Specific antibody and application thereof

Abstract

The invention provides a specific antibody and application thereof. Specifically, the antibody has a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 8. The antibody provided by the embodiment of the invention can be specifically combined with a TrkA receptor in a targeting manner, and is used for treating, preventing or relieving xerophthalmia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular, the present invention relates to the use of anti-TrKA antibodies or antibody fragments in treating, preventing or alleviating dry eye. Background Art

[0002] With the advancement of science and technology, there are more and more types of electronic products. Electronic products are not only used as entertainment tools, but also as indispensable tools for work, study and life. Therefore, the time of using electronic devices is getting longer and longer, and the time of using eyes is getting longer and longer. Long-term use of electronic products can easily cause eye fatigue, soreness and dryness, leading to the occurrence of dry eyes. Dry eyes can be called keratoconjunctivitis sicca (KCS) or dry eyes. The instability of tear film and damage to the ocular surface caused by abnormal quantity or quality of tears and fluid dynamics lead to eye discomfort symptoms and visual dysfunction. Long-term development can cause corneal epithelial damage. Common symptoms are dry eyes, foreign body sensation, and dry eyes cause syndrome. The patient's conjunctival goblet cell density is reduced, the nuclear-cytoplasmic ratio is increased, the epithelial cells are squamous, the corneal epithelium is conjunctivalized, and damaged, and the cornea and conjunctiva are urgently needed. At present, the treatment of dry eyes mostly adopts palliative therapy, focusing on the use of artificial tears to alleviate symptoms. Although these palliative therapies can benefit patients in the short term, they have limited effectiveness in the long-term control of dry eye. Cyclosporine is another type of drug commonly used in the treatment of dry eye in clinical practice. However, they also have the disadvantages of a long onset of action and are prone to causing local discomfort. Dry eye is now becoming younger, that is, due to the use of computers and mobile phones among young people, a large number of actual clinical needs have been formed that require prevention or further drug intervention and clinical treatment. There is an urgent need for innovative drugs for the prevention and clinical treatment of dry eye.

[0003] Studies have shown that TrKA small molecule agonists can stimulate conjunctival goblet cells to secrete mucin and show therapeutic effects in a rat dry eye model.

[0004] However, there is no TrKA antibody or antigen-binding fragment in the prior art for treating, preventing or alleviating dry eye. Summary of the invention

[0005] In order to solve this problem, on the one hand, the present invention provides a use of an antibody or an antigen-binding fragment thereof capable of specifically recognizing TrkA in the preparation of a drug for treating dry eye, wherein the antibody comprises a light chain variable region,

[0006] It comprises the VL-CDR1 shown in SEQ ID NO: 1, the VL-CDR2 shown in SEQ ID NO: 2, and the VL-CDR3 shown in SEQ ID NO: 3; and

[0007] Heavy chain variable region,

[0008] It comprises VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5 and VH-CDR3 shown in SEQ ID NO:6.

[0009] KASENVGGYVS (SEQ ID NO: 1).

[0010] GASSRHT (SEQ ID NO: 2).

[0011] GQNYIYPFT (SEQ ID NO: 3).

[0012] NYWLG (SEQ ID NO:4).

[0013] DFYPRTGNTFYNENFKG (SEQ ID NO: 5).

[0014] AGTGFDY (SEQ ID NO: 6).

[0015] SEQ ID NOs: 1 to 6 are defined according to the Kabat numbering scheme.

[0016] According to an embodiment of the present invention, the antibody contains at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0017] According to an embodiment of the present invention, the antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0018] EIVMTQSPATLSLSVGERVTLSCKASENVGGYVSWYQQKPDQSPKLLIYGASSRHTGV PDRFTGSGSETDFTLTISSVQAEDLAAYHCGQNYIYPFTFGGGTKLEIK (SEQ ID NO: 7).

[0019] According to an embodiment of the present invention, the antibody heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0020] EVQLLESGGGLVQPGGSLKLSCKASGYAFTNYWLGWMKQRPGHGLEWIGDFYPRTG NTFYNENFKGKVTLTADKSSNTAYMQLSSLTSEDSAVYLCARAGTGFDYWGQGTTLTVSS (SEQ ID NO: 8).

[0021] According to an embodiment of the present invention, the antibody contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

[0022] Preferably, the light chain constant region and heavy chain constant region of the antibody are both derived from human IgG antibody or a mutant thereof.

[0023] Preferably, the light chain constant region and heavy chain constant region of the antibody are both derived from human IgG4.

[0024] Preferably, the full-length sequence of the antibody constant region is as shown in SEQ ID NO: 9 or 10.

[0025] Specifically, the antibody or antigen-binding fragment also includes the full-length light chain constant region shown in SEQ ID NO:9.

[0026] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9).

[0027] Specifically, the antibody or antigen-binding fragment also includes the full-length heavy chain constant region shown in SEQ ID NO:10.

[0028] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:10).

[0029] According to an embodiment of the present invention, the antibody light chain has the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:11.

[0030] EIVMTQSPATLSLSVGERVTLSCKASENVGGYVSWYQQKPDQSPKLLIYGASSRHTGVPDRFTGSGSETDFTLTISSVQAEDLAAYHCGQNYIYPFTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:11).

[0031] According to an embodiment of the present invention, the antibody heavy chain has the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12.

[0032] EVQLLESGGGLVQPGGSLKLSCKASGYAFTNYWLGWMKQRPGHGLEWIGDFYPRTGNTFYNENFKGKVTLTADKSSNTAYMQLSSSLTSEDSAVYLCARAGTGFDYWGQGTT LTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ IDNO:12).

[0033] Furthermore, the antibody or antigen-binding fragment also includes a heavy chain constant region CH1 region.

[0034] Specifically, the heavy chain constant region described in the present invention is the CH1 region (such as the amino acid sequence shown in SEQ ID NO: 13); the full-length heavy chain constant region (or the heavy chain full-length constant region) includes the heavy chain constant region (CH1 region) and the Fc region; wherein the Fc region includes the hinge region, the CH2 region and the CH3 region.

[0035] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV (SEQ ID NO: 13).

[0036] Specifically, the Fc portion has the amino acid sequence shown in SEQ ID NO:14, 15, 16, 17 or 18.

[0037] Furthermore, the Fc portion has an amino acid sequence that has at least 80%, 85%, 90%, 95%, 99% sequence identity with the sequence shown in SEQ ID NO:14, 15, 16, 17 or 18.

[0038] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCSVMHEALHNRFTQKSLDRSPGK(SEQ ID NO:14)。

[0039] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPFRPEVHLLPPSREEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISLSPGK(SEQ ID NO:15)。

[0040] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:16)。

[0041] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:17).

[0042] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSLG(SEQ ID NO:18).

[0043] The Fc mutant is obtained by replacing, deleting or adding one or more amino acids to the Fc wild type, and the obtained Fc mutant still retains the biological properties of the Fc wild type.

[0044] Among them, the mutation sites of the Fc part include at least S228, F234, L235, R409 and K447.

[0045] Furthermore, the mutation sites of the Fc part include at least S228, F234, L235, S354, T366, R409 and K447.

[0046] Specifically, the mutation sites of the Fc part include at least S228P, F234A, L235A, S354C, T366W, R409K and K447 deletion.

[0047] Furthermore, the mutation sites of the Fc portion include at least S228, F234, L235, Y349, T366, L368, Y407, R409, H435, Y436 and K447.

[0048] Specifically, the mutation sites of the Fc part include at least S228P, F234A, L235A, Y349C, T366S, L368A, Y407V, R409K, H435R, Y436F and K447 deletion.

[0049] The numbering of mutation sites is defined according to the EU index.

[0050] According to an embodiment of the present invention, the antibody is a single-chain antibody, a multimeric antibody or a CDR-grafted antibody.

[0051] According to an embodiment of the present invention, the single-chain antibody (scFv) includes a light chain variable region of an amino acid sequence shown in SEQ ID NO: 7 and a heavy chain variable region of an amino acid sequence shown in SEQ ID NO: 8, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the light chain variable region through a connecting peptide linker, or the C-terminus of the light chain variable region is connected to the N-terminus of the heavy chain variable region through a connecting peptide linker. It should be noted that the "connecting peptide linker" of the single-chain antibody described in the present application is a connecting peptide for connecting the heavy chain variable region and the light chain variable region of the antibody, which can be a commonly used connecting peptide linker for preparing a single-chain antibody, or a connecting peptide linker modified by scientific researchers. In certain embodiments, the connecting peptide can be a G-rich polypeptide, for example, it can be selected from (G) 3-S (ie, "GGGS"), (G) 4-S (ie, "GGGGS") and (G) 5-S (ie, "GGGGGS"), such as GGGGSGGGGSGGGGS.

[0052] GGGS (SEQ ID NO: 19).

[0053] GGGGS (SEQ ID NO: 20).

[0054] GGGGGS (SEQ ID NO: 21).

[0055] GGGGSGGGGSGGGGS (SEQ ID NO:22).

[0056] Furthermore, the antibody or antigen-binding fragment also includes a heavy chain variable region and a light chain variable region connected in series; the heavy chain variable region and the light chain variable region are connected by a linker; and the linker is selected from the amino acid sequence shown in SEQ ID NO:22.

[0057] According to an embodiment of the present invention, the antigen binding fragment includes Fab, Fab', F(ab)2, F(ab')2, Fv, scFv-Fc fusion protein or scFv-Fv fusion protein.

[0058] According to an embodiment of the present invention, the present invention provides a nucleic acid molecule; the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described in the present invention; according to an embodiment of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can specifically target and bind to the TrkA receptor, blocking the binding of NGF and TrkA.

[0059] Furthermore, the nucleic acid molecule is DNA.

[0060] According to an embodiment of the present invention, the nucleic acid molecule has a nucleotide sequence described in SEQ ID NO:23 or SEQ ID NO:24.

[0061] GAGATCGTGATGACCCAGTCCCCAGCCACACTGAGCCTGTCTGTGGGAGAGAGGGTGACCCTGTCTTG CAAGGCTTCCGAGAACGTGGGCGGCTACGTGAGCTGGTATCAGCAGAAGCCCGACCAGTCTCCTAAGCTGCTGATC TACGGAGCCTCCAGCAGGCACACAGGAGTGCCAGACCGGTTCACCGGATCCGGAAGCGAGACAGACTTCACCCTGA CAATCTCTTCCGTGCAGGCTGAGGATCTGGCCGCTTATCATTGTGGCCAGAATTACATCTATCCCTTCACCTTTGG CGGCGGCACAAAGCTGGAGATCAAG CGGACCGTGGCTGCCCTCTCCGTGTTCATCTTCCCCCCTCCGACGAGCAGCTGAAGTCCGGCACCGCTAGCGTGGTGTGTTTACTGAACAACTTCTACCCTCGTGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCTTTACAGTCCGGCAACTCCCAAGAA TCCGTGACCGAGCAAGATTCCAAGGACTCCACCTACTCTTTATCCTCCACTTTAACTTTATCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCTTGTGAGGTGACCCATCAAGGTTTATCCTCCCCGTGACCAAGTCCTTCAATCGTGGCGAGTGC(SEQ ID NO:23).

[0062] GAGGTGCAGCTGCTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCTCTCTGAAGCTGTCCTGCAA GGCCAGCGGCTACGCTTTCACCAACTATTGGCTGGGATGGATGAAGCAGAGGCCAGGACACGGACTGGAGTGGATC GGCGACTTTTACCCTCGGACCGGCAACACATTCTATAACGAGAACTTCAAGGGCAAGGTGACCCTGACAGCCGATA AGTCCAGCAATACCGCTTACATGCAGCTGTCTTCCCTGACATCCGAGGACTCCGCCGTGTACCTGTGCGCTAGGGC TGGAACCGGATTCGATTATTGGGGCCAGGGCACCACACTGACAGTGAGCTCTGCCAGCACCAAGGGCCCCAGCGTGTTCCCTCTGGCTCCTTGTAGCCGGTCCACCTCCGAGTCCACAGCTGCTCTGGGCTGCCTCGTGAAGGACTACTTTCCCGAACCCGTTACCGTGAGCTGGAATAGCGGCGCTTTAACCTCCGGAGTGCACACCTTCCCCGCTGTGCTCCAGTCCTCCGGTTTATACTCTTTATCCTCCGTGGTGACCGTGCCTTCCTCCAGCCTCGGCACCAAGACCTACACTTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGTCCAAGTACGGACCTCCTTGTCCCCCTTGCCCCGCCCCCGAGGCCGCTGGCGGACCCTCCGTGTTCCTCTTCCCCCCCAAACCCAAGGACACTTTAATGATCTCCCGGACCCCCGAAGTGACTTGTGTGGTGGTGGACGTGTCCCAAGAAGACCCCGAGGTGCAGTTTAACTGGTACGTGGATGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTAGGGAGGAACAGTTCAACTCCACCTACCGGGTGGTGTCCGTGCTCACCGTGCTGCATCAAGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGACTGCCCAGCTCCATCGAGAAGACCATCAGCAAGGCCAAAGGCCAGCCCCGGGAACCTCAAGTTTATACACTGCCCCCCAGCCAAGAAGAGATGACCAAGAACCAAGTTTCTTTAACTTGTTTAGTGAAGGGCTTCTACCCTAGCGACATCGCTGTGGAGTGGGAGTCCAATGGCCAGCCCGAAAACAATTATAAGACCACCCCCCCCGTGCTGGACTCCGATGGTTCTTTTTTTTTATACTCCAAGCTGACAGTGGACAAGTCTCGTTGGCAAGAAGGCAACGTGTTCTCTTGTAGCGTGATGCACGAGGCTTTACACAACCACTACACCCAGAAGTCTTTATCTCTGTCTTTAGGC(SEQ ID NO:24)。

[0063] Among them, the nucleotide sequences shown in the above SEQ ID NOs: 23 to 24 respectively encode the light chain and heavy chain described in the present invention, wherein the underlined parts respectively encode the light chain variable region and heavy chain variable region described in the present invention.

[0064] Among them, DYG047 consists of a light chain with an amino acid sequence shown in SEQ ID NO:11 and a heavy chain with an amino acid sequence shown in SEQ ID NO:12.

[0065] Among them, the nucleotide sequence shown in SEQ ID NO:23 encodes the light chain of the DYG047 test antibody; the nucleotide sequence shown in SEQ ID NO:24 encodes the heavy chain of the DYG047 test antibody.

[0066] According to an embodiment of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. After the expression vector according to an embodiment of the present invention is introduced into a suitable recipient cell, the aforementioned antibody or antigen-binding fragment thereof that specifically recognizes TrkA can be effectively expressed under the mediation of a regulatory system, thereby achieving a large amount of in vitro acquisition of the antibody or antigen-binding fragment.

[0067] According to an embodiment of the present invention, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically recognize TrkA, a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof of the present invention, or an expression vector carrying the nucleic acid molecule in the preparation of a drug for treating, preventing or alleviating dry eye.

[0068] According to an embodiment of the present invention, the drug is used to increase tear secretion and relieve dry eye symptoms.

[0069] The present invention provides an anti-TrKA antibody or antigen-binding fragment that can upregulate the phosphorylation level of ERK by activating the downstream MAPK signaling pathway of TrKA, thereby:

[0070] 1) Stimulate the secretion of conjunctival mucin and stabilize the tear film.

[0071] 2) Improve corneal healing and possibly reduce ocular surface damage,

[0072] 3) Supporting neuronal survival and differentiation and potentially improving sensory responses, thereby treating, preventing or alleviating dry eye disease,

[0073] Anti-TrKA antibodies or antigen-binding fragments can promote the secretion of mucin and improve corneal healing and sensory response in the treatment, prevention or relief of dry eye.

[0074] Details of one or more embodiments of the present invention are set forth in the following description. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of several embodiments and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is a graph showing the results of evaluating the in vitro activity of a test compound using the NIH-3T3-TrKA cell model;

[0076] Figure 2 It is a graph showing the results of evaluating the pharmacodynamic effect of a test antibody using a mouse dry eye model induced by benzalkonium chloride;

[0077] Figure 3 It is a graph showing the results of evaluating the pharmacodynamic effect of the left eye of a test antibody using a mouse dry eye model induced by scopolamine;

[0078] Figure 4 It is a graph showing the results of evaluating the pharmacodynamic effect of the right eye of a test antibody using a mouse dry eye model induced by scopolamine. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present disclosure.

[0080] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0081] The "plural" in the "one or more" generally refers to more than 1. "One or more" may include, for example, 1 to 20, 1 to 10, 1 to 5. Specifically, "one or more" may be 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0082] The term "antibody" refers to a class of proteins or polypeptide sequences derived from immunoglobulin molecules that can specifically bind to an antigen. Antibodies can be monoclonal antibodies or polyclonal antibodies, can be single-chain or multi-chain or complete immunoglobulin molecules, and can be of natural origin or from recombinant technology. The antibodies include conventional antibodies, single-chain antibodies (scFv), multimeric antibodies, and CDR-grafted antibodies. The antibodies also include single-domain antibodies (including monovalent nanobodies (VHH), bivalent nanobodies (two VHHs connected by a linker), and multivalent nanobodies (multiple VHHs connected by a linker)).

[0083] The terms "conventional antibody" and "complete antibody" are used interchangeably herein to refer to antibodies that are substantially similar to natural antibody structures. "Natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH) and three constant domains (CH1, CH2 and CH3). From N-terminal to C-terminal, each light chain has a variable region (VL) and a light chain constant domain (CL). The heavy chain of an antibody can be one of five types, and the five types are α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), and can also be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody can be one of two types, kappa light chains and lambda light chains, based on the amino acid sequence of its constant domain.

[0084] The term "antigen-binding fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen. Examples of antibody fragments include: Fab, Fab', F(ab')2, Fv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, Fd fragments consisting of VH and CH1 domains, linear antibodies, and the like.

[0085] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous - for example, via synthetic coupling - and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0086] In the homodimer of two peptide chains, the heterodimer of two peptide chains, the heterotrimer of three peptide chains, and the heterotetramer of two peptide chains described in the present invention, the peptide chains are bonded to each other via disulfide bonds.

[0087] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, which has been incorporated into the ALIGN program (version 2.0). In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm, using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6, which algorithm has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com).

[0088] The term "variable region" or "variable domain" as used herein refers to the domain of the antibody heavy chain or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0089] The term "variable" as used herein refers to certain segments of the variable domain that are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light chain and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a β-folded configuration, connected by three HVRs, which form a loop connection, and in some cases form a part of the β-folded structure. The HVRs in each chain are closely held together by the FR region, and together with the HVRs of other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, and has other effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

[0090] As used herein, "at least 80% sequence identity" to a sequence can include at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence.

[0091] Example 1 Antibody Preparation

[0092] The DYG047 antibody sequence (the light chain sequence is SEQ ID NO: 23, the heavy chain sequence is SEQ ID NO: 24) was synthesized using the whole gene, constructed into the pcDNA3.1 vector, transiently transfected into HEK293 cells and expressed in the cells, and purified by Protein A medium to obtain an antibody with a purity of more than 95% (the light chain sequence is SEQ ID NO: 11, the heavy chain sequence is SEQ ID NO: 12).

[0093] Or refer to Examples 2 to 5 in Chinese patent application CN114516917A for preparation and detection.

[0094] Example 2 Evaluation of in vitro activity of test compounds using NIH-3T3-TrKA cell model

[0095] Under NGF stimulation, the phosphorylation level of TrKA receptor Y490 on the NIH-3T3-TrKA cell membrane is upregulated, the downstream signaling pathway of TrKA is activated, and the phosphorylation level of ERK is upregulated. Similarly, the test antibody (mAb is the DYG047 test antibody) can bind to the TrKA receptor on the surface of the NIH-3T3-TrKA cell membrane, so that the phosphorylation level of TrKA receptor Y490 is upregulated, the downstream signaling pathway of TrKA is activated, and the phosphorylation level of ERK is upregulated. In the experiment, NGF was used as a positive control, and the HTRF method was used to detect the upregulation of ERK phosphorylation level under the action of the test antibody to measure the in vitro activity of the test antibody. The OD665 / OD616 in the figure reflects the phosphorylation level of ERK. The higher the OD665 / OD616 value, the higher the phosphorylation level of ERK. The results are shown below. Figure 1 .like Figure 1 As shown, the phosphorylation levels of ERK upregulated by the test antibody and NGF were comparable, indicating that the test antibody and NGF had comparable in vitro activities.

[0096] Example 3 Evaluation of the efficacy of the test antibody using the benzalkonium chloride-induced mouse dry eye model

[0097] Benzalkonium chloride eye drops can cause shortened tear film breakup time, ocular epithelial damage, corneal epithelial squamous metaplasia, goblet cell reduction and other changes in ocular surface structure and function in mice. It is a good method for establishing an animal model of dry eye.

[0098] 60 SPF female C57BL / 6 mice aged 6 to 8 weeks were selected. The mice were randomly divided into a normal control group (25 mice) and a dry eye model group (35 mice). The mice in the control group were given normal saline, and 3 μL of 0.2% benzalkonium chloride solution was dripped into each eye of the mice in the dry eye model group twice a day for 28 days to induce a dry eye model in mice. The tear secretion amount was measured by detecting the tear secretion test strip, and the animals were grouped into a blank group (normal saline + solvent), a model group (benzalkonium chloride + solvent), and a drug-treated group (benzalkonium chloride + DYG047). Normal saline and benzalkonium chloride solution were dripped into the eyes twice a day, and the vehicle and 800 μg / ml DYG047 dosage group were dripped into the eyes three times a day for two consecutive weeks. The results are as follows Figure 2 As shown, 28 days after modeling, the tear secretion of mice was significantly reduced (#p<0.05). On the 14th day after model animal administration, 800μg / ml DYG047 test antibody was able to significantly increase the tear secretion of dry eye mice (*p<0.05), indicating that the use of DYG047 may have pharmacological activity in alleviating dry eye.

[0099] Example 4 Evaluation of the efficacy of the test antibody using a scopolamine-induced mouse dry eye model

[0100] Scopolamine hydrobromide was used to block the competitive inhibition of muscarinic acetylcholine receptors in the lacrimal glands, thereby causing a decrease in tear secretion and establishing an animal model of dry eye with insufficient tear secretion, which is more consistent with the pathophysiology of common dry eye patients in clinical practice. The mice were randomly divided into a normal control group, a scopolamine modeling agent + PBS group, a scopolamine modeling agent + 800μg / ml DYG047 group, and a scopolamine modeling agent + 800μg / ml FGF21 group. Except for the normal control group, the other groups were injected with 100μl of 5mg / ml scopolamine hydrobromide solution subcutaneously behind the bilateral ears of each mouse every day, and the corresponding drugs were given every day. On the 0th, 7th, 14th, and 28th days of establishing the dry eye model, phenol red cotton thread was used to detect the basal tear secretion of the mice. The results are shown in Figures 3-4 As shown: After the 7th day of induction, the basal tear secretion of the left and right eyes of the mice in the model group was significantly lower than that of the normal mice (**p<0.01). During the continuous treatment with FGF21 or DYG047, on the 7th, 14th, and 28th days of treatment, the tear secretion of the left eye of the mice in the 800μg / ml FGF21 or DYG047 dose group increased significantly (**p<0.01); on the 14th and 28th days of treatment, the tear secretion of the right eye of the mice in the 800μg / ml FGF21 or DYG047 dose group increased significantly (*p<0.05, **p<0.01). This suggests that DYG047 can significantly increase the tear secretion of dry eye mice and may have pharmacological activity to relieve dry eye syndrome.

[0101] Among them, the FGF21 described in Example 4 has the amino acid sequence shown in SEQ ID NO:25.

[0102] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSSLSLSLGGGGGSGGGGSGGGGSHPIPDSSPLLQFGGQVRQVYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRP DGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLHMVGASQGLSPSYAS(SEQ ID NO:25).

[0103] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0104] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of an antibody or an antigen-binding fragment thereof that can specifically recognize TrkA in the preparation of a drug for treating dry eye, characterized in that: The antibody comprises a light chain variable region comprising VL-CDR1 shown in SEQ ID NO: 1, VL-CDR2 shown in SEQ ID NO: 2, and VL-CDR3 shown in SEQ ID NO: 3; and The heavy chain variable region comprises VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5 and VH-CDR3 shown in SEQ ID NO:

6.

2. The use according to claim 1, characterized in that The antibody contains at least one of a heavy chain framework region sequence and a light chain framework region sequence, and at least a portion of at least one of the heavy chain framework region sequence and the light chain framework region sequence is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.

3. The use according to claim 1, characterized in that The antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

7.

4. The use according to claim 1, characterized in that The antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

8.

5. The use according to claim 1, characterized in that: The antibody contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof; Preferably, the light chain constant region and heavy chain constant region of the antibody are both derived from human IgG antibody or a mutant thereof; Preferably, the light chain constant region and heavy chain constant region of the antibody are both derived from human IgG4; Preferably, the full-length sequence of the antibody constant region is as shown in SEQ ID NO: 9 or 10.

6. The use according to claim 1, characterized in that: The antibody light chain has the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

11.

7. The use according to claim 1, characterized in that The antibody heavy chain has the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

12.

8. The use according to claim 1, characterized in that The antibody is a single-chain antibody, a polymeric antibody or a CDR-grafted antibody.

9. The use according to claim 1, characterized in that The antigen binding fragment is Fab, Fab', F(ab')2, Fv, scFv-Fc fusion protein or scFv-Fv fusion protein.

10. The use according to claim 1, characterized in that The drug is used to increase tear secretion and relieve dry eye symptoms.

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