Immunotherapeutic for prostate cancer treatment

Abstract

To develop a more efficient and cost-effective method to produce therapeutics for treating prostate cancer.SOLUTION: Provided is a GnRH therapeutic for neutralizing GnRH levels in a subject, which can reduce testosterone levels to attenuate or eliminate prostate cancer cell growth and / or metastasis. The therapeutic is produced synthetically. The GnRH therapeutic comprises a hapten carrier (hC) including a monomeric peptide (MP) synthesized separately from the GnRH peptide, and after self-assembly of the hC, GnRH is covalently coupled to form a GnRH-hC conjugate that can serve as a therapeutic. The MP includes heptad repeats following a specific pattern. The hC may include a GnRH peptide attached to a monomeric peptide prior to self-assembly to form a therapeutic. Optionally, the GnRH-hC conjugate further comprises one or more T cell epitopes at the N- and / or C-terminus of one or more amphipathic alpha-helices. Also provided are compositions including immunogenic compositions comprising the therapeutic.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 899,553, filed September 12, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence listing information The sequence listing for the present application is contained in a computer readable text file entitled "H197-0005PCT_ST25.txt," created on or about September 9, 2020, having a file size of approximately 22 KB, and is incorporated herein by reference in its entirety.

[0003] Technical Field This disclosure describes the synthesis of peptide-based prostate cancer therapeutics. [Background technology]

[0004] background In patients with advanced prostate cancer, minimizing the amount of testosterone in the body (to prevent the progression and spread of prostate cancer cells) is very important. Several FDA-approved drugs are on the market that reduce testosterone in men: Lupron Injection (leuprorelin acetate, a gonadotropin-releasing hormone (GnRH) agonist) and Farmagon (degarelix, a GnRH antagonist). Both of these potent drugs have significant adverse side effects, which in some cases contribute to further serious health problems for patients. Therefore, there is a need to develop improved drugs with reduced side effects for treating patients with prostate cancer.

[0005] Recombinant protein expression in hosts such as bacteria (primarily E. coli), yeast, insect cells, and mammalian cells is currently the most common method for producing subunit therapeutics. Recombinant protein expression has been very successful and remains an important method for therapeutic production. Typically, target proteins are identified through genomic analysis, functional assays, in silico analysis (e.g., function prediction, structural analysis, epitope identification, etc.), or a combination of the three. Expression trials are initiated to evaluate yield and solubility for immunogenicity trials. Subunits that produce high-titer antibodies against the disease target are then advanced for functional evaluation, where the therapeutic is tested for its ability to protect the host against disease manifestations and progression. Subunits that meet all these criteria are then advanced for therapeutic production optimization, stability, and toxicity / safety / dosage studies. Expression optimization studies are also important for determining production scale and feasibility. The entire process is notoriously time-consuming, labor-intensive, and very costly. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need to develop more efficient and cost-effective methods for producing therapeutic drugs to treat prostate cancer. [Means for solving the problem]

[0007] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used solely as an aid in determining the scope of the claimed subject matter.

[0008] The present disclosure describes hMP polypeptides comprising a hapten (h) bound to a monomeric peptide (MP). The disclosure also describes conjugates comprising a hapten conjugated to a hapten carrier (hC). The hapten can be a target protein or target antigen. In some embodiments, the hapten is a GnRH peptide, the hMP polypeptide comprises GnRH (G) and MP (GMP), and the conjugate is GnRH-hC. In some embodiments, because hMP can adopt the same secondary, tertiary, or quaternary structure, hMP can function as an oligomeric hC, such as hC or HhC (hexameric hC), after self-assembly. In some embodiments, when a T cell epitope is attached to MP in addition to a hapten, such as GnRH, GMP can function similarly to a GnRH-hC conjugate, but without the conjugated GnRH. In some embodiments, the GnRH peptide comprises the amino acid sequence set forth in SEQ ID NO:28-SEQ ID NO:34.

[0009] The hC described herein comprises a monomeric peptide that is an amphipathic alpha helix containing two or more heptad repeats that self-assemble into a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Each heptad comprises an amino acid sequence set forth in SEQ ID NO: 1-18. In some embodiments, the monomeric peptide self-assembles into a hexameric hapten carrier (HhC). The hC may also comprise a target antigen, such as GnRH, in which case the hC is a GMP that self-assembles into an oligomer (GhC oligomer). In some embodiments, the GMP self-assembles into a hexamer, e.g., an hC comprising a GnRH peptide bound to an HhC (GHhC). In some embodiments, the conjugate described herein comprises GnRH conjugated to an HhC (GnRH-HhC).

[0010] Furthermore, the present disclosure describes conjugates of GhC oligomers or GnRH-hC that contain T cell epitopes at the N- and / or C-termini of the amphipathic alpha helix of hC that are part of a monomeric peptide or covalently linked to either GhC or GnRH-hC.

[0011] In some embodiments, the present disclosure describes a composition comprising a GnRH-hC conjugate or GhC oligomer described herein and an excipient. In some embodiments, the composition may be a pharmaceutical composition, and the composition may be used to treat a subject in need thereof. The pharmaceutical composition may be an immunogenic composition. The subject may be suffering from a disease or condition, such as prostate cancer. The subject may need to have reduced testosterone levels to prevent prostate tumor cells from growing or metastasizing. In some embodiments, the pharmaceutical composition or GnRH-hC conjugate or GhC oligomer may be used as a therapeutic agent to treat prostate cancer.

[0012] In some embodiments, the present disclosure describes methods for using the GnRH-hC conjugates or GhC oligomers described herein as therapeutic agents, such as immunogens, to induce a robust and sustained immune response in subjects in need of reduced testosterone levels. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the synthesis of a GnRH-hC conjugate. Lysines were first activated with a heterobifunctional crosslinker, which reacted with hC lysines and subsequently with the C-terminal cysteine ​​of GnRH to form the GnRH-hC conjugate. The reaction was carried out with a large excess (greater than 10 molar equivalents) of crosslinker and GnRH to ensure complete loading of hC (12 covalently linked GnRH peptides are shown in the figure). Tryptophan in GnRH allows for quantification of coupling efficiency by fluorescence spectroscopy. The N- and C-termini of HhC contain T cell epitopes added during solid-phase peptide synthesis (SPPS). [Figure 2] Figure 1 shows the immunogenicity of hC (alone) and GnRH-hC conjugates. hC and GnRG-hC conjugates were first modeled and then subjected to in silico conformational B cell epitope prediction. The analysis used three web-based servers (BEPro, ElliPro, and DiscoTope 2.0) and one stand-alone Linux program (PTools, which predicts antigenic regions on the protein surface by electrostatic desolvation analysis). The results from all four analyses were normalized and averaged, and the results are shown in the figure. [Figure 3] FIG. 1 shows mouse anti-GnRH IgG titers at d14 (day 14), d28, and d42 induced by exemplary GnRH-HhC conjugates and GHhC oligomers compared to controls. [Figure 4] FIG. 1 shows mouse anti-GnRH IgG titers and testosterone (T) levels at d14 induced by exemplary GnRH-HhC conjugates and GHhC oligomers compared to controls. [Figure 5] FIG. 1 shows mouse anti-GnRH IgG titers and T levels at d28 induced by exemplary GnRH-HhC conjugates and GHhC oligomers compared to controls. [Figure 6] FIG. 1 shows mouse anti-GnRH IgG titers and T levels at d42 induced by exemplary GnRH-HhC conjugates and GHhC oligomers compared to controls. DETAILED DESCRIPTION OF THE INVENTION

[0014] Haptens are small molecules that lack antigenic determinants due to their small size. To become antigenic, haptens must be coupled to a larger carrier protein to become immunogenic. As used herein, the term "hapten" refers to any molecule that lacks an antigenic determinant until it is covalently or noncovalently coupled to a larger carrier protein, i.e., a molecule whose antigenicity is increased by covalent or noncovalent coupling to a larger carrier protein. Similar to haptens, small peptides (i.e., usually less than 5,000 daltons) also lack antigenic determinants that would induce a robust immune response and therefore must be coupled to a larger carrier protein to become immunogenic. Thus, the term "hapten" refers to molecules that are not good immunogens by themselves, but become immunogenic when coupled to a larger molecule. Haptens can be, for example, small organic molecules, monosaccharides, disaccharides, oligosaccharides, lipids, nucleic acids, peptides, or polypeptides. Although haptens may be capable of binding to antibodies, immunization with haptens usually does not elicit a strong antibody response. However, immunogenicity can be achieved when haptens are covalently linked by linking or conjugating them to larger carrier molecules, such as hapten-carrier conjugates greater than 5,000 daltons.

[0015] This disclosure describes a hapten carrier (hC) for small peptides such as GnRH. When GnRH is conjugated to the hC described herein, GnRH can induce a robust immune response.

[0016] GnRH is a 10-residue peptide produced in the hypothalamus of vertebrates. GnRH induces the pituitary gland to synthesize and secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In males, these hormones cause the testes to produce testosterone, and in females, these hormones cause the ovaries to produce estrogen and progesterone. Therefore, antibodies induced by vaccination with GnRH neutralize GnRH, preventing it from binding to its receptor and blocking testosterone production.

[0017] The present disclosure also describes therapeutic agents comprising GnRH-hC conjugates containing GnRH covalently linked to hC as described herein, as well as GnRH oligomers containing GnRH and monomeric peptides. GhC oligomers may also contain T cell epitopes. In this way, GnRH therapeutic agents containing both GnRH-hC and GhC can induce robust and sustained immune responses via adaptive immune response pathways, generating high-potency, high-affinity antibodies targeting endogenous GnRH, preventing the release of LH (luteinizing hormone), and dramatically reducing testosterone production. For example, GnRH can induce robust and sustained immune responses via T cell activation, dendritic cell maturation, B cell activation, proliferation, and maturation, the establishment of robust memory responses, and other pathways. This ultimately results in the neutralization of GnRH before it leaves the hypothalamus and enters the portal vein toward the pituitary gland, where it can control adrenal and gonadal testosterone production.

[0018] After an initial prime / boost in humans, testosterone levels are expected to be less than 0.7 (ng / dL) and maintained below that level with annual or twice-yearly "booster injections." Side effects of the therapeutic agent are minimal due to the presence of a precise number of well-characterized T cell epitopes, the absence of immunodominant epitopes on hC, and the completely synthetic (non-biological) production of the therapeutic agent. The precise spatial and stoichiometric arrangement of multiple conformational and linear GnRH B cell epitopes on the carrier results in a potent therapeutic agent capable of attenuating testosterone better than existing drugs.

[0019] Furthermore, the present disclosure describes a novel method for producing GnRH therapeutics, including GnRH-hC conjugates or GhC oligomers. The method eliminates many of the costly and time-consuming steps of traditional subunit or protein carrier-based therapeutic development. Instead of producing carrier proteins in recombinant expression hosts, the hC and GnRH components are synthetically produced by solid-phase peptide synthesis (SPPS), a flexible and modular system. The method described herein involves designing hC components containing monomeric peptides that self-assemble into amphipathic alpha-helices to form carrier complexes large enough to induce robust immune responses after one or more GnRH peptides are conjugated to the hC. In some embodiments, the monomeric peptides self-assemble into a hexameric hC (HhC) core, and the GnRH peptide can be covalently attached to the HhC core at either the N- or C-terminus of the monomeric peptide before self-assembly into the hexamer, or can be conjugated to the HhC core after self-assembly into the hexamer. In some embodiments, the HhC core can also contain T cell epitopes at the N- and / or C-termini of the amphipathic alpha-helix.

[0020] As an example, Figure 1 shows the components of the GnRH-HhC conjugate described herein. There is a central region that forms the core after hydration, and lysines in this region function to conjugate GnRH. The size of HhC can vary depending on the length of the T cell epitope. Upon hexamer formation, the unconjugated hexamer is 38.5 kDa (Figure 1). The conjugated hexamer can vary depending on the length and size of the conjugated hapten. For example, GnRH (1,200 daltons) loaded onto the hexamer increases in size from 38.5 kDa to approximately 53 kDa.

[0021] The present disclosure includes peptides at least 14 amino acid residues in length, each heptad having the pattern hwxhxyz (SEQ ID NO: 1) (In the formula, h is a hydrophobic or non-polar residue; w is a positively charged, negatively charged, polar uncharged, or nonpolar aliphatic residue; x is a negatively charged, positively charged, non-polar aliphatic, polar uncharged residue, or any natural or non-natural residue for epitope coupling to a hapten or any other molecule; y is any natural or non-natural residue for epitope coupling to a hapten or any other molecule; z is a negatively charged, positively charged, polar uncharged, non-polar aliphatic residue, or any natural or non-natural residue for epitope coupling to a hapten or any other molecule. We describe a core region of hC that contains at least two heptad repeats having the following structure:

[0022] In some embodiments, the hC core region has the pattern (hwxhxyz)n (SEQ ID NO: 2) (In the formula, h is I, L, V, F, W, Y, M, W, G, or A; w is G, R, A, N, Q, H, S, D, E, K, or T; x is R, S, N, Q, A, G, T, D, E, K, H, or C; y is K, H, C, D, E, R, W, Y, Q, N, or a non-natural amino acid or molecule containing a reactive group suitable for covalent coupling; z is A, D, H, S, E, R, N, Q, K, or G; n is an integer greater than 1) The peptide comprises:

[0023] In some embodiments, the exemplary heptads described herein have the following amino acid sequence: LRSIGKD (SEQ ID NO: 3), LRSIGRD (SEQ ID NO: 4), IREISRA (SEQ ID NO: 5), IREVAQS (SEQ ID NO: 6), IRDIAKA (SEQ ID NO: 7), IRDIGRA (SEQ ID NO: 8), IRDVGQS (SEQ ID NO: 9), IRDLAKG (SEQ ID NO: 10), VKDVARG (SEQ ID NO: 11), IRDIGNS (SEQ ID NO: 12), IKDLARG (SEQ ID NO: 13), IKKLKKK (SEQ ID NO: 14), IRSIGKE (SEQ ID NO: 15), IRSIGRE (SEQ ID NO: 16), IKSIGRE (SEQ ID NO: 17), or IRSIGRG (SEQ ID NO: 18) It has.

[0024] In some embodiments, the core region of hC comprises one or more heptads described herein (wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11).

[0025] The present disclosure describes a core region of hC comprising a peptide of at least 14 residues. In some embodiments, the peptide comprises 14 to 80 residues in length and comprises 2 to 11 heptad repeats. In some embodiments, the hC core region comprises a peptide comprising 20 to 70 residues, 25 to 60 residues, 28 to 50 residues, 28 to 40 residues, or 28 to 30 residues. Peptides comprising 14 to 80 residues in length are monomeric.

[0026] The terms "monomeric peptide (MP)" and "monomeric hC (MhC) peptide" are used interchangeably to refer to the monomeric peptides described herein. In some embodiments, the exemplary monomeric peptides or monomeric hC peptides described herein have the following amino acid sequence: LRSIGKDLRSIGKDLRSIGKDLRSIGKD (SEQ ID NO: 19), LRSIGKDLRSIGKDLRSIGKDLRSIGKDS (SEQ ID NO: 20), LRSIGKDLRSIGRDLRSIGKDLRSIGRD (SEQ ID NO: 21), IREISRAIREVAQSIRDIAKAIREIGKS (SEQ ID NO: 22), IRDIGRAIRDVGQSIRDLAKGIRDISKG (SEQ ID NO: 23), VKDVARGIRDIGNSIKDLARGIRDIGRG (SEQ ID NO: 24), IRSIGKEIRSIGREIKSIGREIRSIGRG (SEQ ID NO: 25), IRSIGKEIRSIGREIRSIGKEIRSIGRE (SEQ ID NO: 26), or IRSIGKEIRSIGREIRSIGREIRSIGRE (SEQ ID NO: 27) Includes.

[0027] The peptides described herein can be modified to contain one or more substitutions, insertions, and / or deletions to maintain the above-described hwxhxyz (SEQ ID NO: 1) pattern. Modifications at each position within the heptad repeat or peptide must maintain the amphipathic alpha-helical structure, stability, and oligomerization state of the peptide.

[0028] In some embodiments, the peptides described herein include peptides comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (SEQ ID NO:3)n, (SEQ ID NO:4)n, (SEQ ID NO:5)n, (SEQ ID NO:6)n, (SEQ ID NO:7)n, (SEQ ID NO:8)n, (SEQ ID NO:9)n, (SEQ ID NO:10)n, (SEQ ID NO:11)n, (SEQ ID NO:12)n, (SEQ ID NO:13)n, (SEQ ID NO:14)n, (SEQ ID NO:15)n, (SEQ ID NO:16)n, (SEQ ID NO:17)n, or (SEQ ID NO:18)n, where n is an integer between 2 and 11. In some embodiments, the peptides described herein include peptides comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. Sequence identity refers to the degree of match between two sequences in an alignment and is often expressed as a percentage. Differences between two sequences can be determined by methods routinely practiced in the art for determining identity, designed to give the largest match between the sequences examined. Methods for determining sequence identity can be determined using publicly available computer programs. Computer program methods for determining identity between two sequences include BLASTP. The BLASTP family of programs is publicly available from NCBI and other sources.

[0029] In some embodiments, one or more residues may be added to the N- or C-terminus of the monomeric peptides described herein to increase the stability of the peptide in vivo. For example, V (valine), M (methionine), G (glycine), I (isoleucine), D (aspartic acid), or P (proline), or a combination of these residues, may be added to the N- or C-terminus of the peptide. Additionally, protecting groups may be added to residues to increase the stability of the peptide. Examples of such protecting groups include acetyl, acryl, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl, and PEG (polyethylene glycol), and amide on the C-terminus.

[0030] The peptides described herein can be monomeric hC peptides, but as the monomeric hC peptides self-assemble, they can self-assemble into oligomeric hCs composed of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers. In some embodiments, the monomeric peptides self-assemble into hexamers having six amphipathic alpha-helices. In some embodiments, the hC is a hexamer oligomer.

[0031] In some embodiments, the present disclosure describes hCs containing one or more residues for conjugating a hapten, such as GnRH. The optimal site on hC for conjugating a hapten is the y residue in the heptad repeat; however, if the w, x, and z residues contain reactive side chains, GnRH coupling can also occur at the w, x, and z residues because they are solvent accessible and any residue capable of covalently binding GnRH to the hC, including HhC, can be used. In some embodiments, the y residue is K, H, C, D, E, R, W, Y, Q, N, or a non-natural amino acid containing a reactive group suitable for covalent coupling. In some embodiments, there are two to four y residues on each side of each of the six amphipathic alpha-helices to provide coupling sites. In some embodiments, the y residue is lysine (K).

[0032] In some embodiments, one or more GnRH peptides can be conjugated to MP using y residues during SPPS or after MP has assembled into an oligomer, such as a hexamer. GnRH conjugated to hC is a conjugate, referred to as a GnRH-hC conjugate or a GnRH-oligomer conjugate. In some embodiments, hC is linked to 1-100, 10-90, 20-80, 30-70, 40-60, or 50 GnRH peptides. In some embodiments, the hC is HhC, and the conjugate is GnRH-HhC.

[0033] In some embodiments, GnRH peptides can be added to the N- and / or C-termini of monomeric peptides during SPPS (prior to self-assembly) to form GMPs, which can then self-assemble into oligomers such as GnRH oligomers, or more specifically GnRH HhCs (GnRH hexameric hCs or GHhCs).

[0034] In some embodiments, hMP (hapten conjugated to a monomeric peptide) can self-assemble into hhC (hapten conjugated at the N- or C-terminus of a hapten carrier). When the hapten is GnRH (G), the hMP is GMP, which self-assembles into GhC oligomers, e.g., GHhc (GnRH conjugated to a hexameric hapten carrier).

[0035] GnRH peptides include GnRH1 and GnRH2. GnRH peptides have various names, including gonadotropin-releasing hormone (GnRH or GRH), LHRH (luteinizing hormone-releasing hormone), and gonadotropin-releasing factor (GRF or GnRF).

[0036] GnRH peptides can be found in various vertebrates. Examples of vertebrates include mammals, fish, birds, reptiles, and amphibians. Examples of mammals include primates, rodents, rabbits and hares, dogs, horses, pigs, whales, dolphins, bats, perissodactyla and artiodactyla, and other placental animals. Mammals also include monotremes and marsupials. Examples of fish include sharks, rays, bony fish, dogfish, sturgeon, scorpionfish, and coelacanth. Examples of birds include poultry, chickens, and turkeys. Examples of reptiles include snakes. Examples of amphibians include frogs, toads, and caecilians. Therefore, vertebrates are a good source of GnRH.

[0037] Furthermore, the amino acid sequences for GnRH peptides in vertebrates are highly conserved. They share the following consensus sequence: X1HWSX2GX3X4PG (SEQ ID NO: 28) wherein X1 is a polar or charged amino acid, X2 is a polar amino acid, X3 is a hydrophobic or amphipathic amino acid, and X4 is a hydrophobic, amphipathic, charged, or polar amino acid.

[0033] The amino acid sequence may be represented by including: Charged amino acids include R, K, D, or E. Polar amino acids include Q, N, H, S, T, Y, or C. Amphipathic amino acids include W, Y, or M. Hydrophobic amino acids include A, I, L, M, F, V, P, or G. In some embodiments, X1 is Q or E. In some embodiments, X2 is Y or H. In some embodiments, X3 is L or W. In some embodiments, X4 is L, Y, R, or Q. In some embodiments, X1 is Q or E, X2 is Y or H, X3 is L or W, and X4 is L, Y, R, or Q.

[0038] In some embodiments, the GnRH peptide is a GnRH1 peptide comprising the amino acid sequence: QHWSYGLRPG (SEQ ID NO: 29). In vivo, the first residue of GnRH is pyroglutamic acid (pE) caused by enzyme-catalyzed deamidation followed by cyclization of the side chain of Q. Vertebrate sources of GnRH peptides comprising the amino acid sequence SEQ ID NO: 29 include primates (including humans), rodents, rabbits and hares, placental mammals, bats, perissodactyls, whales, dolphins, artiodactyls, frogs, toads, marsupials, and caecilians.

[0039] In some embodiments, the GnRH peptide is a GnRH1 peptide comprising the amino acid sequence: QHWSHGWLPG (SEQ ID NO: 30). Vertebrate sources of GnRH peptides comprising the amino acid sequence SEQ ID NO: 30 include the spiny dogfish, the toothed catshark, and the coelacanth.

[0040] In some embodiments, the GnRH peptide is a GnRH1 peptide comprising the following amino acid sequence: EHWSYGLRPG (SEQ ID NO: 31). Vertebrate sources of GnRH peptides comprising the amino acid sequence SEQ ID NO: 31 include Russian sturgeon and Chinese hamster.

[0041] In some embodiments, the GnRH peptide is a GnRH1 peptide comprising the following amino acid sequence: QHWSYGWYPG (SEQ ID NO: 32). Vertebrate sources of the GnRH peptide comprising the amino acid sequence SEQ ID NO: 32 include rodents and snakes such as king cobras.

[0042] In some embodiments, the GnRH peptide is a GnRH1 peptide comprising the following amino acid sequence: EHWSYGLQPG (SEQ ID NO: 33).Vertebrate sources of GnRH peptides comprising the amino acid sequence SEQ ID NO: 33 include chickens.

[0043] In some embodiments, the GnRH peptide is a GnRH2 peptide comprising the following amino acid sequence: QHWSHGWYPG (SEQ ID NO: 34). Vertebrate sources of the GnRH peptide comprising the amino acid sequence SEQ ID NO: 34 include primates (including humans), rodents, placental mammals, poultry, turtles, artiodactyla and perissodactyla, bony fish, sharks and rays, and chimaeras.

[0044] In some embodiments, one or more GnRH peptides that can be bound to hC can be the same GnRH peptide or different GnRH peptides. For example, GnRH peptides from different mammalian species can be bound to the same hC. As another example, GnRH1 and GnRH2 can both be bound to the same hC. In some embodiments, two human isoforms of GnRH, GnRH1 and GnRH2, can be bound to the same hC.

[0045] In the context of conjugates or oligomers, the terms "attached" or "linked" or "coupled" are used interchangeably to refer to either conjugated to a self-assembled oligomer (hC) or added to a monomeric peptide during SPPS prior to self-assembly into an oligomeric hC.

[0046] One or more residues may be added to the N- or C-terminus of the GnRH peptide described herein. The one or more residues may make the GnRH peptide more stable. For example, the one or more residues may increase the in vivo half-life of the GnRH peptide. In some embodiments, adding one or more residues to the N-terminus of the GnRH peptide may increase the in vivo half-life of the GnRH peptide by more than 5 times to more than 120 times longer than the half-life of a GnRH peptide without one or more added residues at its N- or C-terminus. In some embodiments, one or more residues may increase the in vivo half-life of the GnRH peptide by 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, or 120-fold greater than the in vivo half-life of the GnRH peptide without the residue(s) added to its N- or C-terminus. In some embodiments, residues such as G (glycine), V (valine), M (methionine), or A (alanine), or combinations thereof, may be added to the N- or C-terminus of the GnRH peptide for stability. For example, adding G to the N-terminus increases the predicted in vivo half-life of the GnRH peptide from 0.8 hours to more than 30 hours, and adding V to the N-terminus increases the half-life of the GnRH peptide from 0.8 hours to 100 hours. In some embodiments, adding one or more residues such as G to the N-terminus of the GnRH peptide increases the half-life of the GnRH peptide by more than 37.5-fold, and adding one or more residues such as V to the N-terminus of the GnRH peptide increases the half-life of the GnRH peptide by more than 125-fold. In some embodiments, the GnRH peptide comprises the amino acid sequence: XQHWSYGLRPG (SEQ ID NO: 35), where X is G or V.

[0047] In some embodiments, the N-terminus of GnRH may also be acetylated for stability.

[0048] Other residues may also be added to the N- or C-terminus of one or more GnRH peptides to aid in conjugation to hC. For example, one or more residues may be added to the N- or C-terminus of the GnRH peptide to sufficiently reduce the pI (isoelectric point) and reduce the electrostatic repulsion with hC. For example, residues GEDC (SEQ ID NO: 36) or DGEGC (SEQ ID NO: 37) may be added to the C-terminus as a linker for conjugation and to modify the pI of the GnRH peptide. Furthermore, residues GEDC (SEQ ID NO: 36) or DGEGC (SEQ ID NO: 37) may also be added to the N-terminus in addition to the other residues described above to improve the stability of the GnRH peptide.

[0049] Optionally, other molecules can be directly conjugated to an hC oligomer, such as HhC, along with the GnRH peptide. Other molecules can also be conjugated to a GnRH peptide and subsequently conjugated to hC. Furthermore, as described herein, GnRH can be conjugated to the N- or C-terminus of an hC monomer peptide during SPPS to form GMP before self-assembly into a GhC oligomer, such as GHhC. One or more other molecules in addition to GnRH can also be conjugated to the N- or C-terminus of an hC monomer (MhC) peptide during SPPS before self-assembly into an hC oligomer.

[0050] Other molecules that can be bound to hC oligomers or MPs include any substance that can induce the production of antibodies useful for treating, preventing, or alleviating symptoms of prostate cancer, or reducing the risk of developing diseases or disorders in subjects caused by elevated levels of testosterone. In addition to GnRH, examples of other molecules include immunomodulators and haptens. Examples of immunomodulators, including adjuvant molecules, include T-cell epitope peptides, nucleic acids, lipids, lipopeptides, lipoproteins, carbohydrates, and short peptides. Peptides containing GnRH and B-cell epitopes that can be used as haptens include synthetically or recombinantly produced, or natural peptides or proteins containing natural or non-natural D- or L-amino acids.

[0051] T cell epitopes that can be used to activate T cell responses (to allow T cells to help B cells) can be found in the extracellular proteins of Clostridium botulinum, Clostridium perfringens, and Staphylococcus aureus, and in the extracellular solute-binding proteins of Mycobacterium and Clostridium tetani. T cell epitopes are also present in Mycobacterium tuberculosis, mumps virus, Plasmodium falciparum, human immunodeficiency virus 1, hepatitis C virus, and influenza A virus. Examples of such T-cell epitopes include the amino acid sequences SEQ ID NO:38 and SEQ ID NO:39 (derived from an extracellular protein of Clostridium botulinum, GenBank: STC78113.1), SEQ ID NO:40 (derived from an extracellular protein of Clostridium perfringens, GenBank: SUY45886.1), SEQ ID NO:41 (derived from an extracellular protein of Staphylococcus aureus, GenBank: SAO03917.1), SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 (derived from extracellular solute binding proteins of various species of Mycobacterium, NCBI Reference Sequence: WP_055398728.1), SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 (derived from extracellular solute binding proteins of Clostridium tetani, GenBank: STC78113.1), SEQ ID NO:49 (derived from an extracellular solute binding protein of Clostridium tetani, GenBank: STC78113.1), SEQ ID NO:40 (derived from an extracellular protein of Clostridium perfringens, GenBank: SUY45886.1), SEQ ID NO:41 (derived from an extracellular protein of Staphylococcus aureus, GenBank: SAO03917.1), SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46 (derived from extracellular solute binding proteins of various species of Mycobacterium, NCBI Reference Sequence: WP_055398728.1), SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 (derived from extracellular solute binding proteins of Clostridium tetani, Gen SEQ ID NO: 50 (derived from the ESAT-6-like protein EsxB of Mycobacterium tuberculosis), SEQ ID NO: 51 (derived from the alpha-crystallin protein of Mycobacterium tuberculosis), SEQ ID NO: 52 (derived from the mumps virus protein of mumps virus), SEQ ID NO: 53 (derived from the DNAJ protein of Plasmodium falciparum), SEQ ID NO: 54 (derived from the Gag-Pol polyprotein of human immunodeficiency virus 1), SEQ ID NO: 55 (derived from the genomic polyprotein of hepatitis C virus), SEQ ID NO: 56 (derived from matrix protein 1 of influenza A virus), and SEQ ID NO: 57 (derived from the hemagglutinin of influenza A virus).

[0052] Lipids that can be bound to hC include those that induce innate immune responses through binding to Toll-like receptors (TLRs). Lipids can also function as adjuvants. Examples of such lipids include monophosphoryl lipid-A, squalene, lipopolysaccharide (LPS), lipoproteins, or lipopeptides. Carbohydrates that can function as haptens include glucose, disaccharides, trisaccharides, and larger sugars, including complex carbohydrates.

[0053] Examples of TLR-binding peptides that can be conjugated to hC include TLR ligands, such as TLR-4 agonist peptides. These peptides act as adjuvant peptides. In some embodiments, the adjuvant peptide comprises the amino acid sequence APPHALS (SEQ ID NO: 58).

[0054] Other molecules may also include haptens, such as B-cell epitopes. B-cell epitopes that can be used as haptens include kisspeptin peptides and epitopes derived from kisspeptin receptors and GnRH receptors. Additional GnRH peptides may also be added as other molecules.

[0055] If the hapten is a small peptide, such as GnRH, the entire peptide can be used as the hapten. If the hapten is a protein, a portion can be used as the hapten. The portion of the protein to use as the hapten can be determined using well-known methods, such as in silico prediction algorithms or peptide-based epitope mapping of the whole protein. Many T-cell and B-cell epitopes have been identified using these methods.

[0056] A hapten that can enhance the immunogenicity of GnRH or enhance the duration or breadth of the immune response to GnRH can be conjugated to hC together with GnRH. For example, a conjugated peptide that functions to bind TLRs can have an adjuvant function and enhance the immunogenicity of GnRH. In some embodiments, a GnRH-hC conjugate can contain one or more different or the same GnRH peptides as well as other haptens or peptides.

[0057] This disclosure describes GnRH immunogens, including GnRH-hC conjugates and GhC oligomers. These conjugates and oligomers may also contain other molecules. Peptides used to generate GnRH immunogens include the monomeric peptides, GnRH peptides, other molecules containing T-cell epitopes, haptens, and adjuvant peptides described herein. They can be chemically synthesized by manual techniques or automated procedures. For example, solid-phase polypeptide synthesis (SPPS) has been practiced since the early 1960s. Over the years, improvements have been made to the early SPPS methods, and many methods have been automated.

[0058] Peptides, particularly the longer peptides described herein, can be generated by native chemical ligation (NCL). Using NCL, large peptides (polypeptides) can be formed by ligating (or coupling) two or more smaller peptides. In some embodiments, a polypeptide comprising a monomeric peptide and two or more haptens can be prepared from two or more smaller peptide fragments and assembled together using NCL technology. As an example, a polypeptide comprising a monomeric peptide and two haptens (one at the N- and one at the C-terminus of the monomeric peptide) can be synthesized from two smaller peptides, which are then covalently linked by NCL. Using NCL, a C (cysteine) is added to the N-terminus of one of the two smaller peptides, and a thioester functional group is added to the C-terminus of the other of the two smaller peptides, and these two peptides are then ligated into a full-length polypeptide. In some embodiments, residues are added to the peptides described herein to facilitate the synthesis of longer polypeptides.

[0059] In some embodiments, a spacer is added between the monomer peptide and the GnRH peptide or one or more other haptens. Examples of one or more residues that can be inserted as a spacer include G (glycine), D (aspartic acid), S (serine), C (cysteine), or a combination thereof. In some embodiments, the spacer can also be D, GD, or GSG.

[0060] The peptides and haptens described herein can also be produced biologically or recombinantly in heterologous expression systems. Any heterologous expression system can be used to produce the peptides described herein. In some embodiments, the expression system comprises Escherichia coli, which lacks machinery for post-translational modification, making the expression system a suitable host for producing the peptides described herein.

[0061] Other molecules, including GnRH, can be conjugated to hC using any known method, including click chemistry, homo- or heterobifunctional cross-linking reagents, or peptide bond formation. In some embodiments, haptens can be conjugated to hC using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide) or NHS / maleimide cross-linking chemistries, which are routinely used in conjugation reactions. The y residue, e.g., lysine, is positioned to provide well-defined hapten geometry and coupling stoichiometry.

[0062] Other molecules, including GnRH, can also be bound to hC via any suitable linker moiety. Examples of linkers include those that form amide bonds, ester bonds, and disulfide bonds. The linker can be a cleavable linker, such as a protease-cleavable peptide linker, a nuclease-sensitive nucleic acid linker, a lipase-sensitive lipid linker, a glycosidase-sensitive carbohydrate linker, a pH-sensitive linker, a hypoxia-sensitive linker, a photocleavable linker, a thermolabile linker, or an enzyme-cleavable linker. The linker can also be a non-cleavable linker. The linker can be bound to hC using any known method, such as click chemistry, passive adsorption, multivalent chelation, high-affinity non-covalent binding, or covalent bond formation. Haptens can also be bound to hC without a linker.

[0063] Furthermore, other molecules, including GnRH, can be conjugated to hC via another molecule. For example, GnRH or another B cell or T cell epitope can be first bound to a carrier for displaying the epitope of interest, and then conjugated to HhC. Examples of such carriers include proteins, peptides, nanoparticles, virus-like particles, or other entities that can function as carriers for displaying GnRH or other epitopes of interest.

[0064] Furthermore, the present disclosure describes GnRH-hC conjugates or GhC oligomers that optionally include one or more other molecules, including those described herein. The one or more other molecules include an immunomodulator and / or a hapten. In some embodiments, the one or more other molecules include a T cell epitope, a B cell epitope, a short peptide, such as a GnRH peptide, or a combination thereof. In some embodiments, the one or more other molecules are linked to the N- and / or C-terminus of one or more helices in the hC core. In some embodiments, the one or more other molecules are linked to the N-terminus of one or more helices in the hC core. In some embodiments, the one or more molecules are linked to the C-terminus of one or more helices in the hC core.

[0065] In some embodiments, T cell epitopes at the N- and / or C-termini of one or more helices in the hC core recruit T helper cells, induce B cells to produce maximal IgG titers to provide a robust immune response, and promote affinity maturation and class switching. Methods for selecting T cell epitope peptides are well known. For example, T cell epitopes can be selected by experimental methods known in the art, identified from the scientific literature, predicted using bioinformatics tools, designed de novo, or a combination thereof. In some embodiments, the T cell epitopes at the N- and C-termini are the same or different. In some embodiments, the T cell epitope is, for example, a CD4+ T cell epitope known to enhance the development of memory B cells and plasma cells that produce high-affinity antibodies. In some embodiments, T cell epitopes that may be included at the N- and / or C-termini of one or more helices of hC include TCE1 (SEQ ID NO:59 or SEQ ID NO:78), TCE2 (SEQ ID NO:60), TCE3 (SEQ ID NO:61), TCE4 (SEQ ID NO:62), or combinations thereof. In some embodiments, T cell epitopes also include peptides comprising the amino acid sequence SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:78. One or more of these T cell epitopes may be bound to hC or GnRH.

[0066] One or more T cell and / or B cell epitopes can also be linked to the GnRH peptide before conjugation to hC. Furthermore, these epitopes recruit T helper cells and induce B cells to produce maximal IgG titers, as well as promote affinity maturation and class switching.

[0067] When a hapten or immunomodulator, such as a T cell or B cell epitope, is linked to GnRH for conjugation to hC or to the N- and / or C-terminus of a monomeric peptide, one or more spacers can be inserted between the hapten and GnRH or between the hapten and the monomeric peptide. The spacer is added for accurate processing of the T cell epitope to ensure proteolytic trimming of the immunomodulator, e.g., a T cell epitope, resulting in a size that fits into the MHC II binding cleft. Examples of such spacers include residues D (aspartic acid), G (glycine), P (proline), S (serine), or combinations thereof. In some embodiments, the spacer includes one or more of D, GD, PGP, GSG, GPGP (SEQ ID NO: 63), GPGPG (SEQ ID NO: 64), GPGPGC (SEQ ID NO: 65), or SGPGPG (SEQ ID NO: 66). In some embodiments, the spacer for correct processing of the T-cell epitope comprises GPGPG (SEQ ID NO: 64).

[0068] The haptens or immunomodulators described herein, which are small peptides, can be linked at the N- and / or C-termini of one or more helices of the hC core. They can be incorporated into monomeric peptides so that they are covalently attached to the N- and / or C-termini of the monomeric peptides using solid-phase synthesis or native chemical ligation (NCL). Haptens can be covalently attached to the N- and / or C-termini using homo- or heterobifunctional crosslinkers or click chemistry reagents, which are well-known reagents for coupling molecules. In some embodiments, the immunomodulator or hapten, e.g., a T cell epitope and / or a B cell epitope, can already be attached to the N- and / or C-termini prior to self-assembly into an hC core, such as an HhC core, and GnRH can be conjugated after self-assembly into the hC core.

[0069] Haptens or immunomodulators at the N- and / or C-terminus can also be linked or conjugated to hC either through reactive small molecules or intermediate functional reagents such as large molecules. Examples of such small molecules include catalysts, stable intermediates, or salts. Examples of such large molecules include multi-antigenic peptides, proteins, or enzymes.

[0070] Furthermore, conjugation of haptens, including GnRH and / or other molecules, to the core of hC can be carried out using any type of linker. The linker can be cleavable or non-cleavable. Cleavable linkers include protease-cleavable peptide linkers, nuclease-sensitive nucleic acid linkers, lipase-sensitive lipid linkers, glycosidase-sensitive carbohydrate linkers, pH-sensitive linkers, enzyme-cleavable linkers, thermolabile linkers, and photocleavable linkers. Crosslinkers can also be used by activating side chain atoms or terminal atoms for covalent reaction with intermediate or final molecular atoms to form covalent bonds.

[0071] The present disclosure describes scaffold peptides comprising an hC monomer peptide and one or more T cell epitopes and / or one or more haptens and / or immunomodulators, such as GnRH, linked to its N- or C-terminus. As described herein, the scaffold peptide may also include one or more spacers, e.g., one or more residues for correct processing of the T cell epitopes or for stabilizing the hapten and / or hC monomer peptide. Table 1 discloses exemplary scaffold peptides.

[0072] [Table 1]

[0073] For example, the GnRH-Hex-TCE2 scaffold peptide comprises a GnRH peptide, an hC monomer peptide (SEQ ID NO: 25), and a T cell epitope 2 (TCE2) peptide. The GnRH-Hex-TCE2 scaffold peptide also comprises one or more stabilizing residues and one or more spacer residues. For example, G is added to the N-terminus of the GnRH peptide to stabilize the peptide, GD is inserted as a spacer between the GnRH peptide and the monomer peptide, and PGP is inserted between the monomer peptide and TCE2. The GnRH peptide and one or more T cell epitopes are bound to the hC monomer peptide prior to self-assembly. In some embodiments, the hC monomer peptide (SEQ ID NO: 25) self-assembles into a hexamer to form the scaffold peptide. Thus, the scaffold peptide GnRH-Hex-TCE2 comprises a hexameric (Hex) core.

[0074] The present disclosure also describes peptide immunogens for conjugation to hC to form hapten-hC conjugates for the preparation of therapeutic agents. The peptide immunogens include one or more haptens, e.g., GnRH peptides, and optionally one or more immunomodulatory agents, e.g., T cell epitopes. As described herein, the peptide immunogens can also include one or more other residues to stabilize the hapten, e.g., to stabilize the GnRH peptide, or for correct processing of the T cell epitope. Table 2 discloses exemplary immunogens for conjugation to hC.

[0075] [Table 2]

[0076] Additionally, the present disclosure describes hapten-hC conjugates. Exemplary hapten-hC conjugates include GnRH-Hex-TCE2+GnRH-TCE1, TCE3-Hex-TCE4+GnRH, TCE3-Hex-TCE4+GnRH-TCE1, and GnRH-Hex-GnRH+GnRH-TCE1. The "+" indicates that it is a conjugate and that an immunogen is conjugated to a scaffold peptide. For example, the GnRH-Hex-TCE2+GnRH-TCE1 conjugate contains the immunogen GnRH-TCE1 peptide (hapten) conjugated to the GnRH-Hex-TCE2 (GHhC) scaffold peptide.

[0077] In some embodiments, the scaffold peptides (hapten-hC oligomers) described herein can also be immunogens. Examples of such oligomeric scaffold peptides include GnRH-Hex-TCE2, TCE2-Hex-GnRH, GnRH-Hex-GnRH, and GnRH-Hex. These exemplary hapten-hC oligomers can serve as both peptide immunogens and scaffold peptides. Optionally, additional haptens, such as GnRH, can be conjugated to the oligomeric core of these scaffold peptides.

[0078] The hapten-hC conjugates and hapten-hC oligomers described herein are used to prepare compositions, such as pharmaceutical compositions. Pharmaceutical compositions containing one or more hapten-hC conjugates and one or more hapten-hC oligomers can be used as therapeutic agents or can be used to prepare therapeutic or therapeutic compositions. The pharmaceutical compositions described herein are also immunogenic compositions that contain immunomodulators, since they enhance the immunogenicity of haptens, such as GnRH peptides. The pharmaceutical compositions described herein are also therapeutic compositions, since they can be used to treat patients in need thereof.

[0079] The present disclosure describes compositions comprising the hC described herein and one or more excipients. In some embodiments, the hC is conjugated to one or more GnRH peptides and / or haptens or modulators (hapten-hC conjugates or GhC oligomers), and optionally to one or more T cell epitopes at one or more N- and / or C-termini of the core amphipathic helices of the hC. In some embodiments, the composition is a pharmaceutical composition, and the excipient is a pharmaceutically acceptable excipient. In some embodiments, the hC is HhC.

[0080] The term "excipient" refers to a diluent, adjuvant, or vehicle with which hC is administered. Examples of adjuvants include complete and incomplete Freund's adjuvant, which are used with animals, especially research animals. Pharmaceutically acceptable excipients can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred excipient when the pharmaceutical composition is administered intravenously. Saline solution and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. Pharmaceutically acceptable adjuvants include those based on monophosphoryl lipid-A mixed with oil, such as squalene.

[0081] The compositions or pharmaceutical compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Such formulations contain a therapeutically effective amount of purified hC, together with appropriate amounts of excipients to provide the form for proper administration to the subject.

[0082] Administration of the pharmaceutical compositions described herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can also be administered to a subject orally, topically, intranasally, enterally, rectally, buccally, vaginally, sublingually, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenously, intracranially, intraperitoneally, or combinations thereof. Administration of the pharmaceutical compositions can be by any mode effective to deliver a therapeutically and / or prophylactically effective amount of the conjugates described herein to a subject in need thereof.

[0083] The compositions described herein include immunogenic compositions. In some embodiments, the compositions described herein are therapeutic agents. The present disclosure describes peptide scaffolds for producing GnRH immunogenic therapeutic agents. The present disclosure also describes methods for preparing therapeutic agents, including designing and preparing monomeric peptides for the core of the hC described herein, oligomerizing the monomeric peptides, and conjugating one or more GnRH peptides to the oligomerized hC to obtain, for example, a GnRH + hC (GnRH-hC) conjugate. Furthermore, the present disclosure describes methods for preparing therapeutic agents, including designing and preparing monomeric peptides for the core of the hC described herein, covalently attaching a hapten, such as a GnRH peptide, to the monomeric peptide, and oligomerizing the monomeric peptide to obtain a hapten-HC oligomer, such as a GnRH oligomer. As described above, monomeric peptides can be synthesized by SPPS, which involves providing prepared monomeric peptides in lyophilized form. Hydration of the lyophilized monomeric peptides results in oligomerization. PBS containing salt and buffering capacity can be used to hydrate the lyophilized monomeric peptides. In some embodiments, the oligomerized hC is HhC.

[0084] The methods described herein include increasing the immunogenicity of GnRH. The methods include conjugating GnRH to the hC described herein. The methods may further include synthesizing a monomeric peptide with one or more other haptens or immunomodulators, for example, T cell or B cell epitopes at the N- and / or C-termini of one or more helices of the hC core. In some embodiments, the monomeric peptide is synthesized with a T cell and / or B cell epitope present at the N- and / or C-termini. The increased immunogenicity of GnRH is compared to the immunogenicity of GnRH itself, for example, not linked to or combined with hC or excipients. Furthermore, the methods described herein also include conjugating one or more other haptens or immunomodulators in addition to GnRH to increase the immunogenicity of GnRH. Examples of such haptens and immunomodulators include small molecules, lipids, lipoproteins, and TLR-4 agonists.

[0085] In some embodiments, the present disclosure describes an immunogenic composition comprising a GnRH-hC conjugate as described herein. The GnRH-hC conjugate optionally comprises one or more T cell and / or B cell epitopes and / or one or more additional haptens or immunomodulators other than GnRH. In some embodiments, the hC is HhC. The immunogenic composition comprises one or more pharmaceutically acceptable excipients. The excipient may be an adjuvant used to enhance or potentiate the immune response to the GnRH-hC conjugate in a therapeutically effective manner. The immunogenic composition may be administered to a subject in need thereof by any of the routes described herein for delivering an effective amount of the GnRH immunogenic therapeutic to cancer patients or any subject in need of reduced testosterone levels.

[0086] Dosages for administering the pharmaceutical and immunogenic compositions described herein to a subject will vary depending on the exact nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration will depend on a variety of factors and can be performed by a physician according to art-accepted practices.

[0087] The pharmaceutical or immunogenic compositions described herein may be a formulation. In some embodiments, the pharmaceutical or immunogenic compositions may be formulated for immediate release or for sustained or extended release. Such formulations may be prepared using well-known techniques. Sustained release formulations may contain the conjugates described herein dispersed in an excipient matrix and / or contained within a reservoir surrounded by a rate-limiting membrane. Excipients for use within such formulations are biocompatible and / or biodegradable. The formulation provides a relatively constant level of active ingredient release. The amount of conjugate contained within a sustained release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[0088] The present disclosure also describes kits having unit doses of the conjugates described herein. Such kits may include a container containing the unit dose, an information package insert with instructions for using the kit to treat or prevent a disease or disorder of interest, and optionally, an appliance or device for delivery of the composition.

[0089] The present disclosure further describes a method for enhancing the immunogenicity of a hapten, such as GnRH. In some embodiments, the method includes obtaining a monomeric peptide described herein, allowing the monomeric peptide to self-assemble into an oligomer (hC), such as a hexamer, and conjugating a hapten, such as a GnRH peptide, to the oligomer (hexamer-hC) to obtain a hapten-hC, such as GnRH-HhC. Immunomodulators can also be conjugated to the oligomer. In some embodiments, the method also includes synthesizing a monomeric peptide (MP) containing a GnRH (G) peptide on the N- and / or C-terminus, and allowing the GnRH-MP (GMP) to self-assemble into an oligomer, such as a hexamer, to obtain a hapten-hC oligomer, such as a GnRH-HhC oligomer. As described herein, the hapten-hC conjugate or oligomer can comprise one or more additional haptens or immunomodulators, such as GnRH, one or more T cell epitopes, or B cell epitopes. As described herein, the hapten-hC conjugate or oligomer can further comprise one or more residues to stabilize the hapten, one or more residues for proper processing of the T cell epitope, and / or one or more spacers inserted between the hapten and the monomeric peptide. The methods described herein can be used to prepare therapeutic agents or therapeutic compositions, such as GnRH immunogenic therapeutic compositions, for administration to a subject in need thereof to prevent or treat the subject.

[0090] The present disclosure also describes the use of the conjugates, oligomers, pharmaceutical compositions, therapeutic agents, therapeutic compositions, and vaccines described herein to treat a subject in need thereof. Similar to the compositions, vaccines include the conjugates or oligomers described herein. The present disclosure also describes methods for treating a subject in need thereof.

[0091] The methods described herein include treating subjects such as humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cows, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). A subject in need of treatment (or in need thereof) is a subject with a disease or disorder that needs to be treated with a GnRH therapeutic composition or immunogenic composition that induces an immune response in the subject that is sufficient or therapeutically effective to treat the subject with the disease or disorder. Thus, a subject in need thereof may be a subject that has been diagnosed with or is suffering from prostate cancer.

[0092] For example, antibodies induced by vaccination with the GnRH-hC conjugates described herein can neutralize GnRH, preventing it from binding to its receptor and blocking testosterone production. Blocking testosterone production can inhibit cells, such as prostate tumor cells, that require testosterone from growing or metastasizing. In some embodiments, the methods described herein can be used to treat subjects diagnosed with prostate cancer.

[0093] Treatment includes administering an effective amount of a conjugate described herein or a composition comprising an effective amount of a described conjugate. An "effective amount" is the amount of an active agent, such as a conjugate or composition described herein, required to effect a desired physiological change in vivo or in vitro. A therapeutically effective amount encompasses an amount that provides an effective amount.

[0094] Effective therapeutic agents contain components capable of inducing both innate and adaptive immune responses after immunization. While innate immunity is induced using adjuvants, in some embodiments, the therapeutic agents described herein comprise a GnRH-hC conjugate containing adaptive B and T cell epitopes, as shown in FIG. 1 . After GnRH conjugation, the GnRH-hC conjugate contains minimal foreign sequences for a more focused and robust immune response against the GnRH B cell epitopes. In some embodiments, for CD4+ T cell activation, the N- and C-termini of each of the six helices and / or core of HhC contain species-specific CD4+ T cell epitopes required to recruit T-cell help, produce long-lived plasma cells and high-titer / high-affinity antibodies, and induce a robust immune memory response. These epitopes are located at the termini of HhC, so that they do not interfere with hapten coupling. These epitopes are selected to lack lysine and cysteine ​​residues so that they are not haptenized or uncontrollably crosslinked during the B cell epitope coupling process. Lysine haptenization in T cell epitopes has been shown to greatly reduce their activity and function. T cell epitopes from a wide variety of species can be obtained from the IEDB database and selected based on positive T and B cell assays, including MHC ligand binding assays, the ability to recruit T cell help, and induction of B cell proliferation. The modular nature of the therapeutic technology described herein simplifies transferring therapeutic constructs between species, as it is simply a matter of replacing T cell epitopes and modifying B cell epitopes when different diseases or conditions are targeted.

[0095] A distinct advantage of the HhC core region described herein is its reduced immunogenicity, particularly after hapteconjugation (Figure 2), which minimizes the presentation of non-productive immunodominant epitopes. Thus, the combination of presentation of multiple GnRH B cell epitopes and presentation of multiple T cell epitopes with the reduction of non-productive immunodominant epitopes produces a highly effective therapeutic.

[0096] The advantages of using a fully synthetic GnRH-hC conjugate therapeutic scaffold or GhC oligomer scaffold are numerous. Modern SPPS routinely produces peptides up to 70–75 residues in length. The HhCs described herein range in size from 55–65 residues, with the length of the T cell epitope defining how much longer the HhC is than the 28–30 residue core region. GnRH peptides are 10 residues long and may contain additional amino acids for spacing or unique chemistry, making fully synthetic construction of therapeutics feasible. Producing kilogram quantities of therapeutic peptides in a cGMP facility eliminates the costly, time-consuming, and resource-intensive industrial production and purification of recombinant proteins and does not require subsequent virus clearance, endotoxin removal, or experimentation for the presence of infectious agents. Peptide synthesis is typically recognized as prohibitively expensive for large-scale therapeutic manufacturing. However, where high nanogram to low μg doses can be used, peptide therapeutics are several times more cost-effective than GnRH conjugated to recombinant subunit therapeutics.

[0097] The terms "residue" and "amino acid" are used interchangeably throughout this disclosure to refer to an "amino acid."

[0098] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specific recited elements, steps, ingredients, or components. Accordingly, the terms "comprise" or "comprising" should be interpreted as describing "comprising, consisting, or consisting essentially of." The transitional phrases "comprise" or "comprise" mean the inclusion of, but are not limited to, unspecified elements, steps, ingredients, or components, even in major amounts, allowing for their inclusion. The transitional phrase "consisting of" excludes any element, step, ingredient, or component not recited. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the specified element, step, ingredient, or component and to those that do not substantially affect the embodiment. In some embodiments, those that do not substantially affect the embodiment are elements, steps, ingredients, or components that do not reduce in a statistically significant manner the ability of the embodiment to perform its function in vitro or in vivo, e.g., to provide immunity to a disease. In some embodiments, components of the conjugates and oligomers described herein, e.g., GnRH, hC, or T-cell epitopes, can consist essentially of, or consist of, particular sequences. In some embodiments, a therapeutic agent or composition can consist essentially of, or consist of, a GnRH-hC conjugate or GhC and an excipient.

[0099] Furthermore, unless otherwise indicated, numbers expressing quantities of ingredients, components, reaction conditions, and the like used in the specification and claims are to be understood as modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective experimental measurements.

[0100] Where further clarity is needed, the term "about," when used in conjunction with a stated numerical value or range, has the meaning reasonably ascribed to the term "about" by one of ordinary skill in the art, i.e., some more or some less than the stated value or range, ±20% of the stated value, ±15% of the stated value, ±10% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, ±1% of the stated value, or any percentage between 1% and 20% of the stated value.

[0101] As used in the context of describing the present invention (particularly in the context of the claims below), "a," "an," "the," and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0102] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise noted herein, each individual value is incorporated into the specification as if it were individually recited herein. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, recitation of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0103] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by circumstances.

[0104] Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to facilitate a better understanding of the invention and do not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0105] The categorization of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Each member of a group may be referred to and claimed individually, or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion is made, the specification is deemed to contain the group as modified, and accordingly executes a written description of all Markush groups used in the appended claims.

[0106] The following exemplary embodiments and examples illustrate exemplary embodiments provided herein. These examples are not intended to, and should not be construed as, limiting the scope of the disclosure. It is clear that the method can be practiced other than as specifically described herein. Numerous modifications and variations are possible in light of the teachings herein and, therefore, are within the scope of the disclosure.

[0107] Illustrative Embodiments 1. A GnRH conjugate (GnRH-hC) or GnRH oligomer (GhC) comprising one or more gonadotropin-releasing hormone (GnRH) peptides covalently bound to a hapten carrier (hC), wherein the hC has the amino acid sequence: (hwxhxyz)n (SEQ ID NO: 2) (In the formula, h is a hydrophobic or non-polar residue; w is a positively charged, negatively charged, polar uncharged, or nonpolar aliphatic residue; x is a negatively charged, positively charged, non-polar aliphatic, or polar uncharged residue; y is a residue for epitope coupling, z is a negatively charged, positively charged, polar uncharged, or non-polar aliphatic residue; n is an integer greater than 1) A GnRH conjugate or GnRH oligomer, comprising an oligomer comprising a monomeric peptide comprising:

[0108] 2. The monomeric peptide has the amino acid sequence SEQ ID NO:2 (In the formula, h is I, L, V, F, W, Y, M, W, G, or A; w is G, R, A, N, Q, H, S, D, E, K, or T; x is R, S, N, Q, A, G, T, D, E, K, H, or C; y is K, H, C, D, E, R, W, Y, Q, N or a non-natural amino acid or molecule containing a reactive group suitable for covalent coupling; z is A, D, H, S, E, R, N, Q, K, or G; n is 2 to 10. 2. The GnRH conjugate or GnRH oligomer of embodiment 1, comprising:

[0109] 3. The GnRH conjugate or GnRH oligomer of embodiment 1 or 2, wherein the monomeric peptide comprises the amino acid sequence SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0110] 4. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 3, wherein the monomeric peptide comprises the amino acid sequence SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0111] 5. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 4, wherein the monomeric peptide further comprises V, M, G, I, D, P, C, S, C, or a combination thereof at the N-terminus and / or C-terminus.

[0112] 6. The conjugate or oligomer of any one of embodiments 1 to 5, wherein the oligomer is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[0113] 7. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 6, wherein the oligomer is a hexamer.

[0114] 8. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 7, wherein the one or more GnRH peptides are obtained from a vertebrate.

[0115] 9. The conjugate of any one of embodiments 1 to 8, wherein the one or more GnRH peptides are obtained from a mammal.

[0116] 10. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 9, wherein the one or more GnRH peptides are obtained from a human.

[0117] 11. The one or more GnRH peptides have the amino acid sequence: X1HWSX2GX3X4PG (SEQ ID NO: 28) (In the formula, X1 is a polar or charged amino acid; X2 is a polar amino acid, X3 is a hydrophobic or amphipathic amino acid; X4 is a hydrophobic, amphipathic, charged or polar amino acid 11. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 10, comprising:

[0118] 12. One or more GnRH peptides have the amino acid sequence SEQ ID NO: 28 (In the formula, X1 is Q or E; X2 is Y or H; X3 is L or W; or X4 is L, Y, R, or Q 12. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 11, comprising:

[0119] 13. The one or more GnRH peptides have the amino acid sequence: QHWSYGLRPG (SEQ ID NO: 29), QHWSHGWLPG (SEQ ID NO: 30), EHWSYGLRPG (SEQ ID NO: 31), QHWSYGWYPG (SEQ ID NO: 32), EHWSYGLQPG (SEQ ID NO: 33), or QHWSHGWYPG (SEQ ID NO: 34) 13. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 12, comprising:

[0120] 14. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 13, wherein the one or more GnRH peptides comprise additional amino acids at the N- and / or C-terminus to stabilize the one or more GnRH peptides and / or to conjugate the one or more GnRH peptides to hC.

[0121] 15. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 14, wherein one or more GnRH peptides comprise G, V, M, A, or a combination thereof at the N- and / or C-terminus, or the N-terminus of one or more GnRH peptides is acetylated.

[0122] 16. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 15, wherein the one or more GnRH peptides further comprise GEDC (SEQ ID NO: 36) or DGEGC (SEQ ID NO: 37) at the N- or C-terminus.

[0123] 17. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 16, comprising two or more GnRH peptides, wherein the GnRH peptides are obtained from different sources and / or comprise different amino acid sequences selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34.

[0124] 18. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 17, wherein one or more GnRH peptides are conjugated to hC via y residues on the monomeric peptides.

[0125] 19. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 18, further comprising one or more immunomodulators or additional haptens.

[0126] 20. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 19, wherein one or more immunomodulatory substances or further haptens are covalently fused (incorporated) to the N- and / or C-terminus of the monomeric peptide or covalently attached to the N- and / or C-terminus of one or more of the helices of the oligomer.

[0127] 21. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 20, comprising one or more spacers between the hapten or immunomodulator and the monomeric peptide.

[0128] 22. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 21, wherein the one or more spacers comprise G (glycine), D (aspartic acid), S (serine), C (cysteine), or a combination thereof.

[0129] 23. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 22, wherein one or more spacers comprise D, GD, and / or GSG.

[0130] 24. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 23, wherein the one or more haptens or immunomodulators comprise a GnRH peptide, one or more T cell epitopes, and / or one or more B cell epitopes.

[0131] 25. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 24, wherein the one or more T cell epitopes comprise a CD4+ T cell epitope.

[0132] 26. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 25, wherein the T cell epitope comprises the amino acid sequence SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:78.

[0133] 27. The GnRH conjugate or GnRH oligomer of embodiment 25 or 26, further comprising one or more residues for correct processing of the one or more T-cell epitopes.

[0134] 28. The GnRH conjugate or GnRH oligomer of embodiment 27, wherein one or more residues comprise D, G, P, or S, or a combination thereof.

[0135] 29. The GnRH conjugate or GnRH oligomer of embodiment 27 or 28, wherein one or more residues comprise D, GD, PGP, GSG, GPGP (SEQ ID NO: 63), GPGPG (SEQ ID NO: 64), GPGPGC (SEQ ID NO: 65), and SGPGPG (SEQ ID NO: 66).

[0136] 30. The GnRH conjugate or GnRH oligomer of any one of embodiments 20 to 29, wherein the one or more immunomodulators or further haptens enhance the immunogenicity of the one or more GnRH peptides or enhance the duration or breadth of the immune response to the one or more GnRH peptides.

[0137] 31. The GnRH conjugate or GnRH oligomer of any one of embodiments 20 to 30, wherein the one or more immunomodulators or further haptens comprise lipids, peptides, nucleic acids, or combinations thereof, and the one or more immunomodulators or further haptens are conjugated to the hC or covalently attached to one or more N- and / or C-termini of the helices of the oligomer.

[0138] 32. The GnRH conjugate or GnRH oligomer of any one of embodiments 20 to 31, wherein the one or more immunomodulators or further haptens comprise monophosphoryl lipid-A, squalene, lipopolysaccharide (LPS), lipoproteins, lipopeptides, APPHALS (SEQ ID NO: 58), kisspeptin peptides, kisspeptin receptors, or GnRH receptors.

[0139] 33. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 32, comprising an oligomeric scaffold peptide comprising the amino acid sequence SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69.

[0140] 34. The GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 33, wherein one or more GnRH peptides comprise the amino acid sequence SEQ ID NO: 70, or one or more GnRH peptides comprise the GnRH-TCE1 epitope comprising the amino acid sequence SEQ ID NO: 71.

[0141] 35. A composition comprising the GnRH conjugate or GnRH oligomer of any one of embodiments 1 to 34 and an excipient.

[0142] 36. The composition of embodiment 35, wherein the composition is a pharmaceutical composition and the excipient is a pharmaceutically acceptable excipient.

[0143] 37. A method for treating a subject having a disease, comprising administering to the subject in need thereof an effective amount of the conjugate of any one of embodiments 1 to 34 or the composition of embodiment 36, wherein the hapten induces an immune response to treat the subject.

[0144] 38. The method of embodiment 37, wherein the subject is a mammal.

[0145] 39. The method of embodiment 37 or 38, wherein the subject is a human.

[0146] 40. The method of any one of embodiments 37 to 39, wherein the disease is prostate cancer.

[0147] 41. A method for enhancing the immunogenicity of a GnRH peptide, comprising: Obtaining a monomeric peptide of any one of embodiments 1 to 34; allowing the monomeric peptides to self-assemble into hC; conjugating the GnRH peptide of any one of embodiments 1 to 34 to said hC to obtain a GnRH-hC conjugate. and

[0148] 42. The method of embodiment 41, wherein the GnRH-hC conjugate is a GnRH hexamer (GnRH-HhC) conjugate.

[0149] 43. A method for enhancing the immunogenicity of a GnRH peptide, comprising: synthesizing a GnRH monomer peptide (GMP), wherein the GMP comprises a monomer peptide of any one of embodiments 1 to 34 and a GnRH peptide of any one of embodiments 1 to 34; and allowing the GMP to self-assemble into GnRH-hC oligomers (GhC). and

[0150] 44. The method of embodiment 42, wherein the GnRH oligomer is a GnRH hexameric oligomer (GHhC).

[0151] 45. A method for preparing a GnRH therapeutic agent, comprising: Obtaining a monomeric peptide of any one of embodiments 1 to 34; allowing the monomeric peptides to self-assemble into hC; conjugating the GnRH peptide of any one of embodiments 1 to 34 to said hC to obtain a GnRH-hC conjugate. and

[0152] 46. ​​The method of embodiment 45, wherein the GnRH-hC conjugate is a GnRH-HhC conjugate.

[0153] 47. A method for preparing a GnRH therapeutic agent, comprising: Synthesizing a GnRH monomer peptide (GMP), wherein the GMP comprises a monomer peptide of any one of embodiments 1 to 34 and a GnRH peptide of any one of embodiments 1 to 34; A step of self-assembling the GMP into GnRH-hC oligomers (GhC) to obtain a GnRH therapeutic agent. and

[0154] 48. The method of embodiment 42, wherein the GnRH-hC oligomer is a GnRH hexameric oligomer (GHhC).

[0155] 49. A peptide comprising a GnRH peptide of any one of embodiments 1 to 34 and a monomeric peptide of any one of embodiments 1 to 34.

[0156] 50. The peptide of embodiment 49, comprising the amino acid sequence SEQ ID NO: 67.

[0157] 51. A peptide immunogen comprising the amino acid sequence SEQ ID NO:70 or SEQ ID NO:71.

[0158] 52. A monomeric peptide comprising a heptad comprising the amino acid sequence SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0159] 53. A monomeric peptide comprising the amino acid sequence SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. [Example]

[0160] Example 1 Development of GnRH therapeutic drugs Although there are 24 coupling sites on each hexameric carrier for hapten conjugation (Figure 1), conjugation is unlikely to occur at all sites due to steric hindrance. It has previously been shown that saturating the carrier with hapten does not necessarily produce the most robust immune response, and there is a trade-off between coupling density, epitope spatial / steric availability for accurate B cell epitope presentation, and antibody titer. Therefore, three separate hexameric conjugation reactions were performed to obtain conjugates with different epitope loading levels. For example, one reaction was performed using 3–5 molar equivalents of GnRH, resulting in the conjugation of only 3–4 peptides; another reaction contained 8–10 molar equivalents to form conjugates with 6–10 peptides; and the third reaction was performed using 25–50 molar equivalents to couple as many epitopes as possible (saturating conditions).

[0161] Figure 1 shows the GnRH conjugation procedure. GnRH was designed with an acetylated N-terminal residue to protect the N-terminal amine from derivatization with crosslinkers. Because native GnRH has a pI of 8.3 and the pI of the hexameric carrier is 10.1, the C-terminus contains an additional residue (GEDC) to adjust the pI, thereby increasing coupling efficiency by imparting a net negative charge on GnRH. The pI of QHWSYGLRPGGEDC (SEQ ID NO: 72) is 5.3. The C-terminal Cys residue was added for specific covalent binding via the cysteinyl sulfhydryl group. A Trp residue was present in native GnRH for fluorescence-based peptide quantification after coupling to determine reaction efficiency.

[0162] Tryptophan fluorescence, gel filtration chromatography, native PAGE, and SELDI-TOF (a MALDI-type MS instrument ideally suited for determining the molecular weight of protein-peptide conjugates) are methods used to quantify peptide epitope coupling efficiency. It is relatively easy to calculate the number of GnRH peptides conjugated to the hexameric carrier and to BSA (GnRH-BSA was used as a coating reagent in ELISA assays). KLH was used as a positive immunization control because it is an antigenic "gold-standard" hapten carrier. However, because KLH is so large, it may only be possible to confirm successful conjugation without calculating the exact number of conjugated peptides.

[0163] Example 2 Characterization of GnRH-hC conjugate constructs Adjuvants: Adjuvants were used for all immunizations to enhance adaptive B and T cell responses, modulate the degree of protective immunity, and maximize GnRH-specific antibody responses. The best adjuvants directly stimulate dendritic cell maturation, and the most effective way to achieve this is through TLR-mediated activation. Synthetic TLR-4-based adjuvants are some of the most effective, and therefore, at least two of these were tested. Monophosphoryl lipid A (MPL) is a potent TLR4 agonist that can serve as the primary adjuvant. MPL was emulsified with squalene (Sq) to form MPL-Sq. The emulsion efficiently primed CD4+ T cells, which are important for inducing both memory and long-lived GnRH antibody responses. The adjuvants E6020 and GLA, approved for use in humans, were also tested. All adjuvants can support CD4+-induced GnRH-hC conjugate uptake into dendritic cells and induce GnRH-hC conjugate-specific Th1 CD4+ T cells to bind T cell epitopes. To evaluate adjuvant function, CD4+ T cells and IgG isotype class switching were quantified in the serum of immunized mice. Another important benefit of adjuvants is the high potential for antigen dose-sparing, which is also examined. Dose-sparing can reduce the amount of GnRH-hC conjugate per immunization, increasing the number of doses that can be obtained from a synthetic peptide batch, and is an important factor in reducing the production cost of synthetic GnRH-hC conjugates.

[0164] At least three experiments were performed for each GnRH-hC conjugate. Mice were given a prime-boost immunization (IM), and B and T cell function was measured at several time points after immunization. Three dose levels of the GnRH-hC conjugate were compared to determine the level that yielded the maximum anti-GnRH IgG titer. Hexamers were maximally loaded with GnRH and formulated with MPL-Sq adjuvant before immunization. Three dose levels (e.g., 0.1 μg, 1 μg, and 10 μg of GnRH-hC) were tested and optimized according to the anti-GnRH IgG titer. This experiment also examined anti-GnRH IgG specificity by measuring IgG responses to hC alone, GnRH alone, and GnRH + hC (unconjugated but combined).

[0165] Immunization of mice with GnRH-hC conjugate: Inbred mice (10 mice / group) were primed / boosted with adjuvanted GnRH-hC conjugate or a control (GnRH-KLH conjugate). The first set of studies provided optimal GnRH-hC conjugate doses, and anti-GnRH IgG titers were measured at each dose level. Serum was collected 14 days after both prime and boost (d35) immunizations to measure antibody midpoint titers. Mouse blood was used to perform B and T cell assays.

[0166] B cell function: Treatment efficacy was measured by GnRH-specific antibody titers in collected mouse sera using a standard ELISA. ELISA plates were coated with GnRH-BSA conjugate and incubated with eight serial 10-fold dilutions of sera (1:10) in blocking buffer. 3 ~1:10 10 ) was prepared and added to ELISA plate wells. HRP-labeled anti-mouse secondary antibody was added, and the plate was developed with colorimetric substrate and read on an ELISA plate reader. Data were plotted, curve-fitted, and statistically analyzed using Prism Graph Pad software to calculate midpoint and endpoint titers.

[0167] T cell function: T cell epitope and adjuvant function were measured by well-established T cell ELISA assays. Commercially available coating reagents and primary / secondary antibodies were purchased and used according to the manufacturer's protocol. IFN-γ, IL-2, IL-4, and TNF-α were quantified in mouse serum as readouts of T cell function in mice immunized with GnRH-hC conjugates. These targets were expanded to include other markers of T cell function, including IL-5, IL-8, IL-10, IL-12p70, and IL-13. GnRH-hC conjugate-induced T cell-dependent isotype class switching and subclass switching were assayed by ELISA using reagents specific for total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA.

[0168] GnRH-hC conjugate safety: An initial safety assessment was performed in a non-GLP setting to ensure that mice did not have adverse reactions to the therapeutic drug components (GnRH-hC, adjuvant). Although more rigorous and detailed safety studies were later performed in GLP studies, this initial assessment provided several important readouts to guide therapeutic drug dose, adjuvant dose, and immunization scheduling. Potential local and systemic toxicity was assessed by observing injection site reactions and signs of inflammation, as well as mouse behavior (e.g., signs of lethargy). If toxicity was observed, different adjuvants and / or T cell epitopes were evaluated.

[0169] Example 3 Preparation of GnRH-hC conjugates and GHhC for use as therapeutic agents To prepare hapten-hC conjugates, peptide immunogens were covalently coupled to hexameric scaffold peptides after reduction of cysteinyl sulfhydryl groups with (tris(2-carboxyethyl)phosphine) and then incubated with the oligomeric scaffolds first activated with a heterobifunctional covalent crosslinker. The peptide immunogens included GnRH peptide and GnRH-TCE1 peptide. The hexameric scaffold peptides included GnRH-Hex-TCE2, TCE3-Hex-TCE4, and GnRH-Hex-GnRH. Their sequences are provided in Tables 1 and 2. The following four GnRH constructs (four GnRH-hC conjugates) were prepared from the peptide immunogens and hexameric scaffold peptides in Tables 1 and 2 for use as therapeutic agents. 1. GnRH-Hex-TCE2+GnRH-TCE1 2. TCE3-Hex-TCE4+GnRH 3. TCE3-Hex-TCE4+GnRH-TCE1 4. GnRH-Hex-GnRH+GnRH-TCE1 GnRH construct 5 (GHhC) is a scaffold peptide obtained by attaching GnRH peptide and TCE2 to the N- and C-termini of a monomeric peptide (see Table 1) during SPPS, followed by assembly of the monomeric peptide. 5. GnRH-Hex-TCE2

[0170] Keyhole limpet hemocyanin (KLH) was chosen for use as a control because it is routinely used as a carrier protein for haptens in the production of antibodies. The following two constructs (conjugates) were prepared for use as controls: 6. KLH+GnRH 7. KLH+GnRH-TCE1

[0171] Constructs 1-7 (1 μg and 10 μg) were adjuvanted with a monophosphoryl lipid A / squalene-based adjuvant and injected into BALB / cJ mice. Each construct was injected into five mice (n=5 per construct). After 14, 28, and 42 days, mouse sera were obtained and analyzed using 10 -3 ~10 -9 Anti-GnRH IgG endpoint titers were measured by ELISA using dilutions (serial 10-fold dilutions). To confirm therapeutic function, testosterone was measured in aliquots of serum from three groups: the two constructs with the highest antibody titers (TCE3-Hex-TCE4 + GnRH and GnRH-Hex-GnRH + GnRH-TCE1 (construct 4)), a positive KLH control (KLH + GnRH), and a PBS-negative control (PBS + adjuvant). Testosterone was extracted from serum, derivatized, and separated and quantified by LC-MS / MS.

[0172] Figure 3 shows antibody endpoint titers in mice immunized with five different GnRH therapeutics (constructs 1-5), three positive controls (constructs 6 and 7 and ReproBloc, a GnRH subunit vaccine used in veterinary applications), and one negative control (PBS + adjuvant) 14, 28, and 42 days after prime immunization. The solid line in the group of dots represents the geometric mean, and the error bars represent the geometric standard deviation. Two doses were selected for this study: 1 μg and 10 μg. IgG titers reached maximum levels 42 days after prime immunization. Three of the five GnRH constructs reached maximum titers at 1 μg. One construct had equivalent antibody titers at both 1 μg and 10 μg (TCE3-Hex-TCE4 + GnRH (construct 2)). This same construct induced titers at approximately equal levels between days 28 and 42 (with no significant increase seen at day 42).

[0173] Construct 2 (TCE3-Hex-TCE4 + GnRH), construct 4 (GnRH-Hex-GnRH + GnRH-TCE1), and construct 5 were selected because they had the highest titers or induced high titers more quickly, and their therapeutic function was confirmed by measuring testosterone levels (Figure 3). Testosterone (T) levels in mice immunized with construct 2 or construct 4, one positive control (KLH + GnRH (construct 6)), and one negative control (PBS control) were analyzed and quantified using LC-MS / MS. Figures 4-6 show the testosterone levels in each mouse and the corresponding antibody titer data (shown in Figure 3). IgG titers and T levels for each mouse are represented individually. T levels were measured on d14, d28, and d42 to determine the time points at which T castrate levels were observed. The castrate T levels are indicated by the dotted line. Figure 6 shows that by day 42, anti-GnRH IgG titers for both constructs induced attenuated levels of T, resulting in antibody titers and T levels similar to the KLH+GnRH positive control.

[0174] Figures 4 to 6 confirm that GnRH-HhC conjugates, such as TCE3-Hex-TCE4+GnRH and GnRH-Hex-GnRH+GnRH_TCE1, are nearly equivalent to KLH+GnRH (positive control) in inducing IgG titers and attenuated levels of T, and are useful as therapeutic agents for treating diseases such as prostate cancer.

[0175] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that skilled artisans will employ such variations as appropriate, and the present inventions intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by circumstances.

[0176] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, those skilled in the art will recognize that various modifications, substitutions, omissions, and changes may be made without departing from the spirit of the invention.

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Claims

1. a scaffold peptide comprising one or more gonadotropin-releasing hormone (GnRH) peptides covalently attached to a monomeric peptide; or A GnRH conjugate (GnRH-hC) or GnRH oligomer (GhC) comprising one or more gonadotropin-releasing hormone (GnRH) peptides covalently bound to a hapten carrier (hC), wherein the hC comprises an oligomer comprising a monomeric peptide. And, The monomeric peptide has the following amino acid sequence: (hwxhxyz)n (SEQ ID NO: 2) (In the formula, h is I, L, V, F, W, Y, M, W, G, or A; w is G, R, A, N, Q, H, S, D, E, or T; x is R, S, N, Q, A, G, T, D, E, H, or C; y is K, C, D, E, or R; z is A, D, H, S, E, R, N, Q, or G; n is 2 to 10) and / or the monomeric peptide comprises the amino acid sequence SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:18; and / or the monomeric peptide comprises the amino acid sequence SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:25; peptides, or GnRH conjugates (GnRH-hC) or GnRH oligomers (GhC).

2. The one or more GnRH peptides have the amino acid sequence: X 1 HWSX 2 GX 3 X 4 PG (SEQ ID NO: 28) (In the formula, X 1 is a polar or charged amino acid, X 2 is a polar amino acid, X 3 is a hydrophobic or amphipathic amino acid, X 4 is a hydrophobic, amphipathic, charged or polar amino acid and / or The one or more GnRH peptides have the amino acid sequence SEQ ID NO:28 (In the formula, X 1 is Q or E, X 2 is Y or H, X 3 is L or W, or X 4 is L, Y, R, or Q) and / or The one or more GnRH peptides have the amino acid sequence: QHWSYGLRPG (SEQ ID NO: 29), QHWSHGWLPG (SEQ ID NO: 30), EHWSYGLRPG (SEQ ID NO: 31), QHWSYGWYPG (SEQ ID NO: 32), EHWSYGLQPG (SEQ ID NO: 33), or QHWSHGWYPG (SEQ ID NO: 34) Including, 2. A peptide according to claim 1, or a GnRH conjugate (GnRH-hC) or a GnRH oligomer (GhC) according to claim 1.

3. one or more GnRH peptides contain G, V, M, A, or a combination thereof at the N- and / or C-terminus; and / or one or more GnRH peptides further comprising GEDC (SEQ ID NO: 36) or DGEGC (SEQ ID NO: 37) at the C-terminus; 3. A peptide according to claim 1 or 2, or a GnRH conjugate (GnRH-hC) or a GnRH oligomer (GhC) according to claim 1 or 2.

4. A peptide described in any one of claims 1 to 3, or a GnRH conjugate (GnRH-hC) or GnRH oligomer (GhC) described in any one of claims 1 to 3, wherein the peptide comprises two or more GnRH peptides, the GnRH peptides being obtained from different sources and / or comprising two or more different amino acid sequences selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO:

34.

5. 5. The peptide of claim 1, wherein the peptide further comprises an immunomodulator or hapten; and / or the immunomodulator or hapten is a T-cell epitope or a B-cell epitope.

6. A peptide described in any one of claims 1 to 5, or a GnRH conjugate (GnRH-hC) or GnRH oligomer (GhC) described in any one of claims 1 to 4, wherein the peptide comprises the amino acid sequence SEQ ID NO: 67 or SEQ ID NO:

69.

7. 7. The GnRH conjugate (GnRH-hC) or GnRH oligomer (GhC) according to any one of claims 1 to 4 and 6, wherein the oligomer comprises a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

8. 8. A GnRH conjugate or GnRH oligomer according to any one of claims 1 to 4, 6 and 7, wherein one or more GnRH peptides are conjugated to hC via y residues on the monomeric peptides.

9. the GnRH conjugate or GnRH oligomer further comprises one or more immunomodulatory substances or haptens; and / or the GnRH conjugate or GnRH oligomer comprises one or more spacers between the hapten or immunomodulator and the monomeric peptide, the spacers comprising G (glycine), D (aspartic acid), S (serine), C (cysteine), or a combination thereof; and / or the one or more haptens or immunomodulators comprise a GnRH peptide, one or more T cell epitopes, and / or one or more B cell epitopes; optionally, the one or more T cell epitopes include a CD4+ T cell epitope; and / or the GnRH conjugate or GnRH oligomer further comprises one or more residues for correct processing of one or more T cell epitopes; and / or one or more residues include D, G, P, S, GD, PGP, GSG, GPGP (SEQ ID NO: 63), GPGPG (SEQ ID NO: 64), GPGPGC (SEQ ID NO: 65), or SGPGPG (SEQ ID NO: 66); A GnRH conjugate or GnRH oligomer according to any one of claims 1 to 4 and 6 to 8.

10. 10. The GnRH conjugate or GnRH oligomer of claim 9, wherein the one or more additional haptens or immunomodulators comprise lipids, peptides, nucleic acids, or combinations thereof, and the one or more additional haptens or immunomodulators are conjugated to the hC or covalently attached to one or more N- and / or C-termini of the helices of the oligomer.

11. the GnRH conjugate or GnRH oligomer comprises a scaffold peptide comprising the amino acid sequence SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69; and / or The GnRH conjugate or GnRH oligomer comprises a peptide immunogen comprising the amino acid sequence SEQ ID NO: 70 or SEQ ID NO: 71; A GnRH conjugate or GnRH oligomer according to any one of claims 1 to 4 and 6 to 10.

12. A composition comprising a peptide, GnRH conjugate or GnRH oligomer according to any one of claims 1 to 11 and an excipient.

13. 13. The composition of claim 12, wherein the composition is a pharmaceutical composition and the excipient is a pharmaceutically acceptable excipient.

14. 14. A peptide according to any one of claims 1 to 6, a GnRH conjugate or GnRH oligomer according to any one of claims 1 to 4 and 6 to 11, or a composition according to claim 13, for use in a method for treating a subject having a disease, the method comprising administering to a subject in need thereof an effective amount of the peptide, GnRH conjugate, GnRH oligomer, or composition, wherein a hapten induces an immune response to treat the subject.

15. the subject is a mammal, optionally the mammal is a human; or the disease is prostate cancer, 15. The peptide, GnRH conjugate, GnRH oligomer, or composition of claim 14.

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