparation of Quercitin-Glut~hione Protein Conjugate 40 mg of quercitin-glutathione was added to 100 mg o~ Woodward's Reagent and mixed for 10 minutes at room temperature. The pH was adjusted to 6.2 with 0.3 M cocodylate buf~-er (pH 6.8).
100 mg o~ HSA was added while maintaining the pH
above 6.2. The mixture ~ras ailowed to remain at room tem-pera~ure for 10-15 minutes and then overnight at 4C. The solution was concentrated under negative pressure. Dialysis removes unbound quercitin-glutathione from its conjugate with the protein.
The conjugate with other proteins and peptides may be prepared in identical fashion.
The following example illustrates the allergic reaction inhibiting capabilities of the complexes and methods of the present invention.

Histamine release from monkcy lung tisSue passively sensitized ~ith the serum of allergic patients is determined in accordance with the method described by Malley et al, J. Immunol. 100, 915 (1968). The sera employed to sensitize the monkey l~mg tissue ~as from a pool of sera rom 20 Timothy-sensi~ive patients ~ith an averagc allergic anti-- body titer of 1:750. The monkey lung tissue is then .

,~

incubated for two hours at 37~C~ with the serum, Prior to c~allenge with the alle.rgen9 the sensitized tissue is incubated for ten minutes at 37C with ~he inhibi~ing agen~. The antigenic challenge is carried out by in-S cubation with the optimum concentration of crude pollen extract (WST) for 15 minutes at 37C.
The results are set forth in Table 1 wherein:
- WST represents an aqueous extract of whole Timothy grass pollen dialyzed to remove low molecular weight frag-ments:
Dl represents the Timothy antigen D fragements having a molecular weight of about 5000;
D2 represents the Timothy antigen D fragment having a molecular weight of about 2500;
D3 represents the Timothy antigen D fragment having a molecular weight of less than 1000 and comprising the - :
complex note~d above ~herein T represents threonine;
HSA Tepresents human serum albumin;
- dGL represents a d-amino acid copolymer of glutanic acid and lysine having a molecular weight of about 40,000;
Rhlg represents rhesus IgG.

25~

.
O l r~ ~ O ~

d~ ,CI ~ ~ ~n IXI . , Cl~ r~ ul . . .' .
Q~ .
r~ ~ ~D r~ o ~
0 ~ 5) ~ N.~1 t~ o o tr~ 1` ~1 ,., ~ ~ I _ ~ ~J r l ri Q~¢ . .

, E~ ~ U ~ o o o o o o o o o o ~r ~ ~ r ~ r ., . .- ., ,~o ~ o o o ~ ~ ~ ~ ~ ~
~D ~ O U~ r` o o ~1U~r~ ~ c~ ) 1~ ~ ~ r 00 ~ . . . ~ .
. .
.
l ~ ~ ~1 ~ o.

., ~ ~

O ~ ~ K
Orl . ~ O
~-1 '1~ ~0 o 3 ~
O ~ l ~1 ~ ~1 ~ rl ~ ~1 I:q a) ~ 1~ IJ O ~;
1~ ~Z; ~ ~ ~ ~rl O aJ

3~ ~ '~ X~
O O

td E~ u~ ~3 S~ U~ 5: 0; ~ ~ ~ ~1 ,a :~ O .IJ
~ ~ 0~ ~¢

--19-- .

~L6;3 ~$~

As is apparent from the results in Tabl~ 1>
the complexes of the presen~ in~enti~n are potent allergic reaction inhibitors~
The following example further illustrates the allerglc reaction inhibi~ing capabilities of the complexes of the înventio~
XA~IPLE 5 The passive transer skin reactivity of ~he com-plexès oE the invention is compared t~ith the antigens pre-sent in Timothy pollen as well as complexes p~epared by con~ugating the peptide or protein to the antigen -fragments through moie~ies other than the glucose moiety.
0~1 ml of sera containing allergic antibodies ~from a pool of sera of 20 Timothy-sensiti~e patients-mean P-K titer of 750) were intradermally injected into the skin of a non-allergic volunteer. 48 hours later each site previously sensitized with sera were challenged with a variety of antigens and complexes. The allergic antibodies in the implanted sera interact with allergenic antigen materials to cause a localized allergic reaction.
If the antigenic material does no~ initiate allergic reactions, no localized reaction l-ill be observed.
The results of various tests involving the com-plexes of the present invention as ~ell as the an~igen material in Timothy pollen are set forth in Table 2.

-2n-,, ',; ,` . .;~ ' ~ .',c~ . ~ . ~ n .
. ` ~ ~ . ~ . ` Q,~ N ~ . ~r o al a~ ` .
~ ~4 C~. -:: - . ~ z; . z; z ~; æ:. ~ . z z . ~ ~ ~ ~ ~ ~ ~~ ~ ~ ~ ~.
a a a a a ~L a a ~ a ~ a O ~ O O O O O OU~ U~ O ~ O ~D
o o CO o o o~ ~ o . o ~
r¢ U~ Lr In In ~ U~ ~ ~
. : .::
o . ~ ,~ ~ ~ ~ ~ ~ . ~ ~ ' ~ .. .. " , N ~r O N 00 ~ ~~r ~1 r-l N O
O ~d l. ~. . . . ~ N
UK l ~o r ~ N rl N
1:~1 m E~O O ul ~n ~
,~ U~ ~ ~ ~ o- ~ - X
. ~ ~ ~ U~ O~rl O 0 ~~1 ~ S~
r~ ~ Sl 0 0 ~ 0 ~ ~ O ~ ~-~1 0 ~ 0 m ~ ~ a) 3 a) ~rlo o ~ra 3 ~ 3 a r~ l Z ,~ ~ ~ ~C~ S~
V ~ 0 0 0 0 ~ W -1 ~C O 0 0 td o c~ ~ O ~ t) X O ~ o a C~ ~ ~ o ~

rlrlrl ~ ~ a O ~ ~ H H ~ ~ f:I~ ~ C~ V V
~) E~ u~ u~ IJ
~0 ~ ~ ~ . , P~ l l l l l l I I
o a) o a 5~ ~ .
Q L:) ~ a ~ ol ~ o bm ~

,, ~ , .

. The above results indicate that the complexes of the present invention do not initiate allergic reactionsO
I~ should ~urther be noted ~ha~ those complexes o~ the antigen D fragments conjugated with the protein through moieties other than the glucose moiety are potent initiators of allergic reac~ions ~hereby indicating the criticality o conjugation through the glucose moiety of the antigen D ~ragment.
EX~PLE 6 This example illustrates the ability of the com-plexes of the inven~ion to suppress the formation of allergic antibodies.
Preliminary studies demonstrated that LAFl mice developed good IgE ~allergic antibodies) responses against Timothy pollen.
Animals are injected iop~ with 10 ~g IYST in alum and three weeks later they receive a second i.p. injection of 10 ~g WST in alum. IgE titers, after the initial immuni-zation, rise to titers between 1/100 to 1/200 as measured by PCA. IgE titers, af~er the second injection of antigen, rise to values bet~een 1/600 to 1/800 as measured by PC~.
Passive cutaneous anaphylaxis ~PCA) Is measured by injection o 0.025 ml of serum dilution into the side of a mouse or rat. Sevent~r-t~o hours (mouse) or three hours , (rat) later the animals are challenged ~ith ~YST in Evans blue dye injected intravenously. Interaction of the circulating ' ' ~ " " ' ' ' , ', antigen and tissue fixed I~E results in a localized allergic reaction and the site is made visual by the leakage of the blue dye -in-to the reacted area. The IgE
titer is defined as the lowest dilution giving a positive PCA reaction.
Mouse IgE in these animals is made against the same antigenic determinant as that expressed on the antigen D fragments.
The two complexes of the invention chosen for test purposes were:
1) Antigen D2-dGL ~D2-dGl): a non-immunogenic copolymer of d-glutamic acid and d-lysine (MW 40,000) with an average of 20 8 antigen D2 groups/mole of dGL attached.
Conjugation was by modiflcation of sugar moiety with C~Br prior to addition to dGL.
2) Quercitin-glutathione-dGL (Q-G-dGL): prepared by conjugation of Quercitin-glutathione to dGL with an average of 12-20 Q-G groups/moles of dGL.
Administration of Q-G-dGL or D2-dGL via i.p. route ]O and given 3 days prior to secondary immunization of WST~
day: ~ 18 ~ 21 28 injection: WST D2dGI, WST Bleed in or in alum Q-G-dGL alum Results: Control animals given saline on day 18 have the following IgE titer-600; average response in 18 animals.

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, Animals trea~ed with D2-dGL:
Animals PCA Titer group low dose (0.12 mg AgD2) 350 6 med~ dose (0O6 mg AgD2) 300 hi~h dose (l.~ mg AgD2) < 100 6 Animals treated ~ith Q-G-dGL:
PCA Titer Animals - low dose (0.24 mg Quercitin) 600 6 med. dose (lo 8 mg ~uercitin~ 300 6 high dose (3.6 mg Quercitin) 100 6 The above data indicates that both conjugates significantly reduce IgE level when administered 3 days prior to secondary antigen injection, thereby indicating antibody neutralization and suppression. Similar tests have indicated that administrations up to 7 days prior to secondary antigen in~ection will give essentially the same results.

. -24-~ .
-

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Process for preparing a multivalent antigen-protein complex, suitable for blocking allergic reactions, selected from the group consisting of:
(1) at least one Timothy antigen D fragment having the antigenic determinant structure:

wherein: G represents glucose; and T represents threonine or a peptide linked to said structure through a threonine molecule;
covalently bonded through at least one glucose moiety to a peptide or protein;
(2) a conjugate having the structure:
;

and (3) a conjugate comprising at least one conjugate of the structure:

covalently bonded to a protein or peptide by a peptide linkage through the glutathione moiety; said process comprising, respectively:
(1) activating the glucose moieties of a compound having the structure:
wherein: G represents glucose; and T represents threonine or a peptide linked to said structure through a threonine molecule;
by reaction with an activating agent and subsequently reacting the activated compound with a peptide or protein;
(2) reacting a compound having the structure:
with glutathione in the presence of an oxidizing agent; and (3) conjugating the conjugate of preceding step (2) with a peptide or protein following activation of the carboxyl groups of the glutathione moiety.

2. A process for preparing a multivalent antigen-protein complex, suitable for blocking allergic reactions, comprising at least one Timothy antigen D fragment having the antigenic determinant structure:

wherein: G represents glucose; and T represents threonine or a peptide linked to said structure through a threonine molecule;
covalently bonded through at least one glucose moiety to a peptide or protein;
said process comprising activating the glucose moieties of a compound having the structure:

wherein: G represents glucose; and T represents threonine or a peptide linked to said structure through a threonine molecule;
by reaction with an activating agent and subsequently reacting the activated compound with a peptide or protein.

3. The process of Claim 2 wherein said activating agent is a cyanogen halide.

4. The process of Claim 2 wherein said protein or peptide is human serum albumin, a d-amino acid copolymer of lysine and glutamic acid or human IgG.

5. The process of Claim 4 wherein T represents threonine.

6. The process of Claim 4 wherein T is a threonine containing peptide such that the molecular weight of said fragment is about 5000.

7. The process of Claim 4 wherein T is threonine containing peptide such that the molecular weight of said fragment is about 2500.

8. The process of Claim 6 wherein said fragment is covalently bonded to a d-amino acid copolymer of lysine and glutamic acid.

9. The process of Claim 6 wherein said fragment is covalently bonded to human IgG.

10. The process of Claim 6 wherein said fragment is covalently bonded to human serum albumin.

11. The process of Claim 7 wherein said fragment is covalently bonded to a d-amino acid copolymer of lysine and glutamic acid.

12. The process of Claim 7 wherein said fragment is covalently bonded to human IgG.

13. The process of Claim 5 wherein said fragment is covalently bonded to human serum albumin.

14. A process for preparing a multivalent antigen-protein complex, suitable for blocking allergic reactions, comprising a conjugate having the structure:
said process comprising reacting a compound having the structure:

with glutathione in the presence of an oxidizing agent.

15. A process for preparing a multivalent antigen-protein complex, suitable for blocking allergic reactions, comprising a conjugate comprising at least one conjugate of the structure:

covalently bonded to a protein or peptide by a peptide linkage through the glutathione moiety, said process comprising reacting a compound having the structure:

with glutathione in the presence of an oxidizing agent, and thereafter conjugating with a peptide or protein following activation of the carboxyl groups of the glutathione moiety.

16. The process of Claim 15 wherein said protein or peptide is human serum albumin, d-amino acid copolymer of lysine and glutamic acid or human IgG.

17. The process of Claim 15 wherein peptide is a d-amino acid copolymer of lysine and glutamic acid.

18. The process of Claim 15 wherein protein is human serum albumin.

19. The process of Claim 15 wherein protein is human IgG.

20. A multivalent antigen-protein complex, suitable for blocking allergic reactions, selected from the group consisting of:
(1) at least one Timothy antigen D fragment having the antigenic determinant structure:

wherein: G represents glucose; and T represents threonine or a peptide linked to said structure through a threonine molecule;
covalently bonded through at least one glucose moiety to a peptide or protein;
(2) a conjugate having the structure:

and (3) a conjugate comprising at least one conjugate of the structure:

covalently bonded to a protein or peptide by a peptide linkage through the glutathione moiety; whenever prepared according to the process of Claim 1, or by an obvious chemical equivalent.