CN116926038A - Epitope peptide of TrkA and application thereof - Google Patents

Abstract

The invention relates to an epitope peptide of TrkA and application thereof, wherein the epitope peptide comprises the amino acid sequence shown in SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b). According to the invention, the dominant epitope of the TrkA antigen is obtained through hydrogen-deuterium exchange mass spectrometry, and the anti-TrkA antibody specifically combined with the dominant epitope can block the combination of a TrkA receptor and ligand NGF thereof, inhibit or weaken NGF/TrKA signal transmission participated by the TrkA, thereby effectively relieving pain and hyperalgesia.

Description

Epitope peptide of TrkA and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an epitope peptide of TrkA and application thereof.

Background

The drugs for chronic pain treatment currently used clinically mainly include non-steroidal anti-inflammatory drugs, anticonvulsants, opioids, etc., however, these drugs have many disadvantages, in which the efficacy of the non-steroidal anti-inflammatory drugs is limited and side effects including gastrointestinal bleeding and renal toxicity are involved; whereas opioids have side effects such as addiction. NGF/TrkA is used as an effective target for developing novel analgesic drugs, and provides a possibility for solving the problems.

Tropomyosin receptor kinase a (Tropomyosin Receptor Kinase, trkA, also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, or TRK1 transforming tyrosine kinase protein) belongs to a member of the Tropomyosin Receptor Kinase (TRK) family. Trk receptors are transmembrane receptors consisting of an extracellular region containing a ligand binding region, a transmembrane region, and an intracellular region containing a tyrosine kinase region. TrkA is expressed primarily in neural crest neurons, sympathetic neurons, and cholinergic neurons of the basal forebrain and striatum, as well as in some non-neuronal tissues and cells including B lymphocytes.

Nerve growth factor (Nerve Growth Factor, NGF) is involved in the pathophysiological processes of pain, mainly by binding to TrkA receptor to activate NGF/TrkA signaling pathway, affecting release of inflammatory mediators, opening of ion channels and promoting growth of nerve fibers, thus participating in the processes of occurrence, conduction and sensitization of pain. Studies show that blocking NGF-TrkA signaling pathway can effectively relieve pain and hyperalgesia, and NGF-TrkA signaling pathway is an effective target for developing novel analgesic drugs.

For the development of NGF-TrkA target analgesic drugs, screening of anti-TrKA monoclonal antibodies capable of blocking the binding of NGF and TrKA is one of the effective means. In general, anti-TrKA monoclonal antibodies can be screened by full-length TrKA receptor or extracellular region immunoanimals of TrKA receptor, but most of the anti-TrKA monoclonal antibodies screened are not capable of blocking NGF and TrKA binding. The discovery of the dominant epitope of the TrKA can instruct the design of a proper immunogen to prepare an anti-TrKA monoclonal antibody capable of specifically blocking the TrKA receptor from combining with ligand NGF thereof, and the discovery efficiency and success rate of the TrKA monoclonal antibody medicine with blocking activity are improved. However, related reports are still fresh.

Disclosure of Invention

The invention aims to provide an epitope peptide of TrkA and application thereof, and an antibody specifically binding to the epitope peptide can effectively block the binding of a TrkA receptor and ligand NGF thereof, inhibit or weaken NGF/TrKA signal transmission participated by TrkA, thereby effectively relieving pain and hyperalgesia.

To this end, in a first aspect, the invention provides an epitope peptide of TrkA comprising the amino acid sequence of SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

In some embodiments, the amino acid sequence of the epitope peptide of TrkA is SEQ ID NO:4.

in some embodiments, the epitope peptide of TrkA is as set forth in SEQ ID NO:4 and is based on the amino acid sequence of the TrkA extracellular region to SEQ ID NO:4, or a polypeptide extending at both the amino terminus, the carboxy terminus, or both the amino terminus and the carboxy terminus.

Wherein the amino acid sequence of the extracellular region of TrkA is SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3.

in some embodiments, the epitope peptide of TrkA is as set forth in SEQ NO:4 and is based on SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3 to SEQ ID NO:4, or a polypeptide extending at both the amino terminus, the carboxy terminus, or both the amino terminus and the carboxy terminus.

Further, the sequence described in SEQ ID NO:4, or the number of amino acids extending from the amino terminus, the carboxy terminus, or both the amino terminus and the carboxy terminus is each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

In some embodiments, the epitope peptide of TrkA is as set forth in SEQ NO:4 and is based on SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3 to SEQ ID NO:4, said m being selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

In some embodiments, the epitope peptide of TrkA is as set forth in SEQ NO:4 and is based on SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3 to SEQ ID NO:4, said n being selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

In some embodiments, the epitope peptide of TrkA is as set forth in SEQ NO:4 and is based on SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3 to SEQ ID NO:4 and q amino acids toward the carboxy terminus, each of said q and q being independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

In some embodiments, the amino acid sequence of the epitope peptide of TrkA is SEQ ID NO: 5-18.

In a second aspect of the invention, there is provided a fusion protein formed by fusing an epitope peptide of the invention with a carrier protein.

Further, the carrier protein is selected from one of Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), ovalbumin (OVA), thyroglobulin and human serum albumin.

In a third aspect of the invention, there is provided a nucleic acid molecule encoding an epitope peptide of the invention or a fusion protein of the invention.

In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the invention.

In a fifth aspect of the invention, a host cell is provided which expresses an epitope peptide according to the invention, and/or expresses a fusion protein according to the invention, and/or comprises a nucleic acid molecule according to the invention, and/or comprises a vector according to the invention.

In a sixth aspect of the invention there is provided the use of an epitope peptide according to the invention, and/or a fusion protein according to the invention, and/or a nucleic acid molecule according to the invention, and/or a vector according to the invention, and/or a host cell according to the invention in the following (a), (b), (c), (d) or (e):

(a) For the preparation of anti-TrkA antibodies;

(b) For preparing aptamer, vaccine, nanoparticle for TrkA;

(c) For preparing a medicament for preventing and/or treating diseases related to TrkA;

(d) For the preparation of a medicament for inhibiting or attenuating the signalling involved in TrkA;

(e) Can be used for preparing analgesic.

In a seventh aspect of the invention, there is provided an antibody which recognizes and binds to an epitope peptide of the invention.

Further, the antibodies are capable of inhibiting or attenuating the signaling involved in TrkA.

Further, the antibody is a monoclonal antibody or a polyclonal antibody, and the species source can be mammals such as human, mouse, rabbit, monkey, cow, sheep, alpaca and the like.

In an eighth aspect of the present invention, there is provided a method for producing an anti-TrkA antibody, comprising: (1) Immunizing animals with the epitope peptide or the fusion protein to obtain hybridoma cells, and collecting culture supernatant; (2) And (3) screening, identifying and purifying the supernatant collected in the step (1) to obtain the anti-TrkA antibody.

In a ninth aspect of the invention, there is provided a vaccine comprising an epitope peptide, fusion protein, nucleic acid molecule, vector or host cell of the invention.

In a tenth aspect of the invention, there is provided a composition comprising an epitope peptide, fusion protein, nucleic acid molecule, vector, host cell, antibody or vaccine of the invention, and a pharmaceutically acceptable carrier and/or adjuvant.

The pharmaceutically acceptable carrier and/or adjuvant should be compatible with the epitope peptide, fusion protein, nucleic acid molecule, carrier, host cell, antibody, vaccine, i.e. capable of being blended therewith without substantially reducing the efficacy of the drug. Some examples of pharmaceutically acceptable carriers or excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; isotonic saline solutions, and the like.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

(1) According to the invention, the dominant epitope of the TrkA antigen is obtained through hydrogen-deuterium exchange mass spectrometry, the anti-TrkA antibody specifically combined with the dominant epitope can block the TrkA receptor from combining with ligand NGF thereof, and the epitope exists in TrkA of human, mice, rats and cynomolgus monkeys.

(2) The epitope peptide provided by the invention has immunogenicity, can induce immune response, can be used for preparing antibodies, nucleic acid aptamer, vaccine, trkA-targeted nano-particles and the like, and can be expected to effectively block the binding of TrkA receptor and ligand NGF thereof, inhibit or weaken NGF/TrKA signal transmission participated by TrkA, thereby effectively relieving pain and hyperalgesia.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

fig. 1: SDS-PAGE electropherograms of specific anti-TrKA monoclonal antibodies;

fig. 2: SDS-PAGE electrophoresis of Human-TrKA receptor extracellular region protein;

fig. 3: ELISA detection results of the binding capacity of the specific anti-TrKA antibody to the extracellular region of the Human-TrKA receptor;

fig. 4: the interaction epitope analysis result of the specific anti-TrKA antibody and the extracellular region of the Human-TrKA receptor;

fig. 5: analysis results of interaction epitope between Human-TrKA receptor extracellular region and specific anti-TrKA antibody;

fig. 6: flow cytometry detection results of Human-NGF signals bound to the extracellular region of Human-TrKA protein on HEK293T-HumanTrkA cells under the action of specific anti-TrKA antibodies;

fig. 7: flow cytometry detection of the binding capacity of a specific anti-TrKA antibody to the Mouse-TrKA receptor;

fig. 8: ELISA detection results of the binding capacity of the specific anti-TrKA antibody and the Rat-TrKA receptor extracellular region;

fig. 9: ELISA detection results of the binding capacity of a specific anti-TrKA antibody to a TrKB, trKC, P75 receptor;

fig. 10: SDS-PAGE identification result of Cynomolgus-TrKA protein;

fig. 11: the Fortebio method evaluates the affinity results of the test antibodies with the Cynomolgus-TrKA protein;

fig. 12: ELISA method for detecting the binding capacity of the tested antibody and polypeptide;

fig. 13: ELISA method for detecting the binding capacity of the tested antibody and polypeptide;

fig. 14: ELISA method detects the blocking effect result of the polypeptide immune rabbit serum on the binding of the Human-TrKA receptor and the ligand thereof, namely Human-NGF.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Unless defined otherwise, 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. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Determination of epitope by hydrogen deuterium exchange mass spectrometry:

hydrogen deuterium exchange mass spectrometry (HDX-MS) epitope localization has proven to be an effective method for rapidly providing epitope structural integrity information. The principle is that hydrogen on protein amide bond can be controllably exchanged with deuterium in heavy water, protein is immersed in the heavy water, hydrogen on protein surface is closely contacted with the heavy water, and the exchange rate is fast; and the hydrogen inside the protein and forming hydrogen bonds is difficult to contact heavy water, so that the exchange rate is slow. And determining the deuteration number of the peptide sequences under different reaction time by utilizing mass spectrum, and further calculating the hydrogen deuterium exchange rate of each peptide sequence, so that the spatial structure information of the protein can be judged. This technique uses mass spectrometry to measure the solvent accessibility of amino acid residues in proteins to determine the interaction site of an antigen-antibody complex in its natural solution, and does not introduce any modifications to the antigen or antibody. During the analysis, both the antigen itself and the antigen-antibody complex are incubated in deuterated solvents to exchange hydrogen for deuterium. Since antibodies provide additional protection to the residues of the binding site, epitopes can be determined by comparing the exchange behavior of unbound antigen and antigen-antibody complexes.

EXAMPLE 1 expression of specific anti-TrKA monoclonal antibodies

Construction of the expression vector: and constructing an expression vector of the specific anti-TrKA monoclonal antibody by adopting a molecular cloning method, and recombining and expressing the specific anti-TrKA monoclonal antibody in a CHO expression system. The light chain nucleotide sequence (SEQ ID NO: 19) and the heavy chain nucleotide sequence (SEQ ID NO: 20) of the coding specific anti-TrKA monoclonal antibody are obtained by the company of the Austenite biotechnology limited through chemical synthesis, and the obtained sequences are inserted between the same enzyme cutting sites of the eukaryotic expression vector after double enzyme cutting, so as to construct the recombinant specific anti-TrKA monoclonal antibody expression vector. The verified correct expression vector was then extracted using the Invitrogen plasmid extraction kit and recovered by linearization with restriction enzymes followed by purification and storage at-20 ℃. Transfection of expression vectors: after resuscitating the CHO host cells with CD CHO medium, the cell density was about 8X 10 ⁵ cells were collected at cell/mL for transfection. Transfected cells were approximately 1X 10 ⁷ cell, vector about 40. Mu.g, was transfected by electric shock method (Bio-Rad, gene pulser Xcell). Cells were cultured in 20mL CD CHO medium after electric shock. The following day of culture, cells were harvested by centrifugation and resuspended in 20mL of CD CHO medium with final MSX concentration of 50. Mu.MCulturing. When the cell density is about 0.6X10 ⁶ At cell/mL, the obtained mixed clone strain was passaged with CD CHO medium, and the passaged cell density was 0.2X10 ⁶ cell/mL. When the cell viability was about 90%, the cell culture broth was collected.

EXAMPLE 2 purification and identification of specific anti-TrKA monoclonal antibodies

Specific anti-TrKA monoclonal antibodies were tested at the translational level.

The cell culture liquid collected in the example 1 is purified by using a Protein A chromatographic column, absorption peaks are collected, mass spectrum detection is carried out, the molecular weight of the mass spectrum detection antibody is about 150KD, and the molecular weight is consistent with the theoretical molecular weight, and the antibody is in a dimer form. Meanwhile, the collected sample is subjected to reduction and non-reduction and then is detected by 10% SDS-PAGE electrophoresis, the reduced SDS-PAGE electropherogram shows two bands, the non-reduced SDS-PAGE electropherogram shows a single band at about 25KD and about 50KD respectively, and the size of the electropherogram band is consistent with theory at about 150KD, as shown in figure 1. After purification the samples were dialyzed overnight at 4℃against 0.01M PBS buffer, pH 7.5.

Example 3expression of the extracellular region of the Human-TrKA receptor

Construction of the expression vector: an expression vector of a His-tagged Human-TrKA receptor extracellular region (nucleotide sequence SEQ ID NO: 21) is constructed by adopting a molecular cloning method, and the Human-TrKA receptor extracellular region is expressed in a recombination mode in a HEK293F expression system. The nucleotide sequence of the extracellular region of the Human-TrKA receptor with the His tag is entrusted with Jin Weizhi biotechnology limited company and is obtained through chemical synthesis, and the obtained sequence is inserted between the same enzyme cutting sites of the eukaryotic expression vector after double enzyme cutting, so that the recombinant Human-TrKA receptor extracellular region expression vector with the His tag is constructed. The verified correct expression vector was then extracted using the Invitrogen plasmid extraction kit and recovered by linearization with restriction enzymes followed by purification and storage at-20 ℃. Transfection of expression vectors: taking density of 3X 10 ⁶ cell/mL, 30mL of 96.44% viable blank HEK293F cells were transfected with vector at about 30. Mu.g by liposome method (GIBCO Expifectamine 293Transfection Kits). 400. Mu.g/mL of G418 pressure screening, when fineCell density of about 1X 10 ⁶ At cell/mL, the obtained mixed clone was passaged with Exip293Expression Medium medium, and the passaged cell density was 0.5X10 ⁶ cell/mL. When the cell viability was about 90%, the cell culture broth was collected.

Example 4 purification and characterization of the extracellular region of the Human-TrKA receptor

Detection of the extracellular domain of the Human-TrKA receptor was performed at the translational level.

The cell culture liquid collected in the example 3 is purified by an NI column, absorption peaks are collected, mass spectrum detection is carried out, and mass spectrum detection shows that a large amount of glycosylation modification exists in the extracellular region of the Human-TrKA receptor after the expression of the HEK293F expression system, and the molecular weight is about 75 KD. Meanwhile, the collected sample is reduced and then detected by 10% SDS-PAGE electrophoresis, and the electrophoresis pattern shows a band which is about 75KD, as shown in figure 2. After purification the samples were dialyzed overnight at 4℃against 0.01M PBS buffer, pH 7.5.

Example 5 detection of binding Capacity of specific anti-TrKA antibodies to extracellular region of Human-TrKA receptor Using ELISA method

In the test of this example, the binding of the specific anti-TrKA antibody to the extracellular region of the Human-TrKA receptor was evaluated by ELISA method at various concentrations (20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.313. Mu.g/mL, 0.156. Mu.g/mL, 0.078. Mu.g/mL, 0.039. Mu.g/mL, 0.019. Mu.g/mL). The results are shown in FIG. 3. At a certain concentration of antibody, OD ₄₅₀ The value reflects the strength of binding of the antibody to the protein, the greater the reading the greater the binding of the antibody to the protein. According to the experimental results of FIG. 3, it was shown that the concentration of the specific anti-TrKA antibody was increased from 0. Mu.g/mL to 20. Mu.g/mL, OD ₄₅₀ The values gradually increased until approaching stability, approaching around 3, with an EC50 of 0.01739 μg/mL. This shows that the specific anti-TrKA antibody can specifically bind to the extracellular region of the Human-TrKA receptor and has good binding capacity.

Example 6 determination of epitope of Human-TrKA receptor and specific anti-TrKA antibody by Hydrogen-deuterium exchange Mass Spectrometry

In this example, the epitope between the Human-TrKA receptor and the specific anti-TrKA antibody was determined by hydrogen deuterium exchange mass spectrometry (HDX-MS), and the specific experimental method is as follows:

1. preparation of a hydrogen deuterium exchange mass spectrum sample:

5-10. Mu. Mol of antigen or antibody or antigen-antibody complex (1:1 molar ratio) (50mM HEPES,pH 7.4, 150mM NaCl,4mM TCEP) was left at 4℃for 1 hour, respectively, to form a stable state of the complex. After dilution of 5. Mu.L of the sample to 20. Mu. L D at 4 ℃ ₂ O (deuterium) and placed into different HDX time points for mass spectrometry analysis (e.g., 0, 10, 60, 300, 900 seconds). After a period of hydrogen deuterium exchange, the mixture was passed through a 100 MmNaH ice-cold with 25. Mu.L ₂ PO ₄ The reaction was stopped by mixing 1M TCEP. Immediately after stopping the reaction, the sample tube was placed on dry ice until the sample was injected into the HDX LEAP PAL3.0 platform. After injection to the fully automated hydrogen-deuterium exchange platform, the sample was passed through a fixed pepsin column at a flow rate of 120 μl/min and the enzymatically digested peptide fragments were captured on a C18 capture column and desalted. Desalted peptide fragments were separated over 8 minutes using a 2.1mm×5cm C18 column (1.9. Mu.m Hypersil Gold, thermo Fisher) with a linear gradient of 4-40% acetonitrile and 0.3% formic acid. During sample processing, both proteolysis and peptide fragment separation were performed at 4 ℃. An Orbitrap mass spectrometer (Orbitrap Fusion was used ^TM Tribrid ^TM Mass Spectrometer, thermo Fisher) obtained hydrogen deuterium exchanged mass spectrum data, the resolution of the measurement was 65,000 (m/z 400). Each sample had three HDX assays (triplets) at each time point. The mass spectrum peak intensity average m/z centroid value (10 ppm accuracy) for each enzymatic peptide fragment was calculated by HDX Workbench software and then converted to deuterium incorporation percentage. The key amino acid sequences involved in the spatial epitope were calculated and the difference in% Delta D was determined by calculating the difference between the two samples (comparing the variation in percentage deuterium incorporation on the same peptide fragment). Differences in Delta% D outside-5 to 5% are considered significant differences. Furthermore, HDX Workbench detected statistical significance between samples at each time point by student's t test (p<0.05 A) the difference.

2. Identification of antigen-antibody two-dimensional polypeptide spectrogram:

exchange by hydrogen deuteriumMass spectrometry platform LEAP PAL3.0 uses secondary mass spectrometry (MS/MS) in a mass spectrometer (Orbitrap Fusion ^TM Tribrid ^TM Mass Spectrometer, thermo Fisher) to identify proteolytic peptides of the protein. The MS/MS data file was processed by Proteome Discover software for peptide fragment identification.

Experimental results:

the epitope analysis of the interaction between a specific anti-TrKA antibody and the extracellular region of an antigen Human-TrKA receptor is shown in FIG. 4, and the analysis can be obtained: DNPFE is a potential epitope region, which is positioned at 380-384 positions of the Human-TrKA receptor sequence; epitope analysis of the interaction of the extracellular region of the antigen Human-TrKA receptor with a specific anti-TrKA antibody is shown in fig. 5, wherein since P does not have amide hydrogen bonds (amide hydrogen), the software automatically does not calculate its HDX activity, and it is found that RTGNTF of a specific anti-TrKA antibody is a potential epitope binding region.

Example 7 detection of blocking of Human-TrKA receptor binding to its ligand Human-NGF by specific anti-TrKA antibodies Using flow cytometry

The Human-NGF is biotinylated, human-NGF can be used for binding to the extracellular region of Human-TrkA protein on HEK293T-HumanTrkA cells, an anti-TrkA monoclonal antibody can also be used for binding to the extracellular region of Human-TrkA protein on HEK293T-HumanTrkA cells, competition experiments are designed, and the binding situation of Human-NGF and Human-TrkA protein extracellular region on HEK293T-HumanTrkA cells is examined by flow cytometry, wherein the binding situation of the specific anti-TrKA antibody to the extracellular region of Human-NGF and Human-TrkA protein is studied under the action of the specific anti-TrKA antibody (20 mug/mL, 10 mug/mL, 5 mug/mL, 2.5 mug/mL, 1.25 mug/mL, 0.625 mug/mL, 0.313 mug/mL, 0.156 mug/mL, 0.078 mug/mL, 0.039 mug/mL).

The experimental results are shown in FIG. 6. In FIG. 6, the parent% value reflects the Human-NGF signal bound to the extracellular domain of the Human-TrKA protein on HEK293T-HumanTrkA cells, and the lower the reading, the weaker the Human-NGF signal bound to the extracellular domain of the Human-TrkA protein on HEK293T-HumanTrkA cells, and the greater the antibody effect on inhibiting Human-NGF binding to Human-TrkA. As shown in FIG. 6, as the concentration of a particular anti-TrKA antibody increases, the parent% value gradually decreases until it approaches zero, i.e., the Human-NGF signal bound to the extracellular region of the Human-TrkA protein gradually decreases until there is no Human-NGF binding to the extracellular region of the Human-TrkA protein, the binding of Human-NGF to Human-TrkA is totally blocked, and the IC50 is 0.7963. Mu.g/mL; it can be seen that, within a certain concentration range, antibodies that specifically bind to the epitope DNPFE are able to block the binding of Human-NGF to Human-TrkA at the cellular level in a dose-dependent manner. This indicates that DNPFE is the dominant epitope region of the Human-TrkA receptor to which an anti-TrKA antibody specifically binds has the activity of blocking the binding of TrkA to NGF.

Example 8 detection of binding Capacity of specific anti-TrKA antibodies to Mouse-TrKA receptor Using flow cytometry

Specific anti-TrKA antibody samples were diluted to 11 concentration gradients (20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.313. Mu.g/mL, 0.156. Mu.g/mL, 0.078. Mu.g/mL, 0.039. Mu.g/mL, 0.019. Mu.g/mL) with PBS buffer, and the binding of the specific anti-TrKA antibody to the HEK293T-Mouse-TrKA cell surface Mouse-TrKA receptor was examined by flow cytometry, and the binding ability of each humanized antibody to the Mouse-TrKA was evaluated at the cellular level.

The results are shown in FIG. 7. In FIG. 7, EC ₅₀ The (half-maximal binding concentration) value reflects the binding capacity of the antibody to Mouse-TrKA; EC (EC) ₅₀ The smaller the value, the stronger the binding capacity of the antibody to Mouse-TrKA and the higher the affinity of the antibody. It is generally believed that EC of high affinity antibodies ₅₀ The value was below 1.5. Mu.g/mL. The results in FIG. 7 show that the specific anti-TrKA antibody binds to the Mouse-TrKA EC ₅₀ Is 0.1364 mug/mL. The specific anti-TrKA antibody can be specifically combined with the Mouse-TrKA receptor extracellular region, and has good combination capability; in addition, the DNPFE is found to be in 283-287 positions of the Mouse-TrKA receptor sequence through sequence analysis; thus, DNPFE is a common epitope of the Human-TrKA, mouse-TrKA receptor.

Example 9 detection of binding Capacity of specific anti-TrKA antibodies to the extracellular region of Rat-TrKA receptor Using ELISA method

In this example, the binding of specific anti-TrKA antibodies to the extracellular domain of the Rat-TrKA receptor was evaluated by ELISA methods at various concentrations (20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.313. Mu.g/mL, 0.156. Mu.g/mL, 0.078. Mu.g/mL, 0.039. Mu.g/mL, 0.019. Mu.g/mL).

The results are shown in FIG. 8. In FIG. 8, OD at a constant antibody concentration ₄₅₀ The value reflects the strength of binding of the antibody to the protein, the greater the reading the greater the binding of the antibody to the protein. Experimental results show that the concentration of specific anti-TrKA antibody is increased from 0 mug/mL to 20 mug/mL and OD ₄₅₀ The values gradually increased until approaching stability, approaching around 3, with an EC50 of 0.008397 μg/mL. This shows that the specific anti-TrKA antibody can specifically bind with the extracellular region of the Rat-TrKA receptor and has good binding capacity; in addition, the DNPFE is found to be in 283-287 positions of the Rat-TrKA receptor sequence through sequence analysis; thus, DNPFE is a common epitope of the Human-TrKA, mouse-TrKA, rat-TrKA receptors. In conclusion, DNPFE is a dominant epitope of the Human-TrKA receptor to which an anti-TrKA antibody specifically binds has cross-reactivity with Human, rat, and mouse species.

Example 10 detection of binding Capacity of specific anti-TrKA antibodies to TrKB, trKC, P75 receptor Using ELISA method

The TrkA receptor family belongs to Receptor Tyrosine Kinases (RTKs), including TrkA, trkB and TrkC, which have high homology. TrkA is a receptor tyrosine kinase for Nerve Growth Factor (NGF), selectively binds NGF, and is a functional receptor for NGF. In addition to the high affinity receptor TrkA, NGF can also bind to its low affinity receptor p 75.

In this example, the binding of specific anti-TrKA antibodies to Human-TrKB, human-TrKC, human-P75 was evaluated by ELISA method at different concentrations (20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.313. Mu.g/mL, 0.156. Mu.g/mL, 0.078. Mu.g/mL, 0.039. Mu.g/mL, 0.019. Mu.g/mL), respectively.

The results are shown in FIG. 9. In FIG. 9, OD at a constant antibody concentration ₄₅₀ The value reflects the strength of binding of the antibody to the protein, the greater the reading the greater the binding of the antibody to the protein. Experimental results show that specific anti-TrKA antibodies and Human-TrKB, human-TrKC and Human-P75Substantially unbound (increasing the concentration of each test antibody from 0. Mu.g/mL to 20. Mu.g/mL, OD) ₄₅₀ The values did not change substantially, approaching 0). It can be seen that the specific anti-TrKA antibody has good specificity and has no non-specific binding with Human-TrKB, human-TrKC and Human-P75; this shows that DNPFE is a dominant epitope region of the Human-TrKA receptor, and that the anti-TrKA antibody specifically bound to DNPFE has good specificity.

Example 11 preparation and identification of Cynomomolgus-TrKA protein

In the HEK293 expression system, cynomomolgus-TrKA protein (Cynomomolgus-TrKA extracellular region amino acid sequence is shown as SEQ ID NO: 22) is expressed recombinantly, and a protein sample obtained by a nickel column is subjected to SDS-PAGE identification, and an experimental result is shown as figure 10.

Wherein, the full-length amino acid sequence of Cynomomolgus-NTRK 1 is shown in SEQ ID NO: 23.

Example 12 evaluation of the affinity of test antibodies to Cynomolgus-TrKA protein Using the Fortebio method

Cynomomolgus-TrKA Protein samples were diluted to 6 concentration gradients (31.5 nM, 15.7nM, 7.87nM, 3.93nM, 1.97nM, 0.9833 nM) with buffer (100 ml PBS buffer, 0.1gBSA was added and stirred until they were well dissolved, and 20. Mu.L Tween 20 was added, and the binding to the concentration gradient Cynomomolgus-TrKA Protein was automatically detected after binding to the test antibodies by an affinity detection SYSTEM (OCTET RED 96 SYSTEM) using a Protein G sensor (manufacturer: PALL, cat# 18-5082), as shown in FIG. 11 and Table 1.KD is the equilibrium dissociation constant between an antibody and its antigen, inversely proportional to affinity. High affinity antibodies are generally considered to be in the low nanomolar range (10 ^-9 ). The results in table 1 show that the KD values of the test antibodies lie in the low nanomolar range (10 ^-9 ) The test antibody has high affinity with Cynomomolgus-TrKA protein.

Wherein, the light chain nucleotide sequence of the coded test antibody, namely the anti-TrKA monoclonal antibody is shown as SEQ ID NO:19, the heavy chain nucleotide sequence is shown as SEQ ID NO: shown at 20.

TABLE 1

Sample ID

KD(M)

kon(1/Ms)

kon Error

kdis(1/s)

kdis Error

Full R 2

Cynomolgus-TrKA

1.23E-09

4.68E+05

7.33E+03

5.76E-04

5.27E-06

0.999216

Example 13 detection of binding Capacity of test antibodies to Polypeptides Using ELISA method

The test antibodies were diluted to 6 concentration gradients (100. Mu.g/mL, 33.3. Mu.g/mL, 11.11. Mu.g/mL, 3.70. Mu.g/mL, 1.23. Mu.g/mL, 0.41. Mu.g/mL) with PBS buffer, the binding of the test antibodies to the polypeptide DNPFE (shown as SEQ ID NO: 4) was examined by ELISA, and the binding capacity of the test antibodies to the polypeptide DNPFE (shown as SEQ ID NO: 4) was evaluated, as a result, see FIG. 12, in which OD450 values reflect the binding strength of the antibodies to the protein at a given antibody concentration, and the binding strength of the antibodies to the protein was increased as the reading value was increased. The experimental result shows that the concentration of the tested antibody is increased from 0 mug/mL to 100 mug/mL, the OD450 value is gradually increased, and the maximum value is about 3.5. It can be seen that the test antibody has good binding ability with the polypeptide DNPFE (shown in SEQ ID NO: 4).

Example 14 detection of binding Capacity of test antibodies to Polypeptides Using ELISA method

The test antibodies were diluted to 6 concentration gradients (100. Mu.g/mL, 33.3. Mu.g/mL, 11.11. Mu.g/mL, 3.70. Mu.g/mL, 1.23. Mu.g/mL, 0.41. Mu.g/mL) with PBS buffer, the binding of the test antibodies to polypeptide MAAFMDNPFEFNPED (shown as SEQ ID NO: 18) was examined by ELISA method, and the binding capacity of the test antibodies to the polypeptide was evaluated, as a result, see FIG. 13, in FIG. 13, the OD450 value reflects the binding strength of the antibodies to the protein at a certain antibody concentration, and the binding strength of the antibodies to the protein was increased as the reading value was increased. The experimental result shows that the concentration of the tested antibody is increased from 0 mug/mL to 100 mug/mL, the OD450 value is gradually increased, and the maximum value is about 3.5. It can be seen that the test antibody has good binding ability to polypeptide MAAFMDNPFEFNPED (shown in SEQ ID NO: 18).

EXAMPLE 15 detection of polypeptide immunized mice serum Anti-TrKA antibody titres Using ELISA method

Mice were immunized with polypeptide MAAFMDNPFEFNPED-C-KLH 50 μg each, 1 week apart from the first and second, 2 weeks apart from the third and second, 1 week post third immunization, blood was collected and serum was isolated by centrifugation. The immune serum and the blank serum are respectively diluted by PBS buffer solutions according to the proportion of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400 by taking the blank serum as a control, the combination condition of the serum with different dilution proportions and the human TrKA protein (manufacturer: sino Biological, product number: 11073-H08H) is detected by an ELISA method, and the antibody titer of polypeptide MAAFMDNPFEFNPED (shown as SEQ ID NO: 18) immune mouse serum Anti-TrKA is detected, and the result is shown in Table 2. As can be seen, the antibody titer of polypeptide MAAFMDNPFEFNPED (shown as SEQ ID NO: 18) immunized mouse serum is 1:3200; experimental results show that polypeptide MAAFMDNPFEFNPED (shown as SEQ ID NO: 18) has certain immunogenicity and can prepare Anti-TrKA antibodies.

TABLE 2

EXAMPLE 16 detection of polypeptide immunized mouse serum Anti-TrKA antibody titres Using ELISA method

Mice were immunized with the polypeptide DNPFE-C-KLH 50. Mu.g each time, 1 week apart from the first and second, 2 weeks apart from the third and second, 1 week post third immunization, blood was taken and serum was isolated by centrifugation. The immune serum and the blank serum are respectively diluted by PBS buffer solutions according to the proportion of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400 by taking the blank serum as a control, the combination condition of the serum with different dilution proportions and the Human-TrKA protein (manufacturer: sino Biological, product number: 11073-H08H) is detected by an ELISA method, and the Anti-TrKA antibody titer of the immune mouse serum of the polypeptide DNPFE (shown as SEQ ID NO: 4) is detected, and the result is shown in Table 3. As can be seen, the titer of the antibody of the Anti-TrKA antibody in the serum of the immunized mice of the polypeptide DNPFE (shown as SEQ ID NO: 4) is 1:800; experimental results show that the polypeptide DNPFE (shown as SEQ ID NO: 4) has certain immunogenicity and can prepare Anti-TrKA antibodies.

TABLE 3 Table 3

EXAMPLE 17 detection of polypeptide immunized mouse serum Anti-TrKA antibody titres Using ELISA method

Mice were immunized with the polypeptide DNPFE-C-KLH 50 μg each time, 1 week apart from the first and second times, 2 weeks apart from the third and second times, 2 weeks apart from the fourth and third times, blood was taken 5 days after the fourth immunization, and serum was isolated by centrifugation. The immune serum and the blank serum are respectively diluted by PBS buffer solutions according to the proportion of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400 by taking the blank serum as a control, the combination condition of the serum with different dilution proportions and the Human-TrKA protein (manufacturer: sino Biological, product number: 11073-H08H) is detected by an ELISA method, and the Anti-TrKA antibody titer of the immune mouse serum of the polypeptide DNPFE (shown as SEQ ID NO: 4) is detected, and the result is shown in Table 4. As can be seen, the titer of the antibody of the Anti-TrKA antibody in the serum of the immunized mice of the polypeptide DNPFE (shown as SEQ ID NO: 4) is 1:1600; experimental results show that the polypeptide DNPFE (shown as SEQ ID NO: 4) has certain immunogenicity and can prepare Anti-TrKA antibodies.

TABLE 4 Table 4

EXAMPLE 18 detection of binding Capacity of polypeptide immunized mouse serum to TrKB, trKC, P receptor by ELISA method

Mice were immunized with polypeptide MAAFMDNPFEFNPED-C-KLH 50 μg each, 1 week apart from the first and second, 2 weeks apart from the third and second, 1 week post third immunization, blood was collected and serum was isolated by centrifugation. Mice were immunized with the polypeptide DNPFE-C-KLH 50 μg each time, 1 week apart from the first and second times, 2 weeks apart from the third and second times, 2 weeks apart from the fourth and third times, blood was taken 5 days after the fourth immunization, and serum was isolated by centrifugation. The immune serum and the blank serum are respectively diluted by PBS buffer solutions according to the proportion of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200 and 1:6400, and the binding condition of the serum with different dilution proportions and Human-TrKB (manufacturer: sino Biological, product number: 10047-H08H), human-TrKC (manufacturer: sino Biological, product number: 10048-H08H) and Human-P75 (manufacturer: sino Biological, product number: 13184-H08H) is detected by ELISA method, and the results are shown in Table 5, table 6 and Table 7. From Table 5, table 6, table 7, it can be seen that the polypeptide MAAFMDNPFEFNPED-C-KLH and DNPFE-C-KLH immunized mouse serum did not significantly bind to Human-TrKB, human-TrKC, human-P75; experimental results show that antibodies prepared after mice are immunized by the polypeptides MAAFMDNPFEFNPED-C-KLH and DNPFE-C-KLH can not be combined with Human-TrKB, human-TrKC and Human-P75 in a non-specific mode, and have good specificity.

TABLE 5

TABLE 6

TABLE 7

EXAMPLE 19 detection of blocking of Human-TrKA receptor binding to its ligand Human-NGF by polypeptide immunized rabbit serum Using ELISA method

Rabbits were immunized with polypeptide MAAFMDNPFEFNPED-C-KLH 300 μg each, 1 week apart from the first and second, 2 weeks apart from the second and third, 2 weeks apart from the third and fourth, 6 days after immunization, blood was taken and serum was centrifuged. The control was blank serum, the immune serum was diluted with PBS buffer at 1:2, 1:4, 1:8, 1:16, and the inhibition of the binding of Human-NGF and Human-TrKA by the immune serum at different dilution ratios was examined by ELISA, and the experimental results are shown in FIG. 14. In FIG. 14, the OD450 value reflects the intensity of binding of Human-TrkA to Human-NGF, and the lower the reading value, the weaker the binding of Human-TrkA to Human-NGF, and the greater the effect of the antibody in inhibiting the binding of Human-NGF to Human-TrKA; as shown in fig. 14, the signal of binding of Human-TrkA to Human-NGF was significantly reduced with immune serum and 1:2 diluted immune serum compared to the empty serogroup. It can be seen that immunization of rabbits with the polypeptide MAAFMDNPFEFNPED-C-KLH produced specific antibodies which inhibited the binding of the Human-TrKA receptor to its ligand Human-NGF.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (11)

1. An epitope peptide of TrkA comprising SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).

2. The epitope peptide of claim 1, wherein the amino acid sequence of the epitope peptide of TrkA is:

SEQ ID NO:4, a step of; or alternatively

The sequence represented by SEQ ID NO:4 and is based on the amino acid sequence of the TrkA extracellular region to SEQ ID NO:4, or a polypeptide having both amino-terminal, carboxy-terminal or both amino-terminal and carboxy-terminal extensions;

preferably, the amino acid sequence of the TrkA extracellular region is SEQ ID NO: 1. SEQ ID NO: 2. or SEQ ID NO:3.

3. the epitope peptide of claim 2, wherein said peptide is represented by SEQ ID NO:4, or the number of amino acids extending from the amino terminus, the carboxy terminus, or both the amino terminus and the carboxy terminus is each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

4. The epitope peptide of claim 2 or 3, wherein the amino acid sequence of the epitope peptide of TrkA is SEQ ID NO: 5-18.

5. A fusion protein formed by fusing the epitope peptide of any one of claims 1-4 with a carrier protein.

6. The fusion protein of claim 5, wherein the carrier protein is selected from one of keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, thyroglobulin, and human serum albumin.

7. A nucleic acid molecule encoding the epitope peptide of any one of claims 1-4 or the fusion protein of claim 5 or 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell expressing the epitope peptide of any one of claims 1-4, and/or expressing the fusion protein of claim 5 or 6, and/or comprising the nucleic acid molecule of claim 7, and/or comprising the vector of claim 8.

10. A composition comprising the epitope peptide of any one of claims 1-4, and/or the fusion protein of claim 5 or 6, and/or the nucleic acid molecule of claim 7, and/or the vector of claim 8, and/or the host cell of claim 9.

11. Use of the epitope peptide of any one of claims 1-4, and/or the fusion protein of claim 5 or 6, and/or the nucleic acid molecule of claim 7, and/or the vector of claim 8, and/or the host cell of claim 9, and/or the composition of claim 10, in the following (a), (b), (c), (d), or (e):

(a) For the preparation of anti-TrkA antibodies;

(b) For preparing aptamer, vaccine, nanoparticle for TrkA;

(c) For preparing a medicament for preventing and/or treating diseases related to TrkA;

(d) For the preparation of a medicament for inhibiting or attenuating the signalling involved in TrkA;

(e) Can be used for preparing analgesic.