Peptide having CGRP mimotope function and CGRP detection using same

Abstract

Disclosed are a peptide having CGRP mimotope function and CGRP detection using same. The peptide may have the following sequences: DVGSYTKAPDF, YGPGGSQAVDF, CRARAGIGLDF, and YVAKATGPADF. The peptide of the present invention can be used in a rapid and accurate single-step immunoassay for CGRP detection, and thus can be effectively used for diagnosis and treatment monitoring of migraine patients.

Description

Peptide with CGRP mimotopic function and detection of CGRP using the same

[0001] The present invention relates to a peptide having a CGRP mimotope function and to CGRP detection using the same.

[0002] Calcitonin gene-related peptide (CGRP) is a member of the calcitonin peptide family, which also includes calcitonin, amylin, adrenomedullin, and calcitonin receptor-stimulating peptide. CGRP is a neuropeptide composed of 37 amino acid residues and is produced by various peripheral and central neurons, primarily located in the trigeminal ganglion. Of the two forms of CGRP, α-CGRP is found in the peripheral and central nervous systems and has been reported as a primary target for migraine treatment, whereas β-CGRP, found in the enteric nervous system, is less well-studied. Migraine is known to be induced by CGRP, and quantitative analysis of CGRP has been requested for migraine diagnosis prior to clinical treatment with monoclonal antibodies against CGRP. In practice, CGRP levels have primarily been measured using immunoassays that require long sample incubation times of up to 24 hours. However, a barrier to measurement was the degradation of CGRP by proteolytic enzymes after blood collection, necessitating a rapid analytical method. In this study, a single-step immunoassay with a 30-minute analysis time was developed using a chemically mimicked peptide (mimotop). The single-step immunoassay was constructed using anti-CGRP antibodies pre-conjugated with mimotops (peptides with a chemical structure similar to CGRP). When CGRP in a positive sample binds to the antibody, the pre-conjugated fluorescently labeled mimotops are quantitatively released, allowing for an estimation of the concentration of CGRP in the sample. These mimotops were selected from a library of Fv antibodies. Fv antibodies represent the variable region (VH) of immunoglobulin G (IgG) and consist of three complementarity-determining regions (CDRs) (CDR1-3) and framework regions (FRs). The Fv antibody library was generated by site-specific mutation of CDR3 of the VH region, and these Fv antibodies were expressed on the outer membrane of Escherichia coli using autodisplay technology. The Fv-antibody library prepared in this way has a diversity of 10^6 clones per library, and each E. coli contains 10^5 Fv-antibody / E.It has been reported that E. coli express Fv antibodies at a high surface density. Using an Fv antibody library, mimotopes for serotonin, dopamine, and food allergens were screened, and a single-step immunoassay based on the selected mimotopes achieved highly sensitive target analysis without washing steps or additional reagent treatment. A single-step immunoassay for CGRP was validated using mimotopes screened from an Fv antibody library. In this study, four clones were screened using two monoclonal anti-CGRP antibodies. The CDR3 regions of the selected Fv antibodies were synthesized into mimotopes consisting of 15 residues for each monoclonal antibody. The binding affinities (KD) of these mimotopes for antibodies were measured, and their interactions with the monoclonal antibodies were analyzed using docking simulations. Finally, a single-step immunoassay was constructed using the synthesized mimotopes to quantify CGRP. Samples from episodic and chronic migraine patients were analyzed and compared with normal controls. Finally, the single-step immunoassay based on Mimotope (total assay time of less than 30 minutes) was found to be statistically consistent with conventional ELISA with a sample incubation time of 24 hours.

[0003] The present invention aims to improve the accuracy and efficiency of calcitonin gene-related peptide (CGRP) detection. Existing detection methods are time-consuming and suffer from rapid degradation of CGRP by proteolytic enzymes. To address these issues, the present invention aims to develop a rapid and accurate CGRP detection method utilizing a peptide with mimotopic properties. This will contribute to faster migraine diagnosis and better treatment options for patients.

[0004] In one aspect, the present invention provides a peptide having a CGRP (Calcitonin Gene Related Peptide) mimotope function and comprising a peptide sequence having at least one functional sequence selected from the group consisting of functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4.

[0005] [Function sequence number 1]

[0006] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP

[0007] [Function sequence number 2]

[0008] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP

[0009] [Function sequence number 3]

[0010] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP

[0011] [Function sequence number 4]

[0012] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP

[0013] Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys); NP is a non-polar amino acid, one of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), and glycine (Gly); AC is an acidic amino acid, one of aspartic acid (Asp) and glutamic acid (Glu); and BS is a basic amino acid, one of lysine (Lys), arginine (Arg), and histidine (His).

[0014] In one embodiment, the peptide may include a sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and having a similarity of 80% or more to one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0015] [Sequence number 1]

[0016] DVGSYTKAPDF

[0017] [Sequence number 2]

[0018] YGPGGSQAVDF

[0019] [Sequence number 3]

[0020] CRARAGIGLDF

[0021] [Sequence number 4]

[0022] YVAKATGPADF.

[0023] In one embodiment, the peptide may have a mimotopic function capable of binding to an antibody capable of binding to Calcitonin Gene Related Peptide (CGRP).

[0024] In one embodiment, the peptide can competitively bind to the antibody in the presence of Calcitonin Gene Related Peptide (CGRP).

[0025] In another aspect, the present invention provides a nucleic acid encoding the peptide.

[0026] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0027] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0028] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0029] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and may include one or more cells selected from the group comprising Neurospora crassa.

[0030] In another aspect, the present invention provides a composition for detecting CGRP, comprising an amino acid fragment having a peptide sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and at least one functional sequence selected from the group consisting of functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4.

[0031] [Function sequence number 1]

[0032] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP

[0033] [Function sequence number 2]

[0034] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP

[0035] [Function sequence number 3]

[0036] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP

[0037] [Function sequence number 4]

[0038] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP

[0039] Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys); NP is a non-polar amino acid, one of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), and glycine (Gly); AC is an acidic amino acid, one of aspartic acid (Asp) and glutamic acid (Glu); and BS is a basic amino acid, one of lysine (Lys), arginine (Arg), and histidine (His).

[0040] In one embodiment, the composition for detecting CGRP may include an amino acid fragment having a sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and having a similarity of 80% or more to one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0041] [Sequence number 1]

[0042] DVGSYTKAPDF

[0043] [Sequence number 2]

[0044] YGPGGSQAVDF

[0045] [Sequence number 3]

[0046] CRARAGIGLDF

[0047] [Sequence number 4]

[0048] YVAKATGPADF.

[0049] In one embodiment, the composition for detecting CGRP may have a mimotope function capable of binding to an antibody capable of binding to CGRP (Calcitonin Gene Related Peptide).

[0050] In one embodiment, the peptide can competitively bind to the antibody in the presence of Calcitonin Gene Related Peptide (CGRP).

[0051] In one embodiment, the amino acid fragment may further comprise a fluorescent marker.

[0052] In another aspect, the present invention provides a kit for detecting CGRP, comprising: a substrate including at least a conduit; an antibody capable of binding to CGRP (Calcitonin Gene Related Peptide) immobilized within the conduit; and a composition for detecting CGRP bound to the antibody; wherein the composition for detecting CGRP competitively binds to the antibody with respect to CGRP.

[0053] In one embodiment, the composition for detecting CGRP may include the composition for detecting CGRP according to the embodiment of the present invention described above.

[0054] In one embodiment, the amino acid fragment of the composition for detecting CGRP may further comprise a fluorescent marker.

[0055] In one embodiment, the CGRP detection kit may further include a fluorescence analysis unit that performs quantitative analysis of the fluorescent marker from the CGRP detection composition released when the antibody is exposed to CGRP.

[0056] Specifically, migraine is known to be triggered by calcitonin gene-related peptide (CGRP), and quantitative analysis of CGRP is required for clinical treatment with monoclonal antibodies against CGRP. However, because CGRP is degraded by proteolytic enzymes after blood collection, a rapid analytical method is necessary. In this study, we developed a one-step immunoassay for CGRP using a chemically mimicked peptide (Mimotop), which can be analyzed within 30 minutes. Four clones were selected from an Fv-antibody library using two monoclonal antibodies against CGRP. For each monoclonal antibody, two Mimotops were synthesized as peptides consisting of 15 amino acids. The binding affinity (KD) was estimated, and the interaction with the monoclonal antibody was analyzed through docking simulations. Finally, a one-step immunoassay for quantifying CGRP was constructed using Mimotop and migraine patient samples. The one-step immunoassay based on Mimotope (total analysis time of less than 30 minutes) showed statistically similar results to conventional ELISA with a sample incubation time of 24 hours.

[0057] The advantage of the present invention is that it enables rapid and accurate detection of CGRP. This can support the early diagnosis and effective treatment of migraine, and significantly shortens analysis time compared to existing time-consuming analysis methods. Furthermore, the detection method using the mimotope peptide exhibits high sensitivity and specificity, enabling accurate detection of even low concentrations of CGRP.

[0058] Figure 1 is a diagram illustrating a screening process for CGRP mimotopes using an Fv-antibody library. The process of screening mimotopes capable of binding to CGRP monoclonal antibodies (Fremanezumab or Galcanezumab) using magnetic beads from an Fv-antibody library having randomized CDR3 regions expressed on the outer membrane of E. coli is illustrated, thereby selecting mimotopes capable of competitively binding to CGRP.

[0059] Figures 2, 3, 4, 5, 6, and 7 are diagrams showing the analysis results of selected E. coli clones No. 1 and No. 27. It was confirmed through flow cytometry and fluorescence intensity measurements that each clone had high binding affinity for Fremanezumab, and the binding constant (KD) values ​​were 2.65 x 10^-8 M and 1.19 x 10^-8 M, respectively.

[0060] Figures 8, 9, 10, 11, 12, and 13 are diagrams showing the analysis results of selected E. coli clones No. 7 and No. 10. It was confirmed through flow cytometry and fluorescence intensity measurements that each clone had high binding affinity for Galcanezumab, and the binding constant (KD) values ​​were 2.25 x 10^-8 M and 3.44 x 10^-8 M, respectively.

[0061] Figure 14 is a diagram illustrating the process of binding a peptide to a CGRP antibody, followed by the release of the fluorescently labeled peptide through PBS washing. This illustrates the process of binding a fluorescently labeled peptide to a CGRP antibody, then washing with PBS to remove unbound peptide, releasing the bound peptide, and detecting a fluorescent signal.

[0062] Figures 15, 16, 17, and 18 are diagrams showing the results of measuring the binding affinity of each peptide (F1, F27, G7, and G10) through SPR (Surface Plasmon Resonance) analysis. The four types of peptides are shown to bind to CGRP antibody (Fremanezumab or Galcanezumab), and the binding constants (KD) of each are indicated. Peptides F1 and F27 bind to Fremanezumab, and peptides G7 and G10 bind to Galcanezumab, and their binding affinities were quantitatively analyzed through SPR signals.

[0063] Figures 19 and 20 are diagrams showing the binding of CGRP mimotopes (F1, F27) to the VH domain of fremanezumab and between the VH and CH domains. The binding affinity and interacting amino acid residues of each mimotope are indicated, and the results analyzed through docking simulations are visually represented. The F1 mimotope binds to the VH domain of fremanezumab, and the F27 mimotope binds to the bridge region between the VH and CH domains.

[0064] Figures 21 and 22 are diagrams showing the binding of CGRP mimotopes (G7, G10) to the VH domain of galcanezumab and between the VH and CH domains. The binding affinity and interacting amino acid residues of each mimotope are indicated, and the results analyzed through docking simulations are visually represented. The G7 mimotope binds to the VH domain of galcanezumab, and the G10 mimotope binds to the bridge region between the VH and CH domains.

[0065] Figure 23 is a diagram illustrating the fluorescently labeled peptide binding and competitive binding process of a CGRP-antibody complex. CGRP antibodies bind to Mimotop peptides and are immobilized on a Maxisorp plate blocked with BSA. Upon addition of α-CGRP, competitive binding occurs, releasing fluorescently labeled Mimotop. The presence of CGRP can be detected through the emitted fluorescence.

[0066] Figures 24 and 25 are diagrams showing changes in fluorescence intensity according to the concentration of α-CGRP in mice and humans. The changes in fluorescence intensity according to CGRP concentration were measured using four types of peptides (F1, F27, G7, and G10), and a high correlation was observed for both mice and human α-CGRP. The changes in fluorescence intensity that occur when each peptide binds to CGRP can be used to quantitatively analyze CGRP concentration.

[0067] Figures 26 and 27 are diagrams comparing the results of measuring CGRP concentrations in migraine patients and normal subjects using a commercial ELISA kit and a one-step immunoassay. CGRP concentrations in episodic migraine patients, chronic migraine patients, and normal subjects were measured and compared using the two methods, and both methods showed that migraine patients had higher CGRP concentrations than normal subjects.

[0068] Figures 28 and 29 statistically compare the results of a commercial ELISA and a one-step immunoassay. The Bland-Altman plot and Passing-Bablok regression analysis confirm that the results of the two assays are statistically similar, indicating that both methods can accurately measure CGRP concentrations. The Bland-Altman plot shows the confidence interval between the two methods, and the Passing-Bablok regression analysis shows the agreement between the two methods through the correlation coefficient.

[0069] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0070] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof. In the context of this specification, the term "about" or the like can mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of a numerical value described in the specification.

[0071] Additionally, the description of one aspect of the present invention may be applied identically or similarly to the same or similar configurations or terms in the description of other aspects.

[0072] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0073] A peptide according to an embodiment of the present invention may include a peptide sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and having one or more functional sequences selected from the group comprising functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4.

[0074] [Function sequence number 1]

[0075] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP

[0076] [Function sequence number 2]

[0077] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP

[0078] [Function sequence number 3]

[0079] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP

[0080] [Function sequence number 4]

[0081] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP

[0082] Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys); NP is a non-polar amino acid, one of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), and glycine (Gly); AC is an acidic amino acid, one of aspartic acid (Asp) and glutamic acid (Glu); and BS is a basic amino acid, one of lysine (Lys), arginine (Arg), and histidine (His).

[0083] In the context of this specification, the term "functional sequence number" refers to an arrangement of codes that classify the physicochemical properties of each amino acid within a peptide sequence. Rather than a single amino acid sequence, it refers to a generalized representation based on the properties that the sequence may have. Such functional sequence numbers are not limited to a specific sequence and can be commonly applied to various sequences with similar combinations of properties, and thus can be utilized in the design or sequence optimization of peptide libraries. Furthermore, the definition based on functional sequences opens up the possibility of including variant sequences that maintain core functions despite direct mutations in the original amino acid sequence, and can contribute to expanding the scope of application of predicted sequences as well as experimentally confirmed sequences.

[0084] In one embodiment, the peptide may include a sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and having a similarity of 80% or more to one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4. In another embodiment, the peptide may include a CGRP (Calcitonin Gene Related Peptide) mimotope function and having one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0085] [Sequence number 1]

[0086] DVGSYTKAPDF

[0087] [Sequence number 2]

[0088] YGPGGSQAVDF

[0089] [Sequence number 3]

[0090] CRARAGIGLDF

[0091] [Sequence number 4]

[0092] YVAKATGPADF.

[0093] In the context of this specification, the term "similarity of peptide sequences" means a numerical representation of the degree to which identical or similar amino acids are arranged between two peptide sequences, and is generally calculated as the ratio (%) of identical residues to the total sequence length. For example, if the lengths of two sequences are the same, the similarity can be calculated by counting the number of times amino acids are identical at each position, dividing this number by the total length, and converting it to a percentage. If the sequence lengths are different, an optimal alignment can be performed using an alignment algorithm (e.g., Needleman-Wunsch or Smith-Waterman), and then the similarity can be evaluated based on the matching ratio of the aligned portion.

[0094] Peptides with a similarity of 80% or greater are functionally similar and can perform essentially identical functions. This is because most functional properties are determined in limited, core regions, such as the active site or binding interface, and the conservation of some amino acids within the overall sequence plays a crucial role in maintaining function. In particular, structurally stable peptides may exhibit only a small number of non-conservative substitutions without significant changes in three-dimensional structure or binding affinity, and in such cases, functional consistency is highly likely to be maintained. Furthermore, numerous experimental reports have shown that peptides with high sequence similarity exhibit similar binding properties toward the same target molecule, which can serve as a reliable indicator for predicting functional equivalence.

[0095] In the context of this specification, a "mimotope" refers to a fragment that mimics the three-dimensional structural characteristics of a specific antigen and has the ability to mimic binding to an antibody. The peptides provided by the embodiments of the present invention effectively mimic antigenic determinants and can play a significant role in the detection of CGRP. These peptides can competitively bind to antibodies against CGRP, thereby enabling them to be used to detect the presence of CGRP.

[0096] In the context of this specification, the dictionary definition of CGRP (Calcitonin Gene Related Peptide) is calcitonin gene-related peptide, a type of neurotransmitter. CGRP consists of 37 amino acids and is primarily produced by neurons in the trigeminal ganglion. This peptide induces vasodilation and regulates inflammatory responses. It is particularly closely associated with neurological disorders such as migraine, and changes in its concentration can serve as a valuable biomarker for the diagnosis and treatment of these diseases. Accurate detection of CGRP can provide crucial clues for identifying the cause of neurogenic headaches and developing treatments. The peptide proposed in the present invention serves as a tool capable of effectively detecting CGRP, contributing to the implementation of rapid and reliable diagnostic methods.

[0097] In one embodiment, the peptide may have a mimotopic function capable of binding to an antibody capable of binding to calcitonin gene-related peptide (CGRP). This peptide mimics the structural characteristics of CGRP and possesses antibody binding capacity, enabling rapid and accurate detection of the presence of CGRP. This function may be particularly helpful in addressing the problem of rapid degradation of CGRP in the blood. Furthermore, this peptide can be used in a variety of clinical settings and may serve as an important tool in the diagnosis and monitoring of neurological disorders such as migraine.

[0098] In one embodiment, the peptide can competitively bind to the antibody in the presence of calcitonin gene-related peptide (CGRP). This allows for rapid measurement of CGRP concentrations, providing high sensitivity and specificity in diagnostic procedures. These peptides can be used to accurately detect the presence of CGRP in blood, body fluid, or tissue samples, and may be particularly useful for the diagnosis and treatment monitoring of migraine patients. Furthermore, this competitive binding mechanism can help prevent rapid degradation of CGRP, thereby yielding more stable and reproducible results.

[0099] A nucleic acid according to an embodiment of the present invention can encode the peptide. In the context of this specification, the dictionary definition of "nucleic acid" is a biochemical substance that stores and transmits genetic information, including DNA or RNA. These nucleic acids are components of genes and contain information necessary for the biosynthesis of proteins and peptides. In the context of this specification, the dictionary definition of a nucleic acid encoding a peptide means that the nucleic acid (DNA or RNA) contains genetic information that directs the synthesis of a peptide composed of a specific amino acid sequence. This information is converted into a peptide through the processes of transcription and translation. During transcription, the genetic information in DNA is copied into mRNA, and during translation, the code in the mRNA is decoded into an amino acid sequence by the ribosome, thereby synthesizing the peptide. Through this process, the nucleic acid directly determines the structure and function of the peptide.

[0100] As is known in the art, the combination of nucleic acids encoding amino acids contained in a peptide can vary. Therefore, the present invention encompasses not only the above-described peptides, but also nucleic acids encoding the above-described peptides, including theoretically 196,608 nucleic acid sequences encoding SEQ ID NO: 1, theoretically 393,216 nucleic acid sequences encoding SEQ ID NO: 2, theoretically 1,327,104 nucleic acid sequences encoding SEQ ID NO: 3, and theoretically 262,144 nucleic acid sequences encoding SEQ ID NO: 4. This variety of nucleic acid sequences is due to the codon variability of each amino acid. A codon is a sequence of three consecutive bases of a nucleic acid that encodes one amino acid, and multiple codons can encode the same amino acid, allowing for the existence of various nucleic acid sequences encoding the same peptide sequence. This increases the flexibility of the present invention and can aid in selecting nucleic acid sequences optimized for specific biological systems or applications. For example, nucleic acid sequences can be optimized to account for codon usage that is translated more efficiently in a particular species, which may contribute to improved expression levels and stability of the peptide.

[0101] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, the dictionary definition of a recombinant expression vector is a molecule used to introduce genes into other cells and cause them to express proteins or peptides. These vectors may take the form of plasmids, viruses, artificial chromosomes, etc., and contain essential elements for replication and gene expression, such as selectable markers, promoters, and reporter genes. Such vectors enable highly efficient expression of target peptides in specific cell types and can enhance the stability and function of peptides within the cells. For example, these vectors can be utilized not only in laboratory research but also in biomanufacturing processes for mass production. Furthermore, recombinant expression vectors containing appropriate regulatory elements can finely control gene expression, thereby optimizing the production of desired proteins or peptides.

[0102] Meanwhile, cells according to embodiments of the present invention can be transformed with the recombinant expression vector. In the context of this specification, the dictionary definition of transformation is the process of altering the genetic makeup of a cell by introducing DNA from outside. Through this process, the cell acquires new genes, which can be expressed within the cell and produce specific proteins. These transformed cells can be used for various purposes in research and industrial applications. For example, when mass production of a specific protein is required in pharmaceutical development, transformed cells can be used to efficiently produce the protein. Furthermore, these cells can be utilized for disease modeling and gene function studies, making them an important tool in life science research.

[0103] Transformation of these cells with the recombinant expression vector can enable the continuous production of specific peptides or proteins. These transformed cells remain stable throughout the production process and can produce the target protein at high yields. This allows researchers to rapidly obtain large quantities of protein, which can contribute to increased experimental repeatability and reliability. Furthermore, these cells can play a crucial role in the development of protein-based therapeutics and can be utilized effectively in disease modeling and drug screening processes.

[0104] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa. These various cells have different physiological characteristics and culture conditions, allowing for optimal selection based on specific protein production needs. This can enable efficient and economical production of target proteins for research and industrial applications.

[0105] Meanwhile, a composition for detecting CGRP according to an embodiment of the present invention may include an amino acid fragment having a peptide sequence having a CGRP (Calcitonin Gene Related Peptide) mimotope function and having one or more functional sequences selected from the group including functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4.

[0106] [Function sequence number 1]

[0107] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP

[0108] [Function sequence number 2]

[0109] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP

[0110] [Function sequence number 3]

[0111] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP

[0112] [Function sequence number 4]

[0113] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP

[0114] Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys); NP is a non-polar amino acid, one of alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), and glycine (Gly); AC is an acidic amino acid, one of aspartic acid (Asp) and glutamic acid (Glu); and BS is a basic amino acid, one of lysine (Lys), arginine (Arg), and histidine (His).

[0115] In one embodiment, the composition for detecting CGRP may include an amino acid fragment having a CGRP (Calcitonin Gene Related Peptide) mimotope function and a sequence having at least 80% similarity to one or more peptide sequences selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4. In another embodiment, the composition for detecting CGRP may include an amino acid fragment having a CGRP (Calcitonin Gene Related Peptide) mimotope function and at least one peptide sequence selected from the group consisting of the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0116] [Sequence number 1]

[0117] DVGSYTKAPDF

[0118] [Sequence number 2]

[0119] YGPGGSQAVDF

[0120] [Sequence number 3]

[0121] CRARAGIGLDF

[0122] [Sequence number 4]

[0123] YVAKATGPADF.

[0124] In one embodiment, the composition for detecting CGRP may have a mimotopic function capable of binding to an antibody capable of binding to CGRP (Calcitonin Gene Related Peptide). In one embodiment, the peptide can competitively bind to the antibody in the presence of CGRP (Calcitonin Gene Related Peptide). This allows for rapid measurement of CGRP concentrations, providing high sensitivity and specificity in the diagnostic process. Such peptides can be used to accurately detect the presence of CGRP in blood, body fluid, or tissue samples, and may be particularly useful for the diagnosis and treatment monitoring of migraine patients. Furthermore, this competitive binding mechanism may help prevent rapid degradation of CGRP, thereby producing more stable and reproducible results. This technology can be usefully utilized in various clinical and research settings and may contribute to the detection of biomarkers for various diseases, including neurological disorders.

[0125] In one embodiment, the amino acid fragment may further comprise a fluorescent marker. The role of the fluorescent marker is that when the mimotope of the CGRP detection composition is pre-bound to an antibody, if CGRP is present in the sample, the mimotope with the fluorescent marker detaches from the antibody through competitive binding. The detached fluorescent marker can then be analyzed to qualitatively confirm the presence of CGRP, and the intensity of the fluorescent signal can be used to quantitatively analyze the concentration of CGRP. This method provides high sensitivity and specificity, enabling rapid and accurate CGRP detection. Furthermore, the introduction of the fluorescent marker allows for simple analysis without the need for complex washing procedures or additional reagent treatment, significantly improving usability in laboratory and clinical settings. This can serve as an important tool for the early diagnosis and treatment monitoring of neurological disorders such as migraine.

[0126] Meanwhile, a kit for detecting CGRP according to an embodiment of the present invention may include at least a substrate including a conduit; an antibody capable of binding to CGRP (Calcitonin Gene Related Peptide) immobilized within the conduit; and a composition for detecting CGRP bound to the antibody. The function of the conduit and the substrate is to allow CGRP to bind to the antibody when a sample flows through the conduit. If CGRP is contained in the sample, the fluorescently labeled mimotope is detached from the antibody by competitive binding. This detached fluorescent label is detected on the substrate, thereby enabling qualitative and quantitative analysis of the presence of CGRP. This structure simplifies sample handling and analysis, enabling fast and efficient detection. In addition, the design of the kit allows users to easily inject samples and read results, thereby increasing its usability in laboratory and field diagnostic environments.

[0127] In one embodiment, the composition for detecting CGRP may comprise the composition for detecting CGRP according to the embodiment of the present invention described above. In one embodiment, the composition for detecting CGRP may competitively bind to the antibody with CGRP. In one embodiment, the amino acid fragment of the composition for detecting CGRP may further comprise a fluorescent marker.

[0128] In one embodiment, the CGRP detection kit may further include a fluorescence analysis unit that performs quantitative analysis of the fluorescent marker from the CGRP detection composition released when the antibody is exposed to CGRP. The role of the fluorescence analysis unit is to detect the signal of the released fluorescent marker and quantitatively analyze it to accurately measure the concentration of CGRP. Specifically, the fluorescence analysis unit can determine the presence and amount of CGRP in a sample by measuring the intensity of the fluorescent signal. This is based on changes in the fluorescent signal according to changes in the concentration of CGRP, and can quickly and precisely detect fluorescent signals generated during sample processing. This function enables rapid detection of CGRP and can be of great help in the diagnosis and monitoring of diseases such as migraine. In addition, the fluorescence analysis unit can be implemented as an automated system, thereby increasing user convenience and ensuring repeatability and reliability of experiments.

[0129] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0130] Materials and Methods

[0131] ingredient

[0132] Fremanezumab (HY-P99019) and galcanezumab (HY-P99021) were purchased from MedChem Express (Princeton, NJ, USA). Dynabeds™ Protein G for CGRP mimotope screening, black 96-well maxisorp microplates, and PCR-related reagents for Fv-antibody library plasmid construction were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The four screened mimotopes were synthesized as FITC-labeled peptides (purity >95%) by Peptron (Daejeon, Korea). Murine and human CGRP and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Korea (Seoul, Korea). A commercial CGRP ELISA kit was purchased from Bertin Bioreagents (Montigny le Bretonneux, France). Human plasma samples from episodic migraine patients, chronic migraine patients, and normal subjects were provided by Severance Hospital (Seoul, Korea). This study was approved by the Institutional Review Board of Severance Hospital (IRB number 2023-3534-001).

[0133] Screening of CGRP mimotopes using Fv-antibody libraries

[0134] The preparation of the Fv-antibody library with randomized CDR3 regions has been described in detail in a previous study. CGRP mimotopes were screened from the Fv-antibody library using Dynabeads™ Protein G. Monoclonal antibodies against human CGRP (Fremanezumab and Galcanezumab) were immobilized on Dynabeads™ Protein G according to the manufacturer's instructions. CGRP mimotope screening was initially performed by expressing Fv-antibodies with randomized CDR3 regions on the outer membrane of Escherichia coli (E. coli) cells. E. coli cells expressing the Fv-antibody library were cultured overnight at 37°C and 200 rpm for 16 h, and the overnight cultures were inoculated at 37°C and 200 rpm for 2.5 h. IPTG induction for the expression of the CDR3-randomized Fv-antibodies was performed at 30°C and 200 rpm for 2 h. Fv-antibody library cells with an OD600 nm = 1.0 were reacted with magnetic beads immobilized with anti-CGRP monoclonal antibodies (Fremanezumab or Galcanezumab) at room temperature for 1 h. Unbound cells were filtered with an external magnet using two types of buffers (PBS buffer containing 0.1% Tween-20 and PBS buffer). Washing steps using an external magnet were performed 20 times, and the screened Fv-antibody expressing clones that specifically bound to the monoclonal antibody against CGRP (Fremanezumab or Galcanezumab) were plated on agar plates and incubated overnight at 37°C. Finally, clones with high binding affinity to Fremanezumab or Galcanezumab were obtained.

[0135] One-step immunoassay

[0136] For the one-step immunoassay of CGRP, anti-CGRP monoclonal antibodies (Fremanezumab and Galcanezumab) were immobilized in 96-well Maxisorp microplates at 4°C for 16 h. After washing with PBS buffer, 1 mg / mL BSA blocking was performed for 40 min. After washing with PBS buffer, 2 M of four different Mimotops (FITC-labeled synthetic peptides) were added to the wells immobilized with BSA-blocked anti-CGRP monoclonal antibodies (Fremanezumab or Galcanezumab) for 1 h. After washing with PBS buffer, rat or human CGRP at concentrations ranging from 1.37 to 1000 pg / mL was added to each well for 30 min to perform the one-step immunoassay, and the supernatants were transferred to another black 96-well microplate. The fluorescence intensity of fluorescently labeled mimotope released due to the presence of CGRP in the supernatant was measured using a GloMax® Discover Microplate Reader from Promega (Madison, WI, USA). The limit of detection (LOD) was calculated as the mean intensity of negative samples plus three standard deviations (3σ).

[0137] For the measurement of CGRP in human plasma samples (n=75) from episodic migraine patients (n=18), chronic migraine patients (n=39), and normal subjects (n=18), 1 / 10 diluted human plasma samples were added to the aforementioned one-step immunoassay 96-well microplate (96-well microplate immobilized with BSA-blocked anti-CGRP monoclonal antibody) for 30 min, and then the supernatant was transferred to another black 96-well microplate and the fluorescence intensity was measured. CGRP measurement in human plasma samples (n=75) using a commercial CGRP ELISA kit was performed as a reference method following the manufacturer's instructions.

[0138] Results and Discussion

[0139] Mimotop selection

[0140] In this study, Fv antibodies represent the variable region (VH) of the heavy chain of immunoglobulin G (IgG), which consists of three complementarity-determining regions (CDR1-3) and four framework regions (FR1-4). To screen for CGRP mimotopes, the CDR3 region of the Fv antibody was used to prepare an Fv antibody library through site-directed mutagenesis, and this library was expressed on the outer membrane of Escherichia coli using autodisplay technology. The Fv antibody library thus prepared had an expression yield of 10^5 Fv antibodies per E. coli cell and a diversity of 10^6 clones per library. Two monoclonal antibodies against human CGRP, Fremanezumab and Galcanezumab, were used to screen for CGRP mimotopes from the Fv antibody library. These two monoclonal antibodies have been used as anti-migraine agents to block the binding of CGRP to its receptor, the CLR. As shown in Figure 1, the selected mimotopes bound to this monoclonal antibody because the Fv-antibody had an amino acid sequence with a chemical environment similar to that of CGRP.

[0141] To screen for these mimotopes, monoclonal antibodies were immobilized on magnetic beads, and bound E. coli cells were separated using an external magnet. The isolated clones were cultured on agar plates, and the final clones were selected through the following steps: (1) selection of E. coli clones with binding affinity for the monoclonal antibody, and (2) identification of oligonucleotide sequences (CDR3 regions) different from the template sequence. As shown in Figures 2, 3, 4, 5, 6, and 7, two clones were selected using magnetic beads with immobilized Fremanezumab, and flow cytometry revealed that the high fluorescence region had high affinity for Fremanezumab (red box area). In the case of the mutant strain with only the autodisplayed CDR1 and CDR2 regions and the E. coli strain without any autodisplayed protein, almost no fluorescence signal was observed in the same red box area. When these clones were treated with various concentrations of CGRP, a quantitative increase in fluorescence signal was observed. These results showed that the CDR3 region of the selected clone had affinity binding to Fremanezumab, and two 11-amino acid sequences corresponded to the mimotope of CGRP. The same CGRP screening was performed with another monoclonal antibody, Galcanezumab, and two clones were selected, as shown in Figures 8, 9, 10, 11, 12, and 13. The magnetic beads with immobilized Galcanezumab and the control strains (same mutant and original E. coli) did not show any significant fluorescence signal. When these clones were treated with various concentrations of CGRP, a quantitative increase in the fluorescence signal was observed. These results also showed that the CDR3 region of the selected clone had affinity binding to Galcanezumab, and two 11-amino acid sequences corresponded to the mimotope of CGRP.The amino acid sequences and binding affinities of the selected mimotopes are summarized in Table 1, and four mimotopes were synthesized as peptides. The binding affinities (KD) of the four mimotopes to Fremanezumab or Galcanezumab were analyzed using an SPR biosensor. Fremanezumab or Galcanezumab was immobilized on a 50 nm wide Au-coated SPR chip, and the four mimotopes responded in the concentration range of 41.2–3330 nM (Figure 14). The binding affinities for Fremanezumab were estimated to be 605 nM for Mimotop (F1) and 669 nM for Mimotop (F27), and the binding affinities for Galcanezumab were estimated to be 689 nM for Mimotop (G7) and 925 nM for Mimotop (G10) (Figs. 15, 16, 17, and 18). These results indicate that the four synthesized peptide forms of Mimotop bind specifically to monoclonal CGRP antibodies (Fremanezumab or Galcanezumab).

[0142] The interactions between monoclonal antibodies and selected mimotopes were investigated through docking simulations using Autodock Vina software from Scripps Research Institute (La Jolla, CA, USA). The CDR information of fremanezumab and galcanezumab was obtained from the AbYsis database (http: / www.abysis.org), and these CDR sequences were matched to the constant region (CH) of the heavy chain of IgG (summarized in Table 2). As shown in Figures 19 and 20, mimotope (F1) was analyzed to bind to the VH of fremanezumab with a binding affinity of -6.0 kcal / mol, while another mimotope (F27) bound to the bridge region between the VH and CH of the monoclonal antibody with a binding affinity of -5.3 kcal / mol. The binding sites between the heavy chain of fremanezumab and mimotope (F1) were W33, W47, A63, Y101 (hydrophobic interactions), W47, Y62, E64, A65 (hydrogen bonds), and E50 (electrostatic interactions). Another mimotope (F27) formed hydrophobic bonds at V95, L117, P158, and K210, and hydrogen bonds at G115, T160, S162, T174, and P176. The major interacting amino acids between the CDRs of the monoclonal antibody and the first selected mimotope (F1) are summarized in Table 3. For the other mimotope (F27), no interacting amino acids were found in the CDRs of the monoclonal antibody. As shown in Figures 21 and 22, mimotope (G7) was analyzed to bind to the VH of galcanezumab with a binding affinity of -6.6 kcal / mol, and another mimotope (G10) bound to the bridge region between the VH and CH of the monoclonal antibody with a binding affinity of -7.1 kcal / mol. The binding sites between the heavy chain of galcanezumab and mimotope (G7) were G44, W47, L99, F106 (hydrophobic interactions), and W33, Q35, L45, E46, and D101 (hydrogen bonds).Another mimotope (G10) formed hydrophobic bonds with V93, W109, and V169 of the heavy chain of galcanezumab, hydrogen bonds with P41, Q43, Y95, Q111, G112, T114, T157, and T171, and electrostatic interactions with H170. The major interacting amino acids between the CDRs of the mAb and the first selected mimotope (G7) are summarized in Table 3. For the other mimotope (G10), no interacting amino acids were found in the CDRs of the mAb. These results showed that both mimotopes, F1 and G7, are in close proximity to the binding domain of CGRP, which matches the paratope of each mAb.

[0143] One-step immunoassay based on Mimotop

[0144] A one-step immunoassay for CGRP was constructed by synthesizing the selected mimotopes into peptides (11 amino acids). Using an SPR biosensor, the binding affinity of Fremanezumab for CGRP was reported to be 63 pM, and that of Galcanezumab for CGRP was 31 pM. These binding affinities were significantly higher than those of the synthesized mimotopes (see Table 1). As shown in Figure 23, the synthesized mimotopes were reacted with monoclonal antibodies for the one-step immunoassay. When CGRP bound to the monoclonal antibodies, fluorescently labeled mimotopes were quantitatively released from the monoclonal antibodies. By measuring the fluorescent signal, one-step immunoassay for CGRP was possible without washing steps or additional reagent treatment. As shown in Figures 24 and 25, four types of mimotopes were used in the one-step immunoassay for mouse CGRP. When comparing the fluorescence signals according to the mimotope, the mimotopes (F1 and G7) that bound to the paratope of the monoclonal antibody showed higher sensitivity than the other mimotopes (G27 and G10) that bound to the bridge region of VH and CH. The limits of detection (LOD) of the one-step immunoassay using the mimotopes of fremanezumab (F1) and galcanezumab (G7) were estimated to be 19.1 pg / mL and 5.41 pg / mL, respectively. In addition, the sensitivity of the assay using the mimotope of galcanezumab (G7) was higher than that of fremanezumab (F1). When human CGRP was detected with the same one-step immunoassay configuration, the mimotope of galcanezumab (G7) had the highest sensitivity among the other mimotopes and was used for further analysis using patient samples. The limits of detection (LOD) of the one-step immunoassay using mimotope for fremanezumab (F1) and galcanezumab (G7) were estimated to be 7.18 pg / mL and 6.03 pg / mL, respectively.

[0145] A one-step immunoassay for CGRP based on Mimotop (G7) was performed using sera from patients with paroxysmal migraine (n=18) and chronic migraine (n=39). Healthy sera were used as negative samples (n=18) for comparison. The cutoff for migraine-positive samples is generally known to be a mean CGRP concentration of 330 pg / mL, while healthy samples range from 3 to 269 pg / mL. A commercial CGRP assay kit from Bertin Bioreagents (Montigny le Bretonneux, France) was used as the reference method, requiring a sample incubation time of 24 hours. As shown in Figures 26 and 27, migraine patient samples showed significantly higher CGRP concentrations than healthy sera. The Mimotop-based one-step immunoassay required a sample incubation time of 30 minutes, and analytical results were obtained without washing steps or additional reagent treatment. The analysis results also showed that migraine patient samples had significantly higher CGRP concentrations compared to healthy serum.

[0146] To compare the results of the two analytical methods (commercial ELISA and one-step immunoassay), we performed Blunt-Altman plots and Passing-Bablok regression analyses using MedCalc software (Ostend, Belgium). As shown in Figures 28 and 29, the Bland-Altman plots showed that the two methods using the same samples were consistent at the 95% confidence level. The Passing-Bablok regression analysis showed that the two methods using the same samples were not consistent at a probability of less than 1 in 10,000 (P<0.0001) with a Spearman correlation coefficient of 0.814. These statistical analyses indicate that the two analytical methods (commercial ELISA and one-step immunoassay) can provide statistically consistent results when using the same samples.

[0147] Screened clonesAmino acid sequenceOligonucleotide sequenceBinding constant(K D, FACS )Binding constant(K D, SPR )FremanezumabScreened Cloneno. 15'-DVGSYTKAPDF-3'5'-GAT GTG GGC AGC TAT ACC AAG GCG CCT GAT TTC-3'2.65 x 10 -8 M6.05 x 10 -7 MFremanezumabScreened Cloneno. 275'-YGPGGSQAVDF-3'5'-TAT GGG CCT GGT GGG AGT CAG GCG GTA GAT TTC-3'1.19 x 10 -8 M6.69 x 10 -7 MGalcanezumabScreened Cloneno. 75'-CRARAGIGLDF-3'5'-TGT CGC GCC CGA GCT GGG ATT GGT CTA GAT TTT-3'2.25 x 10 -8 M6.89 x 10 -7 MGalcanezumabScreened Cloneno. 105'-YVAKATGPADF-3'5'-TAT GTG GCC AAA GCG ACT GGT CCA GCC GAT TTC-3'3.44 x 10 -8 M9.25 x 10 -7 M

[0148] [Table 1] Binding affinity of screened mimotope sequences to fremanezumab and galcanezumab.

[0149] Heavy chain amino acid sequence of FremanezumabHeavy chain amino acid sequence of GalcanezumabFR1 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 30 1QVQLVQSGAEVKKPGSSVKVSKASGYTFG 30 CDR1 31 NYWIS 35 31 NYWMQ 35 FR2 36 WVRQAPGKGLEWVA 49 36 WVRQAPGQGLEWMG 49 CDR2 50 EIRSESDASATHYAEAVKG 68 50 AIYEGTGKTVYIQKFAD 66 FR3 69 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCLA 100 67 RVTITADKSTSTAYMELSSLRSEDTAVYYCAR 98 CDR3 101 YFDYGLAIQNY 111 99 LSDYVSGFGY 108 FR4 112 WGQGTLVTVSS 122 109 WGQGTTVTVSS 119TailASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0150] [표 2] AbYsis 데이터베이스에서 가져온 프레마네주맙(Fremanezumab), 갈카네주맙(Galcanezumab)의 중쇄 아미노산 서열.

[0151] Binding site of heavy chain of monoclonal antibodies against CGRP (Fremanezumab or Galcanezumab)MimotopesHydrophobic interactionHydrogen bondElectrostatic interactionMimotope-F1W 33 , W 47 , A63 , Y 101 W 47 , Y 62 , E 64 , A 65 E 50 Mimotope-F27V 95 , L 117 , P 158 , K 210 G 115 , T 160 , S 162 , T 174 , P 176 -Mimotope-G7G 44 , W 47 , L 99 , F 106 W 33 , Q 35 , L 45 , E 46 , D 101 -Mimotope-G10V 93 , W 109 , V 169 P 41 , Q 43 , Y 95 , Q 111 , G 112 , T 114 , T 157 , T 171 H 170

[0152] [Table 3] Molecular docking analysis of four mimotopes with monoclonal antibodies against CGRP (fremanezumab or galcanezumab).

[0153] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

1. CGRP (Calcitonin Gene Related Peptide) has mimotope function, A peptide sequence comprising one or more functional sequences selected from the group comprising functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4. Peptides: [Function sequence number 1] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP [Function sequence number 2] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP [Function sequence number 3] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP [Function sequence number 4] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys). NP is a non-polar amino acid, and is one of the following: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), or glycine (Gly). AC is an acidic amino acid, either aspartic acid (Asp) or glutamic acid (Glu). BS is a basic amino acid, one of lysine (Lys), arginine (Arg), or histidine (His).

2. In paragraph 1, A sequence comprising a sequence having at least 80% similarity to at least one peptide sequence selected from the group comprising the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO:

4. Peptides: [Sequence number 1] DVGSYTKAPDF [Sequence number 2] YGPGGSQAVDF [Sequence number 3] CRARAGIGLDF [Sequence number 4] YVAKATGPADF.

3. In paragraph 1, The above peptide has a mimotopic function that can bind to an antibody that can bind to CGRP (Calcitonin Gene Related Peptide). Peptide.

4. In paragraph 3, The above peptide competitively binds to the antibody in the presence of CGRP (Calcitonin Gene Related Peptide). Peptide.

5. Coding a peptide according to any one of claims 1 to 4, Nucleic acid.

6. Containing nucleic acid according to Article 5, Recombinant expression vector.

7. Transformed with a recombinant expression vector according to Article 6, cell.

8. In paragraph 7, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli, and insect cells. cell.

9. In paragraph 8, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp and Containing one or more cells selected from the group comprising Neurospora crassa, cell.

10. CGRP (Calcitonin Gene Related Peptide) has mimotope function, Comprising an amino acid fragment having a peptide sequence having one or more functional sequences selected from the group comprising functional sequence number 1, functional sequence number 2, functional sequence number 3, and functional sequence number 4; Composition for detecting CGRP: [Function sequence number 1] AC-NP-NP-PO-PO-PO-BS-NP-NP-AC-NP [Function sequence number 2] PO-NP-NP-NP-NP-PO-PO-NP-NP-AC-NP [Function sequence number 3] PO-BS-NP-BS-NP-NP-NP-NP-NP-AC-NP [Function sequence number 4] PO-NP-NP-BS-NP-PO-NP-NP-NP-AC-NP Here, PO is a polar amino acid, one of serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), and cysteine ​​(Cys). NP is a non-polar amino acid, and is one of the following: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), or glycine (Gly). AC is an acidic amino acid, either aspartic acid (Asp) or glutamic acid (Glu). BS is a basic amino acid, one of lysine (Lys), arginine (Arg), or histidine (His).

11. In paragraph 10, An amino acid fragment comprising a sequence having at least 80% similarity to one or more peptide sequences selected from the group comprising the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO:

4. Composition for detecting CGRP: [Sequence number 1] DVGSYTKAPDF [Sequence number 2] YGPGGSQAVDF [Sequence number 3] CRARAGIGLDF [Sequence number 4] YVAKATGPADF.

12. In paragraph 10, The above CGRP detection composition has a mimotope function that can bind to an antibody that can bind to CGRP (Calcitonin Gene Related Peptide). Composition for detecting CGRP.

13. In paragraph 12, The above peptide competitively binds to the antibody in the presence of CGRP (Calcitonin Gene Related Peptide). Composition for detecting CGRP.

14. In paragraph 10, The above amino acid fragment further comprises a fluorescent marker, Composition for detecting CGRP.

15. A substrate including at least a conduit; An antibody capable of binding to CGRP (Calcitonin Gene Related Peptide) fixed within the above-mentioned duct; and A composition for detecting CGRP bound to the above antibody; The above CGRP detection composition competitively binds to the antibody with CGRP, The composition for detecting CGRP comprises a composition for detecting CGRP according to any one of claims 10 to 14. Kit for detection of CGRP.

16. In paragraph 15, The amino acid fragment of the composition for detecting CGRP further comprises a fluorescent marker. Kit for detection of CGRP.

17. In paragraph 16, Further comprising a fluorescence analysis unit for performing quantitative analysis of the fluorescent marker from the CGRP detection composition released when the antibody is exposed to CGRP. Kit for detection of CGRP.

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