WO2006020541A2 - Methods for synthesizing polypeptides - Google Patents

Abstract

The present invention provides methods for synthesizing peptides or proteins, products produced thereby, and methods for use of those products.

Description

METHODS FOR SYNTHESIZING POLYPEPTIDES

Related Application Information

This application claims the benefit of United States Provisional Application No. 60/601 ,063; filed August 12, 2005, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention relates to methods of synthesizing polypeptides including polypeptides for use as immunogens.

Background of the Invention

In a conventional peptide synthesis process, a solid support having a chemical linker is placed into a peptide synthesizer. Peptides are then synthesized on the chemical linker one amino acid at a time from the carboxyl terminus of the peptide towards the amino terminus. Peptide vaccines are typically made by chemically conjugating a peptide to a carrier molecule (e.g., keyhole limpet hemocyanin) and the complex is injected. This process requires synthesis and purification of the peptide, activation of carrier (if the linkage is via cystine), reaction of peptide with carrier, dialysis to remove chemical reactants, and lyophilization of the complex. Small peptide antigens are generally poor immunogens, and immunization with the larger complex enhances immune response. In methods of synthesizing antigens for use as vaccines, a cluster of two, four, eight or more identical peptides (peptide clusters) are generally synthesized on a solid support using a conventional peptide synthesis process as described above. In order to be active as an antigen, the synthesized peptide is generally large enough to comprise a minimum of two domains that allow interaction with both T-cells and B-cells and is typically between 15 and 20 amino acids in length. For purposes of immunization, peptide clusters can be used directly. Alternatively, peptide monomers are chemically conjugated to a carrier protein prior to use for immunization. Conventional polypeptide synthetic methods have the disadvantage of requiring chemical conjugation of the polypeptide to the carrier, which limits the choice of carrier and may require additional steps to cleave the polypeptide from the carrier. Accordingly, there is a need in the art for improved methods of synthesizing polypeptides.

Summary of the Invention

The present invention provides a faster and cleaner method of synthesizing polypeptides in which the polypeptide is synthesized directly on the carrier without the need for chemical conjugation through a chemical linker. Further, the methods of the invention allow the peptide to be directly synthesized on any carrier of choice, allowing one to design carriers rather than rely on the large commercially available molecules that are traditionally used (e.g., keyhole limpet hemocyanin for synthesis of peptide immunogens). As other advantages, the inventive methods facilitate the synthesis of multiple copies of the polypeptide of interest simultaneously on the carrier and the end-product will be cleaner and better characterized (e.g., in terms of how many epitopes have been synthesized and their location) than the product of a method requiring chemical conjugation to a large carrier molecule.

Accordingly, as one aspect, the invention provides a method for synthesizing a polypeptide, comprising: immobilizing a first polypeptide on a substrate; and synthesizing a second polypeptide on the first polypeptide.

As a further aspect the invention provides a method for synthesizing a peptide complex, comprising: immobilizing a first polypeptide on a substrate; and synthesizing a second polypeptide on the first polypeptide to produce a peptide complex.

As yet another aspect, the invention provides a method for synthesizing a peptide immunogen/substrate complex, comprising: immobilizing a polypeptide comprising a T cell epitope on a substrate to provide a T cell epitope/substrate complex; and synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope to provide a peptide immunogen/substrate complex.

The invention further provides a method for synthesizing a peptide immunogen/substrate complex, comprising: immobilizing a polypeptide comprising a B cell epitope on a substrate to provide a B cell epitope/substrate complex; and synthesizing a polypeptide comprising a T cell epitope on the polypeptide comprising the B cell epitope to provide a peptide immunogen/substrate complex.

As still another aspect, the invention provides a method for synthesizing a peptide immunogen, comprising: covalently linking a polypeptide comprising a T cell epitope to a substrate to provide a T cell epitope/substrate complex having a covalent linkage; synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope of the T cell epitope/substrate complex to provide a peptide immunogen/substrate complex; and cleaving the covalent linkage between the polypeptide comprising the T cell epitope and the substrate to form a peptide immunogen.

Also provided are polypeptides, peptide complexes, peptide immunogen/substrate complexes and peptide immunogens produced by the methods described above. Further provided are methods of inducing synthesis of an antibody, methods for immunizing an animal subject and methods for desensitizing an animal subject using the polypeptides, peptide complexes, peptide immunogen/substrate complexes and peptide immunogens produced by the methods of the invention. These and other aspects of the invention are set forth in more detail in the following description of the invention.

Detailed Description of the Invention

The present invention relates generally to methods for synthesizing peptides or proteins, products thereof, and methods for use of those products. Methods according to embodiments of the present invention can be particularly suitable for synthesizing peptide immunogens (e.g., for use as a vaccine). By synthesizing peptides and proteins directly on a carrier molecule, the methods of the invention are more efficient and can produce a cleaner end-product than conventional polypeptide synthetic methods. The inventive methods can also allow for the use of a wider array of carrier molecules. Definitions: The terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, the following terms have the meanings indicated: The term 'peptide' refers to a compound formed of 2 to 40 amino acids joined by peptide bonds. The term 'protein' refers to a naturally occurring or recombinant molecule composed of greater than 40 amino acids. The term 'polypeptide' encompasses both peptides and proteins.

The term 'peptide complex¹ refers to two or more polypeptides covalently bound together and includes 'peptide immunogens'. A 'peptide immunogen' comprises one or more epitopes. The peptide complex or peptide immunogen can further be reversibly or irreversibly associated with a substrate to form a 'peptide substrate/complex' or a 'peptide immunogen/substrate complex'. In some embodiments, the first polypeptide is referred to herein as a 'carrier.'

As used herein, the term 'binding domain' refers to an amino acid sequence that is capable of binding to a biological molecule, such as but not limited to a specific antibody (B-cell receptor), class I and class Il major histocompatibility antigens, or a specific T-cell receptor. The term 'epitope' refers to an antigenic site on a polypeptide or a peptide complex against which an antibody or T cell receptor is directed and to which it binds. Those skilled in the art will appreciate that the epitope can be isolated, i.e., it does not have to be present in the native intact peptide(s) or protein(s). In some embodiments, the binding domain is an epitope, such as a B cell epitope or a T cell epitope.

The term 'immunogen¹ refers to an antigen or substance that induces an immune response upon exposure to a subject. The term 'antibody' refers to an immunoglobulin molecule that has a specific amino acid sequence by virtue of which it interacts only with the antigen that induced its synthesis (or mimetics thereof). The term 'allergen' includes naturally-occurring or modified allergens and refers to antigens that can induce immediate and/or delayed types of hypersensitivity. 'Allergenic properties' refer to the ability to induce immediate and/or delayed types of hypersensitivity. The term 'modified allergen' refers to an allergen that has been modified but still retains its allergenic properties although they can be less potent. The term 'adjuvant' refers to any immunomodulating substance that can be combined with peptide immunogens or vaccines to enhance or otherwise modulate an immune response in a subject without deleterious effect on the subject.

As used herein, the term 'mimetic' refers to a synthetic peptide with the same or different amino acid sequence as an epitope, but which is recognized by antibodies or T cell receptors against the defined epitope. The term 'resistance-building' includes inducing an initial resistance as well as enhancing existing resistance.

The term 'sorption' as used herein encompasses absorption, adsorption, and ion exchange.

'Effective amount¹ refers to an amount of a compound or composition that is sufficient to produce a desired effect, which can be a therapeutic effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular biologically active agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an effective amount in any individual case can be determined by one of ordinary skill in the art by reference to pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science And Practice of Pharmacy (20th ed. 2000)). Treat¹ or 'treating' refers to any type of treatment that imparts a modulating effect, which, for example, can be a beneficial effect to a subject afflicted with a disorder, disease or illness, including improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the condition, and/or prevention or delay of the onset of the disorder, change in clinical parameters, disease or illness, and the like. A 'pharmaceutically acceptable' component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the polypeptides and/or peptide complexes of the present invention without rendering them unsuitable for their intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are 'undue¹ when their risk outweighs the benefit provided by the pharmaceutical composition. Non-limiting examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions, adjuvants approved for animal or human use, and various types of wetting agents.

The present invention will now be described in detail including descriptions of particular embodiments. This invention can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Variations on the disclosed general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.

Methods of Synthesizing Peptide Complexes According to representative embodiments of the invention, a method for synthesizing a peptide complex is provided. The method comprises immobilizing a first polypeptide (which is considered to be a carrier) on a substrate and directly synthesizing a second polypeptide on the first polypeptide to produce the peptide complex. The first polypeptide can be immobilized on the substrate to form a first polypeptide/substrate complex by various processes as are known in the art. To illustrate, in some embodiments, the first polypeptide is immobilized on the substrate by sorption. In other embodiments, the first polypeptide is immobilized on the substrate by chemically conjugating the first polypeptide to the substrate using methods known in the art.

The second polypeptide can be synthesized on the first polypeptide by various processes as will be understood by those skilled in the art. For example, the second polypeptide can be synthesized on the first polypeptide by placing the first polypeptide/substrate complex into a peptide synthesizer such as the Rainin

Symphony multiple peptide synthesizer, which is commercially available from Protein Technologies, Inc., Tucson, AZ, and synthesizing (building) the second polypeptide onto the first polypeptide portion of the first peptide/substrate complex using standard peptide synthesis procedures.

The substrate can be any suitable substrate to which a first polypeptide can be immobilized as will be understood by those skilled in the art. For example, the substrate can be a polymeric substrate including, but not limited to, reverse phase and/or ion exchange chromatography resins, PVDF (polyvinyldifluoro) membrane materials, and beaded materials including, but not limited to porous glass, and dextran, agarose, acrylamide, methacrylate, and polystyrene formulations. The substrate can be of various shapes and sizes including, but not limited to, membranes and particles. When the substrate is in particle form, the particles can be between about 3 and about 100 microns in size. In one embodiment, where the substrate is in membrane form, the membrane is PVDF with pore sizes between about 0.2 microns and about 0.45 microns. An affinity resin in which a ligand is covalently attached to a solid or a gel can also be used as a substrate. In various embodiments, the first polypeptide and the second polypeptide are individually a protein or a peptide including, but not limited to, polypeptides comprising T cell epitopes, polypeptides comprising B cell epitopes, synthetic and/or recombinant polypeptides, or naturally occurring and/or recombinant proteins or subunits of proteins. In various embodiments, the first polypeptide is a protein or a peptide. In other representative embodiments, the second polypeptide is a protein or a peptide. In some embodiments, the first polypeptide is a protein and the second polypeptide is a peptide. In other embodiments, the first polypeptide is a protein and the second polypeptide is a protein. In yet other embodiments, the first polypeptide is a peptide and the second polypeptide is a peptide. Polypeptides of any size can be synthesized by the processes of this invention. The peptide complex, the first polypeptide, and the second polypeptide can each comprise various numbers of amino acids. Generally, however, the first polypeptide is larger than (i.e., comprises more amino acids than) the second polypeptide. In representative embodiments, the first polypeptide comprises, consists of or consists essentially of from about 2 to about 2,000 amino acids. In other embodiments, the first polypeptide consists of about 2 to about 1 ,000 amino acids, from about 10 to about 500 amino acids, or from about 20 to about 200 amino acids. In some embodiments, the first polypeptide comprises a number of amino acids ranging from a lower limit of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids, to an upper limit of about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27,

28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 300, 400 or 500 amino acids, in any combination of ranges satisfying the condition that the lower limit is less than the upper limit (lower limit < upper limit).

In other illustrative embodiments, the second polypeptide comprises, consists of, or consists essentially of from about 2 to about 500 amino acids. In some embodiments, the second polypeptide consists of from about 2 to about 100 amino acids. In other particular embodiments, the second polypeptides consists of a number of amino acids ranging from a lower limit of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 21 amino acids to an upper limit of about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28,

29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 or 75 amino acids, in any combination of ranges satisfying the condition that the lower limit is less than the upper limit (lower limit < upper limit). In some embodiments, the second polypeptide consists of from about 3 to about 20 amino acids.

Some embodiments of the invention provide a method for synthesizing small polypeptides, such as those consisting of 35 or fewer amino acids. For example, in some embodiments, the second polypeptide consists of 35 or fewer amino acids.

According to some embodiments of the present invention, the peptide complex comprises one or more binding domains, such as but not limited to binding domains that specifically bind to an antibody (B-cell receptor), class I and class Il major histocompatibility antigens, and/or a specific T-cell receptor. The binding domains are located within the first polypeptide and/or the second polypeptide.

These binding domains can be the same or different within the peptide complex as a whole or within the polypeptides individually. In one illustrative embodiment, a process for synthesizing a polypeptide having at least two binding domains includes immobilizing a first polypeptide having a first binding domain on a substrate and synthesizing a second polypeptide having a second binding domain on the first polypeptide to provide the peptide complex. In some embodiments, the first polypeptide has a first binding domain and can have multiple repeating or unique binding domains. For example, the first polypeptide can have 2, 3, 4, 5 or 6 repeating or unique binding domains. In some embodiments, the second polypeptide has a second binding domain and can have multiple repeating or unique binding domains. For example, the second polypeptide can have 2, 3, 4, 5 or 6 repeating or unique binding domains. In some embodiments, the second polypeptide has a second binding domain that can be the same as or different from a first binding domain of the first polypeptide. In one embodiment, the second binding domain of the second polypeptide is different from a first binding domain of the first polypeptide.

In exemplary embodiments, the binding domain is an epitope, such as a B cell epitope or a T cell epitope. In some embodiments, the peptide complex comprises one or more epitopes. These epitopes can be, but are not limited to, T cell epitopes and/or B cell epitopes. In some embodiments, the first polypeptide comprises one or more epitopes. In some embodiments, the second polypeptide comprises one or more epitopes. The epitope(s) of the first and second polypeptide can each be a B cell epitope(s) and/or T cell epitope(s). Any combination of B cell epitope(s) and/or T cell epitope(s) is possible within the first and second polypeptides individually or within the peptide complex as a whole. In one embodiment, the first polypeptide comprises a T cell epitope and the second polypeptide comprises a B cell epitope. In a further embodiment, the first polypeptide comprises a B cell epitope and the second polypeptide comprises a T cell epitope. Some examples of epitopes suitable for this invention include, but are not limited to, epitopes from allergens, viruses, and bacteria.

As another nonlimiting example, the first polypeptide may comprise horse myoglobin and/or bovine serum albumin or an epitope that is useful for a booster immunization (e.g., a measles, influenza or polio epitope), and the second polypeptide may comprise one or more measles epitopes, influenza epitopes, grass epitopes, such as ragweed epitope, or any combination thereof. Other suitable epitopes are described in more detail below. In some embodiments of the invention, the peptide complex is an immunogen or allergen. In other embodiments, the peptide complex is used in an immunogenic composition, a vaccine, or a vaccine and booster combination. The first and second polypeptides can each individually be immunogens as well, such as when the peptide complex provides a vaccine and booster combination. In some embodiments, the peptide complex is an allergen, such as an allergenic protein or peptide from ragweed. Such allergenic proteins and peptides have been isolated and identified and shown to be useful for desensitizing people who suffer from ragweed allergies. See US Patent No. 5,776,761; US Patent No. 5,698,204; US Patent No. 6,335,019; and US Patent No. 6,335,020 to Rogers et al., which are incorporated herein by reference in their entireties.

In some embodiments, the first polypeptide has a built-in linker, for example, the epsilon amino group of lysine, from which the second polypeptide is built. In other embodiments, the first polypeptide is any suitable carrier as is known in the art, which has been modified by adding lysine to incorporate built-in linkers. For example, an ion exchange resin and carrier are incubated for four hours in dimethylformamide (DMF). The first polypeptide can have many points of attachment (e.g., lysines) and therefore many copies of the second polypeptide are formed during the synthesis. The first polypeptide can be cleaved in vitro or in vivo to form two or more fragments, each comprising a portion of the first polypeptide and a complete or fragmented second polypeptide. In representative embodiments, each portion of the first polypeptide comprises an epitope. In some embodiments, the epitope is a T cell epitope(s) and/or a B cell epitope(s). In some embodiments, the second polypeptide comprises an epitope. In some embodiments, the epitope is a T cell epitope(s) and/or a B cell epitope(s). In further embodiments, both the portion of the first polypeptide and the complete or fragmented second polypeptide comprise an epitope. In one embodiment, the portion of the first polypeptide comprises a T cell epitope(s) and the complete or fragmented second polypeptide comprises a B cell epitope(s).

Irreversible substrates: In some embodiments, a first polypeptide is irreversibly immobilized on the substrate. In those embodiments, the substrate is a substrate to which the first polypeptide can be irreversibly bound such as, but not limited to PVDF membranes and/or resins, porous glass, and/or beaded materials as described above. In some embodiments, the substrate is a polymeric substrate, such as PVDF membrane, porous glass beads, dextran, agarose, methacrylate, and polystyrene resin. In some embodiments, the substrate is Amberchrom™. In some embodiments, the substrate is an affinity resin. In one example, incubation of ion exchange resin (substrate) and the first polypeptide (or carrier) in dimethylformamide (DMF) results in irreversible binding of the first polypeptide to the resin.

Reversible substrates: In some embodiments, the first polypeptide is reversibly immobilized on the substrate. In those embodiments, the substrate is a substrate to which the first polypeptide can be reversibly bound, such as, but not limited to a reverse phase resin, ion exchange resin, or porous glass or beaded materials as described above. Some reverse phase and ion exchange resins are commercially available from TOSOH BIOSCIENCE, Montgomeryville, PA. In some embodiments, the substrate is Amberchrom™. In some embodiments, the substrate is an affinity resin.

In some embodiments, the method of synthesizing a peptide complex further comprises releasing the first polypeptide from the substrate. The polypeptide can be released from the substrate by various means as will be understood by those skilled in the art including, but not limited to, contacting the peptide complex with a solution capable of cleaving the bond between the first polypeptide and the substrate without harming the structure of the peptide complex. In some embodiments, the releasing of the first polypeptide from the substrate comprises contacting the peptide complex with a solution comprising acetonitrile and trifluoroacetic acid. In one embodiment, the solution of acetonitrile/dilute trifluoroacetic acid comprises between 50 and 75 weight percent acetonitrile and a 0.1% aqueous solution of trifluoroacetic acid. Other mechanisms for releasing the first polypeptide from the substrate include, but are not limited to treatment with strong organic acid or weak aqueous acid, weak aqueous base, or a specific enzyme depending upon the linkage of the polypeptide to the substrate, as will be understood by those skilled in the art. Methods of Synthesizing lmmunogens

According to still other embodiments of the present invention, a process for synthesizing a peptide immunogen/substrate complex includes irreversibly binding a polypeptide comprising a T-cell epitope to a substrate to provide a T-cell epitope/substrate complex, and building a polypeptide comprising a B-cell epitope on the T-cell epitope/substrate complex to provide a peptide immunogen/substrate complex. The substrate can be a polymeric material such as, but not limited to PVDF membrane, porous glass bead, or dextran, agarose, methacrylate or polystyrene reverse phase and/or ion exchange resins as described above. The peptide immunogen/substrate complexes provided by this embodiment are suitable for human or veterinary use, particularly when the polymeric material is suitable for use in humans. Polymeric materials that can be suitable for use in humans are known to those skilled in the art and can include methacrylate among others. Other embodiments of the invention provide a method for synthesizing a peptide immunogen/substrate complex comprising immobilizing a polypeptide comprising a T cell epitope on a substrate to provide a T cell epitope/substrate complex and synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope to provide a peptide immunogen/substrate complex. The immobilizing of the polypeptide comprising the T cell epitope on the substrate can comprise covalently linking the polypeptide comprising the T cell epitope to the substrate. The immobilizing of the polypeptide comprising the T cell epitope on the substrate can comprise irreversibly binding the polypeptide comprising the T cell epitope to the substrate. The substrate can be a polymeric substrate, such as PVDF membrane, porous glass beads, dextran, agarose, methacrylate, and polystyrene resin. The substrate can be Amberchrom™. The method can further comprise macerating the peptide immunogen/substrate complex. The method can also further comprise administering the peptide immunogen/substrate complex to an animal subject. In other embodiments, the immobilizing of the polypeptide comprising the T cell epitope on the substrate comprises reversibly binding the polypeptide comprising the T cell epitope to the substrate. In these embodiments, the method can further comprise releasing the polypeptide comprising the T cell epitope from the substrate to form a peptide immunogen. The releasing of the polypeptide comprising the T cell epitope from the substrate can comprise contacting the peptide immunogen/substrate complex with a solution comprising acetonitrile and trifluoroacetic acid. In some embodiments, the substrate is a reverse phase resin substrate. In some embodiments, the reverse phase resin is Amberchrom™. Other embodiments of the invention provide a method for synthesizing a peptide immunogen/substrate complex comprising immobilizing a polypeptide comprising a B cell epitope on a substrate to provide a B cell epitope/substrate complex and synthesizing a polypeptide comprising a T cell epitope on the polypeptide comprising the B cell epitope to provide a peptide immunogen/substrate complex. The immobilizing of the polypeptide comprising the B cell epitope on the substrate can comprise covalently linking the polypeptide comprising the B cell epitope to the substrate. The immobilizing of the polypeptide comprising the B cell epitope on the substrate can comprise irreversibly binding the polypeptide comprising the B cell epitope to the substrate. The substrate can be a polymeric substrate, such as PVDF membrane, porous glass beads, dextran, agarose, methacrylate, and polystyrene resin. The substrate can be Amberchrom™. The method can further comprise macerating the peptide immunogen/substrate complex. The method can also further comprise administering the peptide immunogen/substrate complex to an animal subject. In other embodiments, the immobilizing of the polypeptide comprising the B cell epitope on the substrate comprises reversibly binding the polypeptide comprising the B cell epitope to the substrate. In these embodiments, the method can further comprise releasing the polypeptide comprising the B cell epitope from the substrate to form a peptide immunogen. The releasing of the polypeptide comprising the B cell epitope from the substrate can comprise contacting the peptide immunogen/substrate complex with a solution comprising acetonitrile and trifluoroacetic acid. In some embodiments, the substrate is a reverse phase resin substrate. In some embodiments, the reverse phase resin is Amberchrom™.

According to other embodiments of the invention, a method for synthesizing a peptide immunogen is provided comprising: covalently linking a polypeptide comprising a T cell epitope to a substrate to provide a T cell epitope/substrate complex having a covalent linkage; synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope of the T cell epitope/substrate complex to provide a peptide immunogen/substrate complex; and cleaving the covalent linkage between the polypeptide comprising the T cell epitope and the substrate to form a peptide immunogen. The substrates discussed above and methods for reversibly binding the polypeptide to a substrate can be applied to this process as well..

Further embodiments of the present invention include a process for synthesizing a peptide immunogen by reversibly binding a polypeptide comprising a T cell epitope to a substrate to provide a T cell epitope/substrate complex, building a B-cell epitope on the polypeptide comprising the T cell epitope to provide a peptide immunogen/substrate complex, and releasing the peptide immunogen from the substrate to provide the peptide immunogen. The substrate can be a reverse phase or ion exchange resin such as the Amberchrom family of resins available from TOSOH BIOSCIENCE described above.

The operation of releasing the peptide immunogen from the substrate to provide the peptide immunogen can be performed by various processes as will be understood by those skilled in the art. In some embodiments, the operation of releasing the peptide immunogen from the substrate to provide the peptide immunogen can comprise contacting the peptide immunogen/substrate complex with a solution capable of cleaving the bond between the substrate and the polypeptide comprising the T cell epitope without harming the structure of the peptide immunogen. For example, the peptide immunogen/substrate complex can be contacted with a solution of acetonitrile/dilute trifluoroacetic acid. In one embodiment, the solution of acetonitrile/dilute trifluoroacetic acid comprises between 50 and 75 weight percent acetonitrile and a 0.1% aqueous solution of trifluoroacetic acid. Other mechanisms for releasing the immunogen from the immunogen/substrate complex include, but are not limited to treatment with strong organic acid or weak aqueous acid, weak aqueous base, or a specific enzyme depending upon the linkage of the T-cell epitope containing polypeptide/protein to the substrate as will be understood by those skilled in the art.

Products: Products including polypeptides, peptide complexes, peptide immunogen/substrate complexes, and peptide immunogens can be prepared according to the methods of this invention as discussed above, e.g., by reversibly or irreversibly immobilizing a first polypeptide on a substrate and directly synthesizing a second polypeptide on the first polypeptide. In particular embodiments, the first and/or second polypeptide comprises an epitope(s) selected from the group consisting of a measles epitope, influenza epitope, grass epitope, and a combination thereof or an epitope that is useful for a booster immunization {e.g., a measles, influenza or polio epitope). In some embodiments, the first polypeptide is horse myoglobin and/or bovine serum albumin. In some embodiments, the first polypeptide is horse myoglobin and the second polypeptide is a measles epitope. In some embodiments, the first polypeptide is horse myoglobin and the second polypeptide is a grass epitope (e.g., a ragweed epitope).

The first and second polypeptides can comprise epitopes from any allergen or immunogen, including, but not limited to, vaccine antigens, such as influenza antigens, polio antigens, herpes antigens (e.g., CMV, EBV, HSV, VZV), mumps antigens, measles antigens, rubella antigens, diphtheria toxin or other diphtheria antigens, pertussis antigens, hepatitis (e.g., hepatitis A and hepatitis B) antigens, smallpox antigens, adenovirus antigens or any other vaccine antigen; allergen antigens, such as environmental allergens (e.g., dust mite allergens, grass antigens such as ragweed antigens), plant allergens (e.g., pollen), insect allergens (e.g., bee and ant venom), and animal allergens (e.g., cat dander and saliva allergens); infectious agent antigens, such as antigens or proteins encoded by the genomes of Hepadnaviridae (including hepatitis A, B, C, D, E, F, G, etc., e.g., HBsAg, HBcAg, HBeAg), Flaviviridae (including human hepatitis C virus (HCV), yellow fever virus and dengue viruses), Retroviridae (including human immunodeficiency viruses (HIV), e.g., gp120, gp160, gp41 , an antigenic fragment of gp120, an antigenic fragment of gp160 and/or an antigenic fragment of gp41 , and human T lymphotropic viruses (HTLV1 and HTLV2)), Herpesviridae (including herpes simplex viruses (HSV-1 and HSV-2), Epstein Barr virus (EBV), cytomegalovirus, varicella-zoster virus (VZV), human herpes virus 6 (HHV-6) human herpes virus 8 (HHV-8), and herpes B virus), Papovaviridae (including human papilloma viruses), Rhabdoviridae (including rabies virus), Paramyxoviridae (including measles virus and respiratory syncytial virus),

Reoviridae (including rotaviruses), Bunyaviridae (including hantaviruses), Filoviridae (including Ebola virus), Adenoviridae, Parvoviridae (including parvovirus B-19), Arenaviridae (including Lassa virus), Orthomyxoviridae (including influenza viruses, e.g., NP, HA antigen), Poxviridae (including Orf virus, molluscum contageosum virus, smallpox virus and Monkey pox virus), Togaviridae (including Venezuelan equine encephalitis virus), Coronaviridae (including corona viruses), Picornaviridae (including polioviruses), rhinoviruses, orbiviruses, picodnaviruses, encephalomyocarditis virus (EMV), Parainfluenza viruses, adenoviruses, Coxsackieviruses, Echoviruses, Rubeola virus, Rubella virus, human papillomaviruses, Canine distemper virus, Canine contagious hepatitis virus, Feline calicivirus, Feline rhinotracheitis virus, TGE virus (swine), Foot and mouth disease virus, simian virus 5, human parainfluenza virus type 2, human metapneuomovirus, enteroviruses, and any other pathogenic virus now known or later identified (see, e.g., Fundamental Virology, Fields et al., Eds., 3^rd ed., Lippincott-Raven, New York, 1996, the entire contents of which are incorporated by reference herein); antigens from pathogenic microorganisms, such as Rickettsia, Chlamydia, Mycobacteria, Clostridia, Corynebacteria, Mycoplasma, Ureaplasma, Legionella, Shigella, Salmonella, pathogenic Escherichia co// species, Bordatella, Neisseria, Treponema, Bacillus, Haemophilus, Vibrio, Staphylococcus spp., Streptococcus spp., Campylobacter spp., Borrelia spp., Leptospira spp., Erlichia spp., Klebsiella spp., Pseudomonas spp., Helicobacter spp., and any other pathogenic microorganism now known or later identified (see, e.g., Microbiology, Davis et al, Eds., 4^th ed., Lippincott, New York, 1990, the entire contents of which are incorporated herein by reference); antigens from pathogenic protozoa, such as Plasmodium species (e.g., malaria antigens), Babeosis species, Schistosoma species, Trypanosoma species, Pneumocystis carnii, Toxoplasma species, Leishmania species, and any other protozoan pathogen now known or later identified; and antigens from pathogenic yeast and fungi, such as Aspergillus species, Candida species, Cryptococcus species, Histoplasma species, Coccidioides species, and any other pathogenic fungus now known or later identified; and cancer antigens, such as HER2/neu and BRCA1 antigens for breast cancer, MART-1/MelanA, gplOO, tyrosinase, TRP-1 , TRP-2, NY-ESO-1 , CDK-4, β-catenin, MUM-1 , Caspase-8, KIAA0205, HPVE7, SART-1 , PRAME, and p15 antigens, members of the MAGE family (e.g., MAGE-1 , MAGE-2, MAGE-3, MAGE-4 and MAGE-11 ), the BAGE family (e.g., BAGE-1 ), the DAGE/PRAME family (e.g., DAGE-1), the GAGE family (e.g., GAGE-1 and GAGE- 6), the RAGE family (e.g., RAGE-1), the SMAGE family, NAG, TAG-72, CA125, mutated proto-oncogenes such as p21 ras, mutated tumor suppressor genes such as p53, tumor associated viral antigens (e.g., HPV16 El), the SSX family, HOM-MEL- 55, NY-COL-2, HOM-HD-397, HOM-RCC-1.14, HOM-HD-21 , HOM-NSCLC-11 , HOM-MEL-2.4, HOM-TES-11, RCC-3.1.3, NY-ESO-1, the SCP family, human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), MUC-2, MUC-3, MUC- 18, carcino-embryonic antigen (CEA), the raf oncogene product, CA-125, GD2, GD3, GM2, TF, sTn, gp75, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogene product, β-HCG, gp43, HSP-70 , p17 mel, HSP-70, gp43, HMW, HOJ-1 , melanoma gangliosides, TAG-72, mutated proto- oncogenes such as p21ras, mutated tumor suppressor genes such as p53, estrogen receptor, milk fat globulin, telomerases, nuclear matrix proteins, prostatic acid phosphatase, protein MZ2-E, polymorphic epithelial mucin (PEM), folate-binding- protein LK26, truncated epidermal growth factor receptor (EGFR), Thomsen-

Friedenreich (T) antigen, GM-2 and GD-2 gangliosides, polymorphic epithelial mucin, folate-binding protein LK26, human chorionic gonadotropin (HCG), pancreatic oncofetal antigen, cancer antigens 15-3,19-9, 549, 195, squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA), pancreas cancer associated antigen (PaA), mutant K-ras proteins, mutant p53, chimeric protein P210_BCR-ABL, tumor associated viral antigens (e.g., HPV16 E7), an antibody produced by a B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma; hairy cell leukemia), a fragment of such an antibody containing an epitope of the idiotype of the antibody, a malignant B cell antigen receptor, a malignant B cell immunoglobulin idiotype, a variable region of an immunoglobulin, a hypervariable region or complementarity determining region (CDR) of a variable region of an immunoglobulin, a malignant T cell receptor (TCR), a variable region of a TCR and/or a hypervariable region of a TCR, and any other cancer antigen now known or later identified.

In particular embodiments of the invention, the epitope is a ragweed allergen, a grass allergen, or a measles epitope.

Ragweed, and in particular Short Ragweed (Ambrosia artemisiifolia), is clinically the most important source of seasonal aeroallergens, as it is responsible for both the majority of cases and the most severe cases of allergic rhinitis (Pollart, et al. (1989) J. Allergy CHn. Immunol. 83(5):875-82; Rosenberg, et al. (1983) J. Allergy CHn. Immunol. 71(3):302-10; Bruce, et al. (1977) J. Allergy CHn. Immunol. 59(6): 449-59). Ragweed pollen also contributes significantly to exacerbation of asthma and allergic conjunctivitis. Grass pollen is the most frequent cause of hay fever. Although more than

1 ,000 species of grass grow in North America, only a few produce highly allergenic pollen. These include Timothy grass, Kentucky bluegrass, Johnson grass, Bermuda grass, redtop grass, orchard grass, velvet grass, rye grass and sweet vernal grass. . The measles virus, like many members of the paramyxovirus family, contains six major structural proteins: the matrix protein, hemagglutinin, the fusion protein, large protein, phosphoprotein and nucleocapsid protein. Of these, the two envelope glycoproteins, hemagglutinin and fusion protein, have been shown to be responsible for induction of measles virus-neutralizing antibodies. See Varsanyi et al., J. Gen. Virol. 65: 365 (1984), Giraudon et al., Virology 144: 46 (1985), and Drillien et al., Proc. Natl. Acad. Sci. USA 85: 1252-56 (1988).

Accordingly, in particular embodiments, the allergen or epitope is an allergen or epitope isolated from ragweed, a grass species, or measles virus including, but not limited to, those listed Table I.

TABLE I

N/A, not available In general, the weed and grass allergens provided in Table X are members of the polysaccharide lyase family 1 of proteins (e.g., Amb a 1 and Amb a 2); lipid transfer proteins (e.g., Parj 1 , Parj 2, Par o 1); profilins (e.g., HeI a 2, Mer a 1 , Che a 2, Parj 3, Cyn d 12); polcalcins (e.g., Che a 3), or proteins with homology to trypsin inhibitors (e.g., PhI p 11).

In other embodiments, an allergen or antigenic epitope thereof is a peptide or polypeptide isolated from trees such as oak (e.g., Que a 1 ), mountain cedar (e.g, Jun a 1-Jun a 3), birch (e.g., Bet v 1-Bet v 7), ash (e.g, Fra e 1), alder (e.g., AIn g 1), hazel (e.g., Cor a 1 , Cor a 2, or Cor a δ-Cor a 11), juniper (e.g., Jun o 4), and cypress (e.g., Cup a 1 ); molds such as Alternaria alternate (e.g., Alt a 1-AIt a 4, Alt a 6, Alt a 7, or Alt a 10- Alt a 12), Cladosporium herbarum (e.g., CIa h 1-CIa h 6, or CIa h 12), Aspergillus flavus (e.g., Asp fl 13), A. fumigatus (e.g., Asp f 1-Asp f 13, Asp f 15-Asp f 18, Asp f 22w orAsp f 23), A. niger (e.g., Asp n 14 or Asp n 18), A. oryzae (e.g., Asp o 13 or Asp o 21), Penicillium brevicompactum (e.g., Pen ch 13, Pen ch 18, or Pen ch 20), P. citrinum (e.g., Pen c 3, Pen c 13, Pen c 19, Pen c 22w, or Pen c 24), P. oxalicum (e.g., Pen o 18), Epicoccum purpurascens (e.g., Epi p 1), or Fusaήum culmorum (e.g., Fus c 1 or Fus c 2); egg whites (e.g., GaI d 1-GaI d 5); milk (e.g., Bos d 4-Bos d 8); wheat (e.g., Tri a 18 and Tri a 19); cat (FeI d 1-FeI d 7w); dust mites (e.g., Aca s 13, BIo 1 1 , BIo 13- BIo 1 6, BIo 1 10- BIo 1 13, BIo 1 19, Der f 1-Der f 3, Der f 7, Der f 10, Der f 11 , Der f 14- Der f 18w, Der m 1 ,

Der p 1-Der p 10, Der p 14, Eur m 2, Eur m 14, GIy d 2, Lep d 2, Lep d 5, Lep d 7, Lep d 10, Lep d 13, or Tyr p 2); or bees (e.g., Api m 1 , Api m 2, Api m 4, Api m 6, Api m 7, Bom p 1 , or Bom p 4).

Other exemplary food, animal, tree, insect and mold allergens are found at http://www.allergen.org/List.htm Marsh and Freidhoff. 1992. ALBE, an allergen database. IUIS, Baltimore, MD, Edition 1.0).

Any polypeptide can be synthesized by the processes of this invention, including, but not limited to, therapeutic polypeptides for medical or veterinary use, immunogenic polypeptides, enzymes, hormones, cytokines, and growth factors. Examples include, but are not limited to, polypeptides falling into the following therapeutic categories: ACE-inhibitors; anti-anginal drugs; anti-arrhythmias; anti¬ asthmatics; anti-cholesterolemics; anti-convulsants; anti-depressants; anti-diarrhea preparations; anti-histamines; anti-hypertensive drugs; anti-infectives; anti- inflammatory agents; anti-lipid agents; anti-manics; anti-nauseants; anti-stroke agents; anti-thyroid preparations; anti-tumor drugs; anti-tussives; anti-uricemic drugs; anti-viral agents; acne drugs; alkaloids; amino acid preparations; anabolic drugs; analgesics; anesthetics; angiogenesis inhibitors; antacids; anti-arthritics; antibiotics; anticoagulants; antiemetics; antiobesity drugs; antiparasitics; antipsychotics; antipyretics; antispasmodics; antithrombotic drugs; anxiolytic agents; appetite stimulants; appetite suppressants; beta blocking agents; bronchodilators; cardiovascular agents; cerebral dilators; chelating agents; cholecystokinin antagonists; chemotherapeutic agents; cognition activators; contraceptives; coronary dilators; cough suppressants; decongestants; deodorants; dermatological agents; diabetes agents; diuretics; emollients; erythropoietic drugs; expectorants; fertility agents; fungicides; gastrointestinal agents; growth regulators; hormone replacement agents; hyperglycemic agents; hypnotics; hypoglycemic agents; laxatives; migraine treatments; mineral supplements; mucolytics; narcotics; neuroleptics; neuromuscular drugs; NSAIDS; nutritional additives; peripheral vasodilators; prostaglandins; psychotropics; renin inhibitors; respiratory stimulants; steroids; stimulants; sympatholytics; thyroid preparations; tranquilizers; uterine relaxants; vaginal preparations; vasoconstrictors; vasodilators; vertigo agents; vitamins; and wound healing agents.

Methods of Use

The peptide complexes, peptide immunogens, and any other products produced according to the methods of this invention can be administered to an animal subject. In some embodiments, administration is by injection or through oral administration as will be understood by those skilled in the art. Peptide complexes and peptide immunogens provided by this invention can be particularly useful as human and animal vaccines or to produce antisera for research purposes. In some embodiments, they provide a vaccine and booster combination.

In some embodiments, the invention provides methods for inducing synthesis of an antibody in an animal subject comprising administering to the animal subject an effective amount of the peptide complex produced according to the methods of this invention, an effective amount of the peptide immunogen/substrate complex produced according to the methods of this invention, an effective amount of the peptide immunogen produced according to the methods of this invention, and/or an effective amount of the peptide/substrate complex produced according to the methods of this invention. As discussed below, the products can be administered per se or with additional ingredients as discussed herein. In other embodiments, the invention provides methods for immunizing an animal subject comprising administering to the animal subject an effective amount of the peptide complex produced according to the methods of this invention, an effective amount of the peptide immunogen/substrate complex produced according to the methods of this invention, an effective amount of the peptide immunogen produced according to the methods of this invention, and/or an effective amount of the peptide/substrate complex produced according to the methods of this invention. As discussed below, the products can be administered per se or with additional ingredients as discussed herein.

In some embodiments, the invention provides a method for building resistance to allergens in an animal subject or for desensitizing an animal subject to the effects of allergens comprising administering to the animal subject an effective resistance- building amount of the peptide complex produced according to the methods of this invention, an effective amount of the peptide immunogen/substrate complex produced according to the methods of this invention, an effective amount of the peptide immunogen produced according to the methods of this invention, and/or an effective amount of the peptide/substrate complex produced according to the methods of this invention. As discussed below, the products can be administered per se or with additional ingredients as discussed herein.

According to some embodiments of the present invention, methods of treating a subject in need of such treatment include administering an effective amount of a composition of this invention to the subject. The effective amount will vary somewhat from composition to composition, and subject to subject, and will depend upon factors such as the age, species, gender and/or condition of the subject and the route and mode of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art (see, e.g., Remington, The Science And Practice of Pharmacy (9^th Ed. 1995)). As a general proposition, a dosage from about 0.5 μg to about 500 μg/kg will have efficacy with all weights being calculated based upon the weight of the composition and/or active ingredient (e.g., immunogen). A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration. The frequency of administration can be one, two, or three times per day/week/month/year or as necessary to treat the condition. The duration of treatment depends on the type of condition being treated and can be for as long as the life of the patient.

The amount of immunogen sufficient to confer immunity to pathogenic bacteria, viruses, or other microbes can be determined by methods well known to those skilled in the art. This quantity can determined based upon the characteristics of the vaccine recipient and the level of immunity required. Typically, the amount of vaccine to be administered is determined based upon the judgment of a skilled physician. Where vaccines are administered by subcutaneous or intramuscular injection, a range of about 0.5 to about 500 μg purified protein may be given. As useful in the present invention, such dosages are commonly sufficient to provide about 1 μg, possibly about 10 μg, even about 50 μg, and as much as about 100 μg, up to about 500 μg of immunogenic protein, or immunogenic polypeptide, or immunogenically active fragments thereof. In addition, more than one such active material may be present in the vaccine. Thus, more than one antigenic structure may be used in formulating the vaccine, or vaccine composition to use in the methods disclosed herein. This may include two or more individually immunogenic proteins or polypeptides, proteins or polypeptides showing immunogenic activity only when in combination, either quantitatively equal in their respective concentrations or formulated to be present in some ratio, either definite or indefinite. Thus, a vaccine composition for use in the processes disclosed herein may include one or more immunogenic proteins, one or more immunogenic polypeptides, and/or one or more immunogenically active immunogens comprising antigenic fragments of said immunogenic proteins and polypeptides, the latter fragments being present in any proportions. The exact components, and their respective quantities, making up the vaccines, and vaccine compositions, useful in the methods of the present invention are determined, inter alia, by the nature of the disease to be treated or prevented, the severity of such condition where it already exists, the age, sex, and general health of the recipient, as well the personal and professional experience and inclinations of the researcher and/or clinician utilizing these methods. Subjects suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates, humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the present invention is suitable. Human subjects are used in some embodiments. Human subjects of both genders and at any stage of development (e.g., neonate, infant, juvenile, adolescent, adult) can be treated according to the present invention. Illustrative avians include but are not limited to chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo. The present invention can be used in the treatment of various subjects, but the invention can also be used in animal subjects, particularly mammalian subjects such as mice, rats, dogs, cats, livestock and horses for veterinary purposes, and for drug screening and drug development purposes. The compounds of the present invention may be administered per se as well as in the form of pharmaceutically acceptable esters, salts, and other physiologically functional derivatives thereof. The present invention also contemplates pharmaceutical formulations, both for veterinary and for human medical use, which comprise as the active pharmaceutical ingredient one or more compound(s) of the present invention. In such pharmaceutical and medicament formulations, the active pharmaceutical ingredient can be utilized together with one or more pharmaceutically acceptable carrier(s) therefor and optionally any other therapeutic ingredients. The active pharmaceutical ingredient is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired dose.

The formulations include those suitable for parenteral as well as non- parenteral administration, and specific administration modalities include, but are not limited to, oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, intraperitoneal, transdermal, intrathecal, intraarticular, intraarterial, subarachnoid, bronchial, lymphatic, vaginal, and intrauterine administration.

When a compound of the present invention is utilized in a formulation comprising a liquid solution, the formulation advantageously may be administered orally or parenterally. When a compound of the present invention is employed in a liquid suspension formulation or as a powder in a biocompatible carrier formulation, the formulation may be advantageously administered orally, rectally, or bronchially. When a compound of the present invention is utilized directly in the form of a powdered solid, the compound may advantageously be administered orally.

Alternatively, it may be administered bronchially, via nebulization of the powder in a carrier gas, to form a gaseous dispersion of the powder that is inspired by the patient from a breathing circuit comprising a suitable nebulizer device.

The formulations comprising a compound of the present invention may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing a compound of the present invention into association with a carrier that constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing a compound of the present invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of a compound of the present invention as a powder or granules; or a suspension in an aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a draught.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, with the active compound being in a free-flowing form such as a powder or granules which optionally is mixed with a binder, disintegrant, lubricant, inert diluent, surface active agent, or discharging agent. Molded tablets comprised of a mixture of the powdered active compound with a suitable carrier may be made by molding in a suitable machine.

A syrup may be made by adding a compound of the present invention to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredient(s). Such accessory ingredient(s) may include, for example, flavorings, suitable preservatives, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.

Formulations suitable for parenteral administration conveniently can comprise a sterile aqueous preparation of a compound of the present invention, which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution). Such formulations may include suspending agents and thickening agents or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose form. Nasal spray formulations can comprise purified aqueous solutions of a compound of the present invention with preservative agents and isotonic agents.

Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucus membranes.

Formulations for rectal administration may be presented as a suppository with a suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxylic acid.

Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye. Topical formulations can comprise a compound of the present invention dissolved or suspended in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations. In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and/or adjuvants.

The present invention will now be described with reference to the following examples. It should be appreciated that these examples are for the purposes of illustrating aspects of the present invention, and do not limit the scope of the invention as defined by the claims. EXAMPLE 1

Five milliliters of packed, AF 650M Amino Resin (TOSOH BIOSCIENCE) was washed with pH 6.8, 0.1 M sodium phosphate buffer containing 0.25M NaCI. To this packed resin was added 50 milligrams of horse heart myoglobin (Sigma Chemical) dissolved in 15 milliliters of the same buffer. After gentle vortexing, a solution of 25% glutaraldehyde in 1 milliliter of water was added, followed by additional vortexing and incubation at room temperature for 5 minutes. The resin was extensively washed with buffer, followed by water, followed by methanol, and the peptide/resin complex was dried by vacuum and stored at -20°C. Two hundred milligrams of carrier/resin complex were added to the reaction vessel of a Rainin Multiple Peptide Synthesizer and a hexapeptide mimetic (IFYGRI; SEQ ID NO:1) representing a B-cell epitope from grass group I allergen was synthesized with a carboxyl terminal tail composed of an alanine dipeptide.

The resulting peptide/resin complex was treated with 95% trifluoroacetic acid with appropriate scavenger molecules to remove protecting groups from the synthetic peptide. The peptide/resin complex was washed with methanol and dried under vacuum. Several individual beads were subjected to protein sequence analysis with the expected results and yields.

A peptide/resin complex slurry was prepared in a sterile phosphate/saline buffer and injected intraperitoneal^ into six, 30 gram, female, outbred mice. Serial tail bleedings were taken weekly and evaluated for antibody directed against both horse heart myoglobin as well as for activity against the B-cell epitope and grass group I allergen.

EXAMPLE 2

Five milliliters of packed, TOYOPEARL SuperQ 650M Resin (TOSOH BIOSCIENCE) was washed with 0.15M Tris, pH 8.0 and suspended in 10 milliliters of the same buffer. 50 milligrams of bovine serum albumin (BSA) (Sigma Chemical) were solubilized in 10 milliliters of 0.15M Tris, pH 8.0. While vortexing the resin, the BSA solution was added in a continuous stream. Vortexing continued for one full minute after the last of the BSA was added. The peptide/resin complex was washed with 0.15M Tris, pH 8.0 followed by washing in dimethylformamide and incubation of the complex overnight at room temperature in dimethylformamide. The following morning, the complex was washed with water and then with methanol prior to vacuum drying and storage at -20°C.

Two hundred milligrams of resin/protein were placed in the reaction vessel of a Rainin Symphony Peptide Synthesizer and a hexapeptide (IFYGRI; SEQ ID NO:1) representing a B-cell epitope from grass group I allergen was synthesized with a carboxyl terminal tail composed of an alanine dipeptide.

The peptide/resin complex was treated with 95% trifluoroacetic acid with appropriate scavenger molecules to remove protecting groups from the synthetic peptide. The resin was washed with methanol and dried under vacuum. Several individual beads were subjected to protein sequence analysis with the expected results and yields. In addition, several individual beads (with appropriate controls) were subjected to hydrolysis at 150⁰C in 6N HCI. After aqueous extraction of the hydrolyzed beads, the resulting solution was reacted with ninhydrin at 80°C. An aliquot of untreated beads was ninhydrin negative while all beads with either BSA alone or BSA/peptide were strongly ninhydrin positive.

A slurry of the peptide/resin complex was prepared in a sterile phosphate/saline buffer and injected intraperitoneally into six, 30 gram, female, outbred mice. Serial tail bleedings were taken weekly and evaluated for antibody directed against BSA as well as for activity against the B-cell epitope and grass group I allergen.

EXAMPLE 3

A sheet of PVDF (BioRad SequiBlot) membrane was wetted and soaked overnight in a dilute solution of BSA in 0.1% trifluoroacetic acid. The membrane was extensively washed with water and air dried. A number of % inch diameter circles were cut from the membrane using a metal punch. These circles were placed in the reaction column of a Milligen 9050 peptide synthesizer and a thirteen residue peptide (STFYGKPTGAGPK; SEQ ID NO:2) representing a B-cell epitope from grass group I allergen was synthesized. As a control, circles of untreated membrane were included in the reaction vessel. Following synthesis, the membrane circles were treated with 95% trifluoroacetic acid containing the appropriate scavengers for removal of side chain protecting groups on the synthetic peptide. Individual membrane circles were subjected to Edman degradation in a protein sequencer. Membrane which had not been treated with BSA gave no signal, suggesting that peptide had not been synthesized on those membranes. Membrane which had been incubated with BSA gave a strong sequence of synthetic peptide, followed by a weak sequence of BSA, suggesting that each molecule of BSA served as the attachment point of multiple synthetic peptides.

The invention has been described with respect to specific embodiments described herein. The invention may be embodied in different forms and should not be construed as limited to the specific embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties.

Claims

That which is claimed is:

1. A method for synthesizing a peptide complex, comprising: immobilizing a first polypeptide on a substrate; and synthesizing a second polypeptide on the first polypeptide to produce a peptide complex.

2. The method of claim 1 , wherein the first polypeptide is a protein.

3. The method of claim 1 , wherein the first polypeptide is a peptide.

4. The method of any of claims 1-3, wherein the second polypeptide is a peptide.

5. The method of any of claims 1-3, wherein the second polypeptide is a protein.

6. The method of claim 1 , wherein the first polypeptide is a protein and the second polypeptide is a peptide.

7. The method of claim 1 , wherein the first polypeptide is a protein and the second polypeptide is a protein.

8. The method of claim 1 , wherein the first polypeptide is a peptide and the second polypeptide is a peptide.

9. The method of any of claims 1-8, wherein the second polypeptide consists of 35 or fewer amino acids.

10. The method of any of claims 1-8, wherein the second polypeptide consists of 20 or fewer amino acids.

11. The method of any of claims 1-10, wherein the first polypeptide comprises an epitope.

12. The method of any of claims 1-11 , wherein the second polypeptide comprises an epitope.

13. The method of claim 12, wherein the epitope of the second polypeptide is a B cell epitope.

14. The method of claim 12, wherein the epitope of the second polypeptide is a T cell epitope.

15. The method of claim 12, wherein the second polypeptide comprises a T cell epitope and a B cell epitope.

16. The method of any of claims 11-15, wherein the epitope of the first polypeptide is a T cell epitope.

17. The method of any of claims 11-15, wherein the epitope of the first polypeptide is a B cell epitope.

18. The method of any of claims 11-15, wherein the first polypeptide comprises a T cell epitope and a B cell epitope.

19. The method of any of claims 11-18, wherein the peptide complex is an immunogen.

20. The method of claim 19, wherein the peptide complex is a vaccine and booster combination.

21. The method of any of claims 11-20, wherein the second polypeptide comprises an epitope selected from the group consisting of a measles epitope, influenza epitope, grass epitope, and a combination thereof.

22. The method of any of claims 11-21 , wherein the first polypeptide comprises a immunogen that is useful as a booster immunization.

23. The method of claim 22, wherein the first polypeptide comprises a measles epitope.

24. The method of claim 22 or claim 23, wherein the second polypeptide comprises a grass epitope.

25. The method of claim 24, wherein the grass epitope is a ragweed epitope.

26. The method of any of claims 1-25, wherein the immobilizing of the first polypeptide on the substrate comprises irreversibly binding the first polypeptide to the substrate.

27. The method of any of claims 1-26, wherein the substrate is a polymeric substrate.

28. The method of claim 27, wherein the polymeric substrate is selected from the group consisting of PVDF membrane, porous glass beads, dextran, agarose, methacrylate, and polystyrene resin.

29. The method of claim 26, wherein the substrate is Amberchrom™.

30. The rhethod of any of claims 1-25, wherein the immobilizing of the first polypeptide on the substrate comprises reversibly binding the first polypeptide to the substrate.

31. The method of claim 30, further comprising releasing the first polypeptide from the substrate.

32. The method of claim 31 , wherein the releasing of the first polypeptide from the substrate comprises contacting the peptide complex with a solution comprising acetonitrile and trifluoroacetic acid.

33. The method of any of claims 30-32, wherein the substrate is a reverse phase resin substrate.

34. The method of claim 33, wherein the reverse phase resin is Amberchrom™.

35. The methods of any of claims 1-34, further comprising administering the peptide complex to an animal subject.

36. A method for synthesizing a peptide immunogen/substrate complex, comprising: immobilizing a polypeptide comprising a T cell epitope on a substrate to provide a T cell epitope/substrate complex; and synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope to provide a peptide immunogen/substrate complex.

37. A method for synthesizing a peptide immunogen/substrate complex, comprising: immobilizing a polypeptide comprising a B cell epitope on a substrate to provide a B cell epitope/substrate complex; and synthesizing a polypeptide comprising a T cell epitope on the polypeptide comprising the B cell epitope to provide a peptide immunogen/substrate complex.

38. The method of claim 36, wherein the immobilizing of the polypeptide comprising the T cell epitope on the substrate comprises covalently linking the polypeptide comprising the T cell epitope to the substrate.

39. The method of claim 37, wherein the immobilizing of the polypeptide comprising the B cell epitope on the substrate comprises covalently linking the polypeptide comprising the B cell epitope to the substrate.

40. The method of claim 36 or claim 38, wherein the immobilizing of the polypeptide comprising the T cell epitope on the substrate comprises irreversibly binding the polypeptide comprising the T cell epitope to the substrate.

41. The method of claim 37 or claim 39, wherein the immobilizing of the polypeptide comprising the B cell epitope on the substrate comprises irreversibly binding the polypeptide comprising the B cell epitope to the substrate.

42. The method of any of claims 36-41, wherein the substrate is a polymeric substrate.

43. The method of claim 42, wherein the polymeric substrate is selected from the group consisting of PVDF membrane, porous glass beads, dextran, agarose, methacrylate, and polystyrene resin.

44. The method of any of claims 36-41 , wherein the substrate is Amberchrom™.

45. The method of any of claims 36-44, further comprising macerating the peptide immunogen/substrate complex.

46. The method of any of claims 36-45, further comprising administering the peptide immunogen/substrate complex to an animal subject.

47. The method of claim 36 or claim 38, wherein the immobilizing of the polypeptide comprising the T cell epitope on the substrate comprises reversibly binding the polypeptide comprising the T cell epitope to the substrate.

48. The method of claim 37 or claim 39, wherein the immobilizing of the polypeptide comprising the B cell epitope on the substrate comprises reversibly binding the polypeptide comprising the B cell epitope to the substrate.

49. The method of claims 36, 38, or 47, further comprising releasing the polypeptide comprising the T cell epitope from the substrate to form a peptide immunogen.

50. The method of claims 37, 39, or 48 further comprising releasing the polypeptide comprising the B cell epitope from the substrate to form a peptide immunogen.

51. The method of claim 49, wherein the releasing of the polypeptide comprising the T cell epitope from the substrate comprises contacting the peptide immunogen/substrate complex with a solution comprising acetonitrile and trifluoroacetic acid.

52. The method of claim 50, wherein the releasing of the polypeptide comprising the B cell epitope from the substrate comprises contacting the peptide immunogen/substrate complex with a solution comprising acetonitrile and trifluoroacetic acid.

53. The method of any of claims 47-52, wherein the substrate is a reverse phase resin substrate.

54. The method of claim 53, wherein the reverse phase resin is Amberchrom™.

55. A method for synthesizing a peptide immunogen, comprising." covalently linking a polypeptide comprising a T cell epitope to a substrate to provide a T cell epitope/substrate complex having a covalent linkage; synthesizing a polypeptide comprising a B cell epitope on the polypeptide comprising the T cell epitope of the T cell epitope/substrate complex to provide a peptide immunogen/substrate complex; and cleaving the covalent linkage between the polypeptide comprising the T cell epitope and the substrate to form a peptide immunogen.

56. The method of any of claims 49-55, further comprising administering the peptide immunogen to an animal subject.

57. The method of any of claims 36-56, wherein the peptide immunogen/substrate complex is a vaccine and booster combination.

58. A peptide complex prepared according to the method of any of claims 1-34.

59. A peptide immunogen/substrate complex prepared according to the method of claims 36-45, 47, 48 or 57.

60. A peptide immunogen prepared according to the method of any of claims 49-55.

61. A method for inducing synthesis of an antibody in an animal subject comprising administering to the animal subject an effective amount of the peptide complex of claim 58.

62. A method for inducing synthesis of an antibody in an animal subject comprising administering to the animal subject an effective amount of the peptide immunogen/substrate complex of claim 59.

63. A method for inducing synthesis of an antibody in an animal subject comprising administering to the animal subject an effective amount of the peptide immunogen of claim 60.

64. A method for immunizing an animal subject comprising administering to the animal subject an effective amount of the peptide complex of claim 58.

65. A method for immunizing an animal subject comprising administering to the animal subject an effective amount of the peptide immunogen/substrate complex of claim 59.

66. A method for immunizing an animal subject comprising administering to the animal subject an effective amount of the peptide immunogen of claim 60.

67. A method for desensitizing an animal subject to an allergen comprising administering to the animal subject an effective desensitizing amount of the peptide complex of claim 58.

68. A method for desensitizing an animal subject to an allergen comprising administering to the animal subject an effective desensitizing amount of the peptide immunogen/substrate complex of claim 59.

69. A method for desensitizing an animal subject to an allergen comprising administering to the animal subject an effective desensitizing amount of the peptide immunogen of claim 60.