Peptide immunogens targeting calcitonin gene-related peptide (CGRP) and their formulations for the prevention and treatment of migraines.

Abstract

This disclosure relates to a peptide immunogen construct targeting a portion of the calcitonin gene-related peptide (CGRP), compositions containing the construct, antibodies induced by the construct, and methods for preparing and using the construct and compositions thereof. The disclosed peptide immunogen construct has more than about 30 amino acids and contains (a) a B-cell epitope having about 7 or more consecutive amino acid residues from the CGRP receptor-binding or activating region of the full-length CGRP protein, (b) a heterologous Th epitope, and (c) an optional heterologous spacer region. The disclosed CGRP peptide immunogen construct can stimulate the production of highly specific antibodies against CGRP for the prevention and / or treatment of migraines.

Description

[0001] This application is a PCT international application that claims priority to U.S. Provisional Application Serial No. 62 / 787,102, filed on December 31, 2018 (the entire contents of which are incorporated herein by reference). [Technical Field]

[0002] This disclosure relates to a peptide immunogen construct and its formulation targeting calcitonin gene-related peptide (CGRP) for the prevention and treatment of migraines. [Background Technology]

[0003] Migraine is a common physical condition affecting up to 37 million people in the United States. It is considered a systemic condition, not just a headache. Recent research suggests that changes in the brain can begin as early as 24 hours before migraine symptoms appear. Migraine symptoms vary from person to person but may include severe throbbing headache (usually only on one side of the head), nausea, vomiting, photophobia, phonophobia, or a combination of these symptoms. These symptoms can persist even after the headache has subsided.

[0004] Migraines have many subtypes, and symptoms include weakness, numbness, visual changes or loss, dizziness, and difficulty speaking (some patients may appear to have a stroke). The incapacity caused by this chronic condition can be very serious, leading to disruption of the workday and loss of the ability to participate in family activities.

[0005] Sometimes people may use "abortive" medications, which can stop migraine attacks when taken early. For many patients, preventative medications can reduce the frequency and severity of migraines. However, many medications used to prevent or treat migraines were primarily developed for other conditions such as seizures, depression, high blood pressure, and muscle spasms.

[0006] Researchers have been working for decades to develop targeted preventative therapies specifically for migraines. Calcitonin gene-related peptide (CGRP) is a molecule synthesized in peripheral and central neurons. It is involved in various pain processes, including migraines, and acts as a vasodilator. Aphonic drug treatments for migraines focus on blocking CGRP activation at the onset of a migraine. Small molecule CGRP antagonist drugs have been shown to alleviate migraines based on certain measures, but these antagonists may have serious side effects, including hepatotoxicity.

[0007] Monoclonal antibodies targeting CGRP molecules have an inhibitory effect on the pain process and can be used as abstinence medication. Monoclonal antibodies against CGRP can have a long half-life, meaning they can be administered less frequently compared to conventional daily migraine medications (except for botulinum toxin, which is injected every 90 days). Monoclonal antibodies for migraines can be administered subcutaneously once a month and have so far demonstrated a statistically significant reduction in the number of migraine days. Several different pharmaceutical companies are developing these new antibodies for FDA approval.

[0008] While monoclonal anti-CGRP or anti-CGRP receptor antibodies have demonstrated efficacy in the immunotherapy of migraines, they are expensive and require monthly administration to maintain adequate suppression of serum and humoral CGRP levels and thus the resulting clinical benefits. Cost-effective immunotherapy targeting CGRP molecules via safe and well-tolerated vaccination methods remains an exciting new intervention and development for migraine treatment.

[0009] Typical peptide / hapten-carrier protein immunogen preparation methods have many drawbacks and limitations. For example, these methods involve complex chemical coupling steps, use expensive pharmaceutical-grade KLH or toxoid proteins as T helper cell carriers, and most antibodies induced by these protein immunogens target the carrier protein rather than the target B cell epitopes.

[0010] Given the economic and practical drawbacks and limitations of monoclonal antibody therapy and typical peptide / hapten-carrier protein formulations, there is clearly still an unmet need for effective immunotherapeutic compositions that can elicit a highly specific immune response against functional sites on CGRP. These compositions must be easy for patients to use, be manufactured according to strict Good Manufacturing Practices (GMP), and be cost-effective for global application in the treatment of migraine patients.

[0011] Two literature reviews citing other supporting literature can be found for the statements in the background section above, which are incorporated herein by reference in their entirety. The first review contains the latest comments on CGRP and the CGRP receptor (website: en.wikipedia.org / wiki / Calcitonin_gene-related_peptide), while the second review explores the biological characteristics of CGRP signaling, important clinical evidence for the role of CGRP in migraine (including the efficacy of CGRP-targeted therapy), the role of CGRP in the trigeminal vascular system, and new insights into the important role of the trigeminal ganglion in the pathophysiology of migraine (Edvinsson, et al., 2018).

[0012] References

[0013] 1. CHANG, J.C.C., et al., “Adjuvant activity of incomplete Freund's adjuvant,” Advanced Drug Delivery Reviews, 32(3): 173 - 186 (1998)

[0014] 2. “Calcitonin gene - related peptide,” Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / wiki / Calcitonin_gene - related_peptide (accessed December 30, 2018).

[0015] 3. EDVINSSON, L., et al., “CGRP as the target of new migraine therapies - successful translation from bench to clinic”, Nat.Rev.Neurol., 14(6): 338 - 350 (2018)

[0016] 4. FIELDS, G.B., et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W.H. Freeman & Co., New York, NY, p.77 (1992).

[0017] 5. RUSSELL, F.A., et al., “Calcitonin gene - related peptide: physiology and pathophysiology”, Physiol.Rev., 94(4): 1099 - 1142 (2014)

[0018] 6. TAJTI, J., et al., “Messenger molecules and receptor mRNA in the human trigeminal ganglion”, J.Auton.Nerv.Syst.2(8); 76(2 - 3): 176 - 83 (1999)

[0019] 7.TRAGGIAI, E., et al., "An efficient method to make human monoclonalantibodies from memory B cells: potential neutralization of SARS coronavirus", Nature Medicine, 10: 871-875 (2004).

[0020] 8. WATKINS, HA, et al., "Structure-activity relationships for a-calcitonin gene-related peptide", Br. J. Pharmacol., 170(7): 1308-22 (2013). [Summary of the Invention]

[0021] This disclosure relates to a portion of calcitonin gene-related peptide (CGRP) as a B-cell epitope. This disclosure also relates to a peptide immunogen construct containing a B-cell epitope derived from CGRP, a composition containing this peptide immunogen construct, a method for preparing and using this peptide immunogen construct, and an antibody prepared using this peptide immunogen construct.

[0022] One aspect of this disclosure relates to a portion of CGRP derived from different organisms as a B-cell epitope in peptide immunogen constructs and formulations thereof for the prevention and / or treatment of migraines. The disclosed CGRP peptide immunogen constructs (SEQ ID NOs: 116-127 and 130-180) have 30 or more total amino acids and contain a functional B-cell epitope peptide (the functional B-cell epitope peptide has about 7 to about 30 amino acids derived from CGRP derived from humans, marmosets, or rats / mice (i.e., SEQ ID NOs: 1-3, respectively) (SEQ ID NOs: 4-13, 15-19, and 20-24 of Table 1). The functional B-cell epitope peptide can be linked via an optional heterologous spacer region to a heterologous T helper cell (Th) epitope peptide derived from a pathogen protein (e.g., SEQ ID NOs: 74-115) to form the disclosed peptide immunogen construct.

[0023] The disclosed CGRP peptide immunogen constructs may contain CGRP B-cell epitope peptides having approximately 7 to approximately 30 amino acids. The B-cell epitope peptides may be derived from the CGRP receptor-binding region located at the carboxyl terminus and middle region of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22 shown in Table 1). The B-cell epitope peptides may also be derived from the CGRP receptor activation site near the cyclic C2-C7 ring structure located at the N-terminus and middle region of the CGRP molecule (e.g., SEQ ID NOs: 4, 10-13, and 23-24 shown in Table 1). The designed CGRP B-cell epitope peptides may be linked to heterologous Th epitopes derived from pathogen proteins (e.g., SEQ ID NOs: 74-115 shown in Table 2) at the N-terminus or carboxyl terminus of the CGRP peptide. The B cells and Th epitopes work together to stimulate the production of highly specific antibodies that cross-react with full-length CGRP (SEQ ID NOs: 1-3) from various species.

[0024] In some embodiments, the heterologous Th epitopes used to enhance CGRP B cell epitope peptides are derived from natural pathogens EBV BPLF1 (SEQ ID NO: 112), EBV CP (SEQ ID NO: 109), Clostridium tetani (SEQ ID NOs: 74, 77, 104, 106-108), cholera toxin (SEQ ID NO: 81), and Schistosoma mansoni (SEQ ID NO: 80), as well as idealized artificial Th epitopes derived from measles virus fusion proteins (MVF 1 to 5) and hepatitis B surface antigen (HBsAg 1 to 3), which are present in the form of single sequences or combinations of sequences (e.g., SEQ ID NOs: 75, 82-99).

[0025] The publicly disclosed CGRP peptide immunogen construct contains designed B-cell and Th epitope peptides that work together to stimulate the production of highly specific antibodies against functional sites of CGRP (including the CGRP receptor-binding region located at the carboxyl terminus of the CGRP molecule or the cyclic C2-C7 ring structure involved in receptor activation), providing a therapeutic immune response to patients susceptible to or suffering from migraines.

[0026] Another aspect of this disclosure relates to peptide compositions containing CGRP peptide immunogen constructs. In some embodiments, the composition comprises a single peptide immunogen construct. In other embodiments, the peptide composition comprises a mixture of CGRP peptide immunogen constructs. In some embodiments, the mixture of CGRP peptide immunogen constructs has heterologous Th epitopes derived from various pathogens, which can be used to allow coverage of a broad genetic background in patients, resulting in a higher percentage of response rates after immunization, for the prevention and / or treatment of migraines.

[0027] A synergistic enhancement of the CGRP immunogenic construct can be observed in the peptide compositions disclosed herein. Antibody responses derived from these compositions containing CGRP peptide immunogenic constructs are predominantly (>90%) focused on desired cross-reactivity against CGRP functional sites or receptor-binding domain peptides (SEQ ID NOs: 4-13 and 15-24), with less, if any, targeting of heterologous Th epitopes for immunogenic enhancement. This contrasts sharply with standard approaches using conventional carrier proteins (e.g., KLH, toxoids, or other biological carriers for such peptide antigenic enhancement).

[0028] This disclosure also relates to pharmaceutical compositions and formulations for the prevention and / or treatment of migraines. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex formed by electrostatic binding of a peptide composition containing a mixture of CpG oligomers and a CGRP peptide immunogen construct, to further enhance the immunogenicity of the CGRP peptide, which has desired cross-reactivity with full-length CGRP (e.g., SEQ ID NOs: 1-3).

[0029] In other embodiments, the pharmaceutical composition comprises contact with a mineral salt (including aluminum gel or aluminum phosphate) to form a suspension dosage form or with MONTANIDE as an adjuvant. TM A peptide composition comprising a mixture of CGRP peptide immunogen constructs for contact with ISA 51 or 720 to form a water-in-oil emulsion, for the prevention and / or treatment of migraines.

[0030] In addition, this disclosure provides a method for the low-cost preparation and quality control of CGRP peptide immunogen constructs and formulations thereof, which can be used in animals for the prevention and / or treatment of migraines.

[0031] This disclosure also relates to antibodies against the disclosed CGRP peptide immunogen construct. Specifically, the disclosed CGRP peptide immunogen construct is capable of stimulating the production of highly specific functional antibodies that cross-react with the full-length CGRP molecule. The antibodies of this disclosure utilize highly specific binding to CGRP, and, if any, target heterologous Th epitopes for immunogenicity enhancement, in stark contrast to antibodies prepared using conventional proteins or other biological carriers for enhancing the antigenicity of such peptides. Therefore, compared to other peptide or protein immunogens, the disclosed CGRP peptide immunogen construct can disrupt immune tolerance to autologous CGRP and exhibits a high response rate.

[0032] In some embodiments, when the peptide immunogen construct is administered to an individual, the disclosed antibody targets and specifically binds to the CGRP receptor binding site located at the carboxyl terminus of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22). The highly specific antibodies elicited by these CGRP peptide immunogen constructs can inhibit the binding of CGRP and its receptor, as well as downstream activation events (an increase in cellular cAMP caused by the region near the C2-C7 ring structure of CGRP), leading to effective prevention and / or treatment of migraines.

[0033] In other embodiments, when the peptide immunogen constructs of the present invention (e.g., SEQ ID NOs: 4, 10-13, and 23-24) are administered to an individual, the disclosed antibodies target the N-terminal or intermediate region of CGRP located near the cyclic C2-C7 ring structure responsible for downstream cellular activation events, or the C-terminal and intermediate region of the CGRP receptor binding site. The highly specific antibodies elicited by the CGRP peptide immunogen constructs can inhibit (1) the binding of CGRP to the CGRP receptor, and (2) downstream activation events caused by the region near the cyclic C2-C7 ring structure of CGRP, leading to inhibition of cellular cAMP elevation and thus resulting in effective treatment for migraine patients.

[0034] Based on their unique characteristics and properties, open antibodies induced by CGRP peptide immunogen constructs can provide preventative immunotherapy for patients suffering from migraines.

[0035] In another aspect, the present invention provides human monoclonal antibodies against CGRP, which are induced in patients receiving a composition containing an immunogen construct of the CGRP peptide disclosed herein. Traggiai, E., et al., 2004 describes an efficient method for preparing human monoclonal antibodies from B cells isolated from the blood of human patients, which is incorporated herein by reference.

[0036] This disclosure also relates to methods for preparing the disclosed CGRP peptide immunogen construct, composition, and antibody. The disclosed methods provide low-cost preparation and quality control of the CGRP peptide immunogen construct and compositions containing the construct, which can be used in methods for treating patients suffering from migraines.

[0037] This disclosure also includes methods for prevention and / or treatment in individuals susceptible to or suffering from migraines using the disclosed CGRP peptide immunogenic construct and / or antibodies against the CGRP peptide immunogenic construct. Methods for preventing and / or treating migraines in an individual include administering to the individual a composition containing the disclosed CGRP peptide immunogenic construct. In some embodiments, the composition used in the method contains the disclosed CGRP peptide immunogenic construct, which forms a stable immunostimulatory complex by electrostatic binding to a negatively charged oligonucleotide (e.g., a CpG oligomer), which may be further added with an adjuvant for administration to a patient suffering from migraines.

[0038] The disclosed methods also include dosing regimens, dosage forms, and routes of administration for administering the CGRP peptide immunogen construct to prevent and / or treat migraines in individuals.

[0039] [Simplified Explanation of the Diagram]

[0040] Figure 1 shows CGRP sequence alignments from multiple species, including humans (SEQ ID NO: 1), mice (SEQ ID NO: 3), rats (SEQ ID NO: 3), marmosets (SEQ ID NO: 2), and many other species (including horses, chickens, pigs, sheep, cattle, dogs, opossums, geckos, frogs, pufferfish, halibut, goldfish, salmon, medaka, and zebrafish). This diagram is adapted from Watkins, et al., 2013.

[0041] Figure 2 illustrates the pathway from discovery to commercialization of high-precision CGRP-designed peptide immunogen constructs and their formulations for migraine treatment.

[0042] Figure 3 illustrates the immunogenicity study results of CGRP peptide immunogenic constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-122, 139, 123, 130) in guinea pigs (these CGRP peptide immunogenic constructs have CGRP B cell epitope peptides derived from the N-terminal, intermediate, and C-terminal regions of the CGRP molecule).

[0043] Figure 4 illustrates the neutralizing activity of purified antibodies in CGRP-activated cell cultures in the presence of guinea pig immune serum (collected from 6-week blood samples) from representative CGRP peptide immunogen constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-123, 130, 139, and 150). Neutralizing activity is expressed as IC50 of intracellular cAMP production. 50 .

[0044] Figure 5 shows the immunogenicity of representative CGRP peptide immunogen constructs (SEQ ID NOs: 123, 131, 141, 151) formulated in ADJUPHOS and ISA51 formulations in Balb / C mice. The corresponding antibodies purified from guinea pig immune serum collected at 7wpi, 10wpi, and 13wpi were reactive and cross-reactive with full-length human CGRP molecules.

[0045] Figure 6 shows the inhibitory effect on capsaicin-induced skin blood flow measured in Balb / C mice immunized at 0, 3, and 6 wpi using CGRP peptide immunogen constructs (SEQ ID NOs: 123, 131, 141) formulated in the ADJUPHOS or ISA emulsions as shown, compared with controls alone or saline.

[0046]

Implementation Method

[0047] This disclosure relates to a portion of calcitonin gene-related peptide (CGRP) as a B-cell epitope. This disclosure also relates to a peptide immunogen construct containing a B-cell epitope derived from CGRP, a composition containing this peptide immunogen construct, a method for preparing and using this peptide immunogen construct, and an antibody prepared using this peptide immunogen construct.

[0048] One aspect of this disclosure relates to a portion of CGRP derived from different organisms as a B-cell epitope in peptide immunogen constructs and formulations thereof for the prevention and / or treatment of migraines. The disclosed CGRP peptide immunogen constructs (SEQ ID NOs: 116-127 and 130-180) have 30 or more total amino acids and contain a functional B-cell epitope peptide (the functional B-cell epitope peptide has about 7 to about 30 amino acids derived from CGRP derived from humans, marmosets, or rats / mice (i.e., SEQ ID NOs: 1-3, respectively) (SEQ ID NOs: 4-13, 15-19, and 20-24 of Table 1). The functional B-cell epitope peptide can be linked via an optional heterologous spacer region to a heterologous T helper cell (Th) epitope peptide derived from a pathogen protein (e.g., SEQ ID NOs: 74-115) to form the disclosed peptide immunogen construct.

[0049] The disclosed CGRP peptide immunogen constructs may contain CGRP B-cell epitope peptides having approximately 7 to approximately 30 amino acids. The B-cell epitope peptides may be derived from the CGRP receptor-binding region located at the carboxyl terminus and middle region of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22 shown in Table 1). The B-cell epitope peptides may also be derived from the CGRP receptor activation site near the cyclic C2-C7 ring structure located at the N-terminus and middle region of the CGRP molecule (e.g., SEQ ID NOs: 4, 10-13, 20-22 shown in Table 1). The designed CGRP B-cell epitope peptides may be linked to heterologous Th epitopes derived from pathogen proteins (e.g., SEQ ID NOs: 74-115 shown in Table 2) at the N-terminus or carboxyl terminus of the CGRP peptide. The B cells and Th epitopes work together to stimulate the production of highly specific antibodies that cross-react with full-length CGRP (SEQ ID NOs: 1-3) from various species.

[0050] In some embodiments, the heterologous Th epitopes used to enhance CGRP B cell epitope peptides are derived from natural pathogens EBV BPLF1 (SEQ ID NO: 112), EBV CP (SEQ ID NO: 109), Clostridium tetani (SEQ ID NOs: 74, 77, 104, 106-108), cholera toxin (SEQ ID NO: 81), and Schistosoma mansoni (SEQ ID NO: 80), as well as idealized artificial Th epitopes derived from measles virus fusion proteins (MVF 1 to 5) and hepatitis B surface antigen (HBsAg 1 to 3), which are present in the form of single sequences or combinations of sequences (e.g., SEQ ID NOs: 75, 82-99).

[0051] The publicly disclosed CGRP peptide immunogen construct contains designed B-cell and Th epitope peptides that work together to stimulate the production of highly specific antibodies against functional sites of CGRP (including the CGRP receptor-binding region located at the carboxyl terminus of the CGRP molecule or the cyclic C2-C7 ring structure involved in receptor activation), providing a therapeutic immune response to patients susceptible to or suffering from migraines.

[0052] Another aspect of this disclosure relates to peptide compositions containing CGRP peptide immunogen constructs. In some embodiments, the composition comprises a single peptide immunogen construct. In other embodiments, the peptide composition comprises a mixture of CGRP peptide immunogen constructs. In some embodiments, the mixture of CGRP peptide immunogen constructs has heterologous Th epitopes derived from various pathogens, which can be used to allow coverage of a broad genetic background in patients, resulting in a higher percentage of response rates after immunization, for the prevention and / or treatment of migraines.

[0053] Synergistic enhancement of the CGRP immunogenic construct can be observed in the peptide compositions disclosed herein. Antibody responses derived from these compositions containing CGRP peptide immunogenic constructs are predominantly (>90%) focused on desired cross-reactivity against CGRP functional sites or receptor-binding domain peptides (SEQ ID NOs: 4-13, 15-19, and 20-24), with less, if any, targeting of heterologous Th epitopes for immunogenic enhancement. This contrasts sharply with standard approaches using conventional carrier proteins (e.g., KLH, toxoids, or other biological carriers for such peptide antigenic enhancement).

[0054] This disclosure also relates to pharmaceutical compositions and formulations for the prevention and / or treatment of migraines. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex formed by electrostatic binding of a peptide composition containing a mixture of CpG oligomers and a CGRP peptide immunogen construct, to further enhance the immunogenicity of the CGRP peptide, which has desired cross-reactivity with full-length CGRP (e.g., SEQ ID NOs: 1-3).

[0055] In other embodiments, the pharmaceutical composition comprises contact with a mineral salt (including aluminum gel or aluminum phosphate) to form a suspension dosage form or with MONTANIDE as an adjuvant. TM A peptide composition comprising a mixture of CGRP peptide immunogen constructs for contact with ISA 51 or 720 to form a water-in-oil emulsion, for the prevention and / or treatment of migraines.

[0056] In addition, this disclosure provides a method for the low-cost preparation and quality control of CGRP peptide immunogen constructs and formulations thereof, which can be used in animals for the prevention and / or treatment of migraines.

[0057] This disclosure also relates to antibodies against the disclosed CGRP peptide immunogen construct. Specifically, the disclosed CGRP peptide immunogen construct is capable of stimulating the production of highly specific functional antibodies that cross-react with the full-length CGRP molecule. The antibodies of this disclosure utilize highly specific binding to CGRP, and, if any, target heterologous Th epitopes for immunogenicity enhancement, in stark contrast to antibodies prepared using conventional proteins or other biological carriers for enhancing the antigenicity of such peptides. Therefore, compared to other peptide or protein immunogens, the disclosed CGRP peptide immunogen construct can disrupt immune tolerance to autologous CGRP and exhibits a high response rate.

[0058] In some embodiments, when the peptide immunogen construct is administered to an individual, the disclosed antibody targets and specifically binds to the CGRP receptor binding site located at the carboxyl terminus of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22). The highly specific antibodies elicited by these CGRP peptide immunogen constructs can inhibit the binding of CGRP and its receptor, as well as downstream activation events (an increase in cellular cAMP caused by the region near the C2-C7 ring structure of CGRP), leading to effective prevention and / or treatment of migraines.

[0059] In other embodiments, when the peptide immunogen constructs of the present invention (e.g., SEQ ID NOs: 4, 10-13, and 23-24) are administered to an individual, the disclosed antibodies target the N-terminal or intermediate region of CGRP located near the cyclic C2-C7 ring structure responsible for downstream cellular activation events, or the C-terminal and intermediate region of the CGRP receptor binding site. The highly specific antibodies elicited by the CGRP peptide immunogen constructs can inhibit (1) the binding of CGRP to the CGRP receptor, and (2) downstream activation events caused by the region near the cyclic C2-C7 ring structure of CGRP, leading to inhibition of cellular cAMP elevation and thus resulting in effective treatment for migraine patients.

[0060] Based on their unique characteristics and properties, open antibodies induced by CGRP peptide immunogen constructs can provide preventative immunotherapy for patients suffering from migraines.

[0061] In another aspect, the present invention provides human monoclonal antibodies against CGRP, which are induced in patients receiving a composition containing an immunogen construct of the CGRP peptide disclosed herein. Traggiai, E., et al., 2004 describes an efficient method for preparing human monoclonal antibodies from B cells isolated from the blood of human patients, which is incorporated herein by reference.

[0062] This disclosure also relates to methods for preparing the disclosed CGRP peptide immunogen construct, composition, and antibody. The disclosed methods provide low-cost preparation and quality control of the CGRP peptide immunogen construct and compositions containing the construct, which can be used in methods for treating patients suffering from migraines.

[0063] This disclosure also includes methods for prevention and / or treatment in individuals susceptible to or suffering from migraines using the disclosed CGRP peptide immunogenic construct and / or antibodies against the CGRP peptide immunogenic construct. Methods for preventing and / or treating migraines in an individual include administering to the individual a composition containing the disclosed CGRP peptide immunogenic construct. In some embodiments, the composition used in the method contains the disclosed CGRP peptide immunogenic construct, which forms a stable immunostimulatory complex by electrostatic binding to a negatively charged oligonucleotide (e.g., a CpG oligomer), which may be further added with an adjuvant for administration to a patient suffering from migraines.

[0064] The disclosed methods also include dosing regimens, dosage forms, and routes of administration for administering the CGRP peptide immunogen construct to prevent and / or treat migraines in individuals.

[0065] General Rules

[0066] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application are expressly incorporated herein by reference in their entirety for any purpose.

[0067] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless the context clearly indicates otherwise, the words “a,” “an,” and “the” are included in the plural form. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Thus, “including A or B” means including A, or B, or A and B. It should be understood that all amino acid sizes and all molecular weight or molecular mass values ​​for a given polypeptide are approximate and are provided for descriptive purposes. However, similar or equivalent methods and materials described herein may be used in the practice or testing of the methods, suitable methods, and materials disclosed below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including the interpretation of terms) shall prevail. Furthermore, the materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0068] CGRP peptide immunogen construct

[0069] This disclosure provides a peptide immunogen construct containing a B-cell epitope peptide having an amino acid sequence derived from CGRP (SEQ ID NOs: 1-3) or a fragment thereof. The CGRP peptide immunogen construct may contain a CGRP B-cell epitope peptide having about 7 to about 30 amino acids. The B-cell epitope peptide may be derived from (1) a CGRP receptor-binding region located at the carboxyl terminus / intermediate region of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22 shown in Table 1); or (2) a CGRP receptor activation site located near a cyclic C2-C7 ring structure located at the amino terminus / intermediate region of the CGRP molecule (e.g., SEQ ID NOs: 4, 10-13 and 23-24 shown in Table 1). The B-cell epitope is covalently linked, directly or via an optional heterologous spacer region, to a heterologous T helper cell (Th) epitope derived from a pathogen protein (e.g., SEQ ID NOs: 74-115 in Table 2). These structures (containing designed B-cell and Th-cell epitopes working together) stimulate the production of highly specific antibodies that cross-react with full-length CGRP (SEQ ID NO: 1-3) from various species.

[0070] As used herein, the term “CGRP peptide immunogen construct” or “peptide immunogen construct” refers to a peptide having approximately 30 or more amino acids, containing (a) a B cell epitope having approximately 7 or more consecutive amino acid residues derived from full-length CGRP (SEQ ID NOs: 1-3); (b) a heterologous Th epitope; and (c) an optional heterologous spacer region.

[0071] In some implementations, the CGRP peptide immunogen construct may be represented by the following molecular formula:

[0072] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-X

[0073] or

[0074] (CGRP functional B-cell epitope peptide)-(A) n -(Th) m -X

[0075] or

[0076] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-(A) n -(Th) m -X

[0077] in

[0078] Th is a heterologous T helper cell epitope;

[0079] A represents the heterologous septal region;

[0080] (CGRP functional B-cell epitope peptide) is a B-cell epitope peptide with 7 to 30 amino acid residues derived from CGRP involved in receptor binding or receptor activation.

[0081] X represents α-COOH or α-CONH2 of an amino acid;

[0082] m ranges from 1 to approximately 4; and

[0083] n ranges from 0 to approximately 10.

[0084] The CGRP peptide immunogen constructs disclosed herein are designed and selected based on many theoretical foundations, including:

[0085] i. CGRP B cell epitope peptides are themselves non-immunogenic, thus avoiding the activation of autologous T cells;

[0086] ii. CGRP B cell epitope peptides can be made immunogenic by using protein carriers or effective T helper cell epitopes;

[0087] iii. When CGRP B cell epitope peptides become immunogenic and are administered to the host, the peptide immunogenic construct:

[0088] a. Induces high-titer antibodies that preferentially target CGRP B-cell epitopes (rather than protein carriers or T helper cell epitopes);

[0089] b. In immunized hosts, it disrupts immune tolerance and generates highly specific antibodies that cross-react with CGRP (SEQ ID NOs: 1-3);

[0090] c. To generate highly specific antibodies that can inhibit CGRP and CGRP receptor binding and related downstream events (such as increased intracellular cAMP production); and

[0091] d. Produce highly specific antibodies that can cause capsaicin-induced reduction in skin blood flow in vivo.

[0092] The disclosed CGRP peptide immunogen constructs and their formulations can effectively exert the effects of pharmaceutical compositions to prevent and / or treat individuals who are susceptible to or suffer from migraines.

[0093] The various components of the publicly disclosed CGRP peptide immunogen construct are described in further detail below.

[0094] a. B-cell epitope peptides from CGRP

[0095] This disclosure relates to novel peptide compositions for generating high-titer antibodies that are specific for calcitonin gene-related peptide (CGRP) proteins (e.g., SEQ ID NOs: 1-3) from multiple species. The site specificity of the peptide immunogen construct minimizes antibody production against irrelevant sites located in other regions of CGRP or on carrier proteins, thereby providing a high safety profile.

[0096] As used herein, the term "CGRP" refers to the 37-amino acid neuropeptide α-CGRP, belonging to the calcitonin (CT) peptide family. Human CGRP is derived from UniProtKB: P06881-1 and has the amino acid sequence SEQ ID NO: 1. Marmoset (Callithrix jacchus) CGRP is derived from GenBank Accession No.: AAL35592.1 and has the amino acid sequence SEQ ID NO: 2. Rat (Rattus norvegicus) CGRP is derived from UniProtKB: P01256, while mouse (Mus musculus) CGRP is derived from UniProtKB: Q99JA0, and both rat and mouse CGRP have the amino acid sequence SEQ ID NO: 3. The amino acid sequences of CGRP used in this disclosure are shown in Table 1.

[0097] In humans, CGRP is derived from the gene encoding calcitonin, formed by alternative splicing of the calcitonin / CGRP gene located on chromosome 11. In humans, CGRP has two isoforms: α-CGRP and β-CGRP. The difference between the α- and β-isoforms lies in the amino acids located at positions 3, 22, and 25. At the molecular level within smooth muscle cells, CGRP binds to its receptor via its carboxyl-terminal region and then activates the receptor through its cyclic region. The C2-C7 cyclic structure with disulfide bonds plays a central role in receptor activation and is closely associated with increases in intracellular cAMP. In mammalian plasma, the half-life of CGRP is approximately 10 minutes. In the human trigeminal ganglion, CGRP-responsive neurons account for 50% of all neurons (Tajti, et al., 1999).

[0098] CGRP exhibits widespread activity in the central and peripheral nervous systems. It is primarily associated with small, unmyelinated sensory neurons located close to blood vessels. CGRP is a potent vasodilator, and local application results in a transient increase in blood flow. CGRP is also involved in pain transmission, pain modulation, and neuroinflammation. CGRP can be released from sensory neurons via activation of the transient receptor potential cation channel V1 using capsaicin. Laser Doppler imaging (LDI) has been used to detect changes in skin blood flow induced by CGRP.

[0099] As demonstrated by the weakened response in CGRP knockout mice in various pain models, CGRP is also associated with inflammatory pain. This role in pain perception is consistent with the performance of CGRP in sensory neurons.

[0100] One aspect of this disclosure is the use of CGRP-targeting active immunotherapy to achieve long-term CGRP blocking effects and clinical efficacy for the prevention and / or treatment of CGRP-induced migraines. Therefore, this disclosure relates to peptide immunogen constructs and formulations targeting portions of the full-length CGRP protein (SEQ ID NO: 1-3) for the prevention and treatment of migraines.

[0101] The B-cell epitope portion of the CGRP peptide immunogen construct may contain approximately 7 to approximately 30 amino acids from any portion of the full-length CGRP protein (represented by SEQ ID NOs: 1-3). In some embodiments, the B-cell epitope peptides screened and selected based on design theory contain the amino acid sequences of SEQ ID NOs: 4-13 and 15-24 as shown in Table 1.

[0102] In some embodiments, the B-cell epitope peptide originates from the CGRP receptor-binding region R11-F37 (SEQ ID NO: 9) of the CGRP molecule's carboxyl terminus / intermediate region, or fragments thereof (e.g., SEQ ID NOs: 5-8 and 15-22). In other embodiments, the B-cell epitope peptide originates from the CGRP receptor-activating region (e.g., A1-N25 (SEQ ID NO: 13)) or fragments thereof located near the cyclic C2-C7 ring structure.

[0103] The CGRP B-cell epitope peptides disclosed herein also include immunomodulatory analogs or homologs of CGRP. Immunomodulatory analogs or homologs of the CGRP B-cell epitope peptides include variants that retain substantially the same immunogenicity as the original peptide. Immunomodulatory analogs may have retention substitutions at amino acid positions, changes in total charge, covalent linkages with other functional groups, or additions, insertions, or deletions of amino acids and / or any combination thereof (e.g., the CGRP peptide of SEQ ID NOs: 9 vs 25).

[0104] Antibodies generated from peptide immunogen constructs containing these B-cell epitopes derived from CGRP are highly specific and cross-reactive with full-length CGRP from various species (e.g., SEQ ID NOs: 1-3). Based on their unique characteristics and properties, publicly available antibodies induced by CGRP peptide immunogen constructs can provide prophylactic immunotherapy for the prevention and / or treatment of migraines.

[0105] b. Heterogeneous T helper cell epitopes (Th epitopes)

[0106] This disclosure provides a peptide immunogen construct comprising a B-cell epitope derived from CGRP, the B-cell epitope being directly or covalently linked to a heterologous T helper cell (Th) epitope via an optional heterologous spacer region.

[0107] Heterogeneous Th epitopes in CGRP peptide immunogen constructs can enhance the immunogenicity of CGRP fragments, promoting the generation of specific high-titer antibodies against optimized target CGRP B cell epitope peptides selected based on design theory.

[0108] The term "heterologous" used in this article refers to an amino acid sequence that is not derived from a portion of the wild-type CGRP sequence or a homologous amino acid sequence. Therefore, a heterologous Th epitope is a Th epitope derived from an amino acid sequence not naturally present in CGRP (i.e., the Th epitope is not autologously derived from CGRP). Because the Th epitope is heterologous to CGRP, when a heterologous Th epitope is covalently linked to a CGRP B-cell epitope peptide, the native amino acid sequence of CGRP will not extend towards the N-terminus or C-terminus.

[0109] The heterologous Th epitopes disclosed herein can be any Th epitope that does not possess the amino acid sequence naturally present in CGRP. Th epitopes can also have mixed binding motifs targeting multiple species of class 2 MHC molecules. In some embodiments, the Th epitope contains multiple mixed class 2 MHC binding motifs to allow for maximum activation of T helper cells, thereby leading to the initiation and regulation of an immune response. Preferably, the Th epitope itself is non-immunogenic (i.e., antibodies generated from CGRP peptide immunogen constructs are rarely targeted at the Th epitope, if any), thus allowing for a highly focused immune response against the target B cell epitope peptide of the CGRP molecule.

[0110] The Th epitopes disclosed herein include, but are not limited to, amino acid sequences derived from foreign pathogens, as illustrated in Table 2 (SEQ ID NOs: 74-115). Furthermore, Th epitopes include idealized artificial Th epitopes and combinations of idealized artificial Th epitopes (e.g., SEQ ID NOs: 75 and 82-99). Heterologous Th epitope peptides are presented as combinatorial sequences (e.g., SEQ ID NOs: 85, 91, 94, and 97) comprising a mixture of amino acid residues at specific positions within the peptide backbone, representing variable residues based on homologs of a particular peptide. A collection of combinatorial peptides can be synthesized in a single process using a mixture of selected protected amino acids added at specific positions during the synthesis process, rather than a single amino acid. Such a collection of combinatorial heterologous Th epitope peptides allows for broad Th epitope coverage in animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 85, 91, 94, and 97 as shown in Table 2. The Th epitope peptides of this invention provide broad reactivity and immunogenicity in animals and patients from genetically diverse populations.

[0111] c. Heterogeneous septal region

[0112] The disclosed CGRP peptide immunogen construct optionally includes a heterologous spacer region that covalently links a CGRP B cell epitope peptide to a heterologous T helper cell (Th) epitope.

[0113] As mentioned above, the term "heterologous" refers to an amino acid sequence derived from an amino acid sequence that is not native to or homologous to CGRP. Therefore, when a heterologous spacer region is covalently linked to the CGRP B-cell epitope peptide, the native amino acid sequence of CGRP will not extend toward the amino or carboxyl terminus because the spacer region is heterologous to the CGRP sequence.

[0114] A spacer region is any molecule or chemical structure capable of linking two amino acids and / or peptides together. The length or polarity of the spacer region may vary depending on the application. Spacer region linkages can be via amide or carboxyl groups, but other functional groups are also possible. Spacer regions can include chemical compounds, naturally occurring amino acids, or non-naturally occurring amino acids.

[0115] The spacer region provides structural features for CGRP peptide immunogen constructs. Structurally, the spacer region provides physical separation of Th epitopes from B-cell epitopes of the CGRP fragment. Physical separation via the spacer region disrupts any artificial secondary structures created by linking Th epitopes to B-cell epitopes. Furthermore, physical separation of epitopes via the spacer region eliminates interference between Th cell and / or B-cell responses. In addition, the spacer region can be engineered to generate or modify the secondary structure of the peptide immunogen construct. For example, the spacer region can be engineered as a flexible hinge to enhance the separation of Th and B-cell epitopes. The flexible hinged spacer region also allows for more efficient interaction between the presented peptide immunogen and appropriate Th and B cells to enhance immune responses to Th and B-cell epitopes. Examples of sequences encoding flexible hinges are found in proline-rich immunoglobulin heavy chain hinge regions. The sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71) provides a particularly useful flexible hinge for use as a spacer region, wherein Xaa is any amino acid, preferably aspartic acid.

[0116] Spacer regions can also provide functional characteristics for CGRP peptide immunogen constructs. For example, spacer regions can be designed to alter the total charge of the CGRP peptide immunogen construct, which can affect its solubility. Furthermore, altering the total charge of the CGRP peptide immunogen construct can affect its ability to bind to other compounds and reagents. As discussed in further detail below, CGRP peptide immunogen constructs can form stable immunostimulatory complexes with highly charged oligonucleotides (e.g., CpG oligomers) via electrostatic binding. The total charge of the CGRP peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

[0117] Chemical compounds that can serve as spacer regions include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminohexanoic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini-PEG3), trioxatridecan-succinamic acid (Ttds), 12-aminododecanoic acid, Fmoc-5-amino-3-oxovaleric acid (O1Pen), etc.

[0118] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0119] Non-naturally occurring amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, leucine, valine, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminohexanoic acid (Aca), 3-thiol propionic acid (MPA), 3-nitrotyramine, pyroglutamic acid, etc.

[0120] Spacer regions in the CGRP peptide immunogen construct can be covalently linked to the N-terminus or C-terminus of a Th epitope and a CGRP B-cell epitope peptide. In some embodiments, the spacer region is covalently linked to the C-terminus of the Th epitope and the N-terminus of the CGRP B-cell epitope peptide. In other embodiments, the spacer region is covalently linked to the C-terminus of the CGRP B-cell epitope peptide and the N-terminus of the Th epitope. In some embodiments, more than one spacer region can be used, for example, when more than one Th epitope is present in the CGRP peptide immunogen construct. When more than one spacer region is used, each spacer region can be identical or different from each other. Furthermore, when more than one Th epitope is present in the CGRP peptide immunogen construct, spacer regions can be used to separate the Th epitopes; these spacer regions can be identical or different, and the spacer regions separate the Th epitopes from the CGRP B-cell epitope peptide. There are no restrictions on the arrangement of the spacer regions relative to the Th epitope or the CGRP B-cell epitope peptide.

[0121] In some embodiments, the heterologous spacer region is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer region contains more than one naturally occurring or non-naturally occurring amino acid. In specific embodiments, the spacer region is Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72), or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73).

[0122] d. Specific implementation plan for the CGRP peptide immunogen construct

[0123] In some implementations, the CGRP peptide immunogen construct can be represented using the following molecular formula:

[0124] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-X

[0125] or

[0126] (CGRP functional B-cell epitope peptide)-(A) n -(Th) m -X

[0127] or

[0128] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-(A) n -(Th) m -X

[0129] in

[0130] Th is a heterologous T helper cell epitope;

[0131] A represents the heterologous septal region;

[0132] (CGRP functional B-cell epitope peptide) is a B-cell epitope peptide with 7 to 30 amino acid residues derived from CGRP involved in receptor binding or receptor activation.

[0133] X represents α-COOH or α-CONH2 of an amino acid;

[0134] m ranges from 1 to approximately 4; and

[0135] n ranges from 0 to approximately 10.

[0136] B-cell epitope peptides may comprise about 7 to about 30 amino acids from any portion of the full-length CGRP protein (represented by SEQ ID NOs: 1-3). In some embodiments, the B-cell epitope has an amino acid sequence selected from any of SEQ ID NOs: 4-13 and 15-22 as shown in Table 1. In some embodiments, the B-cell epitope peptide originates from the CGRP receptor-binding region located at the carboxyl terminus / intermediate region R11-F37 (SEQ ID NO: 9) of the CGRP molecule, or a fragment thereof (e.g., SEQ ID NOs: 5-8 and 15-22). In other embodiments, the B-cell epitope peptide originates from the CGRP receptor-activating region located near the cyclic C2-C7 ring structure (e.g., A1-N25 (SEQ ID NO: 13)) or a fragment thereof (e.g., SEQ ID NOs: 4, 10-12, and 23-24).

[0137] The heterologous Th epitopes in the CGRP peptide immunogen construct have amino acid sequences selected from any combination of SEQ ID NOs: 74-115, as shown in Table 2. In some embodiments, the CGRP peptide immunogen construct contains more than one Th epitope.

[0138] The optional heterologous spacer region is selected from Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71), ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73), and any combination thereof, wherein Xaa is any amino acid, but aspartic acid is preferred. In a specific embodiment, the heterologous spacer region is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73).

[0139] In some embodiments, the CGRP peptide immunogen construct has an amino acid sequence selected from any one of SEQ ID NOs: 116-127 and 130-180 as shown in Table 3.

[0140] Immunogen constructs containing CGRP peptides with Th epitopes are generated concurrently with the synthesis of a single solid-phase peptide tandem with a CGRP fragment. Th epitopes may also include immunoanalytes of Th epitopes. Immunoassays include immunoenhancing analogs, cross-reactive analogs, and fragments of any of these Th epitopes sufficient to enhance or stimulate an immune response to CGRP B cell epitope peptides.

[0141] In the CGRP peptide immunogen construct, Th epitopes can be covalently linked to the N-terminus or C-terminus of the CGRP B cell epitope peptide. In some embodiments, the Th epitope is covalently linked to the N-terminus of the CGRP B cell epitope peptide. In other embodiments, the Th epitope is covalently linked to the C-terminus of the CGRP B cell epitope peptide. In some embodiments, more than one Th epitope is covalently linked to the CGRP B cell epitope peptide. When more than one Th epitope is linked to the CGRP B cell epitope peptide, each Th epitope may have the same amino acid sequence or different amino acid sequences. Furthermore, when more than one Th epitope is linked to the CGRP B cell epitope peptide, the Th epitopes can be arranged in any order. For example, Th epitopes may be continuously linked to the N-terminus of the CGRP B cell epitope peptide, or continuously linked to the C-terminus of the CGRP B cell epitope peptide, or when different Th epitopes are covalently linked to the C-terminus of the CGRP B cell epitope peptide, the Th epitope may be covalently linked to the N-terminus of the CGRP B cell epitope peptide. There are no restrictions on the arrangement of Th epitopes relative to CGRP B cell epitope peptides.

[0142] In some embodiments, the Th epitope is directly covalently linked to the CGRP B-cell epitope peptide. In other embodiments, the Th epitope is covalently linked to the CGRP fragment via a heterologous spacer region.

[0143] e. Variants, homologs, and functional analogs

[0144] Variants and analogs of the aforementioned immunogenic peptide constructs may also be used, which can induce and / or cross-react with antibodies targeting preferred CGRP B-cell epitope peptides. Analogs (including alleles, species, and induced variants) typically differ from the naturally occurring peptide at one, two, or several positions, usually due to retaining substitutions. Analogs generally exhibit at least 80 or 90% sequence identity with the natural peptide. Some analogs also include modifications of non-natural amino acids or amino-terminal or carboxyl-terminal amino acids at one, two, or several positions.

[0145] Variants that are functional analogs may have retention substitutions at amino acid positions, changes in total charge, covalent linkages with other functional groups, or additions, insertions, or deletions of amino acids and / or any combination thereof.

[0146] Retentional substitution refers to the replacement of one amino acid residue with another amino acid residue that has similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparaginic acid, and glutamic acid; positively charged (basic) amino acids include arginine, lysine, and histamine; and negatively charged (acidic) amino acids include asparaginine and glutamic acid.

[0147] In one specific embodiment, the functional analog has at least 50% identity with the original amino acid sequence. In another embodiment, the functional analog has at least 80% identity with the original amino acid sequence. In yet another embodiment, the functional analog has at least 85% identity with the original amino acid sequence. In still another embodiment, the functional analog has at least 90% identity with the original amino acid sequence.

[0148] Functional immunological analogs of Th epitope peptides are also effective and are included as part of this invention. Functional immunological Th analogs may contain retention substitutions, additions, deletions, and insertions of 1 to 5 amino acid residues in the Th epitope, substantially without altering the Th-stimulatory function of the Th epitope. As described above for CGRP B-cell epitope peptides, retention substitutions, additions, and insertions can be made using natural or non-natural amino acids. Table 2 identifies another variant of the functional analogs of Th epitope peptides. Specifically, SEQ ID NOs: 75 and 82 of MvF1 and MvF2 Th are functional analogs of SEQ ID NOs: 94 and 98 of MvF4 and MvF5 Th, differing in their amino acid backbones by deleting (SEQ ID NOs: 75 and 82) or inserting (SEQ ID NOs: 94 and 98) two amino acids each at the amino and carboxyl ends. The differences between these two series of similar sequences do not affect the function of the Th epitopes contained in these sequences. Therefore, functional immune Th analogues include multiple versions of Th epitopes derived from measles virus fusion proteins MvF1-4Ths (SEQ ID NOs: 75, 82, 85 and 94) and hepatitis surface proteins HBsAg 1-3Ths (SEQ ID NOs: 91, 97 and 99).

[0149] Composition

[0150] This disclosure also provides compositions comprising the disclosed CGRP immunogenic peptide construct.

[0151] a. peptide complexes

[0152] Compositions containing the disclosed CGRP peptide immunogen constructs may be in liquid or solid / lyophilized form. Liquid compositions may include water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structure or functional properties of the CGRP peptide immunogen construct. Peptide compositions may contain one or more disclosed CGRP peptide immunogen constructs.

[0153] b. Pharmaceutical Composition

[0154] This disclosure also relates to pharmaceutical compositions comprising the disclosed CGRP peptide immunogen construct.

[0155] The pharmaceutical composition may contain a carrier and / or other additives in a pharmaceutically acceptable delivery system. Therefore, the pharmaceutical composition may contain a pharmaceutically effective dose of the CGRP peptide immunogen construct and pharmaceutically acceptable carriers, adjuvants, and / or other excipients (e.g., diluents, additives, stabilizers, preservatives, solubilizers, buffers, etc.).

[0156] The pharmaceutical composition may contain one or more adjuvants that accelerate, prolong, or enhance the immune response against the CGRP peptide immunogen construct without possessing any specific antigenic activity. Adjuvants used in the pharmaceutical composition may include oils, oil emulsions, aluminum salts, calcium salts, immunostimulatory complexes, bacterial and viral derivatives, virosomes, carbohydrates, cytokines, and polymeric microparticles. In some embodiments, the adjuvant may be selected from alum (potassium aluminum phosphate), aluminum phosphate (e.g., ADJU- ), aluminum hydroxide (e.g.) ), calcium phosphate, Freund's incomplete adjuvant (IFA), Freund's complete adjuvant, MF59, adjuvant 65, Lipovant, ISCOM, liposyn, saponins, squalene, L121, EmulsIL- Monophospholipid A (MPL), Quil A, QS21 ISA 35, ISA 50V, ISA50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycans, lipopolysaccharides (LPS), ASO1, ASO2, ASO3, ASO4, AFO3, lipophilic phospholipids (lipid A), gamma inulin, algammulin, dextran, dextran, glucomannan, galactomannan, fructan, xylan, dioctadecyl dimethyl ammonium bromide (DDA), and other adjuvants and emulsifiers.

[0157] In some embodiments, the pharmaceutical composition contains MONTANIDE TM ISA 51 (an oil adjuvant composition consisting of vegetable oil and dimannitol oleate, used to prepare water-in-oil emulsions) 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion with EmulsIL-6n or EmulsIL-6n D as an adjuvant.

[0158] The pharmaceutical composition may also include pharmaceutically acceptable additives or excipients. For example, the pharmaceutical composition may contain antioxidants, binders, buffers, build-up agents, carriers, chelating agents, colorants, diluents, disintegrants, emulsifiers, fillers, gelling agents, pH buffers, preservatives, solubilizers, stabilizers, etc.

[0159] The pharmaceutical composition can be formulated into immediate-release or sustained-release dosage forms. Additionally, the pharmaceutical composition can be formulated for inducing systemic or local mucosal immunity via immunogen encapsulation and co-administration with microparticles. Such delivery systems are readily apparent to those skilled in the art.

[0160] The pharmaceutical composition can be formulated as an injection in the form of a liquid solution or suspension. A liquid carrier containing the CGRP peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition can be administered using any suitable method of administration, such as id, iv, ip, im, intranasal, oral, subcutaneous, etc., and can be administered in any suitable delivery device. In some embodiments, the pharmaceutical composition can be formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other routes of administration, including oral and intranasal application, can also be formulated.

[0161] The pharmaceutical composition can also be formulated in suitable dosage units. In some embodiments, the pharmaceutical composition contains about 0.1 μg to about 1 mg of CGRP peptide immunogen construct per kilogram of body weight. The effective dose of the pharmaceutical composition depends on many different factors, including the route of administration, target, patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is for prophylaxis or treatment. Typically, the patient is human, but treatment can also be given to non-human mammals, including genetically modified mammals. When delivered in multiple doses, the pharmaceutical composition can be conveniently aliquoted into appropriate amounts for each dosage unit. As is well known in the therapeutic field, the administered dose depends on the individual's age, weight, and general health condition.

[0162] In some embodiments, the pharmaceutical composition contains one or more CGRP peptide immunogen constructs. Pharmaceutical compositions containing a mixture of one or more CGRP peptide immunogen constructs allow for synergistic enhancement of the construct's immunogenicity. Pharmaceutical compositions containing one or more CGRP peptide immunogen constructs may be more effective in a larger genetic population due to broad class 2 MHC coverage, thus providing an improved immune response against the CGRP peptide immunogen construct.

[0163] In some embodiments, the pharmaceutical composition contains a CGRP peptide immunogen construct selected from SEQ ID NOs: 120-127 and 130-180 (Table 3), as well as homologs, analogs and / or combinations thereof.

[0164] In some embodiments, CGRP peptide immunogen constructs (SEQ ID NOs: 161-163) having heterologous Th epitopes (SEQ ID NOs: 85, 91, 94 and 97) derived from MVF and HBsAg in combined forms are mixed in an equal molar ratio in the formulation to allow for maximum coverage of host populations with different genetic backgrounds.

[0165] Furthermore, by using CGRP peptide immunogen constructs (e.g., those with SEQ ID NO: 108) 1) The majority (>90%) of the antibody responses elicited are focused on the desired cross-reactivity against the B-cell epitope peptide of CGRP, with little, if any, targeting the heterologous Th epitope used for immunogenic enhancement (Implementation Scheme 6, Table 10). This contrasts sharply with antibodies prepared using conventional proteins (e.g., KLH) or other biological protein carriers for enhancing the antigenicity of such CGRP peptides.

[0166] In other embodiments, a pharmaceutical composition comprising a peptide composition, such as a mixture of CGRP peptide immunogen constructs, is contacted with a mineral salt (including alum gel or aluminum phosphate) as an adjuvant to form a suspension dosage form for administration to a host.

[0167] Pharmaceutical compositions containing CGRP peptide immunogen constructs can be used to induce an immune response and generate antibodies in the host after administration.

[0168] c. Immunostimulatory complexes

[0169] This disclosure also relates to pharmaceutical compositions containing a CGRP peptide immunogen construct that forms an immunostimulatory complex with CpG oligonucleotides. Such immunostimulatory complexes are particularly suitable as adjuvants and peptide immunogen stabilizers. The immunostimulatory complexes are in particulate form, which effectively presents the CGRP peptide immunogen to cells of the immune system to generate an immune response. The immunostimulatory complexes can be formulated as suspensions for parenteral administration. The immunostimulatory complexes can also be formulated as water-in-oil (w / o) emulsions, as suspensions bound to mineral salts or in-situ gel polymers, for the efficient delivery of the CGRP peptide immunogen construct to cells of the host immune system after parenteral administration.

[0170] Stabilized immunostimulatory complexes can be formed by electrostatically binding CGRP peptide immunogen constructs with anionic molecules, oligonucleotides, polynucleotides, or combinations thereof. These stabilized immunostimulatory complexes can be incorporated into pharmaceutical compositions as immunogen delivery systems.

[0171] In some embodiments, the CGRP peptide immunogen construct is designed to include a cationic moiety carrying a positive charge at a pH ranging from 5.0 to 8.0. The net charge of the cationic moiety of the CGRP peptide immunogen construct or mixture of constructs is calculated based on the following: each lysine (K), arginine (R), or histidine (H) carries a +1 charge; each aspartic acid (D) or glutamic acid (E) carries a -1 charge; and the other amino acids in the sequence carry a charge of 0. The charges in the cationic moiety of the CGRP peptide immunogen construct are summed and expressed as a net average charge. Suitable peptide immunogens have a cationic moiety with a net average positive charge of +1. Preferably, the peptide immunogen has a net positive charge in the range of +2. In some embodiments, the cationic moiety of the CGRP peptide immunogen construct is a heterologous spacer region. In some embodiments, when the spacer sequence is (α, ε-N)Lys, (α, ε-N)-Lys-Lys-Lys-Lys (SEQ ID NO: 72) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73), the cationic portion of the CGRP peptide immunogen construct has a charge of +4.

[0172] As used herein, "anionic molecule" refers to any molecule carrying a negative charge at a pH ranging from 5.0 to 8.0. In some embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated based on the fact that each phosphodiester or thiophosphate group in the oligomer carries a charge of -1. Suitable anionic oligonucleotides are single-stranded DNA molecules having 8 to 64 nucleotide bases and a CpG motif repeat number ranging from 1 to 10. Preferably, CpG immunostimulatory single-stranded DNA molecules contain 18 to 48 nucleotide bases and a CpG motif repeat number ranging from 3 to 8.

[0173] More preferably, anionic oligonucleotides can be of the molecular formula 5′X 1 CGX 2 3′ indicates that C and G are unmethylated; and X 1 It is a group composed of A (adenine), G (guanine), and T (thymine); and X 2 It is C (cytosine) or T (thymine). Alternatively, anionic oligonucleotides can have the molecular formula 5′(X). 3 )2CG(X 4 )23′ indicates that C and G are unmethylated; and X 3 It is a group consisting of A, T, or G; and X 4It is C or T. In specific embodiments, the CpG oligonucleotide has the following sequences: CpG1: 5′TCg TCg TTT TgT CgT TTT gTC gTT TTg TCg TT 3′ (complete thiophosphorylation) (SEQ ID NO: 182), CpG2: 5′ phosphate TCg TCg TTT TgT CgT TTT gTC gTT 3′ (complete thiophosphorylation) (SEQ ID NO: 183) or CpG3: 5′TCg TCg TTT TgT CgT TTT gTC gTT 3′ (complete thiophosphorylation) (SEQ ID NO: 184).

[0174] The resulting immunostimulatory complexes are in particulate form, typically ranging in size from 1 to 50 micrometers, and are a function of many factors, including the relative charge stoichiometry and molecular weight of the interacting components. Particulate immunostimulatory complexes offer the advantage of providing adjuvanting and upregulation of specific immune responses in vivo. Furthermore, stabilized immunostimulatory complexes are suitable for preparing pharmaceutical compositions using various methods, including water-in-oil emulsions, mineral salt suspensions, and polymeric gels.

[0175] This disclosure also relates to pharmaceutical compositions for the prevention and / or treatment of migraines, comprising formulations. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex, which is formed by electrostatic binding of a peptide composition comprising a mixture of CpG oligomers and CGRP peptide immunogen constructs (e.g., SEQ ID NOs: 120-127 and 130-180) to further enhance the immunogenicity of the CGRP peptide immunogen constructs and to trigger an antibody that cross-reacts with the CGRP proteins of SEQ ID NOs: 1-3, the antibody targeting the CGRP receptor binding or receptor activation region (Embodiment 6).

[0176] In another embodiment, the pharmaceutical composition contains a mixture of CGRP peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 120-127 and 130-180) that form a stabilized immunostimulatory complex with a CpG oligomer. Preferably, the immunostimulatory complex is mixed with a mineral salt (including ALHYDROGEL or ADJUPHOS) as an adjuvant with a high safety factor to form a suspension dosage form for administration to the host.

[0177] Antibody

[0178] This disclosure also provides antibodies induced by CGRP peptide immunogen constructs.

[0179] This disclosure provides a CGRP peptide immunogen construct and its formulation, which is cost-effective in preparation. Its optimal design can trigger high-titer antibodies targeting the CGRP receptor-binding or receptor-activating regions (SEQ ID NOs: 4-13 and 15-24) on the CGRP molecule, which can disrupt immune tolerance against the self-protein CGRP in the immunized host with a high response rate. The antibodies generated using the CGRP peptide immunogen construct have high affinity for the CGRP receptor-binding or activation regions.

[0180] In some embodiments, the CGRP peptide immunogen construct for inducing antibodies comprises a hybrid of CGRP peptides that target CGRP receptor-binding or receptor-activating regions on the CGRP molecule (SEQ ID NOs: 4-13 and 15-24). The CGRP peptide is linked via an optional spacer region to a heterologous Th epitope derived from pathogen proteins (e.g., derived from measles virus fusion (MVF) proteins and other proteins (SEQ ID NOs: 74-115)). The B-cell epitope and Th epitope peptide of the CGRP peptide immunogen construct work together to stimulate the production of highly specific antibodies that cross-react with the CGRP receptor-binding or activation regions on CGRP proteins (SEQ ID NOs: 1-3).

[0181] Conventional methods for enhancing the immunogenicity of peptides, such as by chemically conjugating carrier proteins (e.g., keyhole cyanin (KLH)) or other carrier proteins (e.g., diphtheria toxoid (DT) and tetanus toxoid (TT) proteins), typically result in the production of large amounts of antibodies against the carrier proteins. Therefore, a major drawback of such peptide-carrier protein compositions is that the majority (>90%) of the antibodies produced using this immunogen are non-functional antibodies against the carrier proteins KLH, DT, or TT that can cause epitope inhibition.

[0182] Unlike conventional methods used to enhance the immunogenicity of peptides, antibodies generated using publicly available CGRP peptide immunogenic constructs (e.g., SEQ ID NOs: 116-127 and 130-180) can bind with high specificity to CGRP B cell epitope peptides (SEQ ID NOs: 4-13 and 15-24), and if any, the antibodies are against heterologous Th epitopes (e.g., SEQ ID NOs: 74-115) or optional heterologous spacer regions.

[0183] method

[0184] This disclosure also relates to methods for preparing and using CGRP peptide immunogen constructs, compositions, and pharmaceutical compositions.

[0185] a. Preparation method of CGRP peptide immunogen construct

[0186] The CGRP peptide immunogen constructs disclosed herein can be prepared using chemical synthesis methods well known to those skilled in the art (see, for example, Fields, et al., 1992). The CGRP peptide immunogen constructs can be synthesized using an automated Merrifield solid-phase synthesis method, utilizing side-chain protected amino acids, with α-NH2 chemically protected by t-Boc or F-moc, on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431. The preparation of CGRP peptide immunogen constructs containing combinatorial database peptides with Th epitopes can be achieved by providing a mixture of alternative amino acids for coupling at a given variable position.

[0187] After the desired CGRP peptide immunogen construct is assembled, the resin is processed according to a standard procedure to cleave the peptide from the resin and remove the functional groups on the amino acid side chains. The free peptide can be purified by HPLC and its biochemical properties can be characterized using, for example, amino acid analysis or sequencing. Methods for peptide purification and characterization are well known to those skilled in the art to which this invention pertains.

[0188] The quality of the peptides produced through this chemical process can be controlled and determined, and the reproducibility, immunogenicity, and yield of the CGRP peptide immunogen constructs can be guaranteed. A detailed description of the preparation of CGRP peptide immunogen constructs by solid-phase peptide synthesis is shown in Embodiment 1.

[0189] It has been found that the range of structural variations that allows for the retention of desired immune activity is more inclusive than the range of structural variations that allows for the retention of specific drug activity in small molecule drugs or the presence of desired activity and non-desired toxicity in macromolecules co-produced with biological drugs.

[0190] Therefore, peptide analogs with similar chromatographic and immunological properties to the desired peptide, whether intentionally designed or unavoidably produced as a mixture of deleted sequence byproducts due to synthetic errors, generally have the same efficacy as the purified desired peptide formulation. As long as rigorous QC procedures are established to monitor the preparation and product evaluation processes and ensure the reproducibility and efficacy of the final products using these peptides, mixtures of designed and unintended analogs are also effective.

[0191] CGRP peptide immunogen constructs can also be prepared using recombinant DNA technology comprising nucleic acid molecules, vectors, and / or host cells. Therefore, nucleic acid molecules encoding CGRP peptide immunogen constructs and their immunomodulatory analogs are also included in this disclosure as part of the invention. Similarly, vectors containing nucleic acid molecules (including expression vectors) and host cells containing vectors are also included in this disclosure as part of the invention.

[0192] Various exemplary embodiments also include methods for preparing CGRP peptide immunogen constructs and their immunomodulatory analogs. For example, the method may include the step of culturing host cells under conditions that express the peptide and / or analog, the host cells containing an expression vector comprising a nucleic acid molecule encoding the CGRP peptide immunogen construct and / or its immunomodulatory analog. Longer synthetic peptide immunogens can be synthesized using known recombinant DNA techniques. These techniques are provided in well-known standard manuals with detailed experimental plans. To construct a gene encoding the peptide of the present invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably using the codons most suitable for the organism having the gene to be expressed. Next, a synthetic gene is typically prepared by synthesizing oligonucleotides encoding the peptide and any regulatory factors (if necessary). The synthetic gene is inserted into a suitable selection vector and transfected into host cells. The peptide is then expressed under suitable conditions for the selected expression system and host. The peptide is purified using standard methods and its properties are described.

[0193] b. Preparation method of immune-stimulating complex

[0194] Various exemplary embodiments also include methods for preparing immunostimulatory complexes comprising a CGRP peptide immunogen construct and a CpG oligodeoxynucleotide (ODN) molecule. The stabilized immunostimulatory complex (ISC) is derived from the cationic portion of the CGRP peptide immunogen construct and the polyanionic CpG ODN molecule. The self-assembly system is driven by the electrostatic neutralization of the charges. The stoichiometry of the molar valence ratio of the cationic portion of the CGRP peptide immunogen construct to the anionic oligomer determines the degree of association. The non-covalent electrostatic binding of the CGRP peptide immunogen construct and the CpG ODN is a fully reproducible process. This peptide / CpG ODN immunostimulatory complex aggregate facilitates presentation to "professional" antigen-presenting cells (APCs) in the immune system, thus further enhancing the immunogenicity of the complex. During preparation, the characteristics of these complexes can be easily characterized to control quality. The peptide / CpG ISC exhibits good tolerability in vivo. This novel microparticle system, incorporating CpG ODN and CGRP peptide immunogen constructs, was designed to leverage the broad B-cell mitogenicity associated with CpG ODN use, but to promote a balanced Th-1 / Th-2 response.

[0195] In the disclosed pharmaceutical composition, CpG ODN binds 100% to the immunogen in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles. This particle form allows for a significant reduction in the dose of CpG derived from conventionally used CpG adjuvants, a lower likelihood of adverse innate immune responses, and promotes alternative immunogen processing pathways, including antigen-presenting cells (APCs). Therefore, this dosage form is conceptually novel and offers potential advantages through an alternative mechanism of stimulation that promotes an immune response.

[0196] c. Methods for preparing pharmaceutical compositions

[0197] Various exemplary embodiments also include pharmaceutical compositions containing CGRP peptide immunogen constructs. In some embodiments, the pharmaceutical composition is a dosage form utilizing a water-in-oil emulsion and a suspension containing mineral salts.

[0198] To ensure the widespread use of pharmaceutical compositions, safety is another crucial factor to consider. Although water-in-oil emulsions have been used in many clinical trials, alum remains the primary adjuvant in formulations due to its safety profile. Therefore, alum or its mineral salt, aluminum phosphate (ADJUPHOS), is frequently used clinically as an adjuvant in pharmaceutical preparations.

[0199] Other adjuvants and immunostimulants include 3De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. This adjuvant may or may not be used with other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) and Theramide). TM Or other bacterial cell wall components. Oil-in-water emulsions containing MF59 (see Van Nest et al. patent application WO 90 / 14837, which is incorporated herein by reference in its entirety), comprising 5% squalene, 0.5% TWEEN 80, and 0.5% Span 85 (optionally containing varying amounts of MTP-PE), formulated into submicron particles using a microfluidic apparatus; SAF, comprising 10% squalene, 0.4% TWEEN 80, 5% pluronic-block copolymer L121, and thr-MDP, formed into submicron emulsions using microfluidization or produced into large-particle emulsions using vortexing; and Ribi... TM An adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.) comprising 2% squalene, 0.2% TWEEN 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimethicone ester (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (detox). TM Other adjuvants include Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage community-stimulating factor (M-CSF), and tumor necrosis factor (TNF-α).

[0200] The choice of adjuvant depends on the stability of the immunogen formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant on the immunized species, and, in humans, pharmaceutically acceptable adjuvants are those that have been approved or are approved for human administration by the relevant regulatory agency. For example, alum alone, MPL, or Freund's incomplete adjuvant (Chang, et al., 1998, which is incorporated herein by reference in its entirety) or optionally all combinations thereof are suitable for human administration.

[0201] The composition may include a pharmaceutically acceptable nontoxic carrier or diluent, defined as a carrier commonly used in the formulation of pharmaceutical compositions for administration to animals or humans. The diluent is chosen to avoid affecting the bioactivity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, glucose solution, and Hank's solution. Furthermore, the pharmaceutical composition or dosage form may also contain other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0202] The pharmaceutical composition may also include large, slowly metabolized macromolecules (e.g., proteins, polysaccharides (e.g., chitosan), polylactic acid, polyglycolic acid, and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, etc.), polymerized amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can serve as immunostimulants (i.e., adjuvants).

[0203] The pharmaceutical compositions of the present invention may further comprise suitable delivery carriers. Suitable delivery carriers include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen microspheres, and cochleates.

[0204] d. Method of using the pharmaceutical composition

[0205] This disclosure also includes methods for using pharmaceutical compositions comprising CGRP peptide immunogen constructs.

[0206] In some embodiments, pharmaceutical compositions containing CGRP peptide immunogen constructs can be used to treat migraines.

[0207] In some embodiments, the method includes administering a pharmaceutically effective dose of a drug composition containing a CGRP peptide immunogen construct to a host in need. In some embodiments, the method includes administering a pharmaceutically effective dose of a drug composition containing a CGRP peptide immunogen construct to a warm-blooded animal (e.g., human, cynomolgus monkey, mouse) to induce a highly specific antibody that cross-reacts with human CGRP protein (SEQ ID NO: 1) or CGRP proteins from other species (e.g., SEQ ID NOs: 2 and 3).

[0208] In some embodiments, pharmaceutical compositions containing CGRP peptide immunogen constructs can be used to treat migraines, as illustrated in an in vivo capsaicin-induced dorsal blood flow model.

[0209] e. In vitro functional analysis and in vivo proof-of-concept studies

[0210] Antibodies elicited in immunized hosts by CGRP peptide immunogen constructs can be used for in vitro functional analysis. These functional analyses include, but are not limited to:

[0211] (1) In vitro binding with CGRP protein (SEQ ID NOs: 1-3);

[0212] (2) In vitro inhibition of CGRP binding to its receptor;

[0213] (3) In vitro inhibition of intracellular cAMP elevation;

[0214] (4) Inhibit capsaicin-induced dorsal blood flow model in mice. Detailed Implementation Plan

[0215] (1) A CGRP peptide immunogen construct having about 30 or more amino acids, represented by the following molecular formula:

[0216] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-X

[0217] or

[0218] (CGRP functional B-cell epitope peptide)-(A) n -(Th) m -X

[0219] or

[0220] (Th) m -(A) n -(CGRP functional B-cell epitope peptide)-(A)n-(Th) m -X

[0221] in

[0222] Th is a heterologous T helper cell epitope;

[0223] A represents the heterologous septal region;

[0224] (CGRP functional B cell epitope peptide) is a B cell epitope peptide having 7 to 30 amino acid residues derived from the CGRP receptor binding or activation region, having SEQ ID NOs: 4-13 and 15-24 of CGRP (SEQ ID NOs: 1-3) as shown in Table 1.

[0225] X represents α-COOH or α-CONH2 of an amino acid;

[0226] m ranges from 1 to approximately 4; and

[0227] n ranges from 0 to approximately 10.

[0228] (2) The CGRP peptide immunogen construct as described in (1), wherein the CGRP receptor binding or activation region is selected from the group consisting of SEQ ID NOs: 4-13 and 15-24.

[0229] (3) The CGRP peptide immunogen construct as described in either (1) or (2), wherein the heterologous T helper cell epitope is selected from the group consisting of SEQ ID NOs: 74-115.

[0230] (4) The CGRP peptide immunogen construct as described in (1), wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120-127 and 130-180.

[0231] (5) A CGRP peptide immunogen construct comprising:

[0232] aB cell epitopes, which contain about 7 to about 30 amino acid residues from CGRP sequences SEQ ID NOs: 1 to 3;

[0233] bT helper cell epitopes comprising an amino acid sequence selected from SEQ ID NOs: 74-115 and any combination thereof; and

[0234] c. An optional heterologous spacer region selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71), and any combination thereof.

[0235] B-cell epitopes are covalently linked to T-helper cell epitopes, either directly or via an optional heterologous spacer region.

[0236] (6) The CGRP peptide immunogen construct as described in (5), wherein the B cell epitopes are selected from the group consisting of SEQ ID NOs: 4-13 and 15-24.

[0237] (7) The CGRP peptide immunogen construct as described in (5), wherein the T helper cell epitopes are selected from the group consisting of SEQ ID NOs: 78, 77, 80-82, 85, 91, 94, 97, 98-99, 106-109, 112 and any combination thereof.

[0238] (8) The CGRP peptide immunogen construct as described in (5), wherein the optional heterologous spacer region is (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73) or Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71), wherein Xaa is any amino acid.

[0239] (9) The CGRP peptide immunogen construct as described in (5), wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope.

[0240] (10) The CGRP peptide immunogen construct as described in (5), wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope via an optional heterologous spacer region.

[0241] (11) A composition comprising the CGRP peptide immunogen construct as described in (1).

[0242] (12) A pharmaceutical composition comprising:

[0243] a. The peptide immunogen construct as described in (1); and

[0244] b. Pharmaceutically acceptable delivery carriers and / or adjuvants.

[0245] (13) The pharmaceutical composition as described in (12), wherein

[0246] a. CGRP functional B-cell epitope peptides are selected from the group consisting of SEQ ID NOs: 4-13 and 15-24;

[0247] b. Heterogeneous T helper cell epitopes are selected from the group consisting of SEQ ID NOs: 74-115, and

[0248] c. The heterologous spacer region is selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72), Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73), and Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71) and any combination thereof; and

[0249] The CGRP peptide immunogen construct is mixed with CpG oligodeoxynucleotides (ODN) to form a stable immunostimulatory complex.

[0250] (14) The pharmaceutical composition as described in (12), wherein

[0251] a. The CGRP peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120-127 and 130-180; and

[0252] The CGRP peptide immunogen construct is mixed with CpG oligodeoxynucleotides (ODN) to form a stable immunostimulatory complex.

[0253] (15) A method for generating an antibody against CGRP in an animal, comprising administering to the animal the pharmaceutical composition as described in (12).

[0254] (16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the CGRP receptor-binding or activated region of SEQ ID NOs: 4-13 and 15-24.

[0255] (17) The isolated antibody or its epitope-binding fragment as described in (16) binds to the CGRP peptide immunogen construct.

[0256] (18) A composition comprising the isolated antibody or epitope-binding fragment thereof as described in (16).

[0257] (19) A method for preventing and / or treating migraines in animals, comprising administering to the animals the pharmaceutical composition as described in (12).

[0258] Example 1. Synthesis of CGRP-related peptides and preparation of their formulations

[0259] a. Synthesis of CGRP-related peptides

[0260] Methods for synthesizing CGRP-related peptides, included in the development of CGRP peptide immunogen constructs, are described. Peptides synthesized on a small scale are used for serological analysis, laboratory tests, and field trials, while peptides synthesized on a large scale (kg) are used for the industrial / commercial production of pharmaceutical compositions. For epitope identification and for screening and selecting optimal peptide immunogen constructs for therapeutic vaccines that effectively target CGRP, a large number of CGRP-related antigenic peptides with sequences of approximately 10 to 70 amino acids in length were designed.

[0261] Representative full-length CGRP (SEQ ID NOs: 1-3), CGRP peptide fragments, and 10-mer peptides used for epitope identification in various serological analyses from human, mouse, rat, and macaque species are listed in Table 1 (SEQ ID NOs: 1-70).

[0262] Selected CGRP B-cell epitope peptides were synthetically linked to meticulously designed T helper cell (Th) epitope peptides (as shown in Table 2 (SEQ ID NOs: 74-115)) derived from pathogen proteins (including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza virus, Clostridium tetani, and Epstein-Barr virus (EBV)) to create CGRP peptide immunogen constructs. The Th epitope peptides were used as single sequences (SEQ ID NOs: 74-84, 86-90, 92-93, 95-96, 98-115) or in combination from databases (SEQ ID NOs: 85, 91, 94, and 97) to enhance the immunogenicity of their respective CGRP peptide immunogen constructs.

[0263] Table 3 (SEQ ID NOs: 116-180) identifies representative CGRP peptide immunogenic constructs selected from hundreds of peptide constructs. All peptides used for immunogenicity studies or related serological tests for anti-CGRP antibody detection and / or measurement were synthesized on a small-scale F-moc chemical process using a Biosystems Peptide Synthesizer 430A, 431, and / or 433. Each peptide was prepared independently on a solid-phase support with F-moc protection at the amino terminus and side-chain protecting groups of the trifunctional amino acids. The intact peptides were cleaved from the solid-phase support, and the side-chain protecting groups were removed with 90% trifluoroacetic acid (TFA). The synthesized peptide products were evaluated using matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry to determine the correct amino acid composition. Reversed-phase HPLC (RP-HPLC) was also used to evaluate each synthesized peptide to confirm the synthetic state and concentration of the product. Despite strict control of the synthesis process (including stepwise monitoring of coupling efficiency), peptide analogs can still be generated due to unforeseen events during extended cycles, including amino acid insertion, deletion, substitution, and premature termination. Therefore, the synthesized products generally include multiple peptide analogs and the target peptide.

[0264] Despite including these unintended peptide analogs, the final synthetic peptide products can still be used for immunological applications, including immunodiagnostics (as antibodies capturing antigens) and pharmaceutical compositions (as peptide immunogens). Generally, provided a rigorous QC procedure is developed to monitor the preparation process and product quality assessment procedures to ensure the reproducibility and efficacy of the final products using these peptides, the peptide analogs, including mixtures of byproducts generated during the intentional design or synthesis process, are usually as effective as the purified product of the desired peptide. Large quantities of peptides, ranging from hundreds to thousands of grams, can be synthesized on a scale of 15 mmol / L to 150 mmol / L using a custom-designed automated peptide synthesizer, the UBI2003, or a similar model.

[0265] For the active ingredient used in the final pharmaceutical composition for clinical trials, the CGRP-related peptide immunogen construct can be purified using a prepared RP-HPLC under a light elution gradient, and the purity and consistency characteristics can be characterized using MALDI-TOF mass spectrometry, amino acid analysis, and RP-HPLC.

[0266] b. Preparation of compositions containing CGRP peptide immunogen constructs

[0267] Dosage forms are prepared using water-in-oil emulsions and suspensions containing mineral salts. Safety is another important factor to consider when designing pharmaceutical compositions for broad patient populations. Although water-in-oil emulsions are used in many clinical trials of pharmaceutical compositions in humans, alum remains a primary adjuvant in pharmaceutical compositions due to its safety profile. Therefore, alum or its mineral salt ADJUPHOS (aluminum phosphate) is frequently used as an adjuvant in clinical formulations.

[0268] In summary, the dosage forms specified in each experimental group described below typically contain all types of specially designed CGRP peptide immunogen constructs. Over 200 specially designed CGRP peptide immunogen constructs were carefully evaluated in guinea pigs for their relative immunogenicity, using corresponding CGRP peptides representing immunogenic B-cell epitope peptides. Epitope identification and serological cross-reactivity in various homologous peptides were analyzed using ELISA assays on well discs coated with peptides selected from SEQ ID NOs: 1-70.

[0269] As specified, use Seppic MONTANIDE, an oil agent approved for human use. TM ISA 51 is formulated in water-in-oil emulsion form, or mixed with mineral salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum), to prepare immunogen constructs of varying amounts. Typically, the CGRP peptide immunogen construct is dissolved in water at a concentration of approximately 20 to 2000 μg / mL and then mixed with MONTANIDE. TM ISA 51 was formulated as a water-in-oil emulsion (1:1 volume) or with mineral salts ADJUPHOS or ALHYDROGEL (1:1 volume) to prepare a composition. The composition was incubated at room temperature for approximately 30 minutes and vortexed for approximately 10 to 15 seconds prior to immunization. Animals were immunized with 2 to 3 doses of the specific composition, administered at time 0 (primary immunization) and 3 weeks after primary immunization (wpi), with a second booster immunization optionally administered at 5 or 6 wpi, via intramuscular route. Serum from immunized animals was then tested using selected B-cell epitope peptides to assess the immunogenicity of various CGRP peptide immunogenic constructs present in the formulation, as well as the cross-reactivity of the corresponding serum with CGRP protein. Based on the functional characteristics of the corresponding serum, those CGRP peptide immunogenic constructs initially identified in guinea pig screening with strong immunogenicity were further tested in vitro. Then, the selected candidate CGRP peptide immunogen constructs were prepared using an oil-in-water emulsion, mineral salt, and alum-based formulation and administered according to the immunization protocol during a specified period.

[0270] In the preparation of clinical trial submissions in patients with migraines following the application for investigational new drug, only the most promising CGRP peptide immunogen constructs will undergo further extensive evaluation before being included in the final dosage form for GLP-guided preclinical studies to investigate immunogenicity, duration, toxicity, and efficacy.

[0271] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0272] Example 2. Serological tests and reagents

[0273] The following details the serological tests and reagents used to evaluate the functional immunogenicity of CGRP peptide immunogen constructs and their formulations.

[0274] a. ELISA assays based on CGRP or CGRP B-cell epitope peptides for immunogenicity and antibody specificity analysis.

[0275] An ELISA assay for evaluating immune serum samples was developed and is described in the following examples. CGRP or CGRP B-cell epitope peptides (SEQ ID NOs: 1 to 70) at a concentration of 2 μg / mL (unless otherwise specified) in 10 mM sodium bicarbonate buffer at pH 9.5 were used to coat the wells of a 96-well disc at 37°C for 1 hour in a 100 μL volume.

[0276] Pores coated with CGRP or CGRP B-cell epitope peptides were reacted with 250 μL of gelatin prepared in PBS at a concentration of 3% by weight at 37°C for 1 hour to block non-specific protein binding sites. Then, a solution containing 0.05% by volume was used... Wash the wells three times with PBS containing 20% ​​normal goat serum, 1% gelatin, and 0.05% PBS. Dilute the serum sample to be tested with PBS at a ratio of 1:20 (unless otherwise specified). Add 100 μL of the diluted sample (e.g., serum, plasma) to each well and incubate at 37°C for 60 minutes. Then, use a 0.05% (v / v) solution of PBS. Wash the wells 6 times to remove unbound antibodies. Use horseradish peroxidase (HRP) conjugated species (e.g., guinea pig or rat) specific goat multiclonal anti-IgG antibodies or protein A / G as labeled tracers to bind to the antibody / peptide antigen complexes formed in the positive wells. Prepare 100 μL of HRP-labeled detection reagent at the pre-titrated optimal dilution with 1 v / v normal goat serum and 0.05 v / v... Add the solution to each well in 20 mL of PBS and react at 37°C for another 30 minutes. Utilize the 0.05% volume percentage of the solution. The wells were washed six times with PBS (20 μL) to remove unbound antibody, and then reacted with a substrate-receptor mixture containing 0.04% (w / v) 3',3',5',5'-tetramethylbenzidine (TMB) and 0.12% (v / v) hydrogen peroxide in sodium citrate buffer for 15 minutes. The peroxidase label was detected using the substrate-receptor mixture by the formation of a colored product. The reaction was terminated by adding 100 μL of 1.0 M sulfuric acid, and the absorbance at 450 nm was measured (A). 450 To determine the antibody titers of animals vaccinated with various peptide vaccine formulations, serum serially diluted 10 times from 1:100 to 1:10,000, or from 1:100 to 1:4.19x10⁻¹, was used. 8 The serum was tested using serially diluted serum at 4-fold concentrations, and A was used. 450 The critical value is set to A at 0.5. 450 Linear regression analysis was used to calculate the titer of the test serum, expressed as Log. 10 express.

[0277] b. Assess antibody reactivity against Th peptides using Th peptide-based ELISA assays.

[0278] A similar ELISA method was used as described above, employing 100 μL of Th peptide at a concentration of 2 μg / mL (unless otherwise specified) in 10 mM sodium bicarbonate buffer at pH 9.5, incubated at 37°C for 1 hour to coat the wells of a 96-well ELISA pan. To determine the antibody titers in animals vaccinated with various CGRP peptide vaccine formulations, serially diluted serum from 10-fold dilutions (1:100 to 1:10,000) was used for testing, and A... 450 The critical value is set to A at 0.5. 450 Linear regression analysis was used to calculate the titer of the test serum, expressed as Log. 10 express.

[0279] c. Detailed and specific analysis of target CGRP B-cell epitope peptides was performed using an ELISA assay based on the 10-mer peptide of B-cell epitope clusters, utilizing epitope identification.

[0280] A fine-grained specificity analysis of anti-CGRP antibodies from hosts immunized with a CGRP peptide immunogen construct was performed using an ELISA assay based on B-cell epitope clusters and 10-mer peptides for epitope identification. In short, following the steps of the antibody ELISA method described above, in a double-replica manner, 0.5 μg of individual CGRP or a related 10-mer peptide (SEQ ID NOs: 26-70) was coated per well of a 96-well plate per 0.1 mL well. Then, 100 μL of serum sample (prepared in PBS, diluted 1:100) was reacted in the 10-mer plate. For specificity confirmation, a fine-grained specificity analysis of anti-CGRP antibodies from the immunized host, correlated with the target B-cell epitope, was performed using the corresponding CGRP peptide or an unrelated control peptide.

[0281] d. Immunogenicity assessment

[0282] Pre-immune and immune serum samples were collected from animals or human individuals according to the experimental vaccination protocol, and heated at 56°C for 30 minutes to inactivate serum complement factors. Following administration of the vaccine formulation, blood samples were obtained according to the protocol, and their immunogenicity against specific targets was assessed using an ELISA assay based on corresponding CGRP B-cell epitope peptides. Serially diluted serum samples were tested, and the logarithm of the reciprocal of the dilution factor was calculated (Logarithm of CGRP). 10 The positive titer is indicated by the ability to elicit a high-titer antibody response specific to the desired epitope within the target antigen and high cross-reactivity with CGRP protein, while maintaining antibody reactivity against the "T helper cell epitope" used to provide enhancement of the desired B cell response at a low to negligible level. The immunogenicity of a particular vaccine formulation is evaluated based on its ability to elicit a high-titer antibody response specific to the desired epitope within the target antigen and high cross-reactivity with CGRP protein, while maintaining antibody reactivity against the "T helper cell epitope" used to provide enhancement of the desired B cell response at a low to negligible level.

[0283] Example 3. Evaluation of the functional properties of antibodies induced by CGRP peptide immunogen constructs and their formulations in an in vitro assay targeting intracellular cAMP production.

[0284] Further testing was conducted to determine the ability of immune serum or purified anti-CGRP antibodies in the immunized vaccine to inhibit CGRP-induced intracellular cAMP production.

[0285] a. Antibody purification

[0286] Follow all antibody purification procedures according to the instruction manual for the antibody purification kit (Thermo Fisher, Cat no. 89953). Carefully calibrate the purified IgG concentrations for each group for in vitro assays.

[0287] b. Cell preparation and maintenance

[0288] L6( CRL-1458 TMThe cell line was purchased from ATCC. The basal medium for this cell line was Dulbecco's Modified Eagle's Medium (catalog number 30-2002) prepared by ATCC. Fetal bovine serum was added to a final concentration of 10% to prepare a complete growth medium. The cell line was then cultured at 37°C in an environment containing 95% air and 5% CO2.

[0289] c. CGRP treatment in the presence / absence of IgGs

[0290] Anti-CGRP IgGs were further screened for in vitro neutralization activity using a cell-based cAMP activation assay. All steps were performed in a 384-well round-bottom low-volume microplate (Mediomics, LLC, catalog number 163301). Five μL of rat α-CGRP (final concentration 10 nM) was incubated at room temperature for 30 minutes in the presence of anti-CGRP IgG (final concentration 1–20 μg / ml). Then, 5 μL of rat L6 muscle cells (5000 cells) prepared in 1X KRB-IBMS buffer were added. The microplate was incubated at room temperature for 30 minutes.

[0291] d. Cell-based CGRP neutralization assay (cAMP level detection)

[0292] Following the reaction, cAMP activation was performed using the MEDIOMICS BRIDGE-IT all-in-one cAMP fluorescence assay (MEDIOMICS, catalog number 122938 / 122939) according to the manufacturer's instructions. 10 μl of the all-in-one cAMP assay solution was added to each well, and the mixture was aspirated from both ends to lyse the cells and begin cAMP detection. The microplate was covered to prevent evaporation and light exposure. The microplate was incubated at room temperature for 30 minutes, and fluorescence intensity was detected using a SPECTRAMAX I3X MULTI-MODE MICROPLATE READER (excitation at 485 nm, detection of divergence at 540 nm). Data were recorded as a percentage. 0% represents L6 cells only, while 100% represents CGRP-treated L6 cells.

[0293] Example 4. Animals used for safety, immunogenicity, toxicity, and efficacy studies.

[0294] a. Guinea pig:

[0295] Mature, not in contact with or stimulated by antigens Immunogenicity studies were conducted on adult male and female Duncan-Hartley guinea pigs (300-350 g / BW). At least three guinea pigs were used in each group. The study was conducted at an animal facility contracted to United Biomedical Inc. (UBI) as the commissioning agency, in accordance with an approved IACUC application involving Duncan-Hartley guinea pigs (8-12 weeks old; Covance Research Laboratories, Denver, PA, USA).

[0296] b. Crab-eating macaque:

[0297] Immunogenicity and repeated-dose toxicity studies were conducted on adult male and female monkeys (cynomolgus monkeys, approximately 3-4 years old; Zhaoyan New Drug Research Center, Suzhou, China) at animal facilities contracted by UBI as the trial commissioner, in accordance with an approved IACUC application.

[0298] c. Mice

[0299] Female Balb / C mice (n=6 / group) were injected five times at 0, 3, 6, 9, and 12 wpi before capsaicin challenge, via intramuscular injection (IM) of either the test vaccine (40 μg / 0.1 ml / dose) or the control (0.1 ml / dose). Animals were housed in the UBI Asia laboratory's animal facilities and acclimatized for one week under fixed temperature (22°C), humidity (72%), and a 12-hour light / 12-hour dark cycle. Mice had free access to food and water. All experimental protocols followed laboratory animal care principles. Blood samples were collected as described in the experimental protocol. Antibody titers against anti-CGRP (mice) were analyzed using ELISA.

[0300] Example 5. A vaccine formulation for evaluating the immunogenicity of CGRP peptide constructs in guinea pigs.

[0301] The pharmaceutical compositions and vaccine formulations used in each experiment are described in more detail below.

[0302] In summary, the dosage forms specified in each experimental group typically contain all types of specially designed CGRP peptide immunogen constructs with CGRP B-cell epitope peptide fragments linked to hybrid T helper cell epitopes via different types of spacer regions (e.g., εLys(εK) or lysine-lysine-lysine(KKK) to enhance peptide construct solubility). These hybrid T helper cell epitopes comprise two sets of artificial T helper cell epitopes derived from measles virus fusion proteins and hepatitis B surface antigen. The CGRP B-cell epitope peptides are linked to the amino or carboxyl terminus of the specially designed peptide construct. Hundreds of specially designed CGRP peptide immunogen constructs were initially evaluated in guinea pigs for their relative immunogenicity to their corresponding CGRP B-cell epitope peptides. As specified, varying amounts of the CGRP peptide immunogen construct are formulated in an oil-in-water emulsion using Seppic MONTANIDE ISA 51, an oil-based formulation approved for human vaccines, or in a suspension using mineral salts (ADJUPHOS) or ALHYDROGEL. Typically, the vaccine formulation is prepared by dissolving the CGRP peptide construct in water at a concentration of approximately 20 to 800 μg / mL and formulating it with MONTANIDE ISA 51 to form an oil-in-water emulsion (1:1 volume), or with mineral salts (ADJUPHOS) or ALHYDROGEL (1:1 volume). The vaccine formulation is incubated at room temperature for approximately 30 minutes and vortexed for approximately 10 to 15 seconds prior to immunization.

[0303] Animals were immunized with 2 to 5 doses of a specific vaccine formulation, administered at time 0 (primary immunization) and 3 weeks after primary immunization (wpi) (booster immunization), with a second booster immunization optionally administered at 5 or 6 wpi, via intramuscular route. The immunogenicity of the corresponding CGRP peptide immunogenic construct used in the individual vaccine formulation, and its cross-reactivity with the corresponding CGRP B-cell epitope peptide or full-length CGRP, were then evaluated in these immunized animals. For dosing regimens specified in the immunization protocol, those CGRP peptide immunogenic constructs that showed strong immunogenicity in the initial screening in guinea pigs were further tested in rhesus monkeys in water-in-oil emulsions, mineral salt formulations, and alum-based formulations.

[0304] Using corresponding mouse CGRP peptide immunogen constructs, only the most promising CGRP peptide immunogen construct candidates were further extensively evaluated to assess their ability to overcome immune tolerance in mice. The CGRP peptide immunogen constructs exhibiting the best immunogenicity in mice elicited anti-CGRP antibody titers against endogenous CGRP; particularly, they demonstrated the ability to inhibit capsaicin-induced skin blood flow in mouse models. The optimized CGRP peptide immunogen constructs were incorporated into the final vaccine formulation, providing a basis for use in GLP-guided studies demonstrating immunogenicity, duration of action, toxicity, and efficacy in preparation for Investigational New Drug (IND) applications, and in clinical trials in migraine patients.

[0305] Example 6. Design rationality, screening, identification, functional property evaluation and optimization of a multi-component vaccine formulation containing a CGRP peptide immunogen construct for the treatment of migraine.

[0306] Based on the scientific information provided in Figures 1 and 2, CGRP was selected as the target molecule for design and the subject of this invention. Figure 1 shows the alignment of CGRP sequences from humans (SEQ ID NO: 1), macaques (SEQ ID NO: 2), mice / rats (SEQ ID NO: 3), and many other species; Figure 2 describes the pathway from vaccine discovery to commercialization based on highly precise, specially designed synthetic peptides. Figure 2 provides a brief overview of these steps, accompanied by a flowchart illustrating the development process of CGRP vaccine formulations from discovery to commercialization (industrialization). Detailed evaluation and analysis of each step, yielding both pleasant and unpleasant surprises, has led to numerous experiments in the past, ultimately resulting in the commercialization of safe and effective CGRP vaccine formulations.

[0307] a. Design History

[0308] Each peptide immunogen construct or immunotherapy product requires its own design focus and methodology, based on its specific disease mechanism and the target protein required for intervention. For migraine treatment, CGRP is selected as the target molecule based on the available scientific information outlined in Figure 1. As shown in Figure 2, the process from discovery to commercialization typically takes one to several decades. Identification of CGRP B-cell epitope peptides associated with the functional site used for intervention is crucial for immunogen construct design. Successive lead immunogenicity studies are conducted in guinea pigs, incorporating various T helper support (carrier proteins or suitable T helper peptides) in various formulations, followed by evaluation of the functional properties of the induced purified antibodies or vaccine formulations using specific CGRP peptide immunogen constructs in specific in vitro functional analyses or in vivo proof-of-concept studies in selected animal models. After extensive serological validation, candidate CGRP B-cell epitope peptide immunogen constructs are then further tested in non-human primates to further validate the immunogenicity and direction of the CGRP peptide immunogen design. The selected CGRP peptide immunogen constructs were then formulated with different mixtures to assess subtle differences in functional properties related to their respective interactions when used in combination. Further evaluation determined the final peptide constructs, peptide compositions, and formulations, as well as the various physical parameters of the formulations, leading to the development of the final product.

[0309] The amino acid sequence of the CGRP peptide immunogen construct was selected based on several design theories. Several of these theories include the use of CGRP B-cell epitope peptide sequences, as detailed below:

[0310] (i) CGRP lacks autologous T helper cell epitopes to avoid autologous T cell activation (as previously used in the treatment of Alzheimer's disease using Aβ-targeting). 1-42 Clinical trials of the AN1792 vaccine have reported that autologous T-cell activation may lead to brain inflammation, which in turn can cause meningococcal encephalitis.

[0311] (ii) It is not immunogenic itself because it is its own molecule;

[0312] (iii) When administered to a host, immunogenicity can be acquired by using a protein carrier or an effective T helper cell epitope;

[0313] (iv) Initiate high-titer antibodies against CGRP peptide sequences (B cell epitopes) that are not against protein carriers or effective T helper cell epitopes.

[0314] (v) Induces high-titer antibodies that inhibit the increase in intracellular cAMP caused by CGRP and CGRP receptor interaction and cell activation; and

[0315] (vi) This vaccine formulation, when administered to animal models (e.g., BALB / c mice), can inhibit capsaicin-induced dorsal blood flow, and can serve as proof-of-concept evidence for the treatment of migraines.

[0316] b. Design and validation of CGRP peptide immunogen constructs for pharmaceutical compositions that have the potential to treat migraine patients.

[0317] To generate the most effective peptide constructs for inclusion in pharmaceutical compositions, libraries of human CGRP B-cell epitope peptides (e.g., SEQ ID NOs: 4-24) and hybrid T-helper cell epitopes derived from various pathogens or artificial T-helper cell epitopes (e.g., SEQ ID NOs: 74-115) were further engineered and fabricated, for example, representative CGRP peptide immunogenic constructs (e.g., SEQ ID NOs: 116-180) to provide initial immunogenicity studies for guinea pigs.

[0318] i) Design by selecting CGRP B-cell epitope peptide sequences from receptor-binding or receptor-activation regions.

[0319] CGRP B cell epitopes were designed by selecting the CGRP receptor-binding region located in the middle / carboxyl terminus of CGRP and the receptor activation region from the C2-C7 cyclic structure / middle region of the CGRP N-terminus. The peptide immunogen constructs were then further prepared to induce immune serum in guinea pigs. The immune serum was initially provided for immunogenicity assay by ELISA on a microplate coated with CGRP B cell epitope peptides, and then provided for in vitro functional analysis evaluation.

[0320] Upon binding to its receptor, CGRP transmits activation signals within the cell, leading to elevated intracellular cAMP levels in other cellular events. Compared to an antibody-deficient control group, as shown in Figure 4 and the table within Figure 3, the purified antibody from guinea pig immune serum against a specific CGRP peptide immunogen construct demonstrated the ability to neutralize CGRP functional properties, with an IC50 rating of inhibiting 50% of the cAMP elevation. 50 .

[0321] Initially, these CGRP peptide immunogenic constructs were formulated using ISA 51 and CpG. Primary immunization was performed in guinea pigs at a dose of 400 μg / 1 mL, followed by booster immunizations at a dose of 100 μg / 0.25 mL (3, 6, and 9 wpi) for immunogenicity studies. To test immunogenicity in guinea pigs, an ELISA assay was used, with guinea pig immune serum from each wpi collection serially diluted 10-fold from 1:100 to 1:10000. Corresponding mouse / rat CGRP B-cell epitope peptides and full-length CGRP peptides were coated onto ELISA microplates at a dose of 0.5 μg peptide per well. Using A... 450 The critical value is set to A at 0.5. 450 Linear regression analysis was used to calculate the titer of the test serum, expressed as Log. 10 As shown in Figure 3, Tables 4, 5, and 6 display the detailed titers of CGRP peptide immunogen constructs derived from representative B-cell epitopes. Although designed short CGRP peptides are generally non-immunogenic due to their lack of endogenous Th epitopes, the addition of exogenous Th epitopes can enhance the immunogenicity of specific CGRP peptide immunogen constructs. Detailed analysis of the reactivity / specificity patterns of various structures from Tables 4, 5, and 6, as well as Figure 3, allows for the assessment of the immunogenicity conferred by certain residues within the CGRP molecule, which aids in the further design of optimal peptide immunogen constructs.

[0322] ii) Lack of autologous T helper cell epitopes within the selected CGRP B cell epitopes to avoid autologous T cell activation.

[0323] As shown in Table 8, representative CGRP B-cell epitopes, such as those with SEQ ID Nos: 5, 6 and 15, do not elicit any antibodies against CGRP when provided to the corresponding peptide immunogen constructs for use in effective vaccine formulations. Therefore, they lack undesired endogenous Ths epitopes within the selected CGRP B-cell epitopes.

[0324] iii) The concentrated antibody response induced by the CGRP peptide immunogen construct targets only CGRP B cell epitopes.

[0325] It is well known that all carrier proteins used to enhance the immune response to target B cell epitope peptides (such as keyhole cyanin (KLH), diphtheria toxoid (DT), and tetanus toxoid (TT) proteins) can induce more than 90% of antibodies to target the enhancing carrier protein and less than 10% of antibodies to target B cell epitopes in the immune host by chemically conjugating the B cell epitope peptide with its respective carrier protein.

[0326] Therefore, it is important to assess the specificity of the CGRP peptide immunogen constructs of the present invention. Two representative CGRP peptide immunogen constructs (SEQ ID NOs: 142 and 143 from Table 9) have B-cell epitopes of different lengths from CGRP 20-37 and 22-37, which are linked to heterologous T helper cell epitopes via spacer sequences. 1 (SEQ ID NO: 98) was prepared for antigenicity assessment. 1 (T helper cell epitope peptides for B cell epitope immune enhancement) was coated on a microplate, and guinea pig immune serum was used to test its effectiveness against those for immune enhancement. Cross-reactivity of peptides. As shown in Table 8, the high immunogenicity of target CGRP B cell epitope peptides corresponding to these structural pairs (even with only one injection, the high titer of antibodies produced against CGRP B cell epitopes is >5 Log). 10 This illustrates the point, and in stark contrast, it was found that most (if not all) immune sera were... One peptide is unreactive.

[0327] In summary, simple immunogen design (comprising a targeted CGRP B-cell epitope peptide linked to a carefully selected T helper cell epitope) can generate a concentrated immune response against the corresponding CGRP B-cell epitope peptide. For pharmaceutical composition design, the more specific the resulting immune response, the greater the safety it provides to the composition. Therefore, the CGRP peptide immunogen construct of this invention is highly specific, yet highly effective against its B-cell target.

[0328] iv) Fine epitope identification using immune serum targeting the selected CGRP peptide immunogen construct.

[0329] As shown in Table 10, in the fine epitope identification study, antibody binding sites were located at specific residues within the target B cell epitope region, resulting in the synthesis of 45 overlapping 10-mer peptides (SEQ ID NOs: 26-70). These peptides covered the sequence from amino acid -9 to amino acid 45, encompassing the full-length region of CGRP before and after CGRP molecule processing, as well as the precursor sequence. These 10-mer peptides were then coated onto 96-well microplates as solid-phase immunosorbents. Guinea pig antiserum, prepared at a 1:100 dilution in sample dilution buffer, was added to the wells of the microplates coated with 2.0 μg / mL of the 10-mer peptides, and the plates were incubated at 37°C for 1 hour. After washing the wells with washing buffer, horseradish peroxidase (HRP) conjugated recombinant protein A / G was added, and the plates were incubated for 30 minutes. After washing again with PBS, the antibody was added to the wells, and the absorbance at 450 nm was measured using an ELISA microdisk analyzer. The samples were analyzed in duplicate. The binding of the immune serum induced by the CGRP peptide immunogen to the wells coated with the corresponding CGRP B cell epitope peptide represented the strongest antibody binding signal.

[0330] Table 10 shows the detailed epitope identification results, indicating that guinea pig serum from the CGRP peptide immunogen constructs SEQ ID NOs: 122, 123, 127, 129, 130, 132, 137, and 139 (containing CGRP B cell epitope peptides from the receptor-binding regions derived from amino acids 11 to 37 and from the antibody-activating regions located near the C2-C7 ring structures of amino acids 1-25, 11-25, 8-18, 11-35, 11-37, 15-37, and 18-37) induces a primary target for antibodies derived from amino acid 1-37 (SEQ ID NOs: 122, 123, 127, 129, 130, 132, 137, and 139). IDNO: 1) is a high-titer antibody containing the 10-mer peptide cluster. It exhibits high cross-reactivity with peptides, primarily with the following amino acids in different forms: 1-13 are associated with B epitopes 1-25; 15-26 are associated with B epitopes 11-25; and mainly with B epitopes 8-18; 22-33 and 26-36 are associated with B epitopes 11-35; 23-33 and 28-37 are associated with B epitopes 11-37; 23-33 and 28-37 are associated with B epitopes 18-37; 22-33 and 26-36 are associated with B epitopes 11-35; and 17-26, 20-30, and 28-37 are associated with B epitopes 15-37. Interestingly, it was found that although the same B-cell epitopes were used to design the peptide immunogen constructs SEQ ID NOs: 123 and 130, SEQ ID NO: 123 differed from SEQ ID NO: 130 only in the spacer region (having an additional KKK spacer region). Additional reactivity against AAs 20-30 and AAs 26-36 was found in the structure SEQ ID NO: 130 (which has a shorter spacer region without KKK residues).

[0331] In summary, the designed synthetic CGRP peptide immunogen construct induced a robust immune response in guinea pigs, generating multiclonal antibodies against different 10-mer peptide clusters in CGRP. These 10-mer peptide clusters are highly proximal to both the CGRP receptor-binding region near their respective carboxyl termini and the receptor activation region near the C2-C7 cyclic structure, potentially providing important medical interventions. Epitope identification and functional analysis will allow for the identification of the optimal peptide immunogen construct for vaccine formulations.

[0332] Example 7. Evaluation of the functional properties of antibodies induced by CGRP peptide immunogen constructs and their formulations in vitro.

[0333] As shown in Tables 4, 5, 6, 7, 8, and 9, after demonstrating that antibodies purified from immune serum of guinea pigs immunized with carefully selected candidate CGRP immunogen constructs possess high immunogenicity and cross-reactivity, the following studies were designed to evaluate whether representative purified IgGs from these immune sera collected from each animal at 6 wpi could inhibit the intracellular increase in cAMP following CGRP binding to its receptor due to activation of the C2-C7 ring structure in CGRP.

[0334] At the molecular level within smooth muscle cells, CGRP binds to its receptor via its carboxyl-terminal region and then activates the receptor using its cyclic region (reference). The C2-C7 cyclic structure with disulfide bonds plays a central role in receptor activation, which is closely associated with an increase in intracellular cAMP. In neutralization assays, various anti-CGRP IgGs were used to characterize their potential anti-CGRP effects. Specifically, the effects were assessed using changes in intracellular cAMP levels via functional pharmacology. This in vitro functional assessment is particularly important for evaluating the anti-CGRP activity of guinea pig immune sera against the CGRP peptide immunogen construct of this invention, and the assay method is detailed in Example 4.

[0335] Inhibiting intracellular cAMP elevation through anti-CGRP antibody in CGRP-activated phosphorylation

[0336] As described in Example 4, immune serum was collected from 6 wpi blood samples from each animal, and antibodies were purified. As shown in Example 6, 21 CGRP peptide immunogen constructs were tested in guinea pigs for their individual immunogenicity. The purified antibodies were classified into three categories based on the individual target B-cell epitope peptides used in the peptide immunogen constructs. These targeted B-cell epitope peptides from the N-terminal, intermediate, and C-terminal regions, respectively. Data were recorded as the percentage of cAMP detected in CGRP-treated L6 cells. 0% represents L6 cells only, while 100% represents CGRP-treated L6 cells. As shown in Figure 4 and the accompanying table, structures containing CGRP B-cell epitope peptides from the N-terminal, intermediate, or C-terminal regions exhibited IC50 values ​​ranging from 0.60 to >20 for cAMP levels. 50 (μg / mL). For practical purposes, we use IC50 concentrations less than 10 μg / mL. 50 As a significant antibody-mediated inhibition of cAMP production. Ranking of representative structures exhibiting effective functional immunogenicity (with IC50 from low to high). 50The CGRP peptide immunogen construct (μg / mL) is SEQ ID NOs: 130>127>150>125>137>123>121>119>124>131>126>133>120>129>135>116~117~118~128~139. With the precision of up to a few residues in the CGRP 37-mer structure, we can depict the optimal design of the CGRP peptide immunogen construct.

[0337] In summary, the relative ranking of the CGRP peptide immunogen constructs in terms of their individual functional properties, as shown above, is valuable for demonstrating functional efficacy in subsequent CGRP vaccine formulations.

[0338] Example 8. As a proof-of-concept study for a CGRP vaccine, representative mouse CGRP peptide immunogen constructs were evaluated in a prophylactic mode using a BALB / c mouse capsaicin-induced skin blood flow model.

[0339] a. Experimental theoretical basis

[0340] Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide widely expressed in the central and peripheral nervous systems. It is primarily associated with small, unmyelinated sensory neurons located close to blood vessels. CGRP is a potent vasodilator, and local application of CGRP results in a transient increase in blood flow. CGRP is also involved in pain transmission, pain modulation, and neuroinflammation. CGRP can be released from sensory neurons via activation of transient receptor potential cation channels using capsaicin.

[0341] Laser Doppler imaging (LDI) has been used to detect changes in skin blood flow, which have been shown to be primarily induced by CGRP. As demonstrated by the weakened responses in CGRP knockout mice in various pain models, CGRP is also associated with inflammatory pain. This role in pain perception is consistent with the manifestation of CGRP in sensory neurons.

[0342] In mammalian plasma, the half-life of CGRP is approximately 10 minutes. In the human trigeminal ganglion, CGRP-immunoreactive neurons account for 50% of all neurons (Tajti, et al., 1999). Targeting CGRP to treat migraine through anti-CGRP vaccination could meet an unmet medical need, with the potential for long-term CGRP blockade to cost-effectively treat migraine in a preventative mode over extended periods.

[0343] This study was designed to test a mouse CGRP peptide immunogen construct, a representative counterpart of the human CGRP peptide immunogen construct, in a Balb / C mouse model that was identified in Example 7 as having the ability to reduce capsaicin-induced skin blood flow. This model serves as a proof-of-concept study to demonstrate the efficacy of anti-CGRP vaccines in treating migraines.

[0344] b. Experimental Design

[0345] Representative test items:

[0346] The following lists the CGRP peptide immunogen construct vaccine formulations used in this trial:

[0347] Group 1: (SEQ ID NO: 123) p4799kb:

[0348] Formulated with ISA51 and CpG3 1-AAl1-37 (Human αCGRP)

[0349] Group 2: (SEQ ID NO: 141) p5154kb:

[0350] Formulated with ISA51 and CpG3 3-AA11-37 (Human αCGRP)

[0351] Group 3: (SEQ ID NO: 151) p5155kb:

[0352] Formulated with ISA51 and CpG3 3-AA11-37 (rat / mouse αCGRP)

[0353] Group 4: (SEQ ID NO: 123) p4799kb:

[0354] Formulated with ADJUPHOS and CpG3 1-AA11-37 (Human αCGRP)

[0355] Group 5: (SEQ ID NO: 131) p5144kb:

[0356] Formulated with ISA51 and CpG3 1-AA11-25 (Human αCGRP)

[0357] Table 2 lists these items and their group information.

[0358] Reference substance:

[0359] Group 6: ISA51 and CpG3

[0360] Group 7: ADJUPHOS and CpG3

[0361] Group 8: Physiological saline

[0362] Groups and dosages:

[0363] The grouping is executed as follows:

[0364] Individual identification:

[0365] The test animals were identified using the picric acid symbol.

[0366] Group identification:

[0367] Properly label the cages for identification. The label should include the trial name, IACUC number, route of administration, observation period, cage number, number of animals per cage, species, strain, sex, date of admission, age at admission, animal number, breeder, and agent.

[0368] Route of administration and injection site:

[0369] Female Balb / C mice (n=6 per group) were injected five times at 0, 3, 6, 9, and 12 wpi before capsaicin induction (challenge). The injection was performed intramuscularly (IM) at one injection site (quadriceps femoris muscle of the hind limb) with either the test vaccine (40 μg / 0.1 ml / dose) or the control (0.1 ml / dose).

[0370] Processing procedure:

[0371] During the treatment process, the control or test item was injected a total of 5 times at 0, 3, 6, 9 and 12 wpi.

[0372] Collection and preparation of biological samples:

[0373] Allow the blood to clot in the test tube to stand at room temperature for at least 30 to 60 minutes to separate the serum from the clot. Remove the clot by centrifuging at 1,000 x g for 10 minutes using a refrigerated centrifuge. Immediately transfer the serum to sterile 1.5 mL microcentrifuge tubes (polypropylene tubes). Keep all samples on moist ice during processing. Unused serum samples should be frozen at -80°C.

[0374] Laser Doppler Imaging (LDI):

[0375] On the day of the experiment, the mice were shaved on their backs and placed on a heating pad located below the LDI instrument. Anesthesia was induced using 25 mg / kg Zoltil, and the animals were stabilized under anesthesia for approximately 20 minutes before scanning. A series of scans followed two baseline scans.

[0376] A 2 μL solution of capsaicin (prepared by dissolving 50 mg of capsaicin in a solution containing 83.4 μL EtOH, 55.6 μL Lween 20, and 27.8 μL pure water in a 3:2:1 ratio) was applied to each of the two O-rings placed on the animal's back. Scans were performed every 2.5 minutes for 10 minutes. Data from the region of interest were analyzed using MoorVMS-LDF software. The mean signal from the region of interest at each time point was calculated using an Excel worksheet. Data were reported as a percentage change from baseline.

[0377] Immunological analysis of antibody titer:

[0378] Serum or CSF samples were collected and coated with full-length human CGRP antigen. Anti-CGRP antibody titers were measured using an ELISA kit prepared as shown in Example 2. Serum samples were serially diluted 3-fold from an initial dilution of 1:1000. Absorbance values ​​(A) were detected using an automated microplate reader. 450 To determine the antibody ELISA titer, using Log 10 express.

[0379] c. Test Results

[0380] Injection site reaction:

[0381] Immunization procedures can sometimes cause mild and temporary swelling at the injection site.

[0382] Antibody titers from immune serum of mice that have received immunization:

[0383] Representative ELISA results showed that two peptide immunogen constructs from mouse counterparts (e.g., SEQ ID NO: 151) and CGRP peptide immunogen constructs (SEQ ID NOs: 132 and 141) not only induced high immunogenic titers against their individual B-cell epitope peptides (SEQ ID NOs: 15 and 9), but also revealed moderate cross-reactivity of antibodies from these two immune sera against their homologous full-length human CGRP, as shown in Figure 5. This study demonstrates that two representative CGRP peptide immunogens can induce specific antibodies that are cross-reactive against human CGRP B-cell epitope peptides and their mouse counterpart peptides. In this POC animal study, mouse / rat CGRP B-cell epitope peptide immunogen construct counterparts were used... 3 (to) 1. More effective combination of Th peptide libraries) as T helper cell peptides to further enhance the immunogenicity of selected structures in mice (e.g., SEQ ID NO: 132, 134, 136, 138, 140, 141 and 151) and connectors (e.g. SEQ ID NO: 72).

[0384] LDI results:

[0385] The capsaicin model for measuring skin blood flow is an in vivo pharmacokinetic model in both animals and humans. It is non-invasive, technically simple, and provides rapid and objective outcome variables. This model is repeatable, and the measurements are sufficiently reproducible. Therefore, this model provides an ideal assessment method for the clinical evaluation of CGRP blocking therapy. Like all other biomarker models, this model also has its limitations. The capsaicin model remains a simulation of the naturally occurring pathophysiological process of the desired investigation. The effect of drug- or vaccine-induced antibodies on capsaicin-induced skin blood flow may provide a trend toward anti-CGRP activity, suggesting their efficacy in inhibiting peripheral skin blood flow.

[0386] As shown in Figure 6, when compared with controls such as saline or various adjuvant formulations alone, the CGRP peptide immunogen constructs containing SEQ ID NOs: 123, 131, 141, and 151 in the CpG-containing ADJUPHOS and ISA51 formulations all formed peptide / CpG complexes, which elicited significant and specific anti-CGRP antibody titers (Log) in vaccinated mice after three immunizations. 10 From 4 to 6). Animals were measured at 8, 12, and 14 wpi, showing inhibition of capsaicin-induced skin microvascular blood flow (Figure 6). This inhibitory effect lasted for 14 weeks within the measured timeframes. Although the titer of the selected antibody preparation in the immunogenicity study or its corresponding IC50 value for the cAMP inhibitory efficacy against individual antibodies was measured in vitro... 50 Differences were found in the s-values, but the three representative CGRP peptide immunogen constructs (e.g., SEQ ID NOs: 123, 131 and 141) and their formulations showed similar ability to reduce capsaicin-induced skin blood flow in these immunized mice, suggesting the efficacy of using these CGRP peptide immunogen constructs in treating migraines.

[0387] Table 1

[0388] Amino acid sequences of α-CGRP and its fragments used in serological assays

[0389]

[0390]

[0391] Table 2

[0392] The amino acid sequence of the pathogen protein-derived Th epitope, which is used to design α-CGRP peptide immunogen constructs, includes an idealized artificial Th epitope.

[0393]

[0394] Table 3

[0395] Amino acid sequence of α-CGRP peptide immunogen construct

[0396]

[0397]

[0398] ＊ Peptides cyclize with the indicated cysteine ​​residues via disulfide bonds (Table 4).

[0399] Immunogenicity assessment of α-CGRP peptide immunogen constructs in guinea pigs

[0400]

[0401]

[0402] Table 6

[0403] Immunogenicity assessment of α-CGRP peptide immunogen constructs in guinea pigs

[0404]

[0405] Table 7

[0406] Cross-reactivity between immune serum and full-length CGRP

[0407]

[0408] Table 8

[0409] Lack of intrinsic Th epitopes within the selected CGRP B epitope sequence

[0410]

[0411] Table 9

[0412] Immunogenicity assessment of the Th epitope region of the selected CGRP peptide immunogen construct in guinea pigs

[0413]

[0414]

[0415]

Claims

1. A CGRP peptide immunogen construct, represented by the following molecular formula: (Th)–(A)–(CGRP functional B-cell epitope peptide)–X or (CGRP functional B-cell epitope peptide)–(A)–(Th)–X or (Th)–(A)–(CGRP functional B-cell epitope peptide)–(A)–(Th)–X in Th is a heterologous T helper cell epitope of SEQ ID NO:98; A is a heterologous spacer region selected from (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72) and Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73); (CGRP functional B-cell epitope peptide) is a B-cell epitope peptide selected from SEQ ID NO: 4-19; And X is α-COOH or α-CONH2 of an amino acid.

2. A CGRP peptide immunogen construct, comprising the following: a. B-cell epitopes selected from SEQ ID NO: 4-19; b. T helper cell epitopes of SEQ ID NO:98; and c. Heterogeneous spacer regions selected from (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72) and Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73). The B-cell epitope is covalently linked to the T-helper cell epitope via the heterologous spacer region.

3. The CGRP peptide immunogen construct according to claim 1 or 2, wherein the B cell epitope is selected from SEQ ID NO: 5-7, 9-13, 15-16 and 18.

4. The CGRP peptide immunogen construct according to claim 1 or 2, wherein the B cell epitope is selected from SEQ ID NO: 6, 8, 9 and 19.

5. The CGRP peptide immunogen construct according to claim 4, wherein the B cell epitope is SEQ ID NO:

6.

6. The CGRP peptide immunogen construct according to claim 1 or 2, wherein the heterologous spacer region is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73).

7. The CGRP peptide immunogen construct of claim 2, wherein the T helper cell epitope is covalently linked to the amino terminus of the B cell epitope via the heterologous spacer region.

8. A CGRP peptide immunogen construct, wherein the amino acid sequence of the CGRP peptide immunogen construct is selected from SEQ ID NO:116-133, 135, 137, 139 and 141.

9. A CGRP peptide immunogen construct, wherein the amino acid sequence of the CGRP peptide immunogen construct is selected from SEQ ID NO: 119-121, 123-127, 130-131, 133, 137 and 139.

10. A CGRP peptide immunogen construct, wherein the amino acid sequence of the CGRP peptide immunogen construct is selected from SEQ ID NO: 120, 121, 122, 123, 125, 127, 129, 130, 131, 132, 137 and 141.

11. A CGRP peptide immunogen construct, wherein the amino acid sequence of the CGRP peptide immunogen construct is selected from SEQ ID NO: 120, 123, 130, 131 and 141.

12. A CGRP peptide immunogen construct, wherein the amino acid sequence of the CGRP peptide immunogen construct is SEQ ID NO:

120.

13. A composition comprising the CGRP peptide immunogen construct as described in any one of claims 1-12.

14. A pharmaceutical composition comprising: a. The CGRP peptide immunogen construct as described in any one of claims 1-12; and b. Pharmaceutically acceptable delivery carriers and / or adjuvants.

15. The pharmaceutical composition of claim 14, wherein... The CGRP peptide immunogen construct is mixed with CpG oligodeoxynucleotides (ODN) to form a stable immunostimulatory complex.

16. The pharmaceutical composition of claim 14, wherein the pharmaceutically acceptable delivery carrier and / or adjuvant is an aluminum salt.

17. The pharmaceutical composition of claim 15, wherein the pharmaceutically acceptable delivery carrier and / or adjuvant is aluminum phosphate.

18. Use of the CGRP peptide immunogen construct according to any one of claims 1-12 in the preparation of a medicament for the prevention and / or treatment of migraine in a subject.

19. The use according to claim 18, wherein the CGRP peptide immunogen construct comprises the B cell epitope of SEQ ID NO:

6.

20. The use according to claim 18, wherein the CGRP peptide immunogen construct comprises the amino acid sequence SEQ ID NO: 120.

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