United States Patent:  6,048,529

Appl. No.:  414174

The invention relates to a procedure for synthesis of well-defined conjugates of peptides to the tolerogenic polymer monomethoxypolyethylene glycol (mPEG) or polyvinyl alcohol (PVA). This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is specifically coupled to one or the other or both of the termini of an otherwise unaltered peptide molecule. A synthetic peptide synthesized using this method and corresponding to a myasthenogenic region of an acetylcholine receptor was conjugated to monomethoxypolyethylene glycol. Injection of animals with the mPEG-conjugate and subsequent immunization with whole receptor suppressed the development of experimental autoimmune myasthenia gravis (EAMG) by electrophysiological criteria. Specifically-conjugated, tolerogenic peptides are also disclosed for diseases as diverse as ragweed pollen allergy and Grave's disease.

SUMMARY OF THE INVENTION

The present invention overcomes at least some of the problems existing in prior art approaches to the construction of reagents for the treatment of autoimmune diseases. In one aspect, the invention broadly discloses a synthetic method for construction of specifically-modified peptides covalently attached to a polymer which renders the synthetic peptide tolerogenic. In another aspect, the invention broadly discloses the use of these specifically-modified synthetic peptides in the treatment of diseases of autoimmunity and other unwanted responses such as allergic reactions and graft rejections. In yet another aspect, the invention provides for the reagents designed to immunosuppress undesirable immune responses. The invention also provides a method of testing such reagents for efficacy as immunosuppressants.

More specifically, a method of producing reagents useful in the treatment of autoimmune diseases is disclosed herein. In certain preferred aspects, the method for producing such reagents entails producing a peptide covalently linked via its carboxy-terminal amino acid to a synthetic resin. It will be understood well by those of skill in the art, however, that due to its ease, coupling of the carboxy terminus to the synthetic resin is only one manner in which to provide a single free, amino terminus for subsequent derivatization. However, the same skilled artisan will also realize that it is possible to use alternative protocols to specifically block the amino terminus and to derivatize the carboxy terminus of such a peptide. Therefore while the preferred technique will involve a carboxy terminus attached to a synthetic resin and a free amino terminus, derivatization of either or both termini is anticipated by the inventors to give equally efficient tolerogenic peptides.

Any of the synthetic resins known to those of skill in the art will be amenable to the methodology. For instance, one may use synthetic methods based on either t-butyloxycarbonyl (t-Boc) derivatized amino acids synthesized on a phenylacetamidomethyl (PAM) resin or by 9-fluorenmethylcarbonyl (Fmoc) derivatized amino acids on a benzyloxybenzyl alcohol resin (McCormick and Atassi 1984; Mulac-Jericevic and Atassi, 1987; Atassi et al., 1991).

The peptides of the invention will typically be protected from inadvertent coupling along the side chains by the presence of side chain-protected amino acids in the peptide. It will be well understood by those of skill in the art that such side chain protecting groups can vary depending upon the nature of the synthetic procedure.

In the preferred embodiment, the peptide may be synthesized beginning with any sized initial peptide fragment attached by its carboxy terminus to the resin. Thus, it will be understood by those of skill in the art that one may obtain presynthesized and derivatized peptides of variable lengths. Alternatively, one may obtain from any number of commercial sources synthetic resins which have one or more derivatized amino acids coupled to the resin by its carboxy terminus. The invention, therefore, is not limited to the use of wholly synthetic peptides and may include peptide fragments derived from native antigens themselves or from antigens obtained using recombinant DNA technology so long as these peptides may be protected along their side chains and covalently bound to a resin at their carboxy or amino terminus.

The peptides produced by the methods of the invention will typically correspond to an epitope which is suspected of inducing an autoimmune response or other undesired responses such as allergic conditions or graft rejections. Such an epitope may be suspected for any number of reasons. There may be empirical data which indicate a specific and relatively restricted epitope as a linear sequence found as an identical sequence in the native antigen known to cause the immune response of the disease. Alternatively, such an epitope may be a non-linear sequence corresponding to an antigenic region of a native antigen but which linear sequence does not exist as such in the native antigen.

Moreover, the peptides produced by the invention may be suspected as epitopes due to a regional localization to a region known to contain the minimally-sized epitope inducing the maximal antigenic response in the immune disease. For instance, it is known by those of skill in the art that many cell membrane-associated antigens chiefly present the extracellular portions of the polypeptide as potential epitopes. Thus, the epitope suspected of inducing the immune response may only be suspected as a battery of potential epitopes which are typically presented in the physiological state. In some cases, therefore, one may wish to test a battery of overlapping peptides representing sequential segments of the exposed extracellular regions of a given native antigen.

The peptide so selected and/or synthesized is attached to a resin by one of its termini, preferably by its carboxy-terminus, and is then derivatized at its other, preferably amino, terminal amino acid with a tolerogenic polymer. Since all side chains will still be protected as they were during the synthetic procedure, and since one terminus is likewise protected by coupling to the resin, the only reactive group will occur at the other terminus, preferably at the growing N-terminal amino acid as the .alpha.-NH₂ of that terminal residue. It is to this terminus that the tolerogenic polymer is attached.

The methods of the invention complete the synthesis of the terminally protected, tolerogenic peptides by deprotecting the side chain-protected amino acids comprising the peptide. Depending on the nature of the synthetic chemistry used to construct the peptide, deprotection will be achieved variously by methods known well to those of skill in the art. Similarly, depending upon the resin used to initiate synthesis, cleaving the peptide from the resin will take various forms. Purification of the peptide will also take various forms depending upon the nature of the resulting peptide. In some cases, more hydrophilic peptides may be amenable to purification schemes depending upon the solubility of the peptide in water based solvents. More hydrophobic peptides may require organic solvents and purification schemes in which the peptides will be most soluble.

Even though methods of the invention relate to any epitope-specific tolerogenic peptide used to construct reagents capable of treating immune diseases, the invention relates more specifically to certain characterized peptide reagents. Thus, the invention discloses the specific construction of any of the peptides shown in Sequence ID Nos. 1-7. Certain of these specific peptide reagents relate to specific immune diseases such as myasthenia gravis, ragweed pollen allergy, and Grave's disease. Moreover, the methods of the invention relate to specific native polypeptides such as a subunit of an acetylcholine receptor, ragweed pollen antigen Ra3, or a polypeptide subunit of the thyroid-stimulating hormone receptor responsible for Grave's disease.

It is preferred that the peptide reagent designed will be directly or indirectly responsible for the major immune response as the principal causative agent of symptoms of the immune disease. However, there may be instances where peptides corresponding to regions of the native antigen responsible for lesser immune responses will be desired. In particular, combinations of reagents, each of which accounts in part for the immune response, may be preferred in certain instances.

The methods of the invention require the covalent coupling of a tolerogenic polymer to the peptide reagents. Such tolerogenic polymers are known well to those of skill in the art. For instance, such a polymer may be polyethylene glycol or a polyethylene glycol derivative. In a preferred embodiment of the invention, the monomethoxy derivative of polyethylene glycol will be used. Alternatively, polyvinyl alcohol or a derivative of polyvinyl alcohol may be used.

In any instance, the basic polymer selected will be treated in a manner as to make the polymer amenable to a coupling reaction. In a preferred embodiment, the method used to derivatize the polymer will involve succinylation of the polymer so as to derivatize the hydroxyl groups of the polymer and to generate any number of reactive carboxyl groups. Complete derivatization is monitored as is availability of the reactive carboxy groups on the surface of the modified polymer.

In another principal aspect of the invention, a method of treating an autoimmune disease is disclosed. The method consists of first producing a tolerogenic polymer-derivatized peptide as described above. The peptide reagent so produced will typically correspond to an epitope which is suspected of inducing an autoimmune response of the disease. After the peptide is so produced, the method treats a patient with the peptide. The patient may be one who has the disease. Alternatively, the patient may be one who is likely to develop the autoimmune disease. For instance, certain autoimmune diseases have long non-symptomatic episodes in which major immune responses are not present. In the autoimmune disease myasthenia gravis, patients typically experience sometimes very lengthy non-symptomatic periods followed by periods of almost complete debilitation due to the ongoing immune response. The patient is thus treated at an optimal time with tolerogenic peptide, preferably prior to onset of a major autoimmune response to the natural antigen from which the epitope was designed.

Reagents useful in the treatment of an autoimmune disease are also disclosed in the present invention. Generally, such a reagent will be a peptide corresponding to an epitope which is suspected of inducing an autoimmune response which peptide is derivatized at an N-terminal amino acid of the peptide with a tolerogenic polymer.

More specifically, the reagent will be one of the group of peptides disclosed in Sequence ID Nos. 1-7. It will be recognized, however, by those of skill in the art that the reagent peptides may contain functionally equivalent amino acid substitutions. The importance of the hydropathic index of amino acids in conferring biological function on a protein has been discussed generally by Kyte and Doolittle (1982). It has been found by these researchers and others that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain similar if not identical biological activity. As displayed in Table I below, amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with the substrate molecule. Similarly, in peptides whose secondary structure is not a principal aspect of the interaction of the peptide, position within the peptide and the characteristic of the amino acid residue determine the interactions the peptide has in a biological system. It is proposed that biological functional equivalence may typically be maintained where amino acids having no more than a +/-1 to 2 difference in the index value, and more preferably within a +/-1 difference, are exchanged.

Claim 1 of 21 Claims

1. A method of producing a peptide reagent useful in the treatment of myasthenia gravis, wherein said peptide reagent is a tolerogenic polymer-linked peptide the peptide of which is known to or suspected of inducing an immune response responsible for symptoms of myasthenia gravis, the method comprising:

conjugating a resin-linked peptide with a tolerogenic polymer having a single reactive end group available for said conjugation, the peptide of said resin-linked peptide having side chain-protected amino acids, and being covalently linked to said resin via a first terminal amino acid of said peptide, and having a second terminal amino acid opposite said first terminal amino acid which is available for said conjugation with said tolerogenic polymer;

deprotecting said side chain-protected amino acids and

cleaving said peptide from said resin to provide a single molecular species of peptide-tolerogenic polymer conjugate;

recovering said peptide-tolerogenic polymer conjugate; and,

purifying said peptide-tolerogenic polymer conjugate to provide said peptide reagent.

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