Peptide immunogens targeting calcitonin gene-related peptide (CGRP) and formulations thereof for prevention and treatment of migraine

Abstract

To provide peptide immunogen constructs targeting portions of the calcitonin gene-related peptide (CGRP), compositions containing the constructs, antibodies elicited by the constructs, and methods for making and using the constructs and compositions thereof.SOLUTION: The disclosed peptide immunogen constructs have more than about 30 amino acids, and contain: (a) a B cell epitope having more than about 7 contiguous amino acid residues from the CGRP receptor binding or activation regions of the full-length CGRP protein; (b) a heterologous Th epitope; and (c) an optional heterologous spacer. The disclosed CGRP peptide immunogen constructs stimulate the generation of highly specific antibodies directed to the CGRP for the prevention and / or treatment of migraine.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a PCT international application claiming the benefit of U.S. Provisional Application No. 62 / 787,102, filed December 31, 2018, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to peptide immunogen constructs and formulations thereof targeting calcitonin gene-related peptide (CGRP) for the prevention and treatment of migraine headaches. [Background technology]

[0003] Migraine is a common medical condition that affects many people, up to 37 million in the United States. It is considered to be a systemic disease, not just a headache. Recent studies have demonstrated that changes in the brain can begin to occur as early as 24 hours before migraine symptoms begin. Migraine symptoms vary depending on the individual affected, but may include a severe throbbing headache (which is often on only one side of the head), nausea, vomiting, sensitivity to light (photophobia), sensitivity to sound (phonophobic), or a combination of these symptoms. These symptoms may persist even after the headache has passed.

[0004] There are various subtypes of migraine, and associated symptoms include weakness, numbness, changes or loss of vision, vertigo, and difficulty speaking (in some patients it may appear as if they are having a stroke). The disability caused by this chronic condition can be significant, resulting in lost work days and an inability to participate in family life.

[0005] People may be able to use "preemptive" medications, which, if taken early, can arrest the migraine process. For many patients, preventive medications can reduce both the frequency and severity of migraines. However, many of the preventive medications used to prevent or treat migraines were originally developed for other conditions, such as epileptic seizures, depression, high blood pressure, and muscle spasms.

[0006] Researchers have been trying to develop a "targeted" preventative therapy specifically for migraine for decades. Calcitonin gene-related peptide (CGRP) is a molecule synthesized in both peripheral and central neurons. This molecule has been implicated in a variety of pain processes, including migraine, and also functions as a vasodilator. Preemptive treatment of migraine has focused on blocking CGRP activation at the onset of a migraine headache. Small molecule CGRP antagonist drugs have been shown to reduce migraine pain based on certain measures, but these antagonists can have serious side effects, including liver toxicity.

[0007] Monoclonal antibodies targeting the CGRP molecule have an inhibitory effect on the pain process and can be used as a preemptive treatment. Monoclonal antibodies against CGRP can have a long half-life, meaning that they can be administered less frequently than typical migraine medications, which are taken daily. (An exception is botulinum toxin, which is injected every 90 days.) Monoclonal antibodies for migraine require monthly subcutaneous injections and have so far demonstrated a statistically significant reduction in the number of migraine days. Several different pharmaceutical companies are developing these novel antibodies for FDA approval.

[0008] Although such monoclonal anti-CGRP or monoclonal anti-CGRP receptor antibodies may prove effective in immunotherapy of migraine, they are expensive and must be administered monthly to maintain sufficient suppression of serum and fluid CGRP levels and the clinical benefit derived therefrom. A cost-effective immunotherapy targeting the CGRP molecule through a safe and well-tolerated vaccination approach remains an exciting new therapeutic intervention and development for migraine treatment.

[0009] Classical peptide / hapten carrier protein immunogen preparation methods are associated with numerous shortcomings and drawbacks, such as the fact that they may involve complex chemical coupling procedures, such as the use of expensive medical grade KLH or toxoid proteins as helper T cell carriers, and most of the antibodies induced by the protein immunogens are directed against the carrier protein and not against the target B cell epitope(s).

[0010] In view of the economic and practical disadvantages and limitations associated with monoclonal therapies and classical peptide / hapten carrier protein preparations, there is clearly an unmet need to develop an effective immunotherapeutic composition that can induce a highly specific immune response against the functional site(s) of CGRP to treat patients suffering from migraine, that is easily administered to patients, can be manufactured under strict Good Manufacturing Practices (GMP), and is cost-effective for global utilization.

[0011] Regarding the statements made in the Background section above, two review articles citing additional supporting literature can be found, which are hereby incorporated by reference in their entirety. The first article contains an updated review of CGRP and CGRP receptors (website: en.wikipedia.org / wiki / Calcitonin_gene-related_peptide). The second article covers the biology of CGRP signaling, which is the key clinical evidence for the role of CGRP in migraine headaches, including new insights into the efficacy of CGRP-targeted therapies, the role of CGRP in the trigeminovascular system, and the central role of the trigeminal ganglion in migraine pathophysiology (Edvinsson, et al, 2018).

[0012] References 1. CHANG, JCC, et al., "Adjuvant activity of incomplete Freund's adjuvant," Advanced Drug Delivery Reviews, 32(3):173-186 (1998) 2. "Calcitonin gene-related peptide," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org / wiki / Calcitonin_gene-related_peptide (accessed December 30, 2018). 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) 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). 5. RUSSELL, F.A., et al., “Calcitonin gene-related peptide: physiology and pathophysiology”, Physiol. Rev., 94(4):1099-1142 (2014) 6. TAJTI, J., et al., “Messenger molecules and receptor mRNA in the human trigeminal ganglion”, J. Auton. Nerv. Syst. 28;76(2-3):176-83 (1999) 7. TRAGGIAI, E., et al., “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus”, Nature Medicine, 10:871-875 (2004). 8. WATKINS, H.A., et al., “Structure-activity relationships for a-calcitonin gene -related peptide”, Br. J. Pharmacol., 170(7):1308-22 (2013)

Summary of the Invention

[0013] The present disclosure relates to portions of calcitonin gene-related peptide (CGRP) that can be used as B cell epitopes. The disclosure also relates to peptide immunogenic constructs that contain B cell epitopes from CGRP, compositions that contain the peptide immunogenic constructs, methods of making and using the peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

[0014] One aspect of the present disclosure relates to portions of CGRP from different species that serve as B cell epitopes in peptide immunogen constructs, and combinations thereof, that can be used to prevent and / or treat migraine. The CGRP peptide immunogen constructs of the present disclosure (SEQ ID NOs: 116-127 and 130-180) contain functional B cell epitope peptides (SEQ ID NOs: 4-13, 15-19, and 20-24 in Table 1) having a total of 30 or more amino acids and having about 7 to about 30 amino acids derived from human, marmoset, or rat / mouse CGRP (i.e., SEQ ID NOs: 1-3, respectively). The peptide immunogen constructs of the present disclosure can be formed by linking the functional B cell epitope peptides to heterologous helper T cell (Th) epitope peptides (e.g., SEQ ID NOs: 74-115) derived from pathogenic proteins through an optional heterologous spacer.

[0015] The CGRP peptide immunogen construct of the present disclosure can contain a CGRP B cell epitope peptide having about 7 to about 30 amino acids. The B cell epitope peptide can be derived from the CGRP receptor binding region located in the C-terminal and central regions of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22 shown in Table 1). The B cell epitope peptide can also be derived from the CGRP receptor activation site around the cyclic C2-C7 loop located in the N-terminal and central regions 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 peptide can be linked to a heterologous Th epitope from a pathogenic protein (e.g., SEQ ID NOs: 74-115 in Table 2) at either the N-terminus or C-terminus of the CGRP peptide. The B cell epitope and Th epitope together act to stimulate the generation of highly specific antibodies that are cross-reactive with full-length CGRP (SEQ ID NOs: 1-3) of various species.

[0016] In certain embodiments, the heterologous Th epitopes used to enhance the 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 (MVF1-5) and hepatitis B surface antigen (HBsAg1-3), either in the form of single sequences or in combination sequences (e.g., SEQ ID NOs:75, 82-99).

[0017] The CGRP peptide immunogen constructs of the present disclosure contain both designed B cell epitope peptides and Th epitope peptides, which together act to stimulate the production of highly specific antibodies directed against CGRP functional sites, including the CGRP receptor binding region located at the C-terminus of the CGRP molecule or the cyclic C2-C7 loop involved in receptor activation, resulting in a therapeutic immune response in patients predisposed to or suffering from migraine.

[0018] Another aspect of the present disclosure relates to a peptide composition comprising a CGRP peptide immunogen construct. In some embodiments, the composition comprises one type of peptide immunogen construct. In other embodiments, the peptide composition comprises a mixture of CGRP peptide immunogen constructs. In certain embodiments, the mixture of CGRP peptide immunogen constructs has heterologous Th epitopes from different pathogens available to allow for a broad range of coverage similar to the genetic background of patients, thereby increasing the percentage of responder rate in immunization for the prevention and / or treatment of migraine.

[0019] Synergistic enhancement of CGRP immunogen constructs can be observed with the peptide compositions of the present disclosure. The antibody responses obtained from such peptide immunogen constructs, including CGRP peptide immunogen constructs, were largely (>90%) focused on the desired cross-reactivity against the CGRP functional site(s) or receptor binding domain peptides (SEQ ID NOs: 4-13 and 15-24), with little, if any, directed against the heterologous Th epitope used for immunogenicity enhancement. This is in sharp contrast to standard methods using traditional carrier proteins (KLH, toxoids, etc.) or other biological carriers used for such peptide antigenic enhancement.

[0020] The present disclosure is also directed to pharmaceutical compositions and formulations for the prevention and / or treatment of migraine. In some embodiments, the pharmaceutical compositions comprise a stabilized immunostimulatory complex formed via electrostatic association by mixing a peptide composition comprising a mixture of CGRP peptide immunogen constructs with a CpG oligomer to further enhance the immunogenicity of the CGRP peptide to obtain a desired cross-reactivity with full-length CGRP (e.g., SEQ ID NOs: 1-3).

[0021] In other embodiments, pharmaceutical compositions that can be used to prevent and / or treat migraine headaches include a peptide composition comprising a mixture of CGRP peptide immunogen constructs contacted with an adjuvant, such as an inorganic salt (including ALHYDROGEL or ADJU-PHOS) to form a suspension formulation, or with an adjuvant, such as MONTANIDE™ ISA 51 or MONTANIDE ISA 720 to form a water-in-oil emulsion.

[0022] Additionally, the present disclosure provides methods for the low cost production and quality control of CGRP peptide immunogen constructs and formulations thereof capable of preventing and / or treating migraine in animals.

[0023] The present disclosure also relates to antibodies directed against the CGRP peptide immunogen constructs of the present disclosure. In particular, the CGRP peptide immunogen constructs of the present disclosure can stimulate the generation of highly specific functional antibodies that cross-react with full-length CGRP molecules. The antibodies of the present disclosure bind to CGRP with high specificity, and few, if any, antibodies directed against heterologous Th epitopes are used for immunogenicity enhancement, which is in stark contrast to the antibodies generated using conventional proteins or other biological carriers used for such peptide immunogenicity enhancement. Thus, the CGRP peptide immunogen constructs of the present disclosure have the ability to break immune tolerance to self-CGRP, and obtain a high responder rate compared to other peptide or protein immunogens.

[0024] In some embodiments, the antibodies of the disclosure are directed against and specifically bind to the CGRP receptor binding site (e.g., SEQ ID NOs: 5-9 and 15-22) in the C-terminal portion of the CGRP molecule when the peptide immunogen constructs are administered to a subject. The highly specific antibodies induced by these CGRP peptide immunogen constructs can inhibit the binding of CGRP to the CGRP receptor and inhibit downstream activation events in the elevation of cellular cAMP caused by the region around the cyclic C2-C7 loop of CGRP, thereby resulting in effective migraine prevention and / or treatment.

[0025] In other embodiments, antibodies of the disclosure are directed against the N-terminus or central region of CGRP around the cyclic C2-C7 loop that contributes to downstream cellular activation events when the peptide immunogen constructs of the invention are administered to a subject, or against the C-terminus and central region of the CGRP receptor binding site (e.g., SEQ ID NOs: 4, 10-13, and 23-24). Highly specific antibodies induced by the CGRP peptide immunogen constructs can inhibit (1) the binding of CGRP to the CGRP receptor, and (2) the downstream activation events triggered by the region around the cyclic C2-C7 loop of CGRP, thereby suppressing cellular cAMP elevation and thus providing an effective treatment for patients suffering from migraine headaches.

[0026] Based on their unique characteristics and properties, the antibodies of the present disclosure induced by the CGRP peptide immunogen constructs can provide a prophylactic immunotherapeutic approach to treat patients suffering from migraine headaches.

[0027] In a further aspect, the present invention provides human monoclonal antibodies against CGRP induced in patients administered compositions containing the CGRP peptide immunogen constructs of the present disclosure. An efficient method for generating human monoclonal antibodies from B cells isolated from the blood of human patients is described by Traggiai, E., et al, 2004, which is incorporated by reference.

[0028] The present disclosure also relates to methods of producing the disclosed CGRP peptide immunogen constructs, compositions, and antibodies, which allow for the low-cost production and quality control of CGRP peptide immunogen constructs and compositions containing the constructs, which can be used to treat patients suffering from migraine headaches.

[0029] The present disclosure also includes methods for preventing and / or treating a subject predisposed to or suffering from migraine using a CGRP peptide immunogen construct of the present disclosure and / or an antibody directed against a CGRP peptide immunogen construct. The method of preventing and / or treating migraine in a subject includes administering to the subject a composition containing a CGRP peptide immunogen construct of the present disclosure. In certain embodiments, the composition used in the method includes a CGRP peptide immunogen construct of the present disclosure in the form of a stable immunostimulatory complex formed with a negatively charged oligonucleotide (such as a CpG oligomer) through electrostatic association for administration to a patient suffering from migraine, and the composition can further be supplemented with an adjuvant.

[0030] The methods of the disclosure also include dosing regimens, dosage forms, and routes for administering a CGRP peptide immunogen construct to prevent and / or treat migraine in a subject. [Brief description of the drawings]

[0031] [Figure 1] Figure 1 shows an alignment of CGRP sequences from multiple species, including human (SEQ ID NO:1), marmoset (SEQ ID NO:2), mouse (SEQ ID NO:3), rat (SEQ ID NO:3), and many others, including horse, bird, pig, sheep, cow, dog, opossum, gecko, frog, pufferfish, flounder, goldfish, salmon, killifish (Medalea), and zebrafish. This figure is adapted from Watkins, et al, 2013. [Diagram 2]FIG. 2 shows the pathway from discovery to commercialization of precision CGRP designer peptide immunogen constructs and their formulations for the treatment of migraine. [Diagram 3] FIG. 3 shows the results of an immunogenicity study in guinea pigs of CGRP peptide immunogen constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-122, 139, 123, 130) using CGRP B cell epitope peptides derived from the N-terminal, central, and C-terminal regions of the CGRP molecule. [Figure 4] FIG. 3 shows the results of an immunogenicity study in guinea pigs of CGRP peptide immunogen constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-122, 139, 123, 130) using CGRP B cell epitope peptides derived from the N-terminal, central, and C-terminal regions of the CGRP molecule. [Diagram 5] FIG. 4 shows the neutralizing activity, expressed as intracellular cAMP generation IC50, of representative CGRP peptide immunogen constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-123, 130, 139, and 150) in CGRP-activated cell cultures in the presence of antibodies purified from guinea pig immune serum (collected from bleeds at 6 wpi). [Figure 6] FIG. 4 shows the neutralizing activity, expressed as intracellular cAMP generation IC50, of representative CGRP peptide immunogen constructs (SEQ ID NOs: 116-118, 124-127, 128, 129, 131, 133, 135, 137, 119-123, 130, 139, and 150) in CGRP-activated cell cultures in the presence of antibodies purified from guinea pig immune serum (collected from bleeds at 6 wpi). [Figure 7]FIG. 5 shows the results of immunogenicity studies in Balb / C mice of representative CGRP peptide immunogen constructs (SEQ ID NOs: 123, 131, 141, 151) for their reactivity and cross-reactivity with their respective full-length human CGRP molecules using corresponding antibodies purified from guinea pig immune sera bled 7 wpi, 10 wpi, and 13 wpi in both ADJUPHOS and ISA51 formulations. [Figure 8] FIG. 6 shows the inhibitory effect of capsaicin-induced skin blood flow measured in Balb / C mice vaccinated at 0, 3, and 6 wpi with CGRP peptide immunogen constructs (SEQ ID NOs: 123, 131, 141) formulated in either the designated ADJUPHOS or ISA emulsion formulations, compared to adjuvant only or saline controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure relates to portions of calcitonin gene-related peptide (CGRP) that can be used as B cell epitopes. The disclosure also relates to peptide immunogenic constructs that contain B cell epitopes from CGRP, compositions that contain the peptide immunogenic constructs, methods of making and using the peptide immunogenic constructs, and antibodies generated by the peptide immunogenic constructs.

[0033] One aspect of the present disclosure relates to moieties in CGRP from different species that serve as B cell epitopes in peptide immunogen constructs, and combinations thereof, that can be used to prevent and / or treat migraine. The CGRP peptide immunogen constructs of the present disclosure (SEQ ID NOs: 116-127 and 130-180) contain functional B cell epitope peptides (SEQ ID NOs: 4-13, 15-19, and 20-24 in Table 1) having a total of 30 or more amino acids and having about 7 to about 30 amino acids derived from human, marmoset, or rat / mouse CGRP (i.e., SEQ ID NOs: 1-3, respectively). The peptide immunogen constructs of the present disclosure can be formed by linking the functional B cell epitope peptides to heterologous helper T cell (Th) epitope peptides (e.g., SEQ ID NOs: 74-115) derived from pathogenic proteins through an optional heterologous spacer.

[0034] The CGRP peptide immunogen construct of the present disclosure can contain a CGRP B cell epitope peptide having about 7 to about 30 amino acids. The B cell epitope peptide can be derived from the CGRP receptor binding region located in the C-terminal and central regions of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22 shown in Table 1). The B cell epitope peptide can also be derived from the CGRP receptor activation site around the cyclic C2-C7 loop located in the N-terminal and central regions 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 peptide can be linked to a heterologous Th epitope from a pathogenic protein (e.g., SEQ ID NOs: 74-115 in Table 2) at either the N-terminus or C-terminus of the CGRP peptide. The B cell epitope and Th epitope together act to stimulate the generation of highly specific antibodies that are cross-reactive with full-length CGRP (SEQ ID NOs: 1-3) of various species.

[0035] In certain embodiments, the heterologous Th epitopes used to enhance the 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 (MVF1-5) and hepatitis B surface antigen (HBsAg1-3), either in the form of single sequences or in combination sequences (e.g., SEQ ID NOs:75, 82-99).

[0036] The CGRP peptide immunogen constructs of the present disclosure contain both designed B cell epitope peptides and Th epitope peptides, which together act to stimulate the production of highly specific antibodies directed against CGRP functional sites, including the CGRP receptor binding region located at the C-terminus of the CGRP molecule or the cyclic C2-C7 loop involved in receptor activation, resulting in a therapeutic immune response in patients predisposed to or suffering from migraine.

[0037] Another aspect of the present disclosure relates to a peptide composition comprising a CGRP peptide immunogen construct. In some embodiments, the composition comprises one type of peptide immunogen construct. In other embodiments, the peptide composition comprises a mixture of CGRP peptide immunogen constructs. In certain embodiments, the mixture of CGRP peptide immunogen constructs has heterologous Th epitopes from different pathogens available to allow for a broad range of coverage similar to the genetic background of patients, thereby increasing the percentage of responder rate in immunization for the prevention and / or treatment of migraine.

[0038] Synergistic enhancement of CGRP immunogen constructs can be observed with the peptide compositions of the present disclosure. The antibody responses obtained from administration of such peptide immunogen constructs, including CGRP peptide immunogen constructs, were largely (>90%) focused on the desired cross-reactivity against the CGRP functional site(s) or receptor binding domain peptides (SEQ ID NOs: 4-13 and 15-19 and 20-24), with little, if any, directed against the heterologous Th epitope used for immunogenicity enhancement. This is in sharp contrast to standard methods using traditional carrier proteins (KLH, toxoids, etc.) or other biological carriers used for such peptide antigenic enhancement.

[0039] The present disclosure is also directed to pharmaceutical compositions and formulations for the prevention and / or treatment of migraine. In some embodiments, the pharmaceutical compositions comprise a stabilized immunostimulatory complex formed via electrostatic association by mixing a peptide composition comprising a mixture of CGRP peptide immunogen constructs with a CpG oligomer to further enhance the immunogenicity of the CGRP peptide to obtain a desired cross-reactivity with full-length CGRP (e.g., SEQ ID NOs: 1-3).

[0040] In other embodiments, pharmaceutical compositions that can be used to prevent and / or treat migraine headaches include a peptide composition comprising a mixture of CGRP peptide immunogen constructs contacted with an adjuvant, such as an inorganic salt (including ALHYDROGEL or ADJU-PHOS) to form a suspension formulation, or with an adjuvant, such as MONTANIDE™ ISA 51 or MONTANIDE ISA 720 to form a water-in-oil emulsion.

[0041] Additionally, the present disclosure provides methods for the low cost production and quality control of CGRP peptide immunogen constructs and formulations thereof capable of preventing and / or treating migraine in animals.

[0042] The present disclosure also relates to antibodies directed against the CGRP peptide immunogen constructs of the present disclosure. In particular, the CGRP peptide immunogen constructs of the present disclosure can stimulate the generation of highly specific functional antibodies that cross-react with full-length CGRP molecules. The antibodies of the present disclosure bind to CGRP with high specificity, and few, if any, antibodies directed against heterologous Th epitopes are used for immunogenicity enhancement, which is in stark contrast to the antibodies generated using conventional proteins or other biological carriers used for such peptide immunogenicity enhancement. Thus, the CGRP peptide immunogen constructs of the present disclosure have the ability to break immune tolerance to self-CGRP, and obtain a high responder rate compared to other peptide or protein immunogens.

[0043] In some embodiments, the antibodies of the disclosure are directed against and specifically bind to the CGRP receptor binding site (e.g., SEQ ID NOs: 5-9 and 15-22) in the C-terminal portion of the CGRP molecule when the peptide immunogen constructs are administered to a subject. The highly specific antibodies induced by these CGRP peptide immunogen constructs can inhibit the binding of CGRP to the CGRP receptor and inhibit downstream activation events in the elevation of cellular cAMP caused by the region around the cyclic C2-C7 loop of CGRP, thereby resulting in effective migraine prevention and / or treatment.

[0044] In other embodiments, antibodies of the disclosure are directed against the N-terminus or central region of CGRP around the cyclic C2-C7 loop that contributes to downstream cellular activation events when the peptide immunogen constructs of the invention are administered to a subject, or against the C-terminus and central region of the CGRP receptor binding site (e.g., SEQ ID NOs: 4, 10-13, and 23-24). Highly specific antibodies induced by the CGRP peptide immunogen constructs can inhibit (1) the binding of CGRP to the CGRP receptor, and (2) the downstream activation events triggered by the region around the cyclic C2-C7 loop of CGRP, thereby suppressing cellular cAMP elevation and thus providing an effective treatment for patients suffering from migraine headaches.

[0045] Based on their unique characteristics and properties, the antibodies of the present disclosure induced by the CGRP peptide immunogen constructs can provide a prophylactic immunotherapeutic approach to treat patients suffering from migraine headaches.

[0046] In a further aspect, the present invention provides human monoclonal antibodies against CGRP induced in patients administered compositions containing the CGRP peptide immunogen constructs of the present disclosure. An efficient method for generating human monoclonal antibodies from B cells isolated from the blood of human patients is described by Traggiai, E., et al, 2004, which is incorporated by reference.

[0047] The present disclosure also relates to methods of producing the disclosed CGRP peptide immunogen constructs, compositions, and antibodies, which allow for the low-cost production and quality control of CGRP peptide immunogen constructs and compositions containing the constructs, which can be used to treat patients suffering from migraine headaches.

[0048] The present disclosure also includes methods for preventing and / or treating a subject predisposed to or suffering from migraine using a CGRP peptide immunogen construct of the present disclosure and / or an antibody directed against a CGRP peptide immunogen construct. The method of preventing and / or treating migraine in a subject includes administering to the subject a composition containing a CGRP peptide immunogen construct of the present disclosure. In certain embodiments, the composition used in the method includes a CGRP peptide immunogen construct of the present disclosure in the form of a stable immunostimulatory complex formed with a negatively charged oligonucleotide (such as a CpG oligomer) through electrostatic association for administration to a patient suffering from migraine, and the composition can further be supplemented with an adjuvant.

[0049] The methods of the disclosure also include dosing regimens, dosage forms, and routes for administering a CGRP peptide immunogen construct to prevent and / or treat migraine in a subject.

[0050] general The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references or portions of references cited in this application are hereby expressly incorporated by reference in their entirety for all purposes.

[0051] 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 belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Thus, the phrase "comprising A or B" means including A, or B, or A and B. It should be further understood that all amino acid sizes and all molecular weight or molecular mass values ​​given for polypeptides are approximate and are provided for illustrative purposes. In practicing or testing the methods of this disclosure, methods and materials similar or equivalent to those described herein can be used, although suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of any conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0052] CGRP peptide immunogen constructs The present disclosure provides peptide immunogen constructs containing B cell epitope peptides having amino acid sequences derived from CGRP (SEQ ID NOs: 1-3) or fragments thereof. The CGRP peptide immunogen constructs can contain CGRPB epitope peptides having about 7 to about 30 amino acids. The B cell epitope peptides can be derived from (1) the CGRP receptor binding region located at the C-terminus / central region of the CGRP molecule (e.g., SEQ ID NOs: 5-9 and 15-22, as shown in Table 1), or (2) the CGRP receptor activation site around the cyclic C2-C7 loop located at the N-terminus / central region of the CGRP molecule (e.g., SEQ ID NOs: 4, 10-13, and 23-24, as shown in Table 1). The B cell epitope can be covalently linked, directly or optionally through a heterologous spacer, to a heterologous helper T cell (Th) epitope derived from a pathogenic protein (e.g., SEQ ID NOs: 74-115, as shown in Table 2). These constructs contain both engineered B cell and Th cell epitopes that act together to stimulate the generation of highly specific antibodies that cross-react with full-length CGRP (SEQ ID NOs: 1-3) from a variety of species.

[0053] The phrase "CGRP peptide immunogen construct" or "peptide immunogen construct" as used herein refers to a peptide having more than about 30 amino acids that includes (a) a B cell epitope having more than about 7 contiguous amino acid residues derived from full-length CGRP (SEQ ID NOs: 1-3), (b) a heterologous Th epitope, and (c) an optional heterologous spacer.

[0054] In certain embodiments, the CGRP peptide immunogen construct can be represented by the formula: (Th) m -(A) n -(CGRP functional B epitope peptide)-X or (CGRP functional B epitope peptide)-(A) n -(Th) m -X or (Th) m -(A) n-(CGRP functional B epitope peptide)-(A) n -(Th) m -X During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (CGRP functional B epitope peptide) is a B cell epitope peptide having 7 to 30 amino acid residues derived from CGRP that is involved in either receptor binding or receptor activation, X is the α-COOH or α-CONH2 of an amino acid; m is from 1 to about 4; and n is 0 to about 10.

[0055] The CGRP peptide immunogen constructs of the present disclosure were designed and selected based on a number of rationales, including the following: The i.CGRP B cell epitope peptide is non-immunogenic by itself, thereby avoiding autologous T cell activation. ii. CGRP B cell epitope peptides can be made immunogenic by using protein carriers or strong helper T cell epitope(s). iii. When the CGRP B cell epitope peptide becomes immunogenic and is administered to a host, the peptide immunogen construct a. induces high titer antibodies selectively directed against CGRP B cell epitope(s) and not against the protein carrier or helper T cell epitope(s); b. breaking immune tolerance in an immunized host and generating highly specific antibodies that are cross-reactive with CGRP (SEQ ID NOs: 1-3); c. Generate highly specific antibodies capable of inhibiting the binding of CGRP to the CGRP receptor and associated downstream events, such as increased intracellular cAMP production; and d. Generate highly specific antibodies capable of reducing capsaicin-induced cutaneous blood flow in vivo.

[0056] The CGRP peptide immunogen constructs and formulations thereof of the present disclosure can effectively function as pharmaceutical compositions to prevent and / or treat subjects predisposed to or suffering from migraine headaches.

[0057] The various components of the CGRP peptide immunogen constructs of the disclosure are described in further detail below.

[0058] a. B cell epitope peptide derived from CGRP The present disclosure relates to novel peptide compositions for generating high titer antibodies with specificity for multiple calcitonin gene-related peptide (CGRP) proteins (e.g., SEQ ID NOs: 1-3). The site specificity of the peptide compositions minimizes the generation of antibodies directed to irrelevant sites in other regions of CGRP or to irrelevant sites on carrier proteins, thus providing a high safety factor.

[0059] The term "CGRP" as used herein refers to the 37 amino acid neuropeptide α-CGRP, which belongs to the calcitonin (CT) family of peptides. Human CGRP is derived from UniProtKB:P06881-1 and has the amino acid sequence of SEQ ID NO:1. Marmoset (Callithrix jacchus) CGRP is derived from GenBank accession number AAL35592.1 and has the amino acid sequence of SEQ ID NO:2. Rat (Rattus norvegicus) CGRP is derived from UniProtKB:P01256 and mouse (Mus musculus) CGRP is derived from UniProtKB:Q99JA0, with both rat and mouse CGRP having the amino acid sequence of SEQ ID NO:3. The amino acid sequences of CGRP used in this disclosure are shown in Table 1.

[0060] In humans, CGRP is derived from the gene encoding calcitonin and is 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 α and β isoforms differ in amino acids located at positions 3, 22, and 25. At the molecular level in smooth muscle cells, CGRP can bind to its receptor via its C-terminal region and then activate the receptor by using its loop region. The cyclic C2-C7 loop with disulfide bridges plays a fundamental role in receptor activation and is closely related to the increase in intracellular cAMP. In mammalian plasma, the half-life of CGRP is about 10 minutes. In the human trigeminal ganglion, CGRP-responsive neurons account for up to 50% of the total neurons (Tajti, et al., 1999).

[0061] CGRP is widely expressed in the central and peripheral nervous systems. It is primarily associated with small diameter unmyelinated sensory neurons in close proximity to blood vessels. CGRP is a potent vasodilator, and local administration of CGRP causes a transient increase in blood flow. CGRP has also been implicated in pain transmission, pain modulation, and neurogenic inflammation. CGRP can be released from sensory neurons by activating the transient receptor potential cation channel V1 with capsaicin. Laser Doppler imaging (LDI) has been used to detect CGRP-induced changes in cutaneous blood flow.

[0062] CGRP has also been linked to inflammatory pain in several pain models, as demonstrated by the attenuated responses in CGRP knockout mice. This role in pain perception is consistent with the expression of CGRP in sensory neurons.

[0063] One aspect of the present disclosure is the prevention and / or treatment of CGRP migraine headaches using active immunotherapy targeting CGRP to exert long-term CGRP blockade and clinical efficacy. That is, the present disclosure relates to peptide immunogen constructs and formulations thereof targeting a portion of the full-length CGRP protein (SEQ ID NOs: 1-3) for the prevention and treatment of migraine.

[0064] The B cell epitope portion of the CGRP peptide immunogen construct can contain about 7 to about 30 amino acids derived from any portion of the full-length CGRP protein represented by SEQ ID NOs: 1 to 3. In certain embodiments, the B cell epitope peptides were screened and selected based on the design rationale and contain the amino acid sequences of SEQ ID NOs: 4 to 13 and 15 to 24 as shown in Table 1.

[0065] In some embodiments, the B cell epitope peptide is derived from the CGRP receptor binding region R11-F37 (SEQ ID NO: 9) located in the C-terminal / central region 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 is derived from the CGRP receptor activating region surrounding the cyclic C2-C7 loop, such as A1-N25 (SEQ ID NO: 13) or a fragment thereof (e.g., SEQ ID NOs: 4, 10-12, and 23-24).

[0066] The CGRP B cell epitope peptides of the present disclosure also include immunologically functional analogs or homologs of CGRP. Immunologically functional analogs or homologs of CGRP B cell epitope peptides include variants that retain substantially the same immunogenicity as the original peptide. Immunologically functional analogs can have conservative substitutions at certain amino acid positions; changes in overall charge; covalent linkage to another moiety; or amino acid additions, insertions, or deletions; and / or any combination thereof (e.g., a CGRP peptide of SEQ ID NO:9 versus a CGRP peptide of SEQ ID NO:25).

[0067] Antibodies generated from peptide immunogen constructs containing these B cell epitopes from CGRP are highly specific and cross-reactive with full-length CGRP (e.g., SEQ ID NOs: 1-3) from various species. Based on their unique characteristics and properties, the antibodies of the present disclosure induced by CGRP peptide immunogen constructs can provide a prophylactic immunotherapeutic approach to prevent and / or treat migraine headaches.

[0068] b. Heterologous helper T cell epitopes (Th epitopes) The present disclosure provides peptide immunogen constructs that include B cell epitopes derived from CGRP and heterologous helper T cell (Th) epitopes covalently linked directly or via an optional heterologous spacer.

[0069] The heterologous Th epitope in the CGRP peptide immunogen construct enhances the immunogenicity of the CGRP fragment, thereby facilitating the production of specific high titer antibodies directed against the optimized target CGRP B cell epitope peptides screened and selected on a design basis.

[0070] The term "heterologous" as used herein indicates that an amino acid sequence is derived from an amino acid sequence that is not part of or is not homologous to the wild-type sequence of CGRP. Thus, a heterologous Th epitope is a Th epitope that is derived from an amino acid sequence that is not naturally found in CGRP (i.e., the Th epitope is not derived from CGRP). Because the Th epitope is heterologous to CGRP, the native amino acid sequence of CGRP is not extended either N-terminally or C-terminally, even though such a heterologous Th epitope is covalently linked to a CGRPB epitope peptide.

[0071] The heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in CGRP. The Th epitope can also have motifs that bind nonselectively to MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple motifs that bind nonselectively to MHC class II molecules, allowing for maximum activation of helper T cells, thereby initiating and regulating immune responses. The Th epitope is preferably itself immunologically static, i.e., very few, if any, antibodies generated by the CGRP peptide immunogen construct will be directed against the Th epitope, thus allowing for a highly focused immune response directed to the target B cell epitope peptide of the CGRP molecule.

[0072] The Th epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens as exemplified in Table 2 (SEQ ID NOs: 74-115). Additionally, the 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 represented as combination sequences (e.g., SEQ ID NOs: 85, 91, 94, and 97) contain a mixture of amino acid residues shown at specific positions within a peptide framework based on the variable residues of homologs to that particular peptide. A collection of combination peptides can be synthesized in one process by adding a mixture of designated protected amino acids at a specific position in place of one particular amino acid during the synthesis process. Such a combination collection of heterologous Th epitope peptides allows for broadening the coverage of Th epitopes to animals with diverse genetic backgrounds. Representative combination sequences of heterologous Th epitope peptides include SEQ ID NOs: 85, 91, 94, and 97 shown in Table 2. The Th epitope peptides of the present invention confer broad reactivity and immunogenicity to animals and patients derived from genetically diverse populations.

[0073] C. Heterologous spacer The CGRP peptide immunogen constructs of the disclosure optionally include a heterologous spacer that covalently links the CGRP B-cell epitope peptide and the heterologous helper T cell (Th) epitope.

[0074] As discussed above, the term "heterologous" indicates that an amino acid sequence is derived from an amino acid sequence that is not part of or is not homologous to the wild-type sequence of CGRP. Thus, even though a heterologous spacer is covalently linked to a CGRP B-cell epitope peptide, the native amino acid sequence of CGRP is not extended either N- or C-terminally because the spacer is heterologous to the CGRP sequence.

[0075] A spacer is any molecule or chemical structure that can link two amino acids and / or peptides together. Spacers can vary in length or polarity depending on the application. Attachment of the spacer can be through an amide bond or a carboxyl bond, but other functional groups can also be used. Spacers can include chemical compounds, natural amino acids, or unnatural amino acids.

[0076] Spacers can impart structural features to CGRP peptide immunogen constructs. Structurally, spacers physically separate the Th epitope from the B cell epitope of the CGRP fragment. The physical separation by the spacer can destroy any artificial secondary structure created by linking the Th epitope to the B cell epitope. Furthermore, physically separating the epitopes by the spacer can eliminate interference between the Th cell response and / or the B cell response. Furthermore, spacers can be designed to create or modify secondary structure of the peptide immunogen construct. For example, spacers can be designed to act as a flexible hinge to improve separation of the Th and B cell epitopes. Flexible hinge spacers can also facilitate the efficiency of interaction between the presented peptide immunogen and the appropriate Th and B cells to enhance immune responses to the Th and B cell epitopes. Examples of sequences encoding flexible hinges are found in immunoglobulin heavy chain hinge regions, where such sequences are often rich in proline. One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 71), where Xaa is any amino acid, preferably aspartic acid.

[0077] Spacers can also confer functional features to CGRP peptide immunogen constructs. For example, spacers can be designed to alter the overall charge of the CGRP peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. Additionally, altering the overall charge of the CGRP peptide immunogen construct can affect the ability of the peptide immunogen construct to associate with other compounds and reagents. As described in more detail below, CGRP peptide immunogen constructs can form stable immunostimulatory complexes with highly charged oligonucleotides (such as CpG oligomers) through electrostatic association. The overall charge of the CGRP peptide immunogen construct is important for the formation of such stable immunostimulatory complexes.

[0078] Compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, miniPEG1), 12-amino-4,7,10-trioxadodecanoic acid (miniPEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (miniPEG3), trioxatridecane-succinic acid (Ttds), 12-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0079] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0080] Unnatural amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitro-tyrosine, pyroglutamic acid, and the like.

[0081] The spacer in the CGRP peptide immunogen construct can be covalently linked to either the N-terminus or C-terminus of the Th epitope and the CGRP B cell epitope peptide. In some embodiments, the spacer 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 is covalently linked to the C-terminus of the CGRP B cell epitope peptide and the N-terminus of the Th epitope. In certain embodiments, multiple spacers can be used, for example, when multiple Th epitopes are present in the CGRP peptide immunogen construct. When multiple spacers are used, each spacer can be the same as or different from each other. Furthermore, when multiple Th epitopes are present in the CGRP peptide immunogen construct, these Th epitopes can be separated by a spacer, which can be the same as or different from the spacer used to separate the Th epitope from the IL-6 B cell epitope peptide. There are no limitations on the placement of the spacer in relation to the Th epitope or IL-6 B cell epitope peptide.

[0082] In certain embodiments, the heterologous spacer is a natural or unnatural amino acid. In other embodiments, the spacer comprises a plurality of natural or unnatural amino acids. In certain embodiments, the spacer 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).

[0083] d. Specific Embodiments of CGRP Peptide Immunogen Constructs In certain embodiments, the CGRP peptide immunogen construct can be represented by the formula: (Th) m -(A) n -(CGRP functional B epitope peptide)-X or (CGRP functional B epitope peptide)-(A) n -(Th) m -X or (Th) m -(A) n -(CGRP functional B epitope peptide)-(A) n -(Th) m -X During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (CGRP functional B epitope peptide) is a B cell epitope peptide having 7 to 30 amino acid residues derived from CGRP that is involved in either receptor binding or receptor activation, X is the α-COOH or α-CONH2 of an amino acid; m is from 1 to about 4; n is 0 to about 10.

[0084] The B cell epitope peptide can contain about 7 to about 30 amino acids derived from any portion of the full-length CGRP protein represented by SEQ ID NOs: 1 to 3. In some embodiments, the B cell epitope has an amino acid sequence selected from any of SEQ ID NOs: 4 to 13 and 15 to 22 shown in Table 1. In certain embodiments, the B cell epitope peptide is derived from the CGRP receptor binding region R11-F37 (SEQ ID NO: 9) located in the C-terminal / central region of the CGRP molecule, or a fragment thereof (e.g., SEQ ID NOs: 5 to 8 and 15 to 22). In other embodiments, the B cell epitope peptide is derived from the CGRP receptor activation region around the cyclic C2-C7 loop, such as A1-N25 (SEQ ID NO: 13) or a fragment thereof (e.g., SEQ ID NOs: 4, 10 to 12, and 23 to 24).

[0085] The heterologous Th epitope in the CGRP peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 74-115, and combinations thereof, as set forth in Table 2. In some embodiments, the CGRP peptide immunogen construct contains multiple Th epitopes.

[0086] The optional heterologous spacer is selected from any of 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, where Xaa is any amino acid, but is preferably aspartic acid. In certain embodiments, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 72) or Lys-Lys-Lys-ε-N-Lys (SEQ ID NO: 73).

[0087] In certain embodiments, the CGRP peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 116-127 and 130-180 as shown in Table 3.

[0088] The Th epitopes that make up the CGRP peptide immunogen constructs are generated simultaneously in a single round of solid-phase peptide synthesis in tandem with the CGRP fragment. The Th epitopes can also include immunological analogs of the Th epitopes. Immunological Th analogs include immunopotentiating analogs, cross-reactive analogs, and segments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to an IL-6 B cell epitope peptide.

[0089] The Th epitope in the CGRP peptide immunogen construct can be covalently linked to either 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 certain embodiments, multiple Th epitopes are covalently linked to the CGRP B cell epitope peptide. When multiple Th epitopes are linked to the CGRP B cell epitope peptide, each Th epitope can have the same amino acid sequence or different amino acid sequences. Also, when multiple Th epitopes are linked to the CGRP B cell epitope peptide, these Th epitopes can be arranged in any order. For example, the Th epitopes can be contiguously linked to the N-terminus of the CGRP B cell epitope peptide, contiguously linked to the C-terminus of the CGRP B cell epitope peptide, or one Th epitope can be covalently linked to the N-terminus of the CGRP B cell epitope peptide while another Th epitope can be covalently linked to the C-terminus of the CGRP B cell epitope peptide. There are no limitations on the arrangement of the Th epitopes in relation to the CGRP B cell epitope peptide.

[0090] In some embodiments, the Th epitope is covalently linked directly to the CGRP B cell epitope peptide, hi other embodiments, the Th epitope is covalently linked to the CGRP fragment through a heterologous spacer.

[0091] e. Variants, homologues, and functional analogues Variants and analogs of the above immunogenic peptide constructs that induce and / or cross-react with antibodies against a suitable CGRP B-cell epitope peptide can also be used. Analogs (including allelic, species, and induced variants) typically differ from the native peptide at one, two, or a few positions, and these differences are often due to conservative substitutions. Analogs typically have at least 80% or at least 90% sequence identity with the native peptide. Some analogs contain non-natural amino acids or N- or C-terminal amino acid modifications at one, two, or a few positions.

[0092] Variants that are functional analogs can have conservative substitutions at certain amino acid positions, alterations in the overall charge, covalent attachment to another moiety, or additions, insertions, or deletions of amino acids, and / or any combination thereof.

[0093] A conservative substitution is the replacement of one amino acid residue with another 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, asparagine, and glutamine, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0094] In certain embodiments, a functional analog is at least 50% identical to the original amino acid sequence. In other embodiments, a functional analog is at least 80% identical to the original amino acid sequence. In yet other embodiments, a functional analog is at least 85% identical to the original amino acid sequence. In yet other embodiments, a functional analog is at least 90% identical to the original amino acid sequence.

[0095] Functional immunological analogs of Th epitope peptides are also useful and are included as part of the present invention. Functional immunological Th analogs can include one to about five conservative substitutions, additions, deletions, and insertions of amino acid residues in the Th epitope that do not essentially alter the Th stimulatory function of the Th epitope. Conservative substitutions, additions, and insertions can be achieved using natural or non-natural amino acids, as described above for the IL-6 B cell epitope peptides. In Table 2, another variation of functional analogs of Th epitope peptides is identified. In particular, SEQ ID NOs: 75 and 82 of MvF1 Th and MvF2 Th are functional analogs of SEQ ID NOs: 94 and 98 of MvF4 and MvF5 in view of the amino acid frames of these sequences differing by the deletion of two amino acids from each of the N-terminus and C-terminus (SEQ ID NOs: 75 and 82) or the inclusion of two amino acids at each of the N-terminus and C-terminus (SEQ ID NOs: 94 and 98). The differences between these two sets of similar sequences are not expected to affect the function of the Th epitopes contained therein. Thus, functional immunological Th analogs include several versions of the Th epitope derived from the measles virus fusion protein (MvF1-4 Th) (SEQ ID NOs: 75, 82, 85, and 94) and several versions of the Th epitope derived from the hepatitis surface protein (HBsAg1-3 Th) (SEQ ID NOs: 91, 97, and 99).

[0096] composition The present disclosure also provides compositions containing the CGRP immunogenic peptide constructs of the disclosure.

[0097] a. Peptide Compositions Compositions comprising the CGRP peptide immunogen constructs of the present disclosure can be in liquid or solid / lyophilized form. Liquid compositions can contain water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the CGRP peptide immunogen constructs. Peptide compositions can include one or more of the CGRP peptide immunogen constructs of the present disclosure.

[0098] b. Pharmaceutical Compositions The present disclosure also relates to pharmaceutical compositions containing the CGRP peptide immunogen constructs of the present disclosure.

[0099] A pharmaceutical composition can include a carrier and / or other additives in a pharma- ceutically acceptable delivery system. Thus, a pharmaceutical composition can include a pharma- ceutically effective amount of a CGRP peptide immunogen construct in combination with a pharma- ceutically acceptable carrier, adjuvant, and / or other excipients, such as diluents, additives, stabilizers, preservatives, solubilizers, buffers, and the like.

[0100] The pharmaceutical composition may include one or more adjuvants that act to promote, prolong, or enhance the immune response to the CGRP peptide immunogen construct, but without having any particular antigenic effect themselves. Adjuvants used in the pharmaceutical composition may include oils, oil emulsions, aluminum salts, calcium salts, immunostimulatory complexes, bacterial and viral agents, virosomes, carbohydrates, cytokines, and polymeric microparticles. In certain embodiments, the adjuvant is alum (potassium aluminum phosphate), aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), calcium phosphate, incomplete Freund's adjuvant (IFA), complete Freund's adjuvant, MF59, Adjuvant 65, Lipovant, ISCOM, liposyn, saponin, squalene, L121, EmulsIL-6n®, monophosphoryl lipid A (MPL), Quil A, QS21, MONTANIDE® ISA 35, ISA 50V, ISA 50V2, ISA 51, ISA 206, ISA 720, liposomes, phospholipids, peptidoglycan, lipopolysaccharide (LPS), ASO1, ASO2, ASO3, ASO4, AF03, lipophilic phospholipids (lipid A), gamma inulin, algammulin, glucan, dextran, glucomannan, galactomannan, levan, xylan, dimethyldioctadecylammonium bromide (DDA), and other adjuvants and emulsifiers.

[0101] In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA 51 (an oil adjuvant composition composed of vegetable oil and mannide oleate for the production of a water-in-oil emulsion), TWEEN® 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 comprising EmulsIL-6n or EmulsIL-6n D as an adjuvant.

[0102] The pharmaceutical composition may also include pharma- ceutically acceptable additives or excipients. For example, the pharmaceutical composition may contain antioxidants, binders, buffers, bulking agents, carriers, chelating agents, colorants, diluents, disintegrants, emulsifiers, excipients, gelling agents, pH buffers, preservatives, solubilizers, stabilizers, etc.

[0103] The pharmaceutical composition can be formulated as an immediate release formulation or a sustained release formulation. Additionally, the pharmaceutical composition can be formulated to induce systemic or local mucosal immunity through encapsulation and co-administration of immunogens with microparticles. Such delivery systems can be readily determined by one skilled in the art.

[0104] The pharmaceutical composition can be prepared as an injection, either as a liquid solution or suspension. A liquid vehicle containing the CGRP peptide immunogen construct can also be prepared before injection. The pharmaceutical composition can be administered by any suitable mode of use (e.g., intradermal, intravenous, intraperitoneal, intramuscular, intranasal, oral, subcutaneous, etc.) or by any suitable delivery device. In certain embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other modes of administration, including oral and intranasal, can also be prepared.

[0105] The pharmaceutical composition may also be formulated into a suitable unit dosage form. In some embodiments, the pharmaceutical composition comprises about 0.1 μg to about 1 mg of the CGRP peptide immunogen construct per kg of body weight. The effective dose of the pharmaceutical composition will vary depending on a variety of factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other administered drugs, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. The pharmaceutical composition, when delivered in multiple doses, can be conveniently divided into appropriate amounts per unit dosage form. The dosage will depend on the age, weight, and general health of the subject, as is well known in the therapeutic arts.

[0106] In some embodiments, the pharmaceutical composition comprises multiple CGRP peptide immunogen constructs. The inclusion of a mixture of multiple CGRP peptide immunogen constructs in a pharmaceutical composition can synergistically enhance the immune efficacy of the constructs. Pharmaceutical compositions comprising multiple CGRP peptide immunogen constructs may have broader MHC class II coverage, which may improve efficacy in a larger genetic population, and therefore improve the immune response to the CGRP peptide immunogen constructs.

[0107] 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.

[0108] In one particular embodiment, CGRP peptide immunogen constructs (SEQ ID NOs: 161-163) containing heterologous Th epitopes derived from MVF and HBsAg in a combined form (SEQ ID NOs: 85, 91, 94, and 97) were mixed in an equimolar ratio and used in the formulation to maximize coverage of host populations with diverse genetic backgrounds.

[0109] Furthermore, the antibody responses elicited by the CGRP peptide immunogen constructs (e.g., UBITh®1 and SEQ ID NO: 108) were largely (>90%) focused on the desired cross-reactivity against the B cell epitope peptides of CGRP, with little, if any, directed against the heterologous Th epitope used for immunogenicity enhancement (Example 6, Table 10). This is in sharp contrast to conventional proteins (e.g., KLH) or other biological protein carriers used for such CGRP peptide immunogenicity enhancement.

[0110] In other embodiments, a pharmaceutical composition comprising a peptide composition, e.g., a composition comprising a mixture of CGRP peptide immunogen constructs, contacted with an inorganic salt as an adjuvant, including ALHYDROGEL or ADJUPHOS, to form a suspension formulation, was used for administration to a host.

[0111] Pharmaceutical compositions containing CGRP peptide immunogen constructs can be used to induce an immune response and produce antibodies in the host upon administration.

[0112] C immunostimulatory complex The present disclosure also relates to pharmaceutical compositions containing CGRP peptide immunogen constructs in the form of immunostimulatory complexes with CpG oligonucleotides. Such immunostimulatory complexes are particularly suitable for acting as adjuvants and peptide immunogen stabilizers. The immunostimulatory complexes are in the form of microparticles that can efficiently present the CGRP peptide immunogen to cells of the immune system to generate an immune response. The immunostimulatory complexes may be formulated as suspensions for parenteral administration. The immunostimulatory complexes may be formulated in the form of water-in-oil (w / o) emulsions, as suspensions combined with inorganic salts, or with in situ gelling polymers for efficient delivery of the CGRP peptide immunogen constructs to cells of the host's immune system after parenteral administration.

[0113] The CGRP peptide immunogen constructs can be complexed with anionic molecules, oligonucleotides, polynucleotides, or combinations thereof via electrostatic association to form a stabilized immunostimulatory complex, which can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0114] In certain embodiments, the CGRP peptide immunogen construct is designed to have a cationic moiety that is positively charged in the pH range of 5.0 to 8.0. The net charge of the cationic moiety of the CGRP peptide immunogen construct or of the mixture of constructs is calculated by assigning a charge of +1 for each lysine (K), arginine (R), or histidine (H), a charge of -1 for each aspartic acid (D) or glutamic acid (E), and a charge of 0 for other amino acids in the sequence. The charges are summed within the cationic moiety of the CGRP peptide immunogen construct and expressed as a net charge average. A suitable peptide immunogen has a cationic moiety with an average net positive charge of +1. Preferably, the peptide immunogen has a net positive charge in the range of more than +2. In some embodiments, the cationic moiety of the CGRP peptide immunogen construct is a heterologous spacer. In certain embodiments, the cationic moiety of the CGRP peptide immunogen construct has a charge of +4 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).

[0115] "Anionic molecule" as used herein refers to any molecule that is negatively charged in the pH range of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules with 8 to 64 nucleotide bases and 1 to 10 repeats of the CpG motif. Preferably, the CpG immunostimulatory single-stranded DNA molecules contain 18 to 48 nucleotide bases and 3 to 8 repeats of the CpG motif.

[0116] More preferably, the anionic oligonucleotide comprises 5′X 1 CGX 2 3', in which C and G are not methylated, and X 1 is selected from the group consisting of A (adenine), G (guanine), and T (thymine); 2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide may have a 5'(X 3 )2CG(X 4 ) 23', where C and G are not methylated, and X 3 is selected from the group consisting of A, T, or G; 4 is C or T. In certain embodiments, the CpG oligonucleotide has the sequence: CpG1: 5'TCg TCg TTT TgT CgT TTT gTC gTT TTg TCg TT 3' (fully phosphorothioated) (SEQ ID NO: 182), CpG2: 5' phosphate TCg TCg TTT TgT CgT TTT gTC gTT 3' (fully phosphorothioated) (SEQ ID NO: 183), or CpG3 5'TCg TCg TTT TgT CgT TTT gTC gTT 3' (fully phosphorothioated) (SEQ ID NO: 184).

[0117] The resulting immunostimulatory complex is in the form of particles with particle sizes typically ranging from 1 to 50 microns, depending on many factors including the relative charge stoichiometry and molecular weight of the interacting species. Particulate immunostimulatory complexes have the advantage of providing adjuvant effects and upregulation of specific immune responses in vivo. Furthermore, the stabilized immunostimulatory complexes are suitable for preparation of pharmaceutical compositions by a variety of processes, including water-in-oil emulsions, inorganic salt suspensions, and polymer gels.

[0118] The present disclosure also relates to pharmaceutical compositions (including formulations) for the prevention and / or treatment of migraine. In some embodiments, the pharmaceutical composition comprises a stabilized immunostimulatory complex to further enhance the immunogenicity of the CGRP peptide immunogen construct and to induce antibodies directed against the CGRP receptor binding domain or receptor activating domain that cross-react with the CGRP proteins of SEQ ID NOs: 1-3, and the stabilized immunostimulatory complex is formed via electrostatic association by mixing a peptide composition containing a mixture of CGRP peptide immunogen constructs (e.g., SEQ ID NOs: 120-127 and 130-180) with a CpG oligomer (Example 6).

[0119] In yet other embodiments, the pharmaceutical composition comprises a mixture of CGRP peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 120-127 and 130-180) in the form of a stabilized immunostimulatory complex formed with CpG oligomers, optionally mixed with inorganic salts (including ALHYDROGEL or ADJUPHOS) as adjuvants with high safety factors, to form a suspension formulation for administration to a host.

[0120] antibody The disclosure also provides antibodies induced by the CGRP peptide immunogen constructs.

[0121] The present disclosure provides CGRP peptide immunogen constructs and formulations thereof that are cost-effective to manufacture, optimally designed in themselves, capable of inducing high titer antibodies targeting the CGRP receptor binding region or receptor activation region (SEQ ID NOs: 4-13 and 15-24) of the CGRP molecule, capable of breaking immune tolerance to the self-protein CGRP, and having a high responder rate in immunized hosts. The antibodies generated by the CGRP peptide immunogen constructs have high affinity for the CGRP receptor binding region or activation region.

[0122] In some embodiments, CGRP peptide immunogen constructs for inducing antibodies comprise a hybrid of a CGRP peptide targeting the CGRP receptor binding or receptor activation region of the CGRP molecule (SEQ ID NOs: 4-13 and 15-24) linked, via an optional spacer, to a heterologous Th epitope (SEQ ID NOs: 74-115) derived from a pathogenic protein such as the measles virus fusion (MVF) protein and others. Together, the B epitope peptide and the Th epitope peptide of the CGRP peptide immunogen construct act to stimulate the generation of highly specific antibodies that cross-react with the CGRP receptor binding or activation region of the CGRP protein (SEQ ID NO: 1).

[0123] Traditional methods for enhancing immune responses to peptides, such as chemical coupling to carrier proteins, e.g., keyhole limpet hemocyanin (KLH) or other carrier proteins, such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins, typically result in the generation of large amounts of antibodies directed against the carrier protein. Thus, a major drawback of such peptide-carrier protein compositions is that most (>90%) of the antibodies generated by the immunogen are non-functional antibodies directed against the carrier protein (KLH, DT, or TT), which may lead to epitopic suppression.

[0124] Unlike traditional methods for enhancing immune responses to peptides, antibodies generated by the CGRP peptide immunogen constructs of the present disclosure (e.g., SEQ ID NOs:116-127 and 130-180) bind with high specificity to CGRP B epitope peptides (e.g., SEQ ID NOs:4-13 and 15-24) with few, if any, antibodies directed against heterologous Th epitopes (e.g., SEQ ID NOs:74-115) or optional heterologous spacers.

[0125] method The disclosure also relates to methods of making and using the CGRP peptide immunogen constructs, compositions, and pharmaceutical compositions.

[0126] a. Methods for Producing CGRP Peptide Immunogen Constructs The CGRP peptide immunogen constructs of the present disclosure can be made by chemical synthesis methods well known to those of skill in the art (see, e.g., Fields, et al, 1992). The CGRP peptide immunogen constructs can be synthesized using automated Merrifield solid-phase synthesis techniques, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431, by using side-chain protected amino acids to protect the α-NH2 by either t-Boc or F-moc chemistry. Preparation of CGRP peptide immunogen constructs, including combinatorial library peptides of Th epitopes, can be accomplished by using a mixture of alternative amino acids for coupling at a given variable position.

[0127] After the desired CGRP peptide immunogen construct is fully assembled, the peptide can be cleaved from the resin and the functional groups on the amino acid side chains can be deprotected by treating the resin according to standard procedures. The free peptide can be purified by HPLC and biochemically characterized, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those of skill in the art.

[0128] The quality of peptides produced by this chemical process can be controlled and defined, thereby ensuring reproducibility of CGRP peptide immunogen constructs, immunogenicity, and yield. A detailed description of the production of CGRP peptide immunogen constructs through solid-phase peptide synthesis is provided in Example 1.

[0129] The range of structural variability that allows for retention of the intended immunological activity has been shown to be much more flexible than the range of structural variability that allows for retention of specific drug activity with small molecule drugs, or the retention of desired activity and undesired toxicity found in large molecules that occur with biologically derived drugs.

[0130] Thus, peptide analogs, whether intentionally designed or inevitably resulting from errors in the synthetic process as a mixture of missing sequence by-products with similar chromatographic and immunological properties as the intended peptide, are often as effective as purified preparations of the desired peptide. Designed analogs and unintentional analog mixtures are effective so long as discriminatory QC procedures are developed to monitor both the manufacturing and product evaluation processes to ensure reproducibility and potency of final products using such peptides.

[0131] CGRP peptide immunogenic constructs can also be produced using recombinant DNA technology, including nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding CGRP peptide immunogenic constructs and immunologically functional analogs thereof are also encompassed by the present disclosure as part of the present disclosure. Similarly, vectors (including expression vectors) containing nucleic acid molecules, as well as host cells containing such vectors, are also encompassed by the present disclosure as part of the present disclosure.

[0132] Various exemplary embodiments also encompass methods for producing CGRP peptide immunogen constructs and immunologically functional analogs thereof. For example, such methods may include incubating a host cell containing an expression vector comprising a nucleic acid molecule encoding a CGRP peptide immunogen construct and / or an immunologically functional analog thereof under conditions in which such peptides and / or analogs are expressed. Longer synthetic peptide immunogens can also be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known manuals with detailed protocols. To construct a gene encoding the peptide of the invention, the amino acid sequence is reverse transcribed to obtain a nucleic acid sequence encoding the amino acid sequence, preferably using optimal codons for the organism in which the gene will be expressed. A synthetic gene is then typically made by synthesizing oligonucleotides encoding the peptide and any control elements as required. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under appropriate conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0133] b. Methods for Producing Immunostimulatory Conjugates Various exemplary embodiments also encompass a method for producing an immunostimulatory complex comprising a CGRP peptide immunogen construct and a CpG oligodeoxynucleotide (ODN) molecule. A stabilized immunostimulatory complex (ISC) is obtained from the cationic portion of the CGRP peptide immunogen construct and a polyanionic CpG ODN molecule. This self-assembly system is driven by electrostatic neutralization of charges. The stoichiometry of the molar charge ratio of the cationic portion of the CGRP peptide immunogen construct to the anionic oligomer determines the degree of assembly. The non-covalent electrostatic association of the CGRP peptide immunogen construct with the CpG ODN is a fully reproducible process. The peptide / CpG ODN immunostimulatory complex assembly promotes presentation to "professional" antigen presenting cells (APCs) of the immune system, thereby further enhancing the immunogenicity of the complex. Such complexes are easily characterized for quality control during production. The peptide / CpG ISC is well tolerated in vivo. This novel particulate system containing CpG ODN and CGRP peptide immunogen constructs was designed to take advantage of the general B cell mitogenicity associated with the use of CpG ODN and further promote balancing of Th-1 / Th-2 type responses.

[0134] The CpG ODN in the pharmaceutical composition of the present disclosure is 100% bound to immunogens in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized microparticles. This microparticle form can significantly reduce the dosage of CpG compared to the conventional use of CpG adjuvants, lowering the possibility of harmful natural immune responses, and promotes alternative immunogen processing pathways involving antigen-presenting cells (APCs). Thus, such formulations are conceptually novel and are expected to bring benefits by promoting the stimulation of immune responses through alternative mechanisms.

[0135] c. Methods for producing pharmaceutical compositions Various exemplary embodiments also encompass pharmaceutical compositions containing the CGRP peptide immunogen constructs. In certain embodiments, the pharmaceutical compositions employ water-in-oil emulsions, and may be in suspension with inorganic salts.

[0136] When a pharmaceutical composition is intended for use by a large population, safety becomes another important consideration. Although water-in-oil emulsions have been used in many clinical trials, alum remains the primary adjuvant used in formulations due to its safety profile. Thus, alum or its inorganic salt, aluminum phosphate (ADJUPHOS), is often used as an adjuvant in formulations for clinical use.

[0137] Other adjuvants and immunostimulants include 3-O-deacylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants may be used with or without other specific immunostimulants. Such other specific immune stimulants include 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-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isogluc-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wall components. Oil-in-water emulsions include MF59 (see WO 90 / 14837 to Van Nest et al., which is hereby incorporated by reference in its entirety) (5% squalene, 0.5% TWEEN 80, and 0.5% Span 85 (and optionally various amounts of MTP-PE) compounded using a microfluidizer to produce submicron particles), SAF (10% squalene, 0.4% TWEEN 80, 5% pluronic block polymer L121, and thr-MDP, either microfluidized to a submicron emulsion or vortexed to produce a larger particle size emulsion), and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Mont.) (2% of squalene, 0.2% TWEEN 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS) (preferably MPL+CWS (Detox™)).Other adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF-α).

[0138] The choice of adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the potency of the adjuvant for the species being immunized; in humans, a pharma- ceutically acceptable adjuvant is one that has been or can be approved for administration to humans by the relevant regulatory agency. For example, Alum, MPL, or incomplete Freund's adjuvant (Chang, et al., 1998), which is hereby incorporated by reference in its entirety), alone or in any optional combination, are all suitable for administration to humans.

[0139] The composition may contain a pharma- ceutically acceptable non-toxic carrier or diluent, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. The pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

[0140] Pharmaceutical compositions can also include large, slowly metabolized macromolecules, such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex-functionalized sepharose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, such carriers can function as immunostimulating agents (i.e., adjuvants).

[0141] The pharmaceutical compositions of the present disclosure may further comprise a suitable delivery vehicle, including, but not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleate vesicles.

[0142] d. Use of the pharmaceutical composition The disclosure also includes methods of using pharmaceutical compositions containing the CGRP peptide immunogen constructs.

[0143] In certain embodiments, pharmaceutical compositions containing a CGRP peptide immunogen construct can be used to treat migraine headaches.

[0144] In some embodiments, the method comprises administering to a host in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of a CGRP peptide immunogen construct. In certain embodiments, the method comprises administering to a warm-blooded animal (e.g., human, cynomolgus monkey, mouse) a pharmaceutical composition comprising a pharmacologically effective amount of a CGRP peptide immunogen construct to induce highly specific antibodies that cross-react with human CGRP protein (SEQ ID NO: 1) or CGRP proteins from other species (e.g., SEQ ID NOs: 2 and 3).

[0145] In certain embodiments, pharmaceutical compositions containing CGRP peptide immunogen constructs can be used to treat migraine headaches, as demonstrated in an in vivo capsaicin-induced skin blood flow model.

[0146] e. In vitro functional assays and in vivo proof-of-concept studies Antibodies induced in hosts immunized with CGRP peptide immunogen constructs can be used in in vitro functional assays, including, but not limited to, the following: (1) In vitro binding to CGRP protein (SEQ ID NOs: 1 to 3) (2) Inhibition of the binding of CGRP to its receptor in vitro (3) Inhibition of intracellular cAMP elevation in vitro (4) In vivo inhibition of capsaicin-induced cutaneous blood flow in mice.

[0147] Specific embodiments (1) having about 30 or more amino acids and having the following formula: (Th) m -(A) n -(CGRP functional B epitope peptide)-X or (CGRP functional B epitope peptide)-(A) n -(Th) m -X or (Th) m -(A) n -(CGRP functional B epitope peptide)-(A) n -(Th) m -X During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (CGRP functional B epitope peptide) is a B cell epitope peptide having 7 to about 30 amino acid residues derived from the CGRP receptor binding or activation region having SEQ ID NOs: 4 to 13 and 15 to 24 in CGRP (SEQ ID NOs: 1 to 3) as shown in Table 1, X is the α-COOH or α-CONH2 of an amino acid; m is from 1 to about 4; and n is 0 to about 10; The CGRP peptide immunogen constructs are represented as follows:

[0148] (2) The CGRP peptide immunogen construct according to (1), wherein the CGRP receptor binding region or activation region is selected from the group consisting of SEQ ID NOs: 4 to 13 and 15 to 24.

[0149] (3) The CGRP peptide immunogen construct according to either (1) or (2), wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 74 to 115.

[0150] (4) The CGRP peptide immunogen construct according to (1), wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120 to 127 and 130 to 180.

[0151] (5) The following: a. a B cell epitope comprising about 7 to about 30 amino acid residues derived from a CGRP sequence of SEQ ID NO: 1 to 3; b. A helper T cell epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 to 115 and any combination thereof; and c. an optional heterologous spacer selected from the group consisting of the 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; CGRP peptide immunogen construct comprising: the B cell epitope is covalently linked to the helper T cell epitope, either directly or through the optional heterologous spacer; The CGRP peptide immunogen construct.

[0152] (6) The CGRP peptide immunogen construct according to (5), wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 4 to 13 and 15 to 24.

[0153] (7) The CGRP peptide immunogen construct according to (5), wherein the helper T cell epitope is selected from the group consisting of SEQ ID NOs: 78, 77, 80 to 82, 85, 91, 94, 97, 98 to 99, 106 to 109, 112, and any combination thereof.

[0154] (8) The CGRP peptide immunogen construct of (5), wherein the optional heterologous spacer 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.

[0155] (9) The CGRP peptide immunogen construct of (5), wherein the helper T cell epitope is covalently linked to the amino terminus of the B cell epitope.

[0156] (10) The CGRP peptide immunogen construct of (5), wherein the helper T cell epitope is covalently linked to the amino terminus of the B cell epitope via the optional heterologous spacer.

[0157] (11) A composition comprising the CGRP peptide immunogen construct described in (1).

[0158] (12) The following: a. A peptide immunogen construct according to (1), and b. a pharma- ceutically acceptable delivery vehicle and / or adjuvant; 13. A pharmaceutical composition comprising:

[0159] (13) a. The CGRP functional B epitope peptide is selected from the group consisting of SEQ ID NOs: 4 to 13 and 15 to 24; b. the Th epitope is selected from the group consisting of SEQ ID NOs: 74-115; and c. the heterologous spacer is selected from the group consisting of the 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 The CGRP peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex. (12) The pharmaceutical composition according to (12).

[0160] (14)a. The CGRP peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120-127 and 130-180; and The CGRP peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex. (12) The pharmaceutical composition according to (12).

[0161] (15) A method for producing an antibody against CGRP in an animal, the method comprising administering to the animal the pharmaceutical composition according to (12).

[0162] (16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the CGRP receptor binding region or activation region of SEQ ID NOs: 4 to 13 and 15 to 24.

[0163] (17) The isolated antibody or epitope-binding fragment thereof according to (16), which binds to the CGRP peptide immunogen construct.

[0164] (18) A composition comprising the isolated antibody or epitope-binding fragment thereof according to (16).

[0165] (19) A method for preventing and / or treating migraine in an animal, comprising administering to the animal the pharmaceutical composition according to (12). EXAMPLES

[0166] Example 1 Synthesis of CGRP-related peptides and preparation of their formulations a. Synthesis of CGRP-related peptides We describe methods for the synthesis of CGRP-related peptides in an effort to develop CGRP peptide immunogen constructs. These peptides were synthesized in small-scale quantities useful for serological assays, laboratory pilot studies, and field trials, as well as in large-scale (kilogram) quantities useful for industrial / commercial production of pharmaceutical compositions. A large repertoire of CGRP-related antigenic peptides with sequences ranging in length from about 10 to 70 amino acids was designed for the purposes of epitope mapping, as well as screening and selection of the most suitable peptide immunogen constructs for use in an effective CGRP-targeted therapeutic vaccine.

[0167] Table 1 shows representative full-length CGRP (SEQ ID NOs: 1-3) from human, mouse, rat, and macaque species, CGRP peptide fragments, and 10-mer peptides used in various serological assays for epitope mapping (SEQ ID NOs: 1-70).

[0168] Selected CGRP B cell epitope peptides were engineered into CGRP peptide immunogen constructs by synthetically linking the peptides to carefully designed helper T cell (Th) epitope peptides based on pathogenic proteins, including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), peptide influenza, Clostridium tetani, and Epstein-Barr virus (EBV), identified in Table 2 (SEQ ID NOs: 74-115). The Th epitope peptides were used either as single sequences (SEQ ID NOs: 74-84, 86-90, 92-93, 95-96, 98-115) or as combinatorial libraries (SEQ ID NOs: 85, 91, 94, and 97) to enhance the immunogenicity of each of the CGRP peptide immunogen constructs in which they were used.

[0169] The identity of representative CGRP peptide immunogen constructs selected from hundreds of peptide constructs is shown in Table 3 (SEQ ID NOs: 116-180). All peptides used in immunogenicity studies or related serological studies to detect and / or measure anti-CGRP antibodies were synthesized on small scale using F-moc chemistry on Applied BioSystems Model 430A Peptide Synthesizer, Model 431 Peptide Synthesizer, and / or Model 433 Peptide Synthesizer. Each peptide was generated by independent synthesis on a solid support with F-moc protection at the N-terminus and side chain protecting groups for trifunctional amino acids. Completely synthesized peptides were cleaved from the solid support and side chain protecting groups were removed with 90% trifluoroacetic acid (TFA). Synthetic peptide preparations were evaluated by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry to ensure correct amino acid content. Each synthetic peptide was also evaluated by reversed-phase HPLC (RP-HPLC) to confirm the synthesis profile and concentration of the preparations. Despite strict control of the synthetic process (including stepwise monitoring of coupling efficiency), peptide analogs were also generated due to unintended events during the elongation cycles, including amino acid insertions, deletions, substitutions, and premature terminations, and thus the synthesized preparations typically contained multiple peptide analogs in addition to the targeted peptide.

[0170] Despite the inclusion of such unintended peptide analogs, the resulting synthetic peptide preparations were nevertheless suitable for use in immunological applications, including immunological diagnostics (as antibody capture antigens) and pharmaceutical compositions (as peptide immunogens). Typically, such peptide analogs, whether intentionally designed or arising via the synthetic process as a mixture of by-products, are often as effective as purified preparations of the desired peptide, so long as identifying QC procedures are developed to monitor both the manufacturing and product evaluation processes to ensure reproducibility and potency of the final product using such peptides. Large-scale peptide synthesis in hundreds to kilogram quantities was performed on a customized automated peptide synthesizer UBI2003 or similar at the 15 mmol to 150 mmol scale.

[0171] For the active ingredient used in the final pharmaceutical composition for clinical trials, the CGRP-related peptide immunogen construct was purified by preparative RP-HPLC with shallow gradient elution and characterized for purity and identity by MALDI-TOF mass spectrometry, amino acid analysis, and RP-HPLC.

[0172] b. Preparation of Compositions Containing CGRP Peptide Immunogen Constructs Formulations using water-in-oil emulsions and suspensions containing inorganic salts have been prepared. Safety is another important consideration when designing pharmaceutical compositions for use by large populations. Despite the fact that water-in-oil emulsions have been used in humans as pharmaceutical compositions in many clinical trials, alum remains the main adjuvant for use in pharmaceutical compositions, due to its safety. Therefore, alum or its inorganic salts (ADJUPHOS (aluminum phosphate)) are often used as adjuvants in preparations for clinical application.

[0173] Briefly, the formulations designated for each of the test groups described below generally included all types of CGRP designer peptide immunogen constructs. Over 200 designer CGRP peptide immunogen constructs were carefully evaluated in guinea pigs for their relative immunogenicity with corresponding CGRP peptides representing the B-cell epitope peptides of those immunogens. Epitope mapping and serological cross-reactivity analysis for different homologous peptides was performed by ELISA assays using plates coated with peptides selected from the list of SEQ ID NOs: 1-70.

[0174] Varying amounts of CGRP peptide immunogen constructs were routinely prepared as water-in-oil emulsions using Seppic MONTANIDE™ ISA 51 as an oil approved for human use, or mixed with inorganic salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (Alum). Typically, compositions were prepared by dissolving the CGRP peptide immunogen construct in water at about 20-800 μg / mL and combined with MONTANIDE™ ISA 51 to form a water-in-oil emulsion (1:1 by volume) or combined with inorganic salts ADJUPHOS or ALHYDROGEL (Alum) (1:1 by volume). Compositions were kept at room temperature for about 30 minutes and mixed by vortexing for about 10-15 seconds before use for immunization. Animals were immunized with 2-3 doses of a particular composition at 0 (prime) and 3 (boost) weeks post immunization (wpi), with an optional second boost at 5 or 6 wpi, administered by intramuscular route. Sera from immunized animals were then tested with selected B cell epitope peptide(s) to assess the immunogenicity of the various CGRP peptide immunogen constructs present in the formulations and the cross-reactivity of the corresponding sera with CGRP protein. CGRP peptide immunogen constructs that were found to have strong immunogenicity in the initial screening in guinea pigs were further tested for functional properties of the corresponding sera in in vitro assays. Selected candidate CGRP peptide immunogen constructs were then prepared as water-in-oil emulsion formulations, mineral salt formulations, and alum-based formulations for dosing regimes over specific time periods as indicated in the immunization protocol.

[0175] Only the most promising CGRP peptide immunogen constructs underwent extensive further evaluation and were then incorporated into a final formulation for GLP-guided preclinical immunogenicity, duration, toxicity, and efficacy studies in preparation for Investigational New Drug application submission, as well as subsequent clinical trials in patients suffering from migraine.

[0176] The following examples are intended to illustrate the present disclosure and are not to be used as limitations thereon.

[0177] Example 2 Serological Assays and Reagents Serological assays and reagents for assessing the functional immunogenicity of CGRP peptide immunogen constructs and formulations thereof are detailed below.

[0178] a. CGRP or CGRP B epitope peptide-based ELISA test for antibody specificity analysis An ELISA assay was developed to evaluate the immune serum samples described in the following Examples, and is described below: Wells of a 96-well plate were each coated with 100 μL of 10 mM NaHCO3 buffer (pH 9.5) (unless otherwise specified) containing CGRP or CGRP B epitope peptides, etc. (SEQ ID NOs: 1 to 70) at 2 μg / mL (unless otherwise specified) at 37° C. for 1 hour.

[0179] The wells coated with CGRP or CGRP B epitope peptide were incubated with PBS (250 μL) containing 3% gelatin by weight at 37° C. for 1 hour to block nonspecific protein binding sites, and then the wells were washed three times with PBS containing 0.05% TWEEN® 20 by volume and dried. The serum to be analyzed was diluted 1:20 (unless otherwise stated) with PBS containing 20% ​​normal goat serum by volume, 1% gelatin by weight, and 0.05% TWEEN® 20 by volume. One hundred microliters (100 μL) of the diluted specimen (e.g., serum, plasma) was added to each well and reacted at 37° C. for 60 minutes. The wells were then washed six times with PBS containing 0.05% TWEEN® 20 by volume to remove unbound antibodies. Horseradish peroxidase (HRP)-conjugated species (e.g., guinea pig or rat)-specific goat polyclonal anti-IgG antibodies or protein A / G were used as labeled tracers to bind to the antibody / peptide antigen complexes formed in the positive wells. 100 microliters of HRP-conjugated detection reagents in PBS containing 1% normal goat serum by volume and 0.05% TWEEN® 20 by volume at the optimal dilution determined by titration were added to each well and incubated for an additional 30 minutes at 37°C. The wells were washed six times with PBS containing 0.05% TWEEN® 20 by volume to remove unbound antibodies, and the wells were reacted with 100 μL of a substrate mixture containing 0.04% 3',3',5',5'-tetramethylbenzidine (TMB) by weight and 0.12% hydrogen peroxide by volume in sodium citrate buffer for an additional 15 minutes. The substrate mixture was used to detect the peroxidase label by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 M H2SO4 and measuring the absorbance (A) at 450 nm. 450 For the determination of antibody titers in vaccinated animals administered the various peptide vaccine formulations, sera were tested in ten-fold serial dilutions ranging from 1:100 to 1:10,000 or in four-fold serial dilutions ranging from 1:100 to 1:4.19 × 10 8 The serum titers (Log10 (shown in A) 450 With a cutoff value of 0.5, A 450 was calculated by linear regression analysis.

[0180] b. Evaluation of antibody reactivity against Th peptides by Th peptide-based ELISA Wells of a 96-well plate were each coated with 100 μL of Th peptide at 2 μg / mL (unless otherwise stated) in 10 mM NaHCO3 buffer (pH 9.5) for 1 h at 37°C and subjected to the same ELISA method (performed as described above). For the determination of antibody titers of vaccinated animals administered the various CGRP peptide vaccine formulations, sera were tested in 10-fold serial dilutions from 1:100 to 1:10,000. The titers (Log 10 ) is A 450 With a cutoff value of 0.5, A 450 was calculated by linear regression analysis.

[0181] Detailed specificity analysis of target CGRP B cell epitope peptides determined by epitope mapping with cB cell epitope cluster 10-mer peptide-based ELISA assay Detailed specificity analysis of anti-CGRP antibodies obtained from hosts immunized with CGRP peptide immunogen constructs was performed by epitope mapping using a B-cell epitope cluster 10-mer peptide-based ELISA test. Briefly, individual CGRP or related 10-mer peptides (SEQ ID NOs: 26-70) were used to coat wells of a 96-well plate at 0.5 μg / 0.1 mL per well. Then, 100 μL of serum samples (1:100 dilution in PBS) were incubated in duplicate in the 10-mer plate wells following the steps of the antibody ELISA method described above. Detailed specificity analysis of the target B-cell epitope-related of anti-CGRP antibodies obtained from immunized hosts was performed using the corresponding CGRP peptides or an irrelevant control peptide to confirm the specificity.

[0182] d. Immunogenicity assessment Pre-immune and immune serum samples were collected from animals or human subjects according to the experimental vaccination protocol and heated at 56°C for 30 min to inactivate serum complement factors. After administration of the vaccine formulation, blood samples were obtained according to the protocol and their immunogenicity against the specific target site(s) was assessed by corresponding CGRP B-cell epitope peptide-based ELISA test. Serially diluted sera were tested and the Log of the reciprocal dilution factor was used to evaluate the immunogenicity. 10 Positive titers were expressed as positive titers. The immunogenicity of a particular vaccine formulation is assessed for its ability to elicit a high titer antibody response specifically directed against a desired epitope within the target antigen, while maintaining low to negligible antibody reactivity to the "helper T cell epitopes" used to boost the desired B cell response, and for its high cross-reactivity with the CGRP protein.

[0183] Example 3 Functional characterization of antibodies induced by CGRP peptide immunogen constructs and their formulations in an in vitro assay for intracellular cAMP generation Immune sera from immunized vaccinees or purified anti-CGRP antibodies were further tested for their ability to inhibit CGRP-induced intracellular AMP production.

[0184] a. Antibody purification All antibody purification procedures were according to the instructions of the antibody purification kit (Thermo fisher, Cat. No. 89953). The concentration of each IgG purification for each group was carefully calibrated for use in the in vitro assays.

[0185] b. Cell Preparation and Maintenance Cell line L6 (ATCC® CRL-1458™) was purchased from ATCC. The base medium for this cell line is ATCC formulated Dulbecco's Modified Eagle's Medium (Cat. No. 30-2002). Fetal bovine serum was added to a final concentration of 10% to make complete growth medium. Atmosphere: air, 95%; carbon dioxide (CO2), 5%, temperature: 37°C.

[0186] c. CGRP treatment with / without IgG Anti-CGRP IgGs were further screened in vitro for neutralizing activity using a cell-based cAMP activation assay. All procedures were performed in 384-well round-bottom small volume plates (Mediomics, LLC, Cat. No. 163301). Five microliters of rat α-CGRP (final concentration 10 nM) was incubated for 30 minutes at room temperature in the presence of anti-CGRP IgG (final concentration 1-20 μg / ml). Five microliters of 1XKRB-IBMS buffer containing 5000 rat L6 smooth muscle cells were then added. Plates were incubated for 30 minutes at room temperature.

[0187] d. Cell-based CGRP neutralization assay (detection of cAMP levels) After incubation, cAMP activation was performed using Mediomics Bridge-It cAMP All-in-One Fluorescence Assay (Mediomics, Cat. No. 122938 / 122939) according to the manufacturer's instructions. Cells were lysed and the cAMP assay was initiated by adding 10 ml of cAMP All-in-One Assay Solution to each well and mixing by pipetting up and down. Plates were covered to prevent evaporation and to protect from light. Plates were incubated at room temperature for 30 min while fluorescence intensity (excitation 485 nm, emission 540 nm) was read using a fluorescence reader (SpectraMax i3x Multi-Mode Microplate Reader). Data are reported as percentages; 0% represents L6 cells alone and 100% represents L6 cells treated with CGRP.

[0188] Example 4 Animals used in safety, immunogenicity, toxicity, and efficacy studies a. Guinea pig: Immunogenicity studies were performed in mature, naive, and adult male and female Duncan-Hartley guinea pigs (300–350 g / BW). Experiments included at least three guinea pigs per group. Protocols involving Duncan-Hartley guinea pigs (8–12 weeks old, Covance Research Laboratories, Denver, PA, USA) were performed in a UBI-sponsored contract animal facility in accordance with an approved IACUC application.

[0189] b. Cynomolgus monkey: Immunogenicity and repeated-dose toxicity studies were performed in adult male and female monkeys (Macaca fascicularis, approximately 3–4 years old; Joint Laboratories, Suzhou, China) in accordance with an approved IACUC application and in a UBI-sponsored contract animal facility.

[0190] C. Mouse: Female Balb / C mice (n=6 / group) were administered 40 μg / 0.1 ml / dose of the test vaccine or 0.1 ml / dose of the control via intramuscular injection (IM). There was a single injection site (quadriceps of the hind leg) and five injections were administered at 0, 3, 6, 9, and 12 wpi prior to capsaicin challenge. Animals were housed at the UBI Asia Laboratory Animal Facility and acclimated for 1 week under constant temperature (22°C), humidity (72%), and a 12-h light / 12-h dark cycle. Mice had free access to chow and water. Principles of Laboratory Animal Care were followed in all protocols. Blood collection was performed as indicated in the protocol. Anti-CGRP (mouse) antibody titers were tested by ELISA assay.

[0191] Example 5 Vaccine formulations for immunogenicity evaluation of CGRP peptide constructs in guinea pigs The pharmaceutical compositions and vaccine formulations used in each experiment are described in more detail below.

[0192] Briefly, the formulations assigned to each test group generally contained all types of designer CGRP peptide immunogen constructs, including segments with CGRP B cell epitope peptides linked via different types of spacers (e.g., εLys (εK) or lysine-lysine-lysine (KKK) to increase the solubility of the peptide constructs) and non-selective helper T cell epitopes (including two sets of artificial helper T cell epitopes derived from measles virus fusion protein and hepatitis B surface antigen). The CGRP B epitope peptides were linked to the N-terminus or C-terminus of the designer peptide constructs. Hundreds of designer CGRP peptide immunogen constructs were initially evaluated in guinea pigs for their relative immunogenicity with the corresponding CGRP B cell epitope peptides. Varying amounts of CGRP peptide immunogen constructs were routinely prepared as water-in-oil emulsions using Seppic MONTANIDE ISA 51 as an oil approved for human vaccine use, or as suspensions using inorganic salts (ADJUPHOS) or ALHYDROGEL (Alum). Vaccine formulations were typically prepared by dissolving the CGRP peptide construct in water at approximately 20-800 μg / mL and combining with MONTANIDE ISA 51 as a water-in-oil emulsion (1:1 by volume) or with inorganic salts (ADJUPHOS) or ALHYDROGEL (Alum) (1:1 by volume). Vaccine formulations were kept at room temperature for approximately 30 minutes and mixed by vortexing for approximately 10-15 seconds before use for immunization.

[0193] Some animals were immunized with 2-5 doses of a particular vaccine formulation at 0 (prime) and 3 (boost) weeks post immunization (wpi), with an optional second boost at 5 or 6 wpi, by intramuscular route. The immunogenicity of the corresponding CGRP peptide immunogen constructs used in each vaccine formulation was then evaluated in these immunized animals for cross-reactivity with the corresponding CGRP B epitope peptide or full-length CGRP. CGRP peptide immunogen constructs that showed strong immunogenicity in the initial screening in guinea pigs were further tested in macaques in water-in-oil emulsion, mineral salt, and alum-based formulations, all for the dosing regimen over the specified time period as indicated by the immunization protocol.

[0194] Only the most promising CGRP peptide immunogen construct candidates were extensively further evaluated using the corresponding mouse CGRP peptide immunogen construct to evaluate their ability to break immune tolerance in mice. The best immunogenic CGRP peptide immunogen construct in mice was the one that elicited anti-CGRP antibody titers against endogenous CGRP, and in particular, had the best immunogenicity in terms of its ability to inhibit capsaicin-induced skin blood flow in a mouse model. The optimized CGRP peptide immunogen construct was incorporated into the final vaccine formulation for GLP-guided immunogenicity, duration, toxicity, and efficacy studies in preparation for the submission of an Investigational New Drug Application, as well as clinical trials in patients with autoimmune rheumatoid arthritis.

[0195] Example 6 Design rationale, screening, identification, functional characterization, and optimization of multicomponent vaccine formulations incorporating CGRP peptide immunogen constructs for the treatment of migraine Based on the scientific findings presented in Figures 1 and 2, and the findings disclosed in Figure 5 (Russel, et al, 2014) and Figures 2(a)-2(c) (Edvinsson, et al, 2018), CGRP is selected as a target molecule for design and as the subject matter of the present invention. Figure 1 shows an alignment of CGRP sequences from human (SEQ ID NO: 1), marmoset (SEQ ID NO: 2), mouse / rat (SEQ ID NO: 3), and many other species. Figure 5 (Russel, et al, 2014) shows the physiology and pathophysiology in which CGRP is relevant. Figures 2(a)-2(c) (Edvinsson, et al, 2018) show why CGRP could be a target for novel migraine treatments. Figure 2 shows the path from discovery to commercialization of a vaccine based on precision designer synthetic peptides. A general overview of each step is set forth in Figure 2 using a flow diagram that identifies the development process from discovery to commercialization (industrialization) of a CGRP vaccine formulation. Detailed evaluation and analysis of each of these steps, with some pleasant and some not so pleasant surprises, has led to numerous experiments that will ultimately result in the commercialization of a safe and effective CGRP vaccine formulation.

[0196] a. Design history Each peptide immunogen construct or immunotherapy product requires a unique design focus and approach based on its specific disease mechanism and the target protein(s) required to intervene therein. For the treatment of migraine, CGRP was selected as the target molecule based on the scientific knowledge available to the inventors as outlined in Figure 1 and Figure 5 (Russel, et al, 2014) and Figures 2(a)-2(c) (Edvinsson, et al, 2018). The discovery-to-commercialization path as shown in Figure 2 typically takes decades or even decades to accomplish. To design an immunogen construct, it is important to identify a CGRP B-cell epitope peptide that correlates with the functional site(s) of interest for intervention. Pilot immunogenicity studies were carried out in succession in guinea pigs incorporating different helper T cell supports (carrier proteins or suitable helper T cell peptides) into different formulations, followed by evaluation of the functional properties of the vaccine formulations using induced antibody purifications or specific CGRP peptide immunogen constructs in specific in vitro functional assays or in vivo proof-of-concept experiments in selected animal models. Extensive serological validation was then performed to further test the candidate CGRP B epitope peptide immunogen constructs in non-human primates to further validate the immunogenicity and design direction of the CGRP B epitope peptide immunogens. Selected CGRP peptide immunogen constructs were then prepared in different mixtures to evaluate the subtle differences in the functional properties associated with the respective interactions between the peptide constructs when used in combination. Additional evaluations were performed to establish the final peptide constructs, peptide compositions, and their formulations along with the respective physical parameters of the formulations, leading to the final product development process.

[0197] The amino acid sequences of the CGRP peptide immunogen constructs were designed and selected based on a number of rationales, including the adoption of the CGRP B epitope peptide sequence as follows: (i) It lacks self-helper T cell epitopes in CGRP to prevent self-T cell activation, which can lead to brain inflammation and, consequently, meningococcal encephalitis. This makes it possible to develop Aβ antagonists for the treatment of Alzheimer’s disease. 1-42 This has been previously reported in clinical trials with the AN1792 vaccine targeting (ii) Being a self molecule, it is non-immunogenic in itself. (iii) capable of being immunogenic when administered to a host, either via the protein carrier or via a strong helper T cell epitope(s). (iv) induces high titer antibodies directed against the CGRP peptide sequence (B cell epitope) and not against the protein carrier or helper T cell epitope(s). (v) Inducing high-titer antibodies capable of inhibiting the interaction between CGRP and the CGRP receptor and the induction of intracellular cAMP elevation due to cell activation; and (vi) As a proof-of-concept study to verify the treatment of migraine, the vaccine formulation is capable of suppressing capsaicin-induced cutaneous blood flow when administered to an animal model, e.g., BALB / C mice.

[0198] b. Design and validation of CGRP peptide immunogen constructs for pharmaceutical compositions with the potential to treat patients suffering from migraine To generate the most potent peptide constructs for incorporation into pharmaceutical compositions, a repertoire of human CGRP B cell epitope peptides (SEQ ID NOs: 4-24), as well as non-selective or artificial helper T cell epitopes derived from various pathogens (SEQ ID NOs: 74-115) were further engineered into representative CGRP peptide immunogen constructs (SEQ ID NOs: 116-180) for initial immunogenicity testing in guinea pigs.

[0199] i) Selection of CGRP B cell epitope peptide sequences derived from the receptor binding domain or receptor activation domain for design The CGRP receptor binding region located in the central / C-terminus of CGRP and the receptor activation region in the N-terminal C2-C7 loop / central region of CGRP were selected for designing CGRP B epitopes, which were then further made into peptide immunogen constructs to evaluate immunogenicity first by performing ELISA on CGRP B epitope peptide-coated plates, followed by eliciting immune sera in guinea pigs for evaluation by in vitro functional assays.

[0200] When CGRP binds to the CGRP receptor, the CGRP receptor transmits an activation signal within the cell that leads to, among other cellular events, an intracellular increase in cAMP levels. The ability of purified antibodies from guinea pig immune serum against specific CGRP peptide immunogen constructs to neutralize the functional properties of CGRP was demonstrated as shown in FIG. 6 and in the table displayed in FIG. 3, with IC50 values ​​that inhibited the increase in cAMP by 50% compared to controls without antibody. 50 Evaluate based on:

[0201] These CGRP peptide immunogen constructs were initially formulated with ISA 51 and CpG. The formulation was 400 μg / lmL for the primary immunization in guinea pigs and 100 μg / 0.25 mL boost (3, 6, and 9 wpi) for immunogenicity testing. To test immunogenicity in guinea pigs, ELISA assays were performed using guinea pig immune sera from bleeds at various times (wpi) and the immune sera were diluted in 10-fold serial dilutions from 1:100 to 1:10000. The corresponding mouse / rat CGRP B epitope peptides and full-length CGRP peptides were used to coat ELISA plates at 0.5 μg peptide per well. The titers (Log 10 As shown in Figure 3, 450 With a cutoff value of 0.5, A 450The titers were calculated by linear regression analysis of the CGRP peptide immunogen constructs. The detailed potencies of representative B epitopes derived from the CGRP peptide immunogen constructs were shown in Tables 4, 5, and 6. Although the designed short CGRP peptides are often non-immunogenic due to lack of endogenous Th epitopes, the addition of exogenous Th epitopes may enhance the immunogenicity of certain CGRP peptide immunogen constructs. A detailed analysis of the reactivity / specificity patterns of the various constructs from Tables 4, 5, and 6, along with Figure 3, can evaluate the immunogenicity conferred by specific residues in the CGRP molecule, which may further facilitate the design of optimal peptide immunogen constructs.

[0202] ii) Deletion of autologous helper T cell epitopes within the selected CGRPB epitopes to prevent autologous T cell activation As shown in Table 7, representative CGRP B cell epitopes, such as those of SEQ ID NOs: 5, 6, and 15, did not by themselves induce any antibodies to CGRP when administered in potent vaccine formulations in which they were used as counterparts in peptide immunogen constructs, and thus, the CGRP B cell epitopes lack undesirable endogenous Th epitopes within the selected CGRP B cell epitopes.

[0203] iii) Focused antibody responses elicited by CGRP peptide immunogen constructs target exclusively CGRP B cell epitopes It is well known that carrier proteins (e.g., keyhole limpet hemocyanin (KLH), diphtheria toxoid (DT) protein, and tetanus toxoid (TT) protein, etc.) are used to enhance immune responses directed against target B cell epitope peptides, each of which is chemically conjugated to the B cell epitope peptide; however, all of these carrier proteins result in more than 90% of the antibodies induced in an immunized host being directed against the enhancing carrier protein, and less than 10% of the antibodies being directed against the target B cell epitope.

[0204] Therefore, it is interesting to evaluate the specificity of the CGRP peptide immunogen constructs of the present invention. Two representative CGRP peptide immunogen constructs (SEQ ID NOs: 142 and 142 in Table 8) in which B cell epitopes of different lengths derived from CGRP 20-37 and 22-37 are linked to the heterologous T cell epitope UBITh®1 (SEQ ID NO: 98) via a spacer sequence were prepared for immunogenicity evaluation. UBITh®1 (the helper T cell peptide used for B cell epitope immunoboost) was coated on a plate, and cross-reactivity with the UBITh®1 peptide used for immunoboost was tested using guinea pig immune serum. The immunogenicity of these constructs was high against the corresponding CGRP B cell epitope peptides (high titers of antibodies generated against CGRP B cell epitope(s) even after only one injection (Log 5 or more). 10 )), most, if not all, of the immune sera were found to be unreactive against the UBITh®1 peptide (shown in Table 8).

[0205] In summary, the simple immunogen design of incorporating a target CGRP B cell epitope peptide linked to a carefully selected helper T cell epitope allows for the generation of a focused immune response that targets only the corresponding CGRP B cell epitope peptide. For the design of pharmaceutical compositions, the more specific the immune response that it generates, the better the safety profile that the composition has. Therefore, the CGRP peptide immunogen construct of the present invention is highly specific and potent for its target.

[0206] iv) Fine epitope mapping using immune sera directed against selected CGRP peptide immunogen constructs For detailed epitope mapping to assign antibody binding site(s) to specific residues in the target B epitope region, 45 overlapping 10-mer peptides (SEQ ID NOs: 26-70) were synthesized, which, together with the full length region of CGRP, cover the sequence from amino acid -9 to amino acid 45, covering the precursor sequence before and after the processed CGRP molecule. These 10-mer peptides were individually coated onto the wells of a 96-well microtiter plate as solid-phase immunoadsorbents. Pooled guinea pig immune sera were added as a 1:100 dilution in sample dilution buffer to these plate wells coated with 2.0 μg / mL of the 10-mer peptides, followed by incubation at 37°C for 1 hour. After washing the plate wells with wash buffer, horseradish peroxidase-conjugated rProtein A / G was added and incubated for 30 minutes. After washing again with PBS, substrate was added to the wells and absorbance was measured at 450 nm by an ELISA plate reader. The analysis was performed in duplicate samples. Maximal antibody binding signals occur when immune sera induced by a CGRP peptide immunogen binds to wells coated with the corresponding CGRP B cell epitope peptide.

[0207] Detailed epitope mapping results, as shown in Table 9, revealed that CGRP is a marker of oxidative stress syndrome that originates from both the receptor binding region at amino acids 11-37 and the receptor activation region around the C2-C7 loop at amino acids 1-25, 11-25, 8-18, 11-35, 11-37, 15-37, and 18-37. Guinea pig serum pools derived from CGRP peptide immunogen constructs of SEQ ID NOs: 122, 123, 127, 129, 130, 132, 137, and 139 containing B cell epitope peptides induced high titer antibodies that were primarily targeted to the 10-mer peptide cluster of amino acids 1-37 (SEQ ID NO: 1) and showed high cross-reactivity, primarily with B epitopes 1-25, 1-37; B epitopes 11-25, 15-26; and B epitopes 8-18. Almost no cross-reactivity; for B epitope 11-35, 22-33 and 26-36; for B epitope 11-37, 23-33 and 28-37; for B epitope 18-37, 23-33 and 28-37; for B epitope 11-35, 22-33 and 26-36; for B epitope 15-37, 17-26, 20-30, and 28-37. Although the same B epitope was employed in the design of the peptide immunogen constructs of SEQ ID NO:123 and 130, and the only difference between them was that the spacer of SEQ ID NO:123 had an extra KKK spacer compared to SEQ ID NO:130, it is of interest to understand why the construct of SEQ ID NO:130, which had a shorter spacer without the KKK residues, showed additional reactivity against amino acids 20-30 and 26-36.

[0208] In summary, the designed synthetic CGRP peptide immunogen constructs induce robust immune responses in guinea pigs that generate polyclonal antibodies targeting distinct 10-mer peptide clusters within CGRP that are in close proximity to both the CGRP receptor binding region near its respective C-terminus and the receptor activation region near the C2-C7 loop, enabling important medical intervention. Epitope mapping, coupled with functional assay evaluation, will enable the identification of optimal peptide immunogen constructs for use in vaccine formulations.

[0209] Example 7 EX-VIVO FUNCTIONAL CHARACTERIZATION OF ANTIBODIES INDUCED BY CGRP PEPTIDE IMMUNOGEN CONSTRUCTS AND COMPOSITIONS THEREOF After demonstrating that the antibodies purified from immune sera of guinea pigs immunized with carefully selected candidate CGRP immunogen constructs were highly immunogenic and cross-reactive as shown in Tables 4, 5, 6, 7, 8, and 9, the next study was designed to evaluate whether representative purified IgG from these immune sera collected at 6 wpi from each animal could inhibit the intracellular rise in cAMP caused by activation of the C2-C7 loop in CGRP upon binding to its receptor.

[0210] At the molecular level in smooth muscle cells, CGRP can bind to its receptor through its C-terminal region and then activate the receptor by using its loop region (references). The cyclic C2-C7 loop with disulfide bridge has a fundamental role in receptor activation and is closely related to the elevation of intracellular cAMP. Various anti-CGRP IgGs were used to characterize their potential anti-CGRP effects in neutralization assays. Specifically, their effects were evaluated by functional pharmacology experiments using changes in intracellular cAMP levels. This in vitro functional evaluation is particularly important in evaluating the anti-CGRP effects of guinea pig immune sera directed against the CGRP peptide immunogen constructs of the present invention. The assay procedure is detailed in Example 4.

[0211] Anti-CGRP antibody inhibits the increase in intracellular cAMP upon CGRP-activated phosphorylation Immune sera from each animal bled at 6 wpi were collected and antibodies were purified as described in Example 4. Twenty-one CGRP peptide immunogen constructs were tested in guinea pigs for their individual immunogenicity as demonstrated in Example 6. The purified antibodies were classified into three groups based on the respective target B cell epitope peptides used in the peptide immunogen constructs, directed with B cell epitope peptides from the N-terminal, central, and C-terminal regions, respectively. Data are reported as a percentage of cAMP detected in CGRP-treated L6 cells. Zero percent represents L6 cells only, and 100% represents CGRP-treated L6 cells. As shown in FIG. 4 together with the accompanying table, the IC of cAMP levels for constructs incorporating CGRP B epitope peptides from the N-terminal, central, or C-terminal regions were 50 (μg / mL) ranges from 0.60 to over 20. For practical purposes, IC values ​​of less than 10 μg / mL are used. 50 The IC score is taken as significant for antibody-mediated inhibition of cAMP production. Representative constructs that show effective functional immunogenicity are ranked (IC 50 The sequence order is: 130>127>150>125>137>123>121>119>124>131>126>133>120>129>135>116~117~118~128~139, which describes the optimal design of a CGRP peptide immunogen construct with a high degree of precision of up to a few residues in the 37-mer structure of CGRP.

[0212] In summary, the CGRP peptide immunogen constructs as presented above in their relative ranking with respect to their respective functional properties are worthy of subsequent use in CGRP vaccine formulations to demonstrate functional efficacy.

[0213] Example 8 Evaluation of a representative murine CGRP peptide immunogen construct in a prophylactic manner in a capsaicin-induced skin blood flow model in BALB / C mice as a proof-of-concept study of a CGRP vaccine A. Rationale for the Study Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide that is widely expressed in the peripheral and central nervous systems. CGRP is primarily associated with small diameter unmyelinated sensory neurons in close proximity to blood vessels. CGRP is a potent vasodilator, and local administration of CGRP causes a transient increase in blood flow. CGRP has also been implicated in pain transmission, pain modulation, and neurogenic inflammation. CGRP can be released from sensory neurons by activating transient receptor potential cation channels with capsaicin.

[0214] Laser Doppler imaging (LDI) has been used to detect the resulting changes in cutaneous blood flow, which have been shown to be primarily caused by CGRP. CGRP is also associated with inflammatory pain, as demonstrated by the attenuated responses in CGRP knockout mice in several pain models. This role in pain perception is consistent with the expression of CGRP in sensory neurons.

[0215] In mammalian plasma, the half-life of CGRP is approximately 10 minutes. In the human trigeminal ganglion, CGRP-responsive neurons account for up to 50% of the total neurons (Tajti., et al, 1999). Treating migraine headaches using an anti-CGRP vaccination approach by targeting CGRP could fulfill the unmet medical need of treating migraine in a preventative manner, with the potential for long-term, cost-effective CGRP inhibition over a long period of time.

[0216] This study is designed to test murine CGRP peptide immunogen constructs representative of the human CGRP peptide immunogen construct counterparts identified in Example 7 for their ability to reduce capsaicin-induced cutaneous blood flow in a Balb / C mouse model as a proof-of-concept study illustrating the efficacy of an anti-CGRP vaccine therapy for migraine.

[0217] b. Test Plan i) Representative test articles: The CGRP peptide immunogen construct vaccine formulation used in this study is as follows: Group 1: UBITh®1-human αCGRP 11-37 (SEQ ID NO: 123) and ISA51 and CpG3 Group 2: UBITh®3-human αCGRP 11-37 (SEQ ID NO: 141) and ISA51 and CpG3 Group 3: UBITh®3-rat / mouse αCGRP 11-37 (SEQ ID NO: 151) and ISA51 and CpG3 Group 4: UBITh®1-human αCGRP 11-37 (SEQ ID NO: 123) and ADJUPHOS and CpG3 Group 5: UBITh®1 - human αCGRP (SEQ ID NO: 131) with ISA51 and CpG3

[0218] These test articles, along with group information, are summarized in Table 2.

[0219] ii) Control: Group 6: ISA51 and CpG3 Group 7: ADJUPHOS and CpG3 Group 8: Saline

[0220] iii) Grouping and Dosage: The groupings will be as follows:

[0221] iv) Individual Identification: Test animals were identified with a picric acid marker.

[0222] v) Group Identification: Cages were appropriately labeled for identification, including study name, IACUC number, route of administration, observation period, cage number, number of animals / cage, species, strain, sex, date of admission, age at admission, animal identification number, caretaker, and assistant caretaker.

[0223] vi) Route of administration and injection site: Female Balb / C mice (n=6 / group) were administered 40 μg / 0.1 ml / dose of the test vaccine or 0.1 ml / dose of the control via intramuscular injection (IM) at a single injection site (hind quadriceps) for 5 injections at 0, 3, 6, 9, and 12 wpi prior to capsaicin challenge.

[0224] vii) Treatment Schedule: A total of five injections of control or test article were administered during the course of treatment at time points 0, 3, 6, 9, and 12 wpi.

[0225] viii) Biological sample collection and preparation: Blood was allowed to clot in tubes at room temperature for a minimum of 30 minutes and a maximum of 60 minutes to separate serum and clot. Clots were removed by centrifugation at 1,000 × g for 10 minutes in a refrigerated centrifuge. Serum was immediately transferred to sterile 1.5 mL Eppendorf polypropylene tubes. All samples were kept on wet ice during handling. Unused serum samples were stored frozen at -80°C.

[0226] ix) Laser Doppler Imaging (LDI): On the day of the experiment, the hind limbs of the mice were shaved and the mice were placed on a heating pad under the LDI device. The animals were anesthetized with 25 mg / kg Zoletil and the animals were kept under anesthesia for 20 minutes before scanning. Sequential scanning began with two baseline scans, followed by

[0227] 2 μL of capsaicin solution (50 mg capsaicin dissolved in 83.4 μL EtOH, 55.6 μL Tween 20, 27.8 μL purified H2O in a 3:2:1 ratio) was applied to each of the two O-rings. The O-rings were placed on the animal's lower back. Scanning continued for an additional 10 minutes, with one scan every 2.5 minutes. Data was analyzed using moorVMS-LDF software for regions of interest. An Excel worksheet was used to average the signals from the regions of interest at each time point. Data are reported as percent change from baseline.

[0228] x) Immunological analysis of antibody titers: Serum or CSF samples were collected and anti-CGRP antibody titers were measured by ELISA kits prepared as shown in Example 2 using full-length human for antigen coating. Serum samples were serially diluted 3-fold with a starting dilution of 1:1000. Antibody ELISA titers (Log 10 (denoted by A) was measured using an automated plate reader. 450nm The absorbance was determined at .

[0229] c. Test results. i) Injection site reactions: Minor, temporary swelling at the injection site was sometimes observed as a result of the immunization procedure.

[0230] ii) Antibody titers of immune sera obtained from immunized mice: Representative ELISA results not only showed that two peptide immunogen constructs derived from the mouse counterparts (e.g., SEQ ID NO: 151) of the CGRP peptide immunogen constructs (SEQ ID NO: 132 and 141) induced high immunogenic titers against the respective B epitope peptides (SEQ ID NO: 15 and 9), but also showed that the antibodies derived from these two immune sera had moderate cross-reactivity to their cognate full-length human CGRP, as shown in Figure 5. This study demonstrated that the two representative CGRP peptide immunogens can induce specific antibodies that are cross-reactive to the human CGRP B epitope peptide and its mouse counterpart peptide. In this POC animal study, the mouse / rat CGRP B epitope peptide immunogen construct counterpart used UBITh®3 (a more potent combinatorial Th peptide library than UBITh®1) as the helper T cell peptide and a linker (e.g., SEQ ID NO: 72) to further enhance the immunogenicity of selected constructs in mice (e.g., SEQ ID NO: 132, 134, 136, 138, 140, 141, and 151).

[0231] iii) LDI results: The capsaicin model for measuring skin blood flow is an in vivo pharmacodynamic model in both animals and humans. It is non-invasive, technically uncomplicated, and provides a rapid and objective endpoint. The model can be tested repeatedly, and the results are fully reproducible. Therefore, the model provides an ideal evaluation for the clinical evaluation of CGRP blocking therapy. As with all other biomarker models, this model has its own limitations. The capsaicin model is only a simulation of the natural pathophysiological process of the desired test. The effect of drugs or vaccine-induced antibodies on capsaicin-induced skin blood flow can result in a trend of anti-CGRP activity, which indicates their efficacy in inhibiting peripheral skin blood flow.

[0232] As shown in FIG. 6, CGRP peptide immunogen constructs of SEQ ID NOs: 123, 131, 141, and 151 induced significant specific titers (Log 100) of anti-CGRP antibodies in vaccinated mice after three vaccinations in both ADJUPHOS and ISA51 formulations containing CpG in peptide / CpG complexes, when compared to saline controls or various adjuvant-only formulations. 10 4-6) were induced by 100 mg / kg / day of capsaicin. The animals showed that capsaicin-induced cutaneous capillary blood flow was suppressed (Figure 6). Measurements were taken at 8 wpi, 12 wpi, and 14 wpi. The duration of such inhibition lasted for 14 weeks during the measurement period. The potency of selected antibody preparations in immunogenicity studies or the corresponding IC in an in vitro assay measuring the cAMP inhibitory potency of each antibody were measured. 50 Although differences were observed, three representative CGRP peptide immunogen constructs (e.g., SEQ ID NOs: 123, 131, and 141) and their formulations shared similar abilities to reduce capsaicin-induced cutaneous blood flow in vaccinated mice, demonstrating the efficacy of using these CGRP peptide immunogen constructs for the treatment of migraine headaches.

[0233] [Table 1]

[0234] [Table 2]

[0235] [Table 3]

[0236] [Table 4]

[0237] [Table 5]

[0238]

Table 6

[0239]

Table 7

[0240]

Table 8

[0241]

Table 9

[0242]

Table 10

[0243]

Table 11

[0244]

Table 12

[0245]

Table 13

Claims

1. having about 30 or more amino acids and having the following formula: (Th) ｍ - (A) ｎ -(CGRP functional B epitope peptide)-X or (CGRP functional B epitope peptide)-(A) ｎ - (Th) ｍ -X or (Th) ｍ - (A) ｎ -(CGRP functional B epitope peptide)-(A) ｎ - (Th) ｍ -X It is expressed as During the ceremony, Th is a heterologous helper T cell epitope; A is a heterologous spacer, (CGRP functional B epitope peptide) is a B cell epitope peptide having 7 to about 30 amino acid residues derived from the CGRP receptor binding region or activation region having SEQ ID NOs: 4 to 13 and 15 to 24 in CGRP (SEQ ID NOs: 1 to 3); X is the α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; and n is 0 to about 10; CGRP peptide immunogen constructs.

2. 2. The CGRP peptide immunogen construct of claim 1, wherein the CGRP receptor binding region or the activation region is selected from the group consisting of SEQ ID NOs: 4-13 and 15-24.

3. 3. The CGRP peptide immunogen construct of claim 1, wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 74-115.

4. 2. The CGRP peptide immunogen construct of claim 1, wherein said peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120-127 and 130-180.

5. a. a B cell epitope comprising about 7 to about 30 amino acid residues from a CGRP sequence of SEQ ID NO: 1-3; b. a helper T cell epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-115, and any combination thereof; c. an optional heterologous spacer selected from the group consisting of the 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; 1. A CGRP peptide immunogen construct comprising: said B cell epitope is covalently linked to said helper T cell epitope, either directly or through said optional heterologous spacer; The CGRP peptide immunogen construct.

6. 6. The CGRP peptide immunogen construct of claim 5, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 4-13 and 15-24.

7. 6. The CGRP peptide immunogen construct of claim 5, wherein the helper T cell epitope is selected from the group consisting of SEQ ID NOs: 68, 71, 74-82, 87, 89-92, 95, and any combination thereof.

8. 6. The CGRP peptide immunogen construct of claim 5, wherein the optional heterologous spacer 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), where Xaa is any amino acid.

9. 6. The CGRP peptide immunogen construct of claim 5, wherein the helper T cell epitope is covalently linked to the amino terminus of the B cell epitope.

10. 6. The CGRP peptide immunogen construct of claim 5, wherein the helper T cell epitope is covalently linked to the amino terminus of the B cell epitope through the optional heterologous spacer.

11. A composition comprising the CGRP peptide immunogenic construct of claim 1.

12. a. a peptide immunogen construct according to claim 1, and b. a pharma- ceutically acceptable delivery vehicle and / or adjuvant; 13. A pharmaceutical composition comprising:

13. a. the CGRP functional B epitope peptide is selected from the group consisting of SEQ ID NOs: 4-13 and 15-24; b. The Th epitope is selected from the group consisting of SEQ ID NOs: 74-115; and c. the heterologous spacer is selected from the group consisting of the 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; The CGRP peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

13. The pharmaceutical composition of claim 12.

14. a. the CGRP peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 120-127 and 130-180; The CGRP peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

13. The pharmaceutical composition of claim 12.

15. 13. A method for producing antibodies against CGRP in an animal, the method comprising administering to the animal a pharmaceutical composition according to claim 12.

16. An isolated antibody or epitope-binding fragment thereof which specifically binds to the CGRP receptor binding or activation domain of SEQ ID NOs: 4-13 and 15-24.

17. 17. The isolated antibody or epitope-binding fragment thereof of claim 16 conjugated to a CGRP peptide immunogenic construct.

18. A composition comprising the isolated antibody or epitope-binding fragment thereof described in claim 16.

19. A method for the prevention and / or treatment of migraine in an animal, comprising administering to said animal a pharmaceutical composition according to claim 12.

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