AU4250393A - Methods and agents for modulating immune response, and uses thereof - Google Patents

Description

METHODS AND AGENTS FOR MODULATING IMMUNE RESPONSE, AND

USES THEREOF

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of immunology and, more particularly, to the modulation of the immune response to a variety of antigens, including the enhancement of host immunocompetence and the preparation and administration of vaccines for prevention and treatment of disease states.

BACKGROUND OF THE INVENTION

In general, antigens are "presented" to the immune system by antigen presenting cells (APCs) , including, for instance, macrophages and B-cells that work in conjuction with major histocompatibility complex molecules (MHCs) which are present on the APC surface. Normally, natural antigens and molecules supplied as immύnogens are thought to be taken up and partially digested by the APCs, so that smaller pieces of the original antigen are then expressed on the cell surface in the context of MHC molecules.

It is also presently understood that T-lymphocytes, in contrast to B-lymphocytes, are relatively unable to interact with soluble antigen. Typically T-lymphocytes require antigen to be processed and then expressed on the cell surface of APCs in the context of MHC molecules as noted above. Thus, T-cells, and more particularly, the so called "T-cell receptors," are able to recognize the antigen in the form of a bimolecular ligand composed of the processed antigen and one or more MHC molecules.

It is also presently understood that T-lymphocytes, in contrast to B-lymphocytes, are relatively unable to interact with soluble antigen. Typically T-lymphocytes require antigen to be processed and then expressed on the cell surface of APCs in conjunction with MHC molecules as noted above. Thus, T-cells, and more particularly, the so called "T-cell receptors," are able to recognize the antigen in the form of a bimolecular ligand composed of the processed antigen and one or more MHC molecules.

Many epitopes on proteins, including both foreign and endogenous proteins, while recognized are so dominated by the reactivity of other more active epitopes as to be relatively unrecognized or only weakly recognized by the immune system. These epitopes therefore appear to elicit little or no antibody or other immune response, and may elicit at most only a weak response. It has therefore been difficult or impossible, for instance, to raise antibodies against these protein epitopes. By contrast, the epitopes that elicit extraordinarily strong immune responses, in some instances, to the exclusion (or partial exclusion) of the former epitopes can be termed "immunodominant." The invention described herein utilizes different methods of modifying antigens and epitopes in order to modulate the otherwise normal immune response to the antigen.

The present invention relates to a method of modifying immune recognition of epitopes by the immune system. This may involve endogenous or foreign molecules (immunogens) and entail altering the antigenicity thereof or altering the immune response thereto, which includes without limitation modulating the immune response to

(non-modified) naturally occuring endogenous or foreign molecules represented by the immunogen. By altering the molecule of interest, one may raise an immune response to the molecule in non-modified form. For instance, altered epitopes may be present on molecules to which the immune system is wholly unresponsive. Likewise, the epitopes may be essentially unrecognized, or may be inactive epitopes on otherwise antigenic molecules. Similarly, such epitopes may otherwise be only weakly recognized or responded to by the immune system under normal conditions. Alternatively, such epitopes may be so strongly recognized by the immune system that other epitopes on the same immunogen molecule do not elicit immune responses.

The invention described herein broadly relates to the modification of molecules and the modulation of such normal antigenic mechanisms. In particular, the present invention thus relates to modifying the antigenicity or immunogenicity of an antigen by, in one embodiment, treating the antigen with an advanced glycosylation endproduct (AGE) or a compound which forms AGEs. It is contemplated that both positive and negative regulation of the antigenicity of epitopes can be achieved. For example, by rendering epitopes recognized, or recognizable, antibodies can be raised to recognize and bind to the antigen. Enhanced antigenicity and the ability to raise antibodies to otherwise weak or ineffective epitopes finds great utility not only, for example, in vaccine applications in animals, including humans, but also in producing antibodies which can be used as reagents for, among other uses, binding, identifying, characterizing and precipitating epitopes and antigens.

Alternatively, this invention contemplates the effective suppression of immune responses to immunodominant epitopes, in order, for instance, to allow immune responses to otherwise "subordinate" epitopes. This additional ability to modulate antigenicity may be useful, for example, in immunizing animals, including humans, and also in producing antibodies which are reactive towards otherwise silent or weakly antigenic epitopes. Such antibodies are also useful for, among other things, binding, identifying, characterizing and precipitating epitopes and antigens in vivo and in vitro.

Consequently, one object of the present invention is to characterize epitopes which have not been recognized to date.

Another object of the present invention is to modify and to utilize modified antigenicity of polypeptides to assist in the characterization of such polypeptides.

Another object of the present invention is to provide a method of activating epitopes which have not previously been identified. These and other objects will be apparent to those of ordinary skill from the teachings herein.

SUMMARY OF THE INVENTION

A method of modifying the immune response to an antigen is disclosed. In a first embodiment, the antigen is reacted with an AGE or a compound which forms AGEs, in a manner which forms AGEs on said antigen in an amount sufficient to induce a modified immune response.

More particularly, the antigen is reacted in a manner which forms AGEs on the antigen in the epitope region of the molecule to which antigenicity is desired. With appropriate levels of AGEs present on the molecule in this region, the molecular region with AGEs present thereon is "activated," in the sense that the normally poor or nil immune response to the naturally occuring epitope represented by the AGE-modified region is enhanced.

In a second preferred embodiment of the invention, AGE modification of a particular molecular region is used to suppress antibody recognition of a portion of the molecule. At levels of AGE-reaction or accumulation which are typically somewhat higher than those necessary to induce antibody recognition, the reaction of AGEs with the molecule suppresses recognition of the naturally occuring epitope represented by the AGE-bearing site. Alternately, an epitope capable of forming or associating with an AGE may be reacted with an inhibitor of advanced glycosylation such as aminoguanidine or its analogs, to inhibit the formation of an AGE and the consequent development of immunodominance. In these ways, suppression of an immunodominant epitope can allow otherwise immunologically silent or recessive epitopes to be recognized and responded to by the immune system.

The inhibitors of advanced glycosylation endproduct formation in proteins as referred to above, are broadly set forth in U.S. Patent No. 4,758,583, the disclosure of which is incorporated herein by reference. These compounds include compounds that react with a carbonyl moiety of an early glycosylation product. Representative of such advanced glycosylation inhibitors are aminoguanidine, lysine and α-hydrazinohistidine.

In addition to these specific compounds, other agents capable of inhibiting the advanced glycosylation or proteins have likewise been identified and are also utilizable to similarly inhibit the advanced glycosylation of lipids. These agents are set forth in U.S. Patent Nos. 4,908,446; 4,983,604; 5,140,048;

5,175,192; 5,114,943; 5,137,916; 5,130,337; 5,100,919; and 5,106,877 to Ulrich et al., the disclosures of which are likewise incorporated herein by reference.

Accordingly, such compounds include a variety of hydrazine derivatives having, for example, a generic formula as follows: wherein R is a group of the formula

and R., is hydrogen or a lower alkyl group of 1-6 carbon atoms, a hydroxyethyl group, or together with R₂ may be a lower alkylene bridge of 2-4 carbon atoms; R₂ is hydrogen or a lower group alkyl of 1-6 carbon atoms or together with R₁ or R₃ is a lower alkylene bridge of 2-4 carbon atoms, a ino, hydroxy, or an aminoalkylene group of the formula

-(CH₂)_n-N-R₆ R₇ wherein n is an integer of 2-7 and R₆ and R₇ are independently a lower alkyl group of 1-6 carbon atoms or together form a part of a cycloalkyl or heterocyclic ring containing from 1 to 2 heteroatoms, of which at least one is nitrogen; and the second of said heteroatoms is selected from the group consisting of nitrogen, oxygen, and sulfur; with the proviso that when the second of said heteroatoms of the heterocyclic ring is nitrogen and forms a piperazine ring; it may be optionally substituted by a substituent that is identical to the portion of the compound on the first nitrogen of the piperazine ring; R₃ is hydrogen, a lower alkyl group of 1-6 carbon atoms, or together with R₂ or R₄ is a lower alkylene bridge of 2-4 carbon atoms; R₄ is hydrogen, a lower alkyl group of 1-6 carbon atoms or together with R₃ is a lower alkylene bridge of 2-4 carbon atoms; or an amino group; R_g is hydrogen, or a lower alkyl group of 1-6 carbon atoms; with the proviso that at least one of R-,, R.,, R₃, R₄ or R_g is other than hydrogen; or R is an acyl or a lower alkylsulfonyl group of up to 10 carbon atoms and R, is hydrogen; and their pharmaceutically acceptable acid addition salts.

Accordingly, where identified herein, the term "inhibitors of advanced glycosylation" is intended to encompass both the compounds such as aminoguanidine, lysine and α-hydrazinohistidine, and other agents as generically expressed hereinabove and as may be contained in other related patent applications and patents issued subsequently to U.S. Patent No. 4,983,604 and having reference thereto.

Another preferred embodiment of the invention utilizes antigen presenting cells (APCs) and the major histocompatibility complex (MHC) present on the surface of such cells. The antigen is reacted initially with AGEs or a compound which forms AGEs thereon as described above. This AGE-modified product is then combined with APCs having MHC present on the cell surface as well as receptors for AGEs, the antigen or the AGE-antigen product, until processing of the antigen is effective for rendering the epitope recognizable. The processed and displayed antigen is then available to react with other components of the immune system which recognize the epitope or the AGE-modified epitope in the context of the APC.

The invention described herein also preferably includes the antibodies produced by the methods described herein or in response to the immunogens, modified as described herein, said antibodies including monoclonal, polyclonal and chimeric antibodies, as well as immortal strains of cells which produce such antibodies, for example hybridomas which produce monoclonal antibodies which recognize the molecules and AGE-molecules of interest.

The invention also encompasses cellular immune system components, e.g., T-lymphocytes raised in response to such AGE-modified antigens or immunogens, pharmaceutical compositions containing the AGE-modified antigens, antibodies or cellular immune system components and various methods of use.

In a preferred aspect of the invention, the immunogen contains a plurality of epitopes. The AGE or compound which forms AGEs is reacted with at least one epitope on the molecule.

In another preferred aspect of the invention, the reaction between the immunogen and AGE or compound which forms AGEs increases the immunogenicity of the immunogen or relatively increases the immunogenicity of a portion thereof. This can be via an AGE-reaction with the epitope to be responded to, or via a reaction between the AGE or compound which forms AGEs and a region of the antigen near the epitope, i.e., the epitope region, or in another portion of the antigen at a distance from the epitope.

Another preferred aspect of the invention involves reducing the antigenicity of an epitope on an antigen or decreases the relative immunogenicity of a portion thereof. This can be via an AGE-reaction with the epitope to be responded to, or via a reaction between the AGE or compound which forms AGEs and a region of the antigen near the epitope, i.e., in the epitope region or in another portion of the antigen at a distance from the epitope. The reaction between the antigen and the AGE or a compound which forms AGEs thus may serve to reduce the recognition of a portion of the molecule, in some cases, in favor of yet another epitope on the antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described herein in detail in connection with the drawings, in which:

FIGURE 1 is three graphs showing thymidine incorporation as a function of substrate concentration for cells with different incubation protocols;

FIGURE 2 is a graph of thymidine incorporation (CPM) as a function of substrate concentration (AGE- or control RNase) in ug/ml wherein the substrate is pre-incubated with 1% trypsin at 37°C for 16 hours;

FIGURE 3 is a graph of thymidine uptake (CPM x 1000) as a function of substrate concentration (AGE- or control RNase) used to prime mice to activate T-cells. T-cells were primed using a 10-fold lower dose of AGE-RNase than the dose necessary to prime T-cells using control RNase.

FIGURE 4 is a graphic presentation of data generated by competitive ELISA using AGE-RNase and control RNase to raise antibodies;

FIGURE 5 is a graphic presentation of data showing cell mediated (T-cell) immunity as affected by the presence of AGEs on antigens;

FIGURE 6 is an epitope scan of antibody no. 11.3;

FIGURE 7 is an epitope scan of antibody 5.8, and

FIGURE 8 is a comparison of the activity of antibody nos. 11.3 and 5.8. DETAILED DESCRIPTION

The following abbreviations are used herein, and have the following meanings unless otherwise specified:

The term "immunogen" refers to any substance, such as a molecule, cell, virus or fragment of such molecule, cell or virus which can be administered to an individual in an effort to elicit an immune response. Preferably the immunogen is recognizable by the immune system or a component thereof in its AGE-modified form, and the molecule is also recognizable by the antibodies raised to the AGE-antigen in non-modified form.

The term "immunogen" thus simply refers to such substances which are or can be administered or otherwise used to raise antibodies or cellular immune system components, such as by "priming".

When used in connection with "immunogen", the term

"molecule" refers to a molecule or molecular fragment of the antigen in non-AGE modified form unless otherwise specified.

Likewise when used to refer to a cell, virus or fragment thereof, the immunogen can be the cell, virus or component thereof, which can be reacted or treated with AGEs or a compound which forms AGEs to modify the immune response thereto. The term "immunogen" therefore encompasses antigenic materials such as cells, viruses, cellular and viral components, antigenic compounds, and foreign proteins, as well as species which are rendered antigenic by the treatment described herein.

Preferred immunogens used herein include proteins and protein fragments. The term "antigen" refers to substances, e.g., molecules which induce an immune response. It thus can refer to any molecule contacted by the immune system, and may include without limitation, proteins, nucleic acids and the like. Each antigen typically comprises one or more epitopes. These antigenic molecules can undergo reaction with reducing sugars to form AGEs. However, these compounds can also be reacted with AGEs directly, or in the presence of bi- or multifunctional coupling reagents, can be chemically linked, causing attachment of AGEs to the molecule.

Preferably the antigens contemplated and/or described herein or epitopes thereon, are those that will induce an immune response or other immunological reaction upon injection or other exposure to a normal, substantially immunocompetent host after treatment in accordance with the invention.

The invention accordingly extends to antigens that, as stated earlier, have epitopes whose recognition by and corresponding reactivity with the immune system is so minimal or masked as to be virtually nil, in the absence of a reaction with AGE or a compound which forms AGEs. Likewise, the preferred aspects of the invention take into account those antibodies which recognize epitopes present on the molecule even in the absence of a reaction with AGE or an AGE forming compound. Most preferably, the reaction between the molecule of interest and the AGE or a compound which forms AGEs renders an epitope on the molecule recognizable by antibodies, and these antibodies recognize thereafter can recognize the epitope on the molecule in non-AGE modified form.

The terra "AGE-" refers to the advanced glycosylation endproduct of the protein or other compound to which it refers. These compounds can be formed by reacting a molecule of interest with one or more AGEs or with compounds which form AGEs, including without limitation, reducing sugars, and in a manner which leads to the formation of AGEs. The "family" of AGEs has some members which are quite stable and others which are unstable or reactive. Thus, AGE-proteins can also be stable, unstable or reactive. When used with reference to endogenous proteins, such AGE-molecules are typically formed non-enzymatically in vivo. Such compounds can also be produced in vitro by, e.g., incubating a mixture of a reducing sugar and a suitable compound, e.g., a protein or nucleic acid, or by other methods in vitro, such as chemical coupling of AGEs and AGE models to biological macromolecules.

The term "protein" refers to synthetically produced and naturally occurring polypeptides, fragments of polypeptides and derivatives thereof which may undergo advanced glycosylation reactions non-enzymatically, either in vitro or in vivo. For convenience, but not by way of limitation, the description below utilizes the term "protein" but these teachings also apply to other compounds which undergo non-enzymatic advanced glycosylation. Oligonucleotides and amine-containing lipids, as well as other AGE reactive biomolecules may be mentioned as non-limiting examples. The teachings contained herein are therefore not to be limited to proteins or fragments thereof.

The term "reducing sugar" refers to any mono- or poly- saccharide which reduces Fehling's reagent or Tollens' reagent. By way of non-limiting example, nearly all monosaccharides and most disaccharides are reducing sugars; sucrose is a notable exception. Preferred reducing sugars include glucose, glucose-6-phosphate

(G6P) , fructose and ribose. Reducing sugars are known to react with primary amino groups, forming Schiff base intermediates in the form of imines. These compounds may subsequently undergo an Amadori rearrangement, thus producing alpha amino carbonyl moieties. This rearrangement which converts the imines to a more stable secondary or disubstituted amine, more permanently links the sugar and protein. These secondary amines may further react, to generate the class of compounds referred to as AGEs. AGEs may ultimately form or cause intermolecular or intramolecular crosslinkages. AGE formation and crosslinking are particularly noteworthy in proteins having a high lysine content, due to the availability of side chain amino groups.

When proteins are exposed to reducing sugars, the sugars may react chemically with the proteins by mechanisms that do not depend on enzymatic catalysis. Ultimately, through a series of complex rearrangements and further reactions, these sugar-modified proteins become advanced glycosylation endproducts or AGE-proteins. While these compounds constantly form in vivo, it is apparent that AGE clearing mechanisms also operate. For example, macrophages and other cells participate in the uptake and degradation of these endogenous AGE-modified proteins. Clearance of AGE-modified proteins can occur through distinct receptors expressed on macrophages and other cells, the so called AGE-receptors. Among mechanisms which are believed to be operative in accordance with the present invention, the presence of AGEs on an antigen helps to target the antigen to the AGE receptor on macrophages, and may thereby lead to more efficient antigen uptake for processing. In addition, since it has been shown that AGE-modified proteins are more resistant to enzymatic proteolysis and CnBr digestion, this increased resistance to proteolysis may alter peptide fragmentation in ways which modulate antigenicity. The terms "immunocompetent", "normal immune system" and like terms refer to the immune response which can be elicited in a normal mammalian host with the antigen of interest, when the compound is administered without the AGE-modifications described herein. The immunogen can simply be administered to the host in unmodified form, and the normal immune response evaluated. Thus, using art recognized methods, this control is readily ascertained without resort to undue experimentation.

The term "antibody" refers to immunoglobulins, including whole antibodies as well as fragments thereof, such as Fab, F(ab*) or F(ab')₂' that recognize or bind to specific epitopes. The term thus encompasses, inter alia. polyclonal, monoclonal and chimeric antibodies, the last mentioned being described in detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, which are incorporated herein by reference. An antibody "preparation" thus contains such antibodies or fragments thereof, which are reactive with an antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen. An antibody preparation is therefore termed "non-reactive" with the antigen when the binding of the individual immunoglobulin molecules to the antigen is not detectable by commonly used methods.

An antibody is said to "recognize" an epitope if it binds to the epitope. Hence, "recognition" involves the antibody binding reaction with an epitope, which may include the typical binding mechanisms and methods. "Binding" is thus used in the conventional sense, and does not require the formation of chemical bonds.

The term "epitope" is used to identify one or more portions of an antigen or an immunogen which is recognized or recognizable by antibodies or other immune system components. The "epitope region", as used herein, refers to the epitope and the surrounding area in the vicinity of the epitope, taking into account three dimensional space. Hence, this may take into account the tertiary and quaternary structure of the antigen. When AGE reactions occur in the epitope region, the epitope may become more readily recognized by the immune system, or reduced immune system recognition may be achieved, as described herein in detail.

"Processing" and "presentation" refer to the mechanisms by which the antigen is taken up, altered and made available to the immune system. Presentation also includes, when appropriate, complexation or binding with MHC. In certain instances, processing entails the uptake and partial proteolytic degradation of the antigen by APCs, as well as display on the APC surface in the context of MHC.

The terms "reaction" and "complex" as well as derivatives thereof, are used in the general sense, and are not to be construed as requiring any particular reaction mechanism or sequence.

"Ribonuclease" (RNase) refers to the bovine derived enzyme. RNase is known to react with reducing sugars and thereby to form advanced glycosylation endproducts upon co-incubation. RNase is used as a sample protein because of its substantial reactivity with reducing sugars, and because it undergoes substantial crosslinking.

The abbreviation "BSA" refers to bovine serum albumin.

The abbreviation "MHC" refers to major histocompatibility complex, a series of compounds which is normally present to a greater or lesser degree on the surface of, among others, antigen presenting cells. MHC functions to "signal" cellular immune system components, e.g., T- lymphocytes, to recognize and react with the antigen presenting cell and/or the antigen bound to said cell and/or the MHCs thereof. The term "signal" is used in the general sense to refer to the initiation of the reaction between T-cells and APCs bearing processed antigen in the context of MHC. As such the "signal" may involve any reaction between these components which causes the antigen to become recognized by antibodies, an antibody preparation or by the cellular immune system components.

The abbreviation "FFI" refers to a synthetic model AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-lH-imidazole. The compound can be produced by chemical synthesis as described in U.S. Patent No. 4,665,192 issued on May 12, 1987 incorporated by reference.

In one embodiment of the present invention, the non- enzymatic reaction of reducing sugars and proteins which bear one or more epitopes to form AGE-proteins augments the presentation of antigen over and above the endogenous level of antigen presentation which can be observed when antigen is made available to APCs in the absence of a prior reaction with reducing sugar to form AGE-antigen.

Without limiting the invention to any particular mechanism, several explanations for AGE modification of the antigenicity of a given immunogen can be considered. AGEs or compounds which form AGEs may alter the conformation or structure of the antigen, causing it to assume a different overall configuration which exposes different epitopes to the immune system.

Also, the presence of AGEs may enhance the uptake of the AGE-antigen, making more of the antigen available for presentation by the APC. At another level, the presence of AGEs on the antigen may alter the presentation of the antigen within the context of MHC on the APC cell surface. Because antigen may be partially digested prior to presentation, it is also possible that AGEs modify the degree or the pattern of protein fragmentation, causing a different pattern of antigen fragments to be expressed on the cell surface. Likewise, AGEs may interact with MHC, thus causing the antigen to be displayed in a modified manner, and epitopes to be differently recognized. Any one of these possible explanations or any combination of these mechanisms may apply; however, the invention is not limited in this capacity, and a wholly different mechanism may be applicable.

By combining the molecule with an AGE or a compound which forms AGEs and using this AGE modified molecule as the immunogen, a "modified immune response" can be achieved. This means that, e.g., the immunogen can be used to raise antibodies which are specific to epitopes not previously recognized. Additionally, the modified immune response may involve non-antibody immune system components, e.g., T-lymphocytes, which may recognize an epitope not previously presented or recognized. Hence, the "modified immune response" is largely directed to the previously unrecognized epitope on the antigen treated as described herein.

The preferred embodiments of the invention utilize an AGE modified antigen as the immunogen, and seek to raise or react said antigen with antibodies which also recognize the same or a different epitope which is present on the molecule even when said molecule is not pre-reacted with AGE or a compound which forms AGEs. In this aspect of the invention, the so-called modified immune responses therefore involves the generation of antibodies which are not otherwise formed or observed in vitro or in vivo. For example, the immunogen can be incubated with AGEs and then used to inoculate a mammal to raise antibodies to the newly recognizable epitope, produce antiserum or vaccine preparations and the like.

Likewise, antibody molecules can be cleaved to form antibody fragments, which can be recombined in vitro to form chimeric antibodies which recognize or bind to newly recognizable epitopes on the antigen. Hence, the "modified immune response" is not limited to a conventional allergic response, or to increases or decreases in the extent or severity thereof.

Another preferred embodiment of the invention involves the conversion of non-antigenic molecules or epitopes into effective antigens or epitopes which are recognizable. This may be accomplished by an in vitro or an in vivo conversion, to produce an antigen with AGEs reacted therewith or attached thereto. The antigen can be combined with AGE or a compound which forms AGEs to induce a modified immune response to the antigen.

Yet another preferred embodiment of the present invention relates to molecules which are weakly antigenic. In this preferred aspect, the weakly antigenic molecule is reacted with or linked to AGEs, or reacted with a compound which forms AGEs, to increase the antigenicity of the molecule and increase the recognition of one or more epitopes thereon. This can likewise be conducted in vitro or in vivo to convert the molecule to a compound with increased antigenicity.

Another preferred embodiment of the invention involves the modulation of antigenicity towards an otherwise weakly antigenic or non-antigenic epitope of an otherwise antigenic molecule. In such cases, reactions between AGEs or compounds which form AGEs and the molecule result in an enhanced immune response to the otherwise "overlooked" epitopes. In this aspect of the invention, the molecule can be reacted in vitro or in vivo with AGEs or a compound which forms AGEs to reveal one or more epitopes not otherwise recognized by the immune system under normal conditions. In this manner, the epitopes which are normally unrecognized can elicit antibody formation, recognition or other immune responses.

In another preferred embodiment of the invention, immunodominancy of the epitopes on the molecule is modified. Certain antigens containing more than one epitope have characteristic immune responses based upon the dominance of one epitope over the other(s) . This aspect of the invention enhances the recognition of the subordinate epitope(s) by either reacting AGEs or a compound which forms AGEs in vitro or in vivo with the subordinate epitope(s) to enhance the recognition thereof, or by reacting AGEs or a compound which forms AGEs in vitro or in vivo with the immunodominant epitope on the molecule in an amount which is effective for suppressing the recognition of this dominant epitope. This allows other silent or recessive epitope(s) to be expressed.

To suppress recognition of a dominant epitope, the level of AGE reaction on said epitope may be somewhat higher than the level of AGE reaction necessary to enable recessive or silent epitopes to be recognized. This level of AGE reaction is that which is effective for suppressing reactivity of that particular epitope in favor of other epitopes. The level necessary to suppress dominant epitopes is a function of the relative strengths of the recognition signals, concentration of the antigen introduced and other factors. Again the determination of the control level does not require undue experimentation for those of ordinary skill in the art. As stated earlier, the reactivity or immunodominance of an epitope may be modulated or downregulated by the administration of an inhibitor of the formation of advanced glycosylation endproducts. Accordingly, inhibitors such as aminoguanidine and the compounds discussed in detail hereinabove may prevent the formation of AGEs and by doing so, prevent the development of immunodominance and the consequent reactivity that can be experienced in certain autoimmune conditions. The invention therefore extends to the use of inhibitors of advanced glycosylation for such purpose.

In yet another preferred embodiment of the invention, the antigen molecule can be modified to enhance its reactivity with AGEs or compounds which form AGEs. One such approach involves the addition of lysine or another AGE-reactive compound to the antigen molecule. Such other compounds may include other alpha amino acids, such as arginine, histidine and other compounds containing amino groups as well as other AGE reactive moieties. As noted above, this lysine modification can be conducted on the epitope to be combined and reacted with the AGEs, to allow the molecule to become antigenic and facilitate recognition of the antigen. Likewise, this modification can be conducted with weakly antigenic molecules to generate a greater immune response to the antigen, or with a recessive or non-recognizable epitope to enhance recognition thereof over other normally dominant epitopes. Hence, in this preferred embodiment of the invention, the antigenicity of the molecule is modified by introducing a moiety into the molecule which is capable of reacting with or forming an AGE.

Preferably the moiety introduced is a lysine group, which is introduced on the epitope or in the epitope region.

However, the lysine group can be included in a portion of the molecule other than the epitope region to increase or decrease immune system recognition of that particular portion of the immunogen, or to have an effect on the recognition of the epitope.

In another preferred aspect of the invention, the lysine is substituted for another amino acid in the polypeptide on the epitope, in the epitope region or outside of the epitope region elsewhere in the molecule.

In an alternative preferred aspect of the invention, the lysine can be attached at the end of a molecular chain.

In another alternative preferred aspect of the invention, when the antigen is a polypeptide, lysine can be inserted into the polypeptide chain without substituting for another amino acid. This, again, can be undertaken on the epitope, in the epitope region or outside of the epitope region, and can be accomplished by adding the appropriate genetic sequence coding for lysine to the sequence coding for the polypeptide of interest in the desired location, inserting the sequence into an expression vector, transfecting the vector into a host and allowing the host to express the polypeptide with lysine added thereto.

The lysine modifications described above can be conducted on the dominant epitope in an amount effective for increasing or decreasing its recognition by the immune system. This can be conducted to increase or reduce its immunodominance over other epitopes, or to allow other normally non-recognized sites in the molecule to become recognizable by antibodies.

While the lysine modification of polypeptides may also be conducted chemically, the preferred method uses recombinant technology to modify the gene sequence which codes for expression of the molecule of interest. When such genetically engineered proteins are expressed, the lysine group(s) can be contained in the molecule in the position(s) coded for, which of course can involve the addition of codons for the insertion of the lysine molecule into the polypeptide, grafting or adding the lysine molecule onto the end of the polypeptide, or replacement of one or more codons in the DNA or RNA molecule to in turn replace certain protein amino acids with lysine upon eventual expression of the molecule of interest.

An expression vector may be prepared that codes for expression of a mutein of the molecule of interest, which vector may be transfected into a host cell, such that the cell is caused to express the mutein. The mutein may comprise the original amino acid sequence, or portions thereof, substituted with or having inserted therein codons for expression of the otherwise non-existing lysine residues in the primary sequence of the mutein. Suitable lysine-containing moieties include lysine alone and polypeptide fragments including lysine.

To insert one or more lysine molecules or lysine- containing fragments into the polypeptide using non- recombinant means, enzymes may also be used to insert or attach lysine molecules into the polypeptide backbone, or to graft lysine or a lysine containing group onto the polypeptide. Likewise, chemical reagents can be used to graft or insert lysine containing fragments on the molecule as described above. Hence, the antigen molecule can be a polypeptide formed by expressing the molecule from a host expression vector containing a coding sequence for the polypeptide. If the coding sequence is modified to include codons for lysine, the polypeptide can be expressed with lysine in the desired location, such as on the epitope, on the epitope region or elsewhere. By molecularly inserting or otherwise reacting lysine with the protein, or coding for expression of modified polypeptides (muteins) containing lysine, the number of primary amino groups or other AGE reactive moieties on the polypeptide may be increased, thus increasing the reactivity of the polypeptide for AGE formation. These methods allow or enable one to place lysine groups in the primary sequence to thus situate AGEs in any desired site, and thus modulate antigenicity on the epitope, on or near the immunodominant epitope or elsewhere in the molecule at a site other than the epitope of interest.

Compounds other than lysine that contain suitable reactive groups or sites, and that react with AGEs or reducing sugars can also be used for modifying reactivity. Preferably these compounds do not otherwise denature or inactivate the protein.

Another preferred method of enhancing antigenicity encompassed herein includes the use of antigen presenting cells (APCs) . Antigen presenting cells, endogenous MHC molecules which are present on the membranes of said antigen presenting cells in several different forms, and receptor molecules on the surface of the APC can be used. In this embodiment of the invention, the invention may . utilize the in vivo or in vitro derived reaction products generated between proteins and AGEs or compounds which form AGEs.

This embodiment may, for example, take advantage of APC receptor proteins which recognize and bind to polypeptide molecules, AGEs or the reaction product which can be formed in vitro or in vivo between an AGE or a compound which forms AGEs and an AGE-reactive compound.

Antigen uptake by the APCs can occur via nonspecific mechanisms, and may be followed by display of the antigen in association with MHC on the cell surface. Processing can be markedly increased by enhancing the initial interaction between antigen and the surface of the APC. For example, opsonization of antigen by IgG or C3b may significantly enhance antigen processing and presentation. This enhanced ability to process antigen may arise in some cases from the increased internalization of antigen. Antigen may be internalized more efficiently by interaction of the opsinized antigen complex with the Fc receptor, complement receptor or antigen is bound to antigen-specific surface receptor immunoglobulins.

Once antigen is internalized by APCs, partial proteolytic degradation occurs in a prelysozomal endosome, and processed peptide fragments of the antigen become associated with MHC molecules. However, while partial proteolytic degradation of antigen may be essential in order to generate appropriate MHC and T-cell binding to the peptide fragments thereof, excessive degradation of antigen has been found to be detrimental to the eventual immune response. Inhibition of proteolysis which is not essential for the processing of a specific antigen has been shown to enhance processing and presentation, suggesting that the interference with inappropriate proteolysis actually enhances antigen presentation.

These two processes, targeting antigen to the surface of APCs, and interfering with non-essential antigen proteolysis, can be used herein to enhance antigen processing and presentation. For example, by treating the antigen with AGE or a compound which forms AGEs, and then combining the AGE-antigen with APCs, different fragmentation and presentation patterns may result. Likewise by fragmenting the antigen prior to AGE reaction, the fragmentation and presentation patterns are modifiable. To facilitate the recognition or presentation of epitopes not previously recognized, the protein molecule may be reacted with a reducing sugar to form an AGE-protein reaction product (AGE-protein) .

If the AGEs and compounds which are known to form AGEs do not react sufficiently to provide the enhanced antigen presentation at the desired levels, one may modify the particular molecule using lysine, in an amount effective for enhancing reactivity of the molecule with AGEs or compounds which form AGEs. The lysine as mentioned above can be directed to a particular portion of the molecule in relatively low levels to cause AGEs to form on or attach to the portion of the molecule to be characterized. Alternatively, the lysine and AGE reaction steps can be directed to a portion of the molecule in higher levels to activate a different portion of the molecule, thus suppressing the dominant epitope and allowing other epitopes to be recognized.

Alternatively, the AGE-protein or fragment thereof can be combined with APCs having MHC on the cell surface until binding occurs.

At high AGE levels, the presence of the AGEs on the dominant epitope, or in a location on the polypeptide which reduces or interferes with antibody or T-cell recognition of the epitope, may shift the dominance to other epitopes. This may be a function of changes in the percentage distribution or a direct effect on the epitope.

The biological processes within the APCs can be controlled to enable one to qualitate or quantitate the binding of AGE-proteins. For example, the incubation time and temperature can be adjusted to achieve complete internalization by APCs, complete binding of the AGEs and protein, and like parameters. By maintaining APCs and AGE-proteins in combination at an appropriate temperature, e.g., about 4°C, for an appropriate time period, e.g., about one hour, binding of AGE-proteins to APC cell surfaces can be quantitated, since internalization can be effectively decreased or shut down. Alternatively, by increasing the APC/AGE-protein incubation temperature and/or time period, e.g., up to about 37°C for about one hour, internalization can be evaluated.

Additionally, one can determine the relative amounts of AGEs present on an epitope which will effectively "shut- off" this portion of the protein and activate a silent epitope which might be associated with a particular medical condition or disease, e.g., cancer, autoimmune diseases and the like. One can measure the level of AGEs present on the reactive epitope, and then evaluate antibody recognition of the silent epitope. When antibodies begin to recognize the silent epitope, the level of and location of AGEs present on the dominant epitope can be measured, thus correlating the AGE presence or modulation of antigenicity with the disease or condition of interest.

The methods described herein can take into account protein fragmentation, by treating the protein to be evaluated with a cleaving compound, e.g., an enzyme, prior to reaction with the AGE or AGE-forming compound.

In accordance with the present invention, conventional molecular biology, microbiology, cloning technology and recombinant DNA techniques may be utilized which are within the level of skill in the art. Such techniques are explained fully in the literature See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982) ; "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984) ; "Nucleic Acid Hybridization" (B.B. Hames & S.J. Higgins ed 1985); "Transcription And Translation" (B.D. Hames & S.J. Higgins eds. 1984); "Animal Cell Culture" (R.I. Freshney ed 1986) ; "Immobilized cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to Molecular Cloning" (1984) , the teachings of which are incorporated herein by reference.

The antibodies used herein are typically those which recognize the epitopes on the antigens which are made recognizable, enhanced or suppressed as described above. By injecting this type of AGE-reacted antigen into a mammal, such as through a hyperimmunization protocol, modulated antibody responses to the epitopes can be achieved.

The antibodies which are useful herein may be polyclonal, monoclonal or chimeric antibodies, and may be raised to recognize the desired epitope and used in a variety of diagnostic, therapeutic and research applications. For example, the antibodies can be used to screen expression libraries to ultimately obtain the gene that encodes proteins bearing the epitope evaluated. Further, antibodies that recognize the antigen presented can be employed or measured in intact animals to better elucidate the biological role that the protein plays, or to assess the state of immune response or immunologic memory more effectively.

Polyclonal, monoclonal and chimeric antibodies to the antigen can be prepared by well known techniques, such as the hyperimmunization protocol, or the hybridoma technique, utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Immortal, antibody- producing cell lines can also be created by techniques other than fusion, such as direct transformation of lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Likewise, chimeric antibody molecules can be produced using an appropriate transfection and hybridoma protocol.

One preferred antibody used herein is an antibody that recognizes a polypeptide in modified form which is expressed from a host expression vector containing a coding sequence for the modified polypeptide. The sequence can simply be modified to include the AGE reactive moieties, such as by substitution or insertion type additions as described above. A particularly preferred antibody recognizes a polypeptide having one or more lysine groups added to the epitope.

Another preferred antibody recognizes the polypeptide with one or more lysine groups added to the epitope region, e.g., outside of the epitope.

Another preferred antibody recognizes a polypeptide as noted above with lysine groups added to the polypeptide outside of the epitope region, such as on another epitope or in a region not otherwise recognized by the immune system.

With reference to the antibodies described above, which can be used as binding partners forantigens presented in accordance with the teachings herein, the antibody which recognizes a modified antigen (e.g., Ab.,) can be evaluated using a second antibody (Ab₂) . This method takes advantage of the antibody characteristic known as idiotypy. Each antibody contains a unique region that is specific for an antigen. This region is called the idiotype. Antibodies, themselves, contain antigenic determinants; the idiotype of an antibody is an antigenic determinant unique to that molecule. By immunizing an organism with antibodies, anti-antibodies can be raised that recognize them, including antibodies that recognize the idiotype. Antibodies that recognize the idiotype of another antibody are called anti-idiotypic antibodies. Some anti-idiotypic antibodies mimic the original antigen that the antibody recognizes, and are said to bear the "internal image" of the antigen.

Hence, a characteristic property of the anti-idiotype antibody Ab₂ is it will react with Ab One method of raising anti-idiotype antibodies involves raising Ab, and Ab₂ in different mammalian species. For example, Ab₂ may be raised in goats using rabbit antibodies as antigens. Ab₂ would therefore be an anti-rabbit antibody raised in goats. When Ab₂ recognizes Ab,, Ab₂ may have the same characteristics as the epitope.

When the modified antigen is used as a ligand, certain anti-idiotype antibodies can bind to that ligand's receptor. Receptor in this context means binding site or binding molecules. Investigators have identified several of these, including anti-idiotypes that bind to receptors for insulin, angiotensin II, adenosine I, β-adrenalin, and rat brain nicotine and opiate receptors.

Taking advantage of this phenomenon, other ligands maybe isolated using anti-idiotypic antibodies. Anti-idiotypes may be used to screen for molecules binding to the original antigen. One may therefore use this technique to identify other ligands which bind to silent epitopes, or screen for molecules which bind to the polypeptide, thus acting as agonists, antagonists and the like.

The present invention also includes the immunogens which are produced and used as described herein in AGE-modified form. Thus, the preferred immunogen is an AGE-modified protein which has at least one epitope, and at least one AGE reacted with or covalently linked to the epitope, the epitope region or elsewhere in the molecule. The immunogen has modified antigenicity due to the presence of, reaction with or linkage to said AGEs. The immunogen induces the formation of antibodies which recognize the protein in its AGE modified form or in its non-modified form.

A particularly preferred immunogen is an antigenic molecule which has at least one AGE reacted with or covalently linked to the epitope region to which an immune response is desired.

Another particularly preferred immunogen is an antigen which has at least one AGE reacted with or covalently linked to a site other than the epitope region. Hence, another preferred immunogen is an antigen molecule which has multiple epitope regions and AGEs reacted with or covalently linked to at least one such epitope region. Said immunogen can induce the formation of antibodies which recognize the antigen molecule in AGE modified or in non-modified form.

The present immunogen complexes and combinations thus may further include AGEs reacted with or attached thereto, and an antibody which recognizes the epitope bearing the AGEs, or an epitope activated by the presence or reaction of AGEs elsewhere in the immunogen. The complexes and combinations therefore include the antibodies described herein.

One such combination contains an APC, an AGE-protein reaction product and an antibody to an epitope which is present on the immunogen and recognizable after AGE reaction. Another such combination contains an APC, an AGE-protein reaction product and an antibody to an epitope which is present on thhe protein, said antibody raised in respoonse to an AGE-antigen challenge, and further recognizing the epitope without the presence or reaction with AGE or a compound which forms AGEs.

When antigen presenting cells are involved, the antigen presenting cell and antigen-AGE reaction product may be co-incubated under conditions which are effective for allowing the antigen-AGE reaction product to bind to the surface of the APC without substantially internalizing said antigen-AGE reaction product.

Likewise, the APC and antigen-AGE reaction product can be combined under conditions which are effective for allowing said antigen-AGE reaction product to become internalized by the APC.

The above described aspects of the invention can also utilize the molecule reacted with AGEs or a compound which forms AGEs and an antigen fragmentation process as mentioned previously. For example, when the immunogen is a cell, virus or polypeptide, the antigen molecule can be reacted with AGEs or a compound which forms AGEs and then fragmented prior to combining with APCs.

It is also contemplated that fragmentation can modify the presentation of the antigen, such that different epitopes may be recognized.

The antigen can be fragmented prior to treatment with the AGE compound, and then presented. This achieves another modification in the fragmentation patern.

Additionally, the antigen and AGE compound can be combined and the antigen-AGE reaction product can be fragmented by the APC and thereafter displayed or presented. This achieves yet a third fragmentation pattern. It is therefore contemplated that the fragmentation pattern can be significantly effected by the.AGE treatment process described herein, since upon presentation and display, a different fragmentation pattern is noted in each instance. All such modifications are included within this invention.

Further, the method of rendering the epitope recognizable can involve the cellular means for taking up any of the AGE, antigen or antigen-AGE reaction product. This is useful in rendering the epitopes recognizable by the immune system, and is particularly preferred in cases where the epitope is substantially non-recognized by the immune system prior to combining the APC and the antigen- AGE reaction product.

The preferred APC for use herein, when APC treatment is desirable, includes macrophage cells, and most particularly, murine peritoneal macrophage cells.

The present invention also contemplates diagnostic and therapeutic applications for these agents. Accordingly, the AGE-proteins or antibodies thereto may be prepared for use in a variety of these methods.

Any of these agents may be labeled or unlabeled as appropriate. Typically the labelled component is the antibody, but it is possible to label the AGE, antigen, MHC or APCs as well.

Thus, both receptors and the binding partners which recognize the antigen presented are used in connection with the various techniques described herein. For example, a radioimmunoassay may be conducted, using for example, a receptor or other ligand to AGEs that may either be labeled or unlabeled. Labelling may be accomplished, e.g., by radioactive addition, reduction with sodium borohydride or radioiodination.

Labels most commonly employed are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light and others.

Suitable radioactive elements may be selected for the group consisting of ³H, ^UC, ³²P, ³³P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. In the instance where a radioactive label, such is presented with one of the above isotopes is used, known currently available counting procedures may be utilized.

In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, thermometric, amperometric or gasometric techniques known in the art. The enzyme may be conjugated to the advanced glycosylation endproducts, their binding partners or carrier molecules by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Also, and in a preferred embodiment of the present invention, the enzymes themselves may be modified into advanced glycosylation endproducts by reaction with sugars as set forth herein.

Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkaline phosphatase, NAD oxidoreductase plus luciferase, phosphofructokinase plus phosphoenolpyruvate carboxylase, aspartate aminotransferase plus phosphoenol pyruvate decarboxylase. and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,8,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternative labeling material and methods.

A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

In an immunoassay, a control quantity of a binding partner to an AGE-antigen reaction product may be prepared and optionally labeled, such as with an enzyme, a compound that fluoresces and/or a radioactive element, and may then be introduced into a tissue or fluid sample taken from a mammal in order to assess, e.g., the amount of antigen present therein. After the labeled material or its binding partner(s) has had an opportunity to react with the sample, the resulting complex may be examined by known techniques, which vary according to the nature of the label attached. In this manner the AGE receptor, the activity and effect of MHC, or the epitope recognized by the antibody can be evaluated.

In each instance, the advanced glycoslation endproduct forms complexes with one or more binding partners and one member may be labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by the detection methods described herein.

One preferred diagnostic method included herein involves the determination of T-lymphocyte levels, function or activity in a sample taken from a mammal. The AGE- treated immunogen is incubated with APCs, after which T- lymphocytes taken from the mammal are added. The level, function or activity of the T-lyphocytes taken from the mammal can then be compared to a standard.

In one preferred embodiment, the APCs can be bound to a solid support.

In another preferred embodiment, the immunogen-AGE reaction product is combined with APCs at a temperature which is effective to cause binding between the APCs and the immunogen-AGE reaction product. This can be accomplished without allowing substantial internalization by the APCs. In this manner, antigen binding to said APCs can be evaluated. Also, by increasing the temperature, APC internalization of AGE-antigens and subsequent cell metabolic processes can be evaluated.

Therapeutic treatments and diagnostic methods can be performed using any or all of the various components and processes described herein. For example, for the diagnosis or treatment of cancer or infection, an isolated protein can be derived from the tumor, abnormal cells or infectious organism, and this protein can be used as an antigen either in its purified form or as an AGE-derivative. Antibodies to this protein or AGE- protein can be elicited using the methods for enhanced antigen presentation disclosed herein and used to identify, characterize, bind, inhibit or inactivate, as desired, previously unknown or ineffective epitopes on the tumor, abnormal cell, bacterial or viral protein.

This information, in turn, is useful for developing drugs which combat such afflictions, such as agonists, antagonists and the like.

Likewise, the antibodies described above can be raised to have direct diagnostic or therapeutic utility, particularly in oncologic, autoimmune and infectious disease treatments.

A preferred use for the AGE-modified antigens described herein is in the form of a vaccine which can be used to immunize mammalian patients in need of such treatment. By administering to such patient an effective amount of the immunogen, antibodies can be raised to the particular immunogen, which are effective for treating disease or preventing the development or spread thereof.

Likewise, the invention described herein also encompasses the administration of antibodies to a mammalian patient in need of such treatment. In this method of treatment, the patient is administered an effective amount of, e.g., monoclonal, polycolonal and/or chimeric antibodies, which recognize the etiologic agent. For example, antibodies raised to a silent or recessive epitope on a protein derived from a tumor or tumor type can be administered to the mammalian patient, thus providing antibodies which recognize an epitope in non-modified form or the modified epitope on the tumor. Thus, the immune system may be supplemented or otherwise used to treat diseases.

The preferred non-cellular components which recognize antigen and which are used to characterize epitopes presented in accordance with the invention include the antibodies raised to an antigen which are not normally elicited in the absence of the methods described herein. Also, as noted above, the most preferred antibodies are raised to AGE-modified antigen, but recognize the non- modified molecule.

The general procedures set forth above are illustrated in the following examples. All of the protocols disclosed herein may be applied to the qualitative and quantitative determination of epitopes activated by the processes set forth herein.

EXAMPLE 1

PRESENTATION OF ANTIGEN BY MACROPHAGES TO SPECIFIC T-CELL HYBRIDOMAS IS ENHANCED BY AGE-MODIFICATION

macrophage + antigen > 37°C X 1 hr > fix in

1% paraformaldehyde —> add antigen specific T-cell hybridoma and incubate for 24 hrs > T-cell stimulation assayed by measuring IL-2 production.

Methods

1) Antigen: Glucose-modified antigens (RNase and hemoglobin) were prepared by incubating for 4 days or 8 days in a 0.1 M phosphate buffer containing either 0.5M glucose or glucose-6-phosphate (G6P) . Control antigens were incubated for the same length of time in phosphate buffer alone. Proteins were then extensively dialyzed against lx phosphate buffered saline (PBS) , and protein concentrations were determined by either the Bradford method or by using the protein's published extinction coefficient.

2) Mice: All experiments were done with CBA/J(H2K) mice from Jackson Labs.

3) T-Cell Hybridoma Stimulation Assay: The assay system examined the ability of macrophages to present glucose-modified or unmodified antigen to antigen-specific T-cell hybridomas. Peritoneal macrophages were obtained from CBA/J mice 7 to 21 days following infection with 3 x 10⁵ Listeria moncytoσenes bacteria. Following adherence of the macrophages to 96-well plates, the wells were washed and various concentrations of antigen were added. The cells were then placed at 37°C for 1 hour, following which they were fixed in 1% paraformaldehyde.

Antigen specific T-cell hybridoma cells were then added to the fixed macrophage monolayers, and incubation was carried out for 24 hours. T-cell stimulation was assayed by quantitating the release of IL-2 by the T-cell hybridomas. This quantitation was measured by ³H-thymidine incorporation by the IL-2 dependent CTLL-2 cell line.

Additional demonstrations were performed in which antigen and antigen specific T-cell hybridomas were added to live macrophages. Incubation was carried out for 24 hours, following which stimulation was assessed by measuring IL-2 production as above. The results are shown in FIGURE 1. The results closely paralleled the enhancement of T-cell activation obtained in Example 2.

Results

Figure 1 shows that modification of RNase and hemoglobin by either glucose or G6P led to an approximately 10-fold enhancement of antigen presentation (top panel) when compared to unmodified antigen (middle panel) . This enhancement in presentation was similar whether antigen was incubated with glucose or glucose-6-phosphate, and the incubation of antigen with glucose for 8 days vs. 4 days did not substantially increase efficacy.

In contrast, the protein lysozyme did not display enhanced presentation following incubation with either glucose or glucose-6-phosphate. Since EPSILON glucose is added nonenzymatically to, e.g., the E-epsilon amino group of lysine residues, the presence of only 3 lysine residues in lysozyme as compared to 10 and 12 for ribonuclease and hemoglobin may provide a partial explanation. EXAMPLE 2

PRESENTATION OF ANTIGEN PULSED MACROPHAGES BY AGE-ANTIGEN AND FFI-ANTIGEN, BUT NOT BY UNMODIFIED ANTIGEN, SUGGESTS THAT ENHANCED PRESENTATION RESULTS FROM BINDING OF AGE- ANTIGEN TO THE MACROPHAGE CELL SURFACE

Macrophage + antigen > 4°C X 1 hr > wash HBSS—> warm 37°C X 1 hr > fix in 1% paraformaldehyde—> add T-cell hybridoma X 24 hours > assay for stimulation as in Example

1.

Methods

1) Antigen: Glucose-modified and control RNase were prepared as described in Example 1. In addition, the model AGE, FFI, was chemically attached to RNase, and this was used as an antigen.

2) Antigen pulsed macrophages: The above antigens were incubated with macrophage cells (obtained as in Example 1) for 1 hour at 4°C. After washing with Hanks Balanced Salt Solution (HBSS) to remove unbound RNase, the cells were warmed to 37°C for 1 hour. The macrophages were then fixed with 1% paraformaldehyde, following which RNase specific T-cell hybridomas were added to the fixed macrophage monolayer. Stimulation of hybridomas was assessed as described in Example 1. The results are shown below in TABLE I.

Results Under these conditions, unmodified ribonuclease was unable to stimulate ribonuclease specific T-cell hybridomas. In sharp contrast, as shown in Table I below, ribonuclease incubated with glucose or glucose-6-phosphate was able to activate T-cell hybridomas. Stimulation also occurred when FFI-ribonuclease was used as the antigen. The ability of both glucose-modified ribonuclease and FFI-ribonuclease to stimulate under these conditions suggests that they are able to bind to the macrophage cell surface. At 4°C, receptor binding of the ligand occurs, but because the cells are metabolically inactive, internalization does not occur. Since washing removes all of the free antigen, only the antigen which is bound to the cell surface is available for internalization, processing, and presentation once the macrophage is warmed to 37°C. Control ribonuclease which does not bind to macrophage cells is not presented under these conditions, since it is present only in an unbound form at 4°C and hence is completely removed with washing.

TABLE I

Antigen

Concentration fu /ml) 100 31.6 10 3.16

10 Antigen

Control RNase-

Experiment 1 (cpm) 423 190

Experiment 2 1567 140

15 4 day D-glucose RNase

Experiment 1 247 167

Experiment 2 38,947 220

20

4 day Glucose- 6-phosphate Experiment 1 21,173 783 160 93 Experiment 2 5,593 1,107 187 117

25

FFI-RNase Experiment 1 49,250 130 100 130

EXAMPLE 3

COMPETITION BY AN IRRELEVANT AGE-PROTEIN SUGGESTS THAT ENHANCED PRESENTATION OF AGE-ANTIGEN IS MEDIATED VIA BINDING OF MODIFIED ANTIGEN TO THE AGE-RECEPTOR

Macrophage > preincubate with competitor

(AGE or control hemoglobin) for 30 minutes at 4°C > add AGE-antigen and incubate for an additional 60 minutes at 4°C > wash > rest of assay as in Example 2.

Methods 1) Antigens: AGE-antigens consisted of RNase or hemoglobin which was incubated for 4 days in 0.5 M D-glucose and was processed as described in Example 1.

2) Competitive Inhibition Assay: The ability of AGE-hemoglobin to compete with AGE-ribonuclease for presentation to RNase specific T-cell hybridomas was assessed. Adherent macrophages were preincubated with either AGE or control hemoglobin for 30 minutes at 4°C, following which AGE-ribonuclease was added. After incubation at 4°C for an additional 1 hour, unbound antigen was washed away. The macrophages were fixed and then assayed as described in Example 2. The results are shown below in Table II.

Results

As shown in Table II, AGE-hemoglobin, but not control hemoglobin, competes with AGE-ribonuclease for presentation. The ability of an irrelevant AGE-protein to compete for antigen processing suggests that the enhanced presentation of AGE-ribonuclease is due to the ability of macrophages to specifically bind to AGE-modified antigen via AGE receptor. It is believed that only AGE-modified antigens are presented, since binding of antigen by macrophages is required for antigen processing.

TABLE II

EXAMPLE 4

ENZYMATIC HYDROLYSIS OF AGE-MODIFIED ANTIGEN IN VITRO IS ASSOCIATED WITH ENHANCED ANTIGEN PRESENTATION

Prefixed macrophages —> trypsinized ribonuclease + ribonuclease specific hybridoma —> Incubate for 24 hours and assay for hybridoma stimulation.

Methods 1) Antigens: Tryptic fragments of either glucose-modified or unmodified ribonuclease were generated by incubating the appropriate antigen with a buffered 1% trypsin preparation at 37°C for 16 hours.

2) Hybridoma stimulation assay: Trypsin-digested preparations AGE-modified or control ribonuclease and ribonuclease specific T-cell hybridomas were added to a prefixed macrophage monolayer. Incubation was carried out for 24 hours, following which hybridoma stimulation was assayed as previously described. Results

The results shown in FIGURE 2 demonstrate that in-vitro enzymatic hydrolysis of AGE-RNase is associated with enhanced antigen presentation. Under these experimental conditions, the necessity for internalization and processing of antigen by the macrophage is bypassed, since processing of the antigen is performed in vitro with trypsin. The tryptic fragments which are generated are able to directly associate with class II MHC molecules on the macrophage cell surface.

Without limiting the invention to a specific mechanism, two possible explanations can be invoked to explain the above results. First, it is possible that AGE-modification of ribonuclease confers partial resistance to proteolysis, and thereby allows the generation of more appropriate peptide fragments which are capable of binding to self-MHC. This may be analogous to the augmented antigen presentation in live macrophages seen when proteases nonessential for antigen presentation are inhibited. Secondly, AGE modification of ribonuclease confers increased stability for the AGE-peptide to bind to MHC. In order to demonstrate whether enhanced presentation results from increased association with MHC molecules, the core synthetic peptide which is recognized by the ribonuclease-specific hybridoma has been synthesized.

To examine whether AGE modification of antigen confers stability to proteolysis, the ability of AGE modification to protect against a protease which is known to extensively degrade antigen can be examined. If AGE modification confers stability against such a protease, an AGE antigen could tolerate a longer incubation with such a protease than could unmodified antigen, while still retaining its stimulatory properties. EXAMPLE 5

LATE GLYCOSYLATION PRODUCTS, AND NOT EARLY PRODUCTS, ARE RESPONSIBLE FOR ENHANCED PRESENTATION

Method

1) Antigens: Glucose modified ribonuclease and FFI-ribonuclease were prepared as described in Example 2. The Amadori product of RNase and glucose was reduced with sodium borohydride to form the corresponding alcohol.

2) The T-cell stimulation assay was performed as in Example 2. The results of the T-cell stimulation assay are shown below in Table III.

Results

Table III shows that reduction of glycosylated antigen with sodium borohydride does not affect the enhanced presentation of glucose-modified antigen. Since reduction eliminates the early glycosylation products, but does not affect the AGE product, these results suggest that the augmentation of presentation is due to the formation of later endproducts. In addition, the ability of the synthetic model AGE, FFI, to augment presentation further supports the role of the more advanced products in the enhancement of antigen presentation.

TABLE III

Antigen Concentration (ug/ml) 100 31 10

Antigen Control RNase 1,567 180 137 140

D-glucose RNase

(4 days) 38,947 15,480 2,993 220 Reduced D-glu

RNase 47,143 19,603 3,043 560

G6P RNase (4days) 5,593 1,070 187 117

Reduced G6P/RNase 3,100 557 170 110 FFI-RNase 49,250 130 100 120

EXAMPLE 6

OVER-MODIFICATION OF ANTIGEN LEADS TO DIMINISHED ENHANCEMENT OF PRESENTATION

Methods:

1) Antigens: AGE-ribonuclease was prepared as in Example 1 except that incubation with 0.5 M glucose and glucose-6-phosphate was extended to 6 weeks. In addition, ribonuclease was incubated for 4 days with various concentrations of D-glucose and glucose-6-phosphate, ranging in concentration from 0.01 to 0.5 M.

2) T-cell hybridoma stimulation was performed by the method described in Example 2. The results are shown below in Table IV. Results

Incubation of ribonuclease with 0.5 M glucose or glucose- 6-phosphate for six weeks did not lead to enhancement of antigen presentation when compared to ribonuclease that was incubated under identical conditions for only four days (See Table IV, results are taken from different experiments) .

In addition, ribonuclease that was incubated for 15 weeks with glucose-6-phosphate displayed less enhancement of antigen presentation then antigen incubated for the same length of time with D-glucose.

TABLE IV

To identify the optimal concentration of glucose with which to incubate antigen, RNase was incubated for 4 days with various concentrations of D-glucose or glucose-6- phosphate. The optimal concentration of D-glucose and glucose-6-phosphate for enhancement of presentation was between 0.01 and 0.1 M. (See Table V for results). This data provides further evidence that only minor AGE- modification of antigen is necessary to achieve optimal results. Finally, in contrast to earlier Examples, glucose-6-phosphate was inferior to D-glucose in augmenting antigen presentation.

TABLE V

Antigen cone. (ug/ml) 100 31 10

AGE-ANTIGEN IS ASSOCIATED WITH ENHANCED PRIMING OF LYMPH

NODE T-CELLS

Methods

1) T-cell proliferation assay: Priming of Balb/C mice with AGE-ribonuclease or control ribonuclease was accomplished by injecting various concentrations of antigen in Complete Freund's Adjuvant ("CFA") into the base of the tail. Eight days following priming, a single cell suspension of inguinal and periaortic lymph nodes was generated. 2 X 10⁵ cells from the single cell suspension were added together with ribonuclease to microtitre wells. Priming of T-cells was assessed by measuring ³H-thymidine incorporation 2-5 days following plating. (T-cells which have been primed in vivo will proliferate in vitro and will therefore incorporate ³H- thymidine.)

2) AGE-ribonuclease consisted of ribonuclease incubated for four days with 0.5 M D-glucose, and processed as previously described.

Results

Mice were successfully primed with AGE-ribonuclease using a 10-fold lower dose than that needed to prime with control ribonuclease as shown in FIGURE 3. The minimum dose of AGE-ribonuclease required to prime the mice was from about 0.1 to about 1.0 ug, while mice injected with control RNase required between 10 and 100 ug to elicit a maximal response. This data confirms and extends the in vitro findings, by demonstrating that an AGE-antigen is more efficient in activating T-cells in vivo when compared to control antigen.

EXAMPLE 8

ENHANCEMENT OF HUMORAL (ANTIBODY) RESPONSE

As an example of the AGE-modification increasing the immunogenicity of a protein, ribonuclease (RNase) was modified by AGEs (AGE-RNase) and injected into a rabbit to cause the production of antibodies. AGE-RNase was prepared by incubating RNase (25 mg/ml) with 1 M glucose in 0.2 M NaP0₄ buffer (pH 7.4) for 90 days.

Figure 4 shows a competitive ELISA with the resultant antiserum. For this assay, native RNase was immobilized onto 96 well microtitre plates. A defined amount of anti-AGE-RNase antiserum was added (50 ul final dilution 1/20,000) followed by the addition of dilutions of either AGE-RNase or unmodified RNase (denatured) . As shown, AGE-RNase was a more effective competitor than native RNase for the binding of antiserum to the immobilized. native RNase. Since this is a competitive assay between immobilized native RNase and added ligands (native RNase or AGE-RNase) , the antibody-AGE interaction is not operative in this system. This experiment shows that the AGE modi ication causes a change in the structure

(epitopes) of RNase, making it a better competitor than native RNase for the interaction of antiserum with immobilized native RNase.

ELISA METHOD

Ninety-six well microtitre plates (Nunc Immunoplate, Gibco, Grand Island, NY) were coated with AGE-BSA by adding 0.1 ml of a solution of AGE-BSA 10 g/ml, dissolved in PBS) to each well and incubating for 2 hr at room temperature. Wells were washed three times with 0.15 ml of a solution containing PBS, 0.05% Tween 20, and 1 mM NaN₃ (PBS-Tween) , Wells were blocked by incubation for 1 hr with 0.1 ml of a solution of PBS containing 2% goat serum, 0.1% BSA, and ImM NaN. After washing with PBS- Tween 50 ul of competing antigen was added, followed by 50 ul of antisera (final dilution, 1/1000) . Plates were incubated for 3 hr. at room temperature. Walls were then washed with PBS-Tween and developed with an alkaline phosphatase linked anti-rabbit IgG utilizing para- nitrophenylphosphate as the colorimetric substrate.

EXAMPLE 9 ENHANCEMENT AND INHIBITION OF CELL-MEDIATED (T-CELL) IMMUNITY

T-cell hybridomas bearing receptors specific for protein/peptide antigens presented in the context of specific major histocompatibility haplotype have been studied in model systems .in vitro. One such T-cell hybridoma, designated 22D.11 recognizes pigeon Cytochrome C when this protein is presented by antigen presenting cells bearing the I-E^k haplotype. T-cells stimulated in this fashion respond by secreting interleukin-2. This response may then be measured by adding the conditioned media (which contain the secreted interleukin-2) to an interleukin-2 growth dependent cell line (CTLL) and measuring H-thymidine incorporation by standard methods.

For this example Cytochrome C was modified by AGEs by incubation with two sugars: 0.2 M glucose for 4 days and 14 days and 0.2 M glucose-6-phosphate (a more reactive) for 4 and 14 days. Increasing amounts of native and AGE- modified Cytochrome C were incubated with 5xl0⁵ T-cells (22D.11) for 24 hr in 0.1 ml of culture media. At the end of this time 50 ul of media was removed and added to 200 ul of media containing 5 x 10³ CTLL cells per well. These were cultured for 24 hr. then pulsed with 1 uCi of ³H-thymidine per well and harvested 12 hours later for cpm determination.

Figure 5 shows that incubation with glucose for 4 days has a stimulatory effect on T-cells (particularly at high concentrations of antigen) which diminishes at 14 days (when more AGEs form) . Glucose-6-phosphate (G6P) , which forms AGEs more rapidly than glucose, has an inhibitory effect at 4 days. The inhibition increases at 14 days.

These data indicate that in a cell-mediated immune response assay system, AGEs affect the reactivity of antigens with T-cells. At low levels of AGE- modifications on protein, this effect can be stimulatory. At high levels of AGE modification this effect is inhibitory.

EXAMPLE 10

During the examination of the antigenicity of T-cells presented herein, larger consideration was given to the role that T-cells might play in the response to the development and presence of AGE-modified proteins in vivo. The comments and findings that follow are reflective of the discoveries that resulted from this work.

Tissue macrophages remove AGE modified proteins and secrete tissue turnover-regulatory cytokines via an AGE -receptor system. This system consists of two proteins (60kD and 90 kD) with unique N-terminal sequences. Reasoning that lymphocytes, important participants in tissue repair, also interact with tissue AGES, we searched for the expression of similar AGE-binding sites on human and rat T-lymphocytes.

Resting T-cells bind AGE-ligands with a Kd of 7.8 X 10^"°M, whereas, after stimulation with 0.01% PHA for 48 hr, specific binding increases to a Kd of 5.8 X 10^"°M. Immunohistochemical staining and FACS analysis of rat T- cells indicated that 38.2% of resting CD5+ T-cells expressed p60, while after stimulation with PHA, expression increased to 89.4% of T-cells. Similarly, expression of p60 on rat CD4+ and CD8+ T-cells was 34.5% and 58.5%, respectively, at rest and increased to 91.9% and 86.4% in each case, after stimulation. A smaller number of T-cells expressed p90 AGE-receptor with minimal response to PHA-stimulation. Exposure of PHA-stimulated T-cells to AGE-Human Serum Albumin

(AGE-HSA) (0.1-10/μg/ml) led to a marked dose dependent IFNγ mRNA expression and mature protein secretion, while no such response was noted in response to unmodified HSA. The data suggest that T-cells in cooperation with macrophages, may contribute to the regulation of normal tissue turnover through T-cell AGE-receptor mediated IFNγ production, known to exert direct anti-proliferative action, thus counteracting cytokine effects. This balance may be offset by excessive AGE accumulation, as in diabetic tissues, leading to damage. Accordingly, a method of recognizing, modulating and/or removing AGEs is included which comprises the stimulation or activiation of T-cells.

EXAMPLE 11

The polypeptide RNase was analyzed for purposes of determining which epitopes are recognized by antisera raised in one instance to native RNase and in the other instance to AGE-RNase. This experiment was undertaken to confirm that immune recognition and response to otherwise dominant epitopes can be suppressed and that immune recognition and response to otherwise silent or recessive epitopes can be enhanced by treating the polypeptide with a compound which forms AGEs. The polypeptide sequence corresponding to bovine RNase has been determined (124 residues) and was used for epitope mapping and analysis.

PROCEDURE

A comprehensive series of polypeptide fragments corresponding to the amino acid sequence for bovine RNase and containing ten amino acids per fragment were synthesized (with an offset of two amino acids) such that sequential fragments overlapped by eight amino acids. Hence, peptide number one contained amino acid nos. 1 through 10 of the bovine RNase sequence; peptide number two contained amino acid nos. 3 through 12, and so forth. Fifty eight peptides were constructed in this manner.

All peptides received N-terminal acetylation, were linked to molecular spacer arms and were covalently attached to pins held in an 8 x 12 microtitre plate format for purposes of conducting separate ELISAs using anti-RNase and anti-(AGE-RNase) antibodies against each peptide fragment. ELISA ASSAY

A typical solid phase ELISA procedure was employed, using two test antisera, the first with antibodies to RNase (designated as antiserum 11.3), and the second with antibodies to AGE-RNase (designated as antiserum 5.8). The test antibody preparations were diluted 1/10,000 in "supercocktail", (ovalbumin 10.Og and bovine serum albumin 10.Og are dissolved in phosphate buffered saline and Tween 20 is added) and the secondary antibody preparations (Swine anti-rabbit Ig HRP conjugate from Dakopatts) which served to detect bound primary or test antibodies were diluted 1/500 also in supercocktail. Peptides PLAQ and GLAQ were used as positive and negative controls, respectively, (Symbols used below are based upon the one letter designations for amino acids recited in J. Biol. Chem.. 243:3552-3559 (1969) incorporated herein by reference) .

Test antibody 11.3 was raised in rabbits using a hyperimmunization protocol with bovine RNase as the immunogen, and test antibody 5.8 was raised in rabbits using a hyperimmunization protocol using AGE-RNase as the immunogen. Each test antibody was reacted with the 58 peptide fragments and the reactivity of each antibody for each peptide was analyzed. The results are shown graphically in FIGURES 6 and 7. The comparison of individual test results is shown graphically in FIGURE 8.

DISCUSSION Antibody 11.3 demonstrated a relatively high background level of about 400 (OD 0.4) for the GLAQ (negative) control peptide in the immunoassay (ELISA) . The peptide number, sequence and ELISA OD are shown below in TABLE VI. The underlined sequences are common to each sequential peptide which gave a positive ELISA signal, and these represent the best estimate of the sequences which are recognized by the antisera, i.e., the functioning antigenic epitopes.

TABLE VI ANTIBODY 11.3

Peptide No. Sequence OD

26 27 28

45 46

54 55 56

61 62 63 64

Antibody 5.8 demonstrated a relatively low background level of about 100 (OD 0.096) for the GLAQ (negative) control peptide in the immunoassay (ELISA) . The peptide number, sequence and ELISA OD are shown below in TABLE VII. The underlined sequences are common to each sequential peptide which gave a positive ELISA signal, and these represent the best estimate of the sequences which are recognized by the antisera, ie, the functioning antigenic epitopes.

TABLE VII

39 CKNGQTNCYQ 0.125

56 TTOANKHIIV 0.323

57 OANKHIIVAC 0.158

62 EGNPYVPVHF 0.493

As shown in FIGURES 6 and 7, numerous differences between the speci icity and reactivity of the antisera for the peptide fragments were noted, which are attributed to the presence (or absence) of AGEs on the immunogen. The immune response to some epitopes which were non-reactive and did not elicit an antibody response when native RNase was supplied as the immunogen was modulated, and antibodies were formed against a different spectrum of native epitopes when AGE-RNase served as the immunogen. For example, at peptide no. 27, antibodies raised to RNase provide an OD of 1.001, about 3 - 4 times the background level of reactivity. In contrast, the AGE- RNase antibodies provide an OD of 0.02, about 5 times lower than the background OD, demonstrating suppression of this particular epitope upon reaction of RNase with AGEs. Conversely, antibodies raised to AGE-RNase represented by peptides 29 - 31 recognize an epitope which was essentially non-recognized by antibodies raised to RNase in non-modified form.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims (26)

WHAT IS CLAIMED IS:

1. A method of inducing a modified immune response to an antigen comprising reacting the antigen with an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts in an amount sufficient to induce a modified immune response to said antigen.

2. A method in accordance with Claim 1 wherein the reaction increases the antigenicity of the epitope region on the antigen.

3. A method in accordance with Claim 1 wherein the reaction reduces the antigenicity of the epitope region on the antigen.

4. A method in accordance with Claim 1 further comprising modifying the antigenicity of the antigen to include a moiety capable of reacting to form an AGE on the antigen.

5. A method in accordance with Claim 4 wherein the moiety capable of reacting to form an AGE on the antigen is at least one member selected from the group consisting of lysine, arginine, histidine and cysteine.

6. A method of modifying an immune response to a polypeptide comprising modifying an expression sequence which codes for the polypeptide to include at least one member selected from the group recited in Claim 5 in the polypeptide upon expression; expressing the modified polypeptide, and reacting the modified polypeptide with an AGE or a compound which forms AGEs in an amount effective for inducing a modified immune response to said polypeptide.

7. An antibody which recognizes an epitope on an antigenic molecule, said antigenic molecule having been reacted with an advanced glycosylation end product or a compound which forms advanced glycosylation end products in an amount effective for modifying the antigenicity of the antigen, said epitope being non-recognizable by the antibody in the absence of such reaction.

8. An antibody in accordance with Claim 7 which is polyclonal, monoclonal or chimeric.

9. An immunogen comprised of an antigenic molecule having at least one epitope and having an advanced glycosylation endproduct reacted with or covalently linked to said molecule, said immunogen having modified antigenicity upon reaction or linkage to said advanced glycosylation endproduct.

10. A method of rendering an epitope on an antigen recognizable by the immune system, which epitope does not substantially induce an immune response under normal conditions, comprising: reacting the antigen molecule with an AGE or a compound which forms AGEs to form an antigen-AGE reaction product, and combining the antigen-AGE reaction product with an antigen presenting cell having MHC on the cell surface.

11. An antigen presentation complex comprised of: (a) an antigen presenting cell having major histocompatibility complex on the cell surface, and (b) an antigen which is comprised of an epitope presented in the context of MHC on the antigen presenting cell and having AGEs or a compound which forms AGEs bound to or reacted with an epitope or a site other than the epitope.

12. An immunogen comprised of an AGE reaction product bound to an antigen, and said antigen-AGE reaction product having an epitope which is recognized by an antibody defined in accordance with Claims 7 or 8.

13. The immunogen of Claim 12 wherein the epitope is recognizable upon reaction between an antigen presenting cell, MHC and said antigen-AGE reaction product.

14. A method of determining T-lymphocyte levels, function or activity in a sample taken from a mammal comprising: (a) incubating antigen presenting cells with an antigen selected from a molecule and the reaction product of an AGE or a compound which forms AGEs and said molecule; (b) combining said antigen presenting cells and antigen with T-lymphocytes taken from said mammal; and (c) comparing the level, function or activity of said T-lymphocytes to a standard.

15. A method of producing T-lymphocytes which recognize an AGE-modified antigen comprising: administering to a mammal a T-lymphocyte priming effective amount of said AGE-modified antigen, and harvesting said primed T-lymphocytes.

16. A pharmaceutical composition comprised of an immunogen comprised of an antigen molecule treated with an advanced glycosylation end product or a compound which forms advanced gylcosylation end products in an amount effective for modifying the antigenicity of said antigen, in combination with a pharmaceutically acceptable carrier.

17. A pharmaceutical composition comprised of antibodies as described in Claim 7 in combination with a pharmaceutically acceptable carrier.

18. A biological composition comprised of T-lymphocytes which are primed to recognize an AGE-modified antigen in accordance with Claim 15.

19. A method of treating or preventing an infectious disease, an autoimmune disease or cancer in a mammalian patient in need of such treatment or prevention, comprising administering to said patient an effective amount of an immunogen comprised of an antigen molecule reacted with an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts in an amount effective for modifying the immune response to said antigen, said immunogen being administered in an amount effective for treating or preventing said infectious disease, autoimmune disease or cancer.

20. A method of treating or preventing an infectious disease, an autoimmune disease or cancer in a mammalian patient in need of such treatment or prevention, comprising administering to said patient an effective amount of antibodies which recognize an an antigen reacted with an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts in an amount effective for modifying the immune response to the antigen, said antibodies being administered in an amount effective for treating or preventing said infectious disease, autoimmune disease or cancer.

21. A method of treating or preventing an infectious disease, an autoimmune disease or cancer in a mammalian patient in need of such treatment or prevention, ^βi comprising administering to said patient an effective amount of T-lymphocytes raised to recognize an antigen, or antibodies which recognize said antigen, said antigen being reacted with an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts in an amount effective for modifying the immune response to the antigen, said T-lymphocytes being administered in an amount effective for treating or preventing said infectious disease, autoimmune disease or cancer.

22. A method of preparing an immunogen which exhibits modified immunogenicity, comprising combining an antigen with an amount of an advanced glycosylation endproduct or a compound which forms advanced glycosylation endproducts which is effective for causing said antigen to exhibit said modified immunogenicity.

23. A method of treating a tumor in a mammal in need of such treatment, comprising administering to the mammal an amount of an AGE-modified protein sufficient to cause macrophages and/or T-lymphocytes to secrete tumor- regulating cytokines.

24. A method in accordance with Claim 23 further comprising administering to said mammal an effective amount of T-lymphocytes which have been treated with a compound which increases the specific binding of T- lymphocytes to AGE-ligands.

25. A method in accordance with Claim 23, wherein the T- lymphocytes are administered in an amount sufficient to secrete a quantity of IFN sufficient to modulate tumor growth.

26. A method of treating a mammal to remove advanced glycosylation endproducts present in a mammal, comprising combining T-lymphocytes with a compound which enhances the binding capacity of said T-lymphocytes to AGE- ligands, and administering the T-lymphocytes to said mammal.