CA1327523C - Antigen immunogenicity - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A new method is described for eliciting IgG
antibody response to proteins or synthetic peptides, particularly those that are weakly immunogenic, without the requirement for the use of adjuvants, thereby making it easier and safer to confer protection against pathogenic organisms. The antigen is coupled to a monoclonal antibody, specific for membrane determinants expressed on certain types of mammalian recipient cells, called antigen presenting cells. The monoclonal antibody acts as a "vector" or "delivery vehicle" for targeting foreign antigens onto such recipient cells.
This targeting facilitates subsequent antigen recognition by helper T-cells, which are pivotal in helping the induction of antigen-specific IgG
responses.

Description

~ 3~75~3 ENHANCEMENT OF ANTIGEN IMMUNOGENICITY
The present invention relates to a novel procedure for immunization against disease, and/or the generation of antibody responses ln vivo without the use of adju-vants and to novel vaccine materials and conjugates usedin the procedure.
In the conventional method of protection against disease by vaccination, the central principle involves the injection or ingestion of a material that will elicit antibodies against said material, such that, when challenged later with a pathogenic organism containing homologous material, the individual to whom the material has been administered is protected against the patho-genic disease. The materials injected or ingested ~hat have this property of eliciting antibodies are called antig~ns.
It ha~ been known for many years that the immun-ogenicity (that is, its ability to elicit antibodies) of an antigen can be improved by the addition of so-called adjuvants. In some cases, materials that apparently have little or no immunogenicity have been made to make high titxes of antibody in in vivo systems by the addition of an adjuvant. Some of these adjuvants are, -i however, highly toxic and can cause ~andesirabl~ side effects or lesions. Aluminum hydroxide or aluminum phosphate are routinely used as adjuvants for human vaccines, but have been known to cause lumps at the site of injection. Freund's complete adjuvant (FCA), a mixture of a light petroleum oil and killed Myobacter-ium tuberçulosis, can often produce excellent titres for materials which do not normally give antibodies ln vivo under any other conditions. Unfortunately lesions will often develop at the site , ~., ~: , ., : ..
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2 1 327523 of injection when using this material, making the procedure unacceptable for human use.
There are a number of other materials which have been investigated for use as adjuvants. In published European Patent Application No. 0149581, the use of muramyl di-peptide is disclosed. In published United Kingdom Patent Application No. 2,053,231, the use of a synthetic adjuvant consisting of tetrapeptides or pentapeptides is disclosed. In earlier patents, the use of oil emulsions is disclosed as having an adjuvanting effectO Unfortunately, it appears that the better a material is at behaving as an adjuvant to antigen immunogenicity, the worse are the side effects. For example, it has been shown ~hat muramyl dipeptide is an excellent adjuvant but appears to have a number of undesirable properties which prevent its use in human vaccines.
In yeneral, all the adjuvants presently in use in mammals fall into two distinct types. The first type involves the so-called "depot" effect and the second type depends on general immunological stimulation of I the system under study. 'rhe adjuvants which rely on ! the depot effect are believed to bring the immune cells to the antigen site, where the depot effect relies on the injected antigen being trapped or insolubilized in a medium, giving sustained circulating levels. The second type of adjuvant, involving general stimulation of the immune system, appears to rely on an inflammatory reaction resulting in a series of cells being stimulated, such that any ; antigen has an improved chance of eliciting antibodies.
Current theories of immunology suggest that, in order to provide a potent antibody response, an antigen must be seen by both B cells, which ; subsequently develop into the antibody producing cells, and also by helper T-cells, which provide growth and differentiation signals to the antigen specific B-cells. Helper T-cells recognize the ,: , , ' ' . .

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antigen on the surface of antigen presenting cells (APC) in association with Class II major histocom-patibility complex (MHC) gene products.
The prasent invention involves a new method of - 5 immunization which does not involve the use of unaccep-table adjuvants. The present invention, in one aspect, provides a novel immunization method and takes advantage of the fact that both B-cells and ~PC of the macrophage lineage express Class II ~C gene products.
Instead of injecting antigen in the presence of ;adiuvant/ the antigen is coupled to a monoclonal antibody specific for a surface structure of antigen-presenting cells, including Class II MHC determinants and is injected in this form. By coupling it to the antigen, the monoclonal antibody is converted into a "vector" or "delivery vehicle" for targeting the antig~n onto khe relevant cells in the immune system.
In effect, the method brings the antigen to the antigen-presenting cells rather than the other way around, as is thought to be the case when antigen is injected in the presence of Freundts complete adjuvant.
,The present invention also provides, in another '~aspect, a nov~l vaccine for utilization in the novel imm~nization method comprising a conjugate of an antigen ~25 and a monoclonal antibody specific for a gene product of ;the major histocompatibility complex or other surface ructure of antigen~presenting cells in a suitable carrier medium. The present invention, in a further aspect, provides the novel conjugate.
The present invention provides a new method of enhancing the immunogenicity of weak antigens, which -~ does not involve the use of unacceptable adjuvants, is therefore much safer and can utilize materials that are not normally very antigenicO An example of such materials are small peptides which are epitopes of larger proteins or are protein subunits of the pathogens :~' :... ~' themselves. Epitopes are parts of a larger antig~n which represent the immunologic~l recognition sites within khe structurQ. The vaccination method of the invention, th~refore, can avoid the injection o killed or attenuated organisms, which can have unfortunate side effects, by the use of such epitopes or protein subunits in the ~orm of conjugates with targeting monoclonal antibodiesO
In the present invention, an antigen, against which it is desired to raise antibodies in the body, is coupled or bonded to a monoclonal antibody, which is specific for a particular surfacP structure of antigen-presenting cells, including a genP product of th~ major histocompatibility complex (MHC~. This arrangement ~ 15 allows for a concentration of the antigen to the ; relevant cells in the immune system upon injection of the coupled material. The derivatis~d antibody, . there~ore, act~ as a "vector" or '9delivery vehicle" for targeting antigenic determillants onto the macrophage cells or B-cells, thereby facilitating their recogni-tion by the T-helper cells. The presentiny cells ; possess a variety of specific cell surface structures or markers which are targeted by the particular monoclonal antibody, including Cla~s I and Class II hi~tocompati-bility gene products. Thus, antigens may be coupl~d to a monoclonal antibody specific for any of the surface structures on the antigen presenting cells, including Class I and Class II MHC gene produc~s.
-. The term "conjugate" as used herein is employed in its normal s nse that the two molecules are joined together in some convenient manner to provide a stable structure suitable for in vivo administration.
The molecules may be joined together to ~orm the conjugate by bonding u~ing t~chniques known to those skilled ih the art, with the bonds being of any , , , ' ' 5 1 3275~3 suitable structure, including covalent bonding or af f inity bondingO
One specific example of a conjugate use~ul in the present invention is that of the antigenic protein avidin and the monoclonal antibody specific for the I-Ak Class II major histocompatibility determinants. It is under-stood that this Pxample is but sne application of the general principle of the present invention discussed abov~O The preparative procedures describ~d below are illustrative for the specific conjugate. The general principles of such procedures, however, may be employed to provide conjugates of any desired antigenic molecule and any desired monoclonal antibody to provide the desired antigen-antibody conjugate molecule for in vivo administration to achieve the desired adjuvant-independent serological response.
In this speci~ic illust:rative example, monoclonal antibodies specific for the I-Ak Class II major histocompatibility dete~minants may be prepared from a suitable hybridoma cell line. To permit easy conjugation with other p:roteins, the monoclonal , antibodies preferably are biotinylated, prior to exposure to the appropriate protein antigen, in this example avidin. Avidin binds with high affinity to the ~5 biotinylat~d antibody, ~nd has previously been charac-terized as an antigen. Varying doses o~ this conjugate were used to treat a strain of mic~ expressing the I-Ak determinants. Mice of a dif~erent strain expressing only I-Ab determinants were used as controls. The re~ults o~ the~e expariments, given in detail in the Examples below, showed that avidin, when presented as an antigen in this manner (i.e., conjugated to tha Mab oP
I-A~ Class II MHC) and in very low ~uantities can be : made highly immunogenic in the absence of a standard adjuvantO I~ ~quivalent quantities oP free avidin were mixed with non-biotinylated antibody (so that no conju- :

gation occurred), no antibody response was elicited, indicating that the result was conjugate dependant.
Recognition of the I-Ak determinants was essential, since avidin conjugated to an antibody of irrelevant specificity was not e~fective in eliciting an antibody response. In other experiments, alsu detailed in the Example below, a monoclonal antibody that is speci~ic ~or the Class I MHC conjugated to avidin also showed an immunogenic response and generated antibodies to avidin.
The conjugates which are employed in the present invention are not limited to conjugates of a single antigen with the mono lonal antibody but include conjugates where two or mor~ antigenic proteins or peptides are conjugated to the monoclonal antibody, to provide a multivalent conjugate which can elicit an antibody response to each of the conjugated antigens.
As specific examples, biotinylated monoclonal antibody may be conjugated to avidin and bovine serum albumin or to avidin and a Herpes synthetic peptide.
These conjugates were formed and used to immunize mice.
The Herpes peptide is part of the Herpes simplex virus glycoprotein ~. In these experiments, reported in -detail in the Examples below, it was found that anti-bodies were raised against bovine serum albumen and the Herpes peptide, as well as against the avidin when the ;lrespective conjugates were tested. Replacement of the ;~avidin by another protein, namely streptavidin, which also has high binding affinity to biotin, provided a ~good response, indicating that the targeting e~fect is ;30 not restricted to avidin and that there was no serological cros~-reaction of anti~streptavidin antibodies with avidin.
It has also been shown in experiments reported in the Examples below that the response of mice to cumula-tive injections of a conjugate consisting of antibody-avidin-peptide is equivalent to the response using ' ' .

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~' 6 a Freund's complete adjuvant. This latter experiment indicates that vaccines can be constructed using peptide antig~ns that elicit antibodi2s that can be neutralising and protective again~t the pathogen from which the 5 peptide i~ extracted, without the necessity for adjuvants.
It is evident from the abo~e that the invention may be used as a method of immunization in vivo and in vitro for making hybridomas, and thus specific monoclonal antibodiesO Such specific antibodies have a wide-range of use both in medicine and immunology.
In the following specific Examples, the efficacy of the invention is demonstrated for three immunologically-unrelated protein antigens, namely avidin, streptavidin and bo~ine serum albumin. In addition, the use o~ the m~thod resulted in generation o~ IgG antibodies against a synthetic peptide from the glycoprotein D of herpes-;, simplex virus. In addition, an immunogenic r~sponse has been achieved using MAbs against Class I and Class II
determinants of MHC. This data demonstrates the gener-ality of the invention in application to the use of any conjugate of an antigen and a monoclonal antibody specific for surface structures on APC.
The inve~tion is illustrated by the following Examples. In the Examples below, reference is made to the accompanying drawings, wherein:

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~` 1 327523 Figure 1 contains graphical representations, labelled A to D, of the anti-avidin response~ in mice in one series of experiments described in the Examples below;
Figure 2 is a graphical representation of secondary anti-avidin responses in mice in another series of experiments described in the Examples below;
Figure 3 contains graphical representations of anti-bovine serum albumin responses in mice in a further series of experiments described in the Examples below;
Figure 4 contains graphical representations of secondary antibody responses to a synthetic Herpes peptide in mice in another series of experiments described in the Exa-mples below;
Figure 5 conkains graphical representations of secondary antibody responses to streptavidin and avidin in mice in a yet further series of experiments described in the Examples below; and Figure 6 contains graphical representations of antibody responses to avidin in mice in onP more series of experiments described in the Examples below.
` Example I~
This Example illustrates the preparation of biotinylat~d monoclonal antibodies and conjugates ; 25 thereof with avidin.
Hybridoma cell lines TIB-92 ~anti-I-Ak) and HB-65 (anti-influenza A NP, the control antibody) were obtained from the American Type Culture Collection (ATCC~, RocXville, Maryland, U.S.A. and were cultured 30 in RPMI 1640 culture medium, supplemented with 10% fetal calf serum, L-glutamine and antibiotics. The antibodies produced by these cell lines are both of the IgG2a subclass, and were purified from culture supernatants by con~entional protein A-Sepharose chromatography.
The purified antibodies then were biotinylated using the general method described by Goding in ~ 3275~3 "Monoclonal Antibodies: Principles and Practice"
(1983) Academic Press, London. For this method, the antibodies were dialysed against O.lM sodium bicarbonate solution at p~ 8.2, and concentrated to . ~
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~` 8 1 327523 1.0 mg/ml immediately before use. Biotin succinimide ester (Sigma Chemical Co~) was dissolved in dime~hyl sulphoxide to a concentration of lmg/ml, immediately before use. 60 ~1 of this ester solution were added per ml of antibody solution at room temperature and left for 1 hour. The biotinylated protein was dialysed overnight against phosphate buffered saline (PBS) and stored at 4C.
; The biotinylated antibody prepared as just described was mixed with avidin in a ratio of 2 moles of antibody to 1 mole of avidin to form a biotinylated antibody-avidin conjugate for each of the antibodiesO
Exam~le II:
This Example illustrates the immunogenic response of the conjugates prepared as described in Example I
in mice.
Two different strains of mice (namely (B6 x C3H)Fl and B6 in groups of three were injected with the conjugates prepared as described in Example I as well as a mixture of antibody and avidin and of avidin alone according to the schedule contained in the following Table I:

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'', TABLE I Immunization Protocol Priming avidin Adjuvant GroupPriming Antigendose ~ug) (FCA) k 5 I ~bio-anti~I-A )- 5 Avidin*
. - do - 50 . k IIanti-I-A + Avidin 5 - do - 50 ~, III (bio-anti-NP)- 5 : Avidin - do - 50 .,' 15 :. IV Avidin 5 +/
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. Notes: * = Bio~inylated : 20~ = In Group III, antigen was injected only into (B6 x C3HjFl mice NP = nucleoprotein '~ FCA = Freund's complete adjuvant -, The injections consisted of subcutaneous i 2S inoculations of 0.2ml of the various solutions and ^, after 21 days mice in all the groups were boosted with an intraperitoneal injection of 5~g of avidin in 0.2ml . of PBS. Nine days later the mice were bled from the retro-bulbar sinus and their sera stored at 4C.
30The immune sera were tested for anti-avidin activity as follows: Avidin solution (50ul) at a concentration of 20ug/ml was added to the wells of a polyvinyl microtitre plate and incubated for 1 hourr ~: after which the plates were washed. The plates then were incubated with a 1~ solution of bovine serum albumin (BSA) in PBS for an additional 1 hour. The BSA-buffer was removed and 50~1 of serial dilutions of the immune sera in 1~ BSA-PBS were added to each well ; and incubated for 1 hour. The plates were then washed ' '' .~ .
, o 1 327523 three times with 1~ BSA PBS and then each well treated with 50ul of I-protein A for 1 hour. The plates then were washed twice with buffer and the individual wells counted in a gamma counter.
The anti-avidin response of immune sera were plotted graphically and appear as Figure 1. In this Figure 1, the anti-avidin responses of the immune sera from groups I, II, III and IV (Table I~ respectively are shown in A, B, C and D. The open circle line is the response in (B6xC3H)Fl mice while the closed circle line is the response in B6 mice.
As may be seen from the data presented in Figure 1, at the 5 ~ug dose of avidin, a significant response was observed in (B6xC3H)Fl mice injec~ed with (anti~I-A )-avidin conjugate (Fig. lA, open circles) whereas the B6 mice (Fig. lA, closed sircles), which do not have the particular surface antigens for which the antibody was made, were not appreciably sensitized Isee Fig. lA~. This result cannot be attributed to an ~ immuno-stimulating effect of the antibody alone, since the mixture of 5 ~g of avidin with unmodified k anti-I-A MAb did not elicit a response (Fig. lB). An equal amount of avidin coupled to the control anti-NP
Ab also failed to generate an appreciable response ~`~ 25 (Fig. lC), indicating that the positive response shown in Fig lA is due to more than a simple conjugation of avidin to an antibody. As expected 5 ~g of avidin injected with Freund's complete adjuvant induced a strong serological response ~Fig. lD). At the 50 ~g of avidin dose, free avidin in the absence of adjuvant failed to stimulate a response (Fig. lB), but in the form of (bio-anti-I-Ak)-avidin, the conjugate sensitized both (B6xC3H)Fl and B6 mice (Fig. lA).
Responses in the B6 mice are likely a reflection of the elevated reactivity of the avidin-MAb conjugate on ~ the B6 targets and may be attributed either to - cross-reactivity of the conjugated MAb or more efficient APC uptake of the MAb-avidin complex.
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-~-`" 11 1 327523 These results demonstrate that a low dose of avidin (i.e., 5 ~g) can be made immunogenic in the absence of adjuvant when presented to the immune system coupled to a monoclonal antibody specific for recipient class II major hisocompatibility determinants, namely the anti-I-Ak MAb.
The test procedure was repeated using a different anti-Class II MAb, in this case an anti-I-E as the targeting MAb. The results obtained were plotted graphically and are reproduced in Figure 2. As may be seen ther~in, individual (B6xC3H)Fl mice (open squares) immunized with the (anti-I-Ek)-avidin conjugate gave a 7 to 9 fold higher response than the B6 mice (closed squares) that received the same dosage of conjugate. Unconjugated mixtures of avidin and the MAb gave only background levels of reactivity and this data is not presented.
Example III-This Example illustrates the use of antibody-biotin-avidin con~ugates to raise antibodies to a third party protein.
A conjugate of anti~ Ak antibody, avidin and bovine serum albumin (B';A) was formed by mixing equimolar amounts of biotinylated monoclonal antibody (prepared as described in Example 1), avidin and biotinylated bovine serum for 20 minutes at 20C. The mixture then was centrifuged for five minutes at 12,000g to remove any precipatable aggregates. Mice were injected subcutaneously with this complex (approx. 30 ~g BSA) in a similar regime to those in Example II, and, after three weeks, were boosted with an intraperitoneal injection of lO~g of free BSA in PBS Sera from the mice were obtained nine days later and assayed in a manner similar to the samples in Example II. The blocking buffer in the radioimmune assay contained 0.1~ ovalbumin in PBS and 65,000 cpm of 125I-Protein A were added to each assay well.
l'he results obtained were plotted graphically and ; appear as Figure 3. The open circle lines define the .j ~' ,:
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No response was observed in mice injected with an equivalent dose of avidin in an unconjugated mixture of non-biotinylated Mab and avidin and this data is not presented. As may be seen in Figure 3~ a considerable response to bovine serum albumin was observed. These data indicate the practicality of obtaining a serological response to any third party protein simply by coupling it to the MAb-avidin complex. This would allow for the preparation of any number of adjuvant free subunit protein vaccines, that would avoid problems connected with the parent organism of the subunit proteinD
lS Example IV~
This Example illustrates the use of antibody-biotin-avidin conjugates to raise antibodies to a small peptide.
A biotin mediated conjugate was made between 2~ anti-I-Ak and avidin to which the Herpes synthetic peptide was coupled. The Herpes peptide used was an ~,N~terminal peptide (12-20) from glycoprotein D of ;,Herpes simplex virus and was photocoupled to avidin using the benzoyl-benzoyl-glycine linker moiety with a molar ratio of the peptide to avidin of 8:1. The peptide was synthesized and consisted ~f nine amino acids. After a course of three weekly injections of 30 lug of the anti-I-A -avidin peptide conjugate into (B6x3H)Fl mice/ the mice were boosted once with avidin-(herpes peptide) intraperitoneally and then sera were collected nine days after the booster injection. The results are shown in Fig. 4. The illustrated results represent the net antibody `response to Herpes peptide after subtracting the ;~35 binding on the control samples and compared to the ~`~response obtained with a single priming in Freund's complete adjuvant ~FCA) followed by the same booster -' injection.
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~ 3~7523 Example V
This Example illustrates the use of antibody-biotin-streptavidin complexes to raise antibodies to streptavidin.
Streptavidin is a protein which was extracted from StrePtomyces avidinii. This protein material was conjugated with the anti-I-Ak biotinylated antibody following the procedure generally described above in Example I and the conjugate was injected into (B6xC3H~F
and B6 mice, in the manner described in Example II.
Se~a from these mice responded to streptavidin but were negative when assayed against avidin. These results appear graphically for individual mice in Figure 5.
These results show that the targeting effect is not restricted to avidin, but is obtained with another serologically non-cross biotin-binding active protein.
Examle VI:
This Example illustrates the enhancement of avidin immunogenicity using monoclonal antibodies specific ~or Class I MHC's.
Using a method similar to Example I, avidin was coupled to biotinylated anti-Kk monoclonal antibody.
This m~terial ~30 ~y) was used to prime female C3H
(H-2k~ or B6 (H-2b) mice. Three weeks later the mice were bled to obtain preboost sera and then subsequently were boosted with avidin. The results appear in Figure 6. As may be seen therein the C3H mice (open triangles) gave a higher anti-avidin response than B6 mice (closed triangles), indicating that there was specific delivery of avidin only in mice expressing the Kk determinants.
These results illustrate that it is possible that any monoclonal antibodies directed against APC determinants can mediate antigen delivery even when these determinants are not restricted only to APC.
In summary o~ this disclosure, the present invention provides ~ novel method of vaccinating ~`

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1 3~7523 ma~mals by the conjugation of antigens, which may be in the form of synthetic epitopes or protein subuni~s, to monoclonal antibodies specific for antigen presenting cells, such that these antigen-antibody con~ugates may be used to elicit a beneficial antibody response without needing to use deleterious adjuvants. Modifications are possible within the scope of this inYentiOn.

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~"'~" ' ' ', SUPPLEME TARY DISCLOSURE
In the parent disclosure, there is described a new method o~ enhancing the immunogenicity of weak antigens by conjugation to a monoclonal an~ibody specific for a particular surface structure of antigen presenting cells.
The surface structures on the antigen presenting cells o~ the immune system which can he recognized and targeted by the monoclonal antibody portion of the immunoconjugate are numerous and the specific such surface antigen structure targeted by the monoclonal antibody depends on the specific monoclonal antibody.
The monoclonal antibody may be provided specific for a gene product of the MHC, and, in particular, may be specific ~or class I molecules of MHC or for class II
molecules of MHC, as specifically described in the principal disclosure. However, the invention is not limited to such ~pecific surface structures and the conjugates containing the corresponding monoclonal antibodies but rather, as de~cribed in the principal disclosure and zs will be apparent to those skilled in the art, the invention is applicable to any other convenient surface structure of antigen presenting cells which can be recognized and targeted by a specific monoclonal antibody to which an immunogenic molecule is conjugated.
In accordance with this Supplementary Disclosure, and as set forth in the additional Examples below, strong adjuvant-independent serological responses to a delivered antigen have been obtained with conjugates formed with dendritic cell-specific monoclonal antibody and CD4 cell-specific monoclonal antibody. In fact, for the latter m4noclonal antibodies responses to the delivered antigen of about 10 to 20 fold greater than observed for the anti-class II MHC anti~odies.
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1 3~75~3 In the present invention, the monoclonal antibody specific for the target structure is provided in the form of a conjugate with an antigen against which it is desired to elicit an immune respons~. The invention is particularly useful with antigen molecules which normally possess a weakly-immunogenic response, since that response is potentiated by the present invention.
The antigen molecule may be in the form of a peptide or protPin, as discussed above, in the principal disclosure7 but is not limited to such materials.
Antigenic carbohydrates and other antigenic materials may be employed in accordance with this Supplement~ry Disclosure.
In the principal ~isclosure, there is described the antibody response of a conjugate of avidin and the MAb to I-Ak Class II MHC. As d~monstrated in th further Examples below, considerably further enhanced antibody response was obtained using a dendritic cell-specific monoclonal antibody and a CD4-specific monoclonal antibody.
The invention is illustrated further by the following additional Example:
Example VII:
This Example illustrates the enhancement of avidin immunogenicity using monoclonal antibodies specific for differing antigen-presenting cell surface makers.
FQ110Wing the procedures generally described in the above Examples, experiments were conducted to compare the adjuvant-independent serological responses obtained ; 30 to the protein antigen avidin when conjugated to ;different biotinylated monoclonal antibodies and injected in saline into mice~
Monoclonal antibodies specific for different cell surface maker~ were purified by affinity chromatography on Protein G-Sepharose ~trademark) from their respective :, , ~ .
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,~ ' ~ 3275~3 cell culture supernatants (all cell lines were obtained from ATCC).
The purified antibodies were biotinylated by reaction with an N-hydroxysuccinimide dexivative of biotin and then conjugated to monovalent avidin, made functionally monoval~nt by adding biotin to fill three out of four biotin binding sites in the tetramer. The con~ugates between the different biotinylated antibodies and monovalent avidin were formed by mixing the two components at equimolar ratios.
Three different mice strains, namely C3H(H-2k), C57B1/6(H-2b) and (C3HxC57Bl/6~Fl~H-2bxH-2k), were injected with the different immunoconjugates according to an immunization protocol of a single suhcutaneous injection with the immunoconjugate (containing 10 ug of avidin), followed 3 weeks later by a single intraperi-toneal boost with 10 ug of avidin only in saline. Mice were bled 1 week post-boost and the sera analyzed for avidin-specific IgG responses~
The responses observed for the different immuno-targ~ting antibodies are set forth in the following Table II, as arbitrary units of response in an enz~me-linked immunoassay relativ~ to a standard curve established with a monorlonal anti-avidin antibody.

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As expected from the findings reported in the previous Examples, the two anti-class II major histo-compatibility complex antibsdies (anti-I-Ak and anti-I-Ab) promote significant responses in the (C3H x B6~ F1 animals, reflecting allele specific immunotargeting.
In addition, two other antibodies promoted strong adjuvant-independent serological responses, namely the dendritic specific monoclonal antibody TlB 227 and the monoclonal antibody TlB 207 recognizing the CD4 marker in the ~ouse. The immune response observed for these two ~ntibodies is approximately 10 to 20 fold greater than that observed for the anti-Class II MHc antibodies.
The data presented in this Example serve to illustrate that immunospecific targeting of a protein antigen to both MHC and non-~HC sur~ace markers or cells in the immune system promotes strong adjuvant independent serological responses to the delivered antigen.

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Claims (30)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A novel conjugate suitable for administration to a mammal to elicit an immune response, comprising an antigen conjugated with a monoclonal antibody specific for a surface structure of antigen-presenting cells.

2. The conjugate of claim 1, wherein said monoclonal antibody is specific for a gene product of the major histocompatibility complex.

3. The conjugate of claim 2, wherein said monoclonal antibody is specific for Class II molecules of the major histocompatibility complex determinants.

4. The conjugate of claim 2, wherein said monoclonal antibody is specific for Class I molecules of the major histocompatibility complex determinants.

5. The conjugate of claim 1, wherein more than one different tandemly-linked antigen molecule is conjugated with said monoclonal antibody, so as to elicit an immune response to each of said antigen molecules upon administration.

6. The conjugate of claim 1 wherein said antigen comprises a weakly-immunogenic molecule.

7. The conjugate of claim 6 wherein said weakly-immunogenic molecule comprises a synthetic peptide representing the immunological recognition site of an antigen molecule of a pathogen.

8. The conjugate of claim 6 wherein said weakly-immunogenic molecule comprises a protein sub-unit of a pathogen.

9. A vaccine suitable for administration to a mammal to elicit an IgG antibody response to an antigen, which consists essentially of a conjugate comprising a normally weakly-immunogenic antigen conjugated to a monoclonal antibody specific for surface structures of antigen presenting cells and a suitable carrier therefor, whereby said antibody response is obtained in the absence of an immunogenicity-enhancing adjuvant.

10. The vaccine of claim 9 wherein said monoclonal antibody is specific for a gene product of the major histocompatibility complex.

11. The vaccine of claim 10 wherein said weakly-immunogenic antigen comprises a weakly-immunogenic peptide or protein.

12. The vaccine of claim 11 wherein said weakly-immunogenic peptide is a synthetic peptide representing the immunological recognition site of an antigen molecule of a pathogen.

13. The vaccine of claim 11 wherein said weakly-immunogenic protein is a protein subunit of a pathogen.

14. The use of an antigen capable of eliciting an antibody response to a pathogenic disease in the form of a conjugate with a monoclonal antibody specific for surface structures of antigen-presenting cells for the immunization of a mammal against the pathogenic disease.

15. The use of claim 14 wherein said monoclonal antibody is specific for a gene product of the major histocompatibility complex so as to elicit IgG antibody response to the antigen.

16. The use of claim 15 wherein said monoclonal antibody is specific for Class II molecules of the major histocompatibility complex determinants.

17. The use of claim 15 wherein said monoclonal antibody is specific for Class I molecules of the major histocompatibility complex determinants.

18. The use of claim 14 wherein said conjugate contains more than one different tandemly-linked antigen molecule, so as to elicit an immune response to each of said antigenic molecules.

19. The use of claim 15 wherein said antigen is a normally weakly-immunogenic peptide or protein whose immunogenicity is significantly enhanced by said conjugation with said monoclonal antibody, whereby said IgG antibody response to said antigen is achieved in the absence of immunogenicity-enhancing adjuvants.

20. The use of claim 19 wherein said normally weakly-immunogenic peptide or protein is a synthetic peptide representing the immunological recognition site of an antigen molecule of the pathogenic organism.

21. The use of claim 19 wherein said normally weakly-immunogenic peptide or protein is a subunit protein containing the immunological recognition site of an antigen protein molecule of the pathogenic organism.

CLAIMS SUPPORTED BY SUPPLEMENTARY DISCLOSURE

22. The conjugate of claim 1 wherein said monoclonal antibody is specific for dendritic cells.

23. The conjugate of claim 1 wherein said monoclonal antibody is specific for CD4.

24. The conjugate of claim 1 wherein said weakly-immunogenic molecule is an antigenic carbohydrate.

25. The vaccine of claim 9, wherein said monoclonal antibody is specific for dendritic cells.

26. The vaccine of claim 9, wherein said monoclonal antibody is specific for CD4.

27. The vaccine of claim 9, wherein said weakly-immunogenic molecule is an antigenic carbohydrate.

28. The use of claim 14, wherein said monoclonal antibody is specific for dendritic cells.

29. The use of claim 14, wherein said monoclonal antibody is specific for CD4.

30. The use of claim 14, wherein said weakly-immunogenic molecule is an antigenic carbohydrate.