TrkA specific binding protein as well as preparation and application thereof

Abstract

The invention relates to the technical field of biology, in particular to ATrkA specific binding protein capable of being specifically bound with human tropomyosin receptor kinase as well as preparation and application of the ATrkA specific binding protein. The invention provides a TrkA specific binding protein with NGF (nerve growth factor) binding TrkA blocking activity. Furthermore, compared with the traditional antibody, the TrkA specific binding protein provided by the invention has stronger affinity and specificity.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular, to a specific binding protein capable of specifically binding to human tropomyosin receptor kinase A, TrkA, and the preparation and application thereof. Background Art

[0002] Tropomyosin Receptor Kinase A (TrkA, also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptortype 1, or Trk1-transforming tyrosine kinase protein) is a member of the tropomyosin receptor kinase (Trk) family and is encoded by the NTrk1 (neurotrophic receptor tyrosine kinase 1) gene. TrkA selectively binds to nerve growth factor (Nerve Growth Factor, NGF) and is a high affinity receptor for NGF. NGF binding to TrkA can activate TrkA kinase activity, thereby causing the activation of multiple signaling pathways, including Ras / MAPK, PI3K / Akt and PLCγ pathways. In addition to TrkA, NGF also binds to the p75 common neurotrophin receptor (p75NTR, also known as the "low affinity" NGF receptor).

[0003] The NGF / TrkA signaling pathway is related to cell differentiation, proliferation, survival and pain. In injured and inflamed tissues, NGF is highly expressed, and TrkA activates nociceptive neurons through a variety of mechanisms and generates pain signals. In humans, NGF levels are elevated in patients with rheumatoid arthritis, interstitial cystitis, pancreatitis, prostatitis, diabetic neuropathy and cancer pain (Aloe et al. (1992) Arthritis and Rheumatism 35: 351-355; Mantyh PW et al. (2011) Anesthesiology 115 (1): 189-204). After NGF injection, healthy people can develop pain sensitivity and localized pain (Petty et al. (1994) Ann Neurol. 36: 244-246), suggesting that an increase in NGF levels is necessary for hyperalgesia. Homozygous missense mutations in the NGFβ gene can cause HSAN5 symptoms in humans, and these patients are insensitive to pain, cold, and heat (Larsson et al. (2009) Neurobiol Dis, 33: 221-228). Genetic studies on patients with congenital insensitivity to pain and anhidrosis (CIPA) have shown that mutations in the extracellular or intracellular regions of the TrkA gene (NTrk1) can cause CIPA, and in these patients, TrkA cannot be activated by NGF, resulting in the loss of pain perception (Mardy S et al. (2001) Human mutation 18: 462-471).

[0004] Pain is the most common and most troubling symptom in clinical practice, involving a variety of diseases such as tumors, osteoarthritis, rheumatoid arthritis, interstitial cystitis, pancreatitis, diabetes, etc. Currently, non-opioid analgesics, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are mainly used for mild to moderate pain; opioid analgesics are mainly used for moderate to severe pain. However, NSAIDs have a "ceiling effect". Opium can basically only relieve pain symptoms in less than 30% of patients with non-tumor chronic pain, and has no therapeutic effect on the disease itself. In addition, 20% of cancer pain patients have opioid resistance (Kalso E et al. (2004) Pain 112 (3): 372-80). In addition, NSAIDs have gastrointestinal and cardiovascular safety risks, which are particularly obvious during long-term medication. Years of drug improvement research have not been able to effectively reduce the addictiveness and many other side effects of opioid analgesics. This field is in urgent need of non-opioid analgesics that can relieve pain, are non-toxic, and prevent abuse. NGF / TrkA, as a new analgesic mechanism, provides a possible solution to this problem.

[0005] So far, there are several anti-human NGF antibodies in the research and development or clinical development stage. Clinical trials have shown that NGF antibodies have significant analgesic effects on joint pain associated with degenerative joint disease, chronic low back pain, and bladder pain associated with interstitial cystitis (Lane NE et al. (2010) N Engl J Med 363:1521 1531). On the other hand, clinical trials of several NGF antibodies have also shown that NGF antibodies increase the risk of accelerated progression of osteoarthritis in patients (Thomas JS et al. (2019) JAMA 322:37 48). They may be restricted for use in seriously ill patients, cannot be used for a long time, and have dosage restrictions, which requires further safety verification for the clinical application of NGF antibodies. Based on this, antibodies targeting TrkA are theoretically a better treatment option than NGF inhibitors. Because anti-TrkA antibodies can selectively target and bind to TrkA receptors, they can not only block the continuous activation of NGF on the NGF-TrkA signaling pathway, effectively relieving pain and hyperalgesia, but also will not block the binding of NGF to P75, effectively avoiding the impact on P75 and neuronal development, as well as the impact on osteoblast differentiation, proliferation, myoblast differentiation, muscle repair and other related normal physiological functions, and have safety advantages (Akiyama Y et al. (2014) Differentiation, 87: 11111; Deponti et al. (2009) Molecular Biology of the Cell, 20: 3620 3627; Mikami et al. (2012) Differentiation, 84: 392 399).

[0006] In summary, TrkA-blocking antibodies are urgently needed for pain treatment. Summary of the invention

[0007] One or more embodiments of the present application provide TrkA specific binding protein and its preparation and application, including the following technical solutions:

[0008] One or more embodiments of the present application provide a TrkA-specific binding protein having a TrkA binding domain, wherein the TrkA binding domain is selected from any one of the following TrkA binding domains 1 to 5:

[0009] In the TrkA binding domain 1, the sequence of CDR-H1 is GYSFT-Xaa1-YT, the sequence of CDR-H2 is Xaa2-NP-Xaa3-NGG-Xaa4, the sequence of CDR-H3 is A-Xaa5-SAYG-Xaa6-Xaa7-YWYFDV, the sequence of CDR-L1 is SSV-Xaa8-Y, the sequence of CDR-L2 is ATS, and the sequence of CDR-L The sequence of 3 is QQW-Xaa9-Xaa10-Xaa11-PPT; wherein Xaa1 is A or G; Xaa2 is I, F or L, Xaa3 is F or Y, Xaa4 is A or T, Xaa5 is I or G, Xaa6 is S or T, Xaa7 is D or N, Xaa8 is S or N, Xaa9 is S or T, Xaa10 is S or T, and Xaa11 is T or N;

[0010] In TrkA binding domain 2, the sequence of CDR-H1 is GYSFT-Xaa12-YT, the sequence of CDR-H2 is INP-Xaa13-NGG-Xaa14, the sequence of CDR-H3 is A-Xaa15-SAYG-Xaa16-Xaa17-YWYFDV, the sequence of CDR-L1 is QSLLNSRTRKN-Xaa18, the sequence of CDR-L2 is WAS, and the sequence of CDR-L3 is KQSYNLFT; wherein Xaa12 is A or G, Xaa13 is H, F or Y, Xaa14 is S or T, Xaa15 is I, G or H, Xaa16 is S or T, Xaa17 is S or N, and Xaa18 is S or Y;

[0011] In TrkA binding domain 3, the sequence of CDR-H1 is GYTFTSYT, the sequence of CDR-H2 is INPRS-Xaa19-YN, the sequence of CDR-H3 is ARLGGWGYYFDY, the sequence of CDR-L1 is KSVS-Xaa20-SGYSY, the sequence of CDR-L2 is LAS, and the sequence of CDR-L3 is QHSRELFT; among them, Xaa19 is N or D, and Xaa20 is S or T;

[0012] In TrkA binding domain 4, the sequence of CDR-H1 is shown in SEQ ID NO.124, the sequence of CDR-H2 is shown in SEQ ID NO.125, and the sequence of CDR-H3 is shown in SEQ ID NO.126; the sequence of CDR-L1 is shown in SEQ ID NO.149, the sequence of CDR-L2 is shown in SEQ ID NO.150, and the sequence of CDR-L3 is shown in SEQ ID NO.104;

[0013] In TrkA binding domain 5, the sequence of CDR-H1 is shown as SEQ ID NO.140, the sequence of CDR-H2 is shown as SEQ ID NO.141, the sequence of CDR-H3 is shown as SEQ ID NO.142, the sequence of CDR-L1 is shown as SEQ ID NO.157, the sequence of CDR-L2 is shown as SEQ ID NO.154, and the sequence of CDR-L3 is shown as SEQ ID NO.117.

[0014] In some embodiments of the present application, the TrkA-specific binding protein has any one of the following TrkA binding domains:

[0015] TrkA binding domain 1-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.107;

[0016] TrkA binding domain 1-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108;

[0017] TrkA binding domain 1-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.138, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.115;

[0018] TrkA binding domains 1-4 have CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.144, CDR-H3 shown in sequence SEQ ID NO.145, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.120;

[0019] TrkA binding domains 1-5 have CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.146, CDR-H3 shown in sequence SEQ ID NO.147, CDR-L1 shown in sequence SEQ ID NO.159, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108;

[0020] TrkA binding domain 2-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.130, CDR-H3 shown in sequence SEQ ID NO.131, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0021] TrkA binding domain 2-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.135, CDR-H3 shown in sequence SEQ ID NO.136, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0022] TrkA binding domain 2-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.143, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.158, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0023] TrkA binding domain 3-1 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.133, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.155, CDR-L2 shown in sequence SEQ ID NO.156, and CDR-L3 shown in sequence SEQ ID NO.114;

[0024] TrkA binding domain 3-2 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.148, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.160, CDR-L2 shown in sequence SEQ ID NO.156 and CDR-L3 shown in sequence SEQ ID NO.114.

[0025] In some embodiments of the present application, the TrkA-specific binding protein is an antibody, an antigen-binding fragment of an antibody, or a small modular immune drug;

[0026] Optionally, the binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a biclonal antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an intrabody, a nanobody, a binding domain immunoglobulin fusion protein or a small module immune drug.

[0027] In some embodiments of the present application, the TrkA-specific binding protein, the heavy chain variable region and the light chain variable region framework regions are independently derived from mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, alpaca, donkey, deer, mink, chicken, duck or goose.

[0028] In some embodiments of the present application, the TrkA specific binding protein has a heavy chain variable region as shown in any one of the sequences SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29, SEQ ID NO.33, SEQ ID NO.37, SEQ ID NO.41 and SEQ ID NO.45.

[0029] In some embodiments of the present application, the light chain variable region of the TrkA specific binding protein is as shown in any one of the sequences SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27, SEQ ID NO.31, SEQ ID NO.35, SEQ ID NO.39, SEQ ID NO.43 and SEQ ID NO.47.

[0030] In some embodiments of the present application, the TrkA-specific binding protein further comprises an antibody constant region;

[0031] Optionally, the sequence of the antibody constant region is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD;

[0032] Optionally, in the antibody constant region, the species of the light chain constant region and the heavy chain constant region are independently mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, alpaca, donkey, deer, mink, chicken, duck or goose;

[0033] Optionally, the type of the light chain constant region in the antibody constant region is κ type or λ type.

[0034] In some embodiments of the present application, the TrkA specific binding protein has a heavy chain as shown in any one of the sequences SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69, SEQ ID NO.71, SEQ ID NO.73 and SEQ ID NO.75.

[0035] In some embodiments of the present application, the light chain of the TrkA specific binding protein is as shown in any one of the sequences SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74 and SEQ ID NO.76.

[0036] One or more embodiments of the present application also provide a nucleic acid molecule encoding the TrkA-specific binding protein.

[0037] One or more embodiments of the present application also provide a vector, which includes the nucleic acid molecule;

[0038] Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or a combination thereof.

[0039] One or more embodiments of the present application also provide a cell, which includes the nucleic acid molecule or the vector;

[0040] Optionally, the cell is a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

[0041] One or more embodiments of the present application further provide a method for preparing the TrkA specific binding protein, which comprises: culturing the cells, and isolating the TrkA specific binding protein from the obtained culture.

[0042] One or more embodiments of the present application also provide a pharmaceutical composition, which includes the TrkA-specific binding protein, the nucleic acid molecule, the expression vector, the cell or the antibody conjugate, and a pharmaceutically acceptable carrier.

[0043] One or more embodiments of the present application also provide the use of the TrkA specific binding protein, the nucleic acid molecule, the vector, the cell or the pharmaceutical composition in the preparation of a drug for treating a disease;

[0044] Optionally, the disease treated by the drug is pain, neuroma or neuronal disorder;

[0045] Optionally, the pain is acute pain;

[0046] Optionally, the pain is chronic pain;

[0047] Optionally, the pain may be associated with any of the following: inflammatory pain, postoperative pain, neuropathic pain and cancer pain;

[0048] Optionally, the pain may be associated with any of the following: pancreatitis pain, kidney stone pain, endometriosis pain, IBD pain, postoperative adhesion pain, gallstone pain, headache, dysmenorrhea, musculoskeletal pain, sprain pain, visceral pain, ovarian cyst pain, prostatitis pain, cystitis pain, interstitial cystitis pain, postoperative pain, migraine, trigeminal neuralgia, burn and / or wound pain, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis pain, osteoarthritis pain, ankylosing spondylitis pain, periarticular lesion pain, tumor-related pain, chemotherapy-induced perineuropathy (CIPN) pain, pain from tumor bone metastasis and HIV infection pain.

[0049] One or more embodiments of the present application also provide use of the TrkA specific binding protein, the nucleic acid molecule, the vector or the cell in preparing a TrkA detection product.

[0050] One or more embodiments of the present application also provide a TrkA detection product, which includes the TrkA specific binding protein;

[0051] Optionally, the TrkA detection product is a detection reagent, a diagnostic reagent, a test strip, a detection plate or a test kit.

[0052] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0054] Figure 1 This is a diagram showing the results of the chimeric antibody binding activity test for TrkA proteins of different species;

[0055] Figure 2 This is a diagram showing the results of the activity test of the chimeric antibody binding to TrkA and its homologous family proteins;

[0056] Figure 3 This is a diagram showing the results of the chimeric antibody binding to cell surface TrkA activity test;

[0057] Figure 4 This is a result diagram of the chimeric antibody blocking the binding activity of human TrkA and ligand NGF;

[0058] Figure 5 This is a diagram showing the results of the neutralization activity test of the chimeric antibody in inhibiting NGF-induced TF-1 cell proliferation;

[0059] Figure 6 This is a graph showing the test results of the chimeric antibody on CFA-induced inflammatory pain in mice;

[0060] Figure 7 This is a diagram showing the results of humanized anti-TrkA antibody binding to TrkA activity of different species;

[0061] Figure 8 This is a graph showing the results of the activity test of humanized anti-TrkA antibody binding to human TrkA homologous family proteins;

[0062] Fig. 9 This is a diagram showing the results of the humanized anti-TrkA antibody blocking the activity of human TrkA binding to its ligand NGF;

[0063] Fig.10 This is a graph showing the results of in vitro neutralization activity test of humanized anti-TrkA antibody in inhibiting NGF-induced TF-1 cell proliferation;

[0064] Fig.11 The figure shows the effect of humanized anti-TrkA antibody on CFA-induced inflammatory pain in mice. DETAILED DESCRIPTION

[0065] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0067] the term

[0068] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0069] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0070] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0071] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0072] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0073] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0074] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0075] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0076] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0077] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0078] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0079] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0080] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0081] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0082] The terms "TrkA-specific binding protein", "anti-TrkA antibody or fragment thereof" or "humanized anti-TrkA antibody or fragment thereof" herein include antibodies or fragments thereof that bind to human Trk, such as isolated human Trk, specifically antibodies or fragments thereof that bind to TrkA, and more specifically antibodies or fragments thereof that bind to the extracellular region of TrkA. Generally, the humanized anti-TrkA antibody or fragment thereof is capable of inhibiting the functional activity of TrkA and / or is capable of blocking or reducing one or more biological activities that can be induced by NGF binding to TrkA.

[0083] The term "anti-NGF antibody" herein refers to an antibody that can bind to NGF, preferably human NGF. Generally, anti-NGF antibodies can inhibit the functional activation of TrkA and / or can block or reduce one or more biological activities of TrkA. Generally, the anti-NGF antibody should have any one or more of the following characteristics: (a) bind to NGF and inhibit NGF biological activity and / or downstream pathways mediated by NGF signaling function; (b) block or reduce NGF receptor activation (including TrkA receptor dimerization and / or autophosphorylation); (c) increase NGF clearance rate; (d) inhibit (reduce) NGF synthesis, production or release. Anti-NGF antibodies are known in the art, see, e.g., PCT Publication Nos. WO 01 / 78698, WO 01 / 64247, US Patent Nos. 5,844,092, 5,877,016, and 6,153,189; Hongo et al., (2000) Hybridoma, 19:215-227; GenBank Accession Nos. U39608, U39609, L17078, or L17077.

[0084] The term "humanized anti-TrkA antibody or fragment thereof capable of inhibiting the functional activation of TrkA" herein refers to a humanized anti-TrkA antibody having any one or more of the following characteristics: (a) binding to TrkA and inhibiting the biological activity of TrkA and / or downstream pathways mediated by binding to NGF or NTF3 / neurotrophin 3 signaling function; (b) preventing, improving or treating any aspect of pain; (c) blocking or reducing TrkA activation or dimerization and / or autophosphorylation; (d) increasing TrkA clearance; (e) inhibiting or reducing TrkA synthesis and / or cell surface expression.

[0085] The term "humanized anti-TrkA antibody or fragment thereof capable of blocking or reducing one or more biological activities of TrkA" herein refers to a humanized anti-Trk antibody capable of directly or indirectly reducing, inhibiting, neutralizing or eliminating the biological activity of TrkA.

[0086] The term "TrkA biological activity" or "biological activity of TrkA" herein refers to but is not limited to any one or more of the following: the ability to bind NGF or other neurotrophic factors; the ability to homodimerize or heterodimerize and / or autophosphorylate; the ability to activate NGF-induced signaling pathways; the ability to promote cell differentiation, proliferation, survival, growth, migration and other cell physiological changes (including (for neurons, including peripheral neurons and central neurons) neuronal morphological changes), synaptogenesis, synaptic function, neurotransmitter and / or neuropeptide release and regeneration after injury; and the ability to relieve pain and tumor pain associated with bone metastasis.

[0087] The term "IC50" herein refers to the half maximal inhibitory concentration (IC50), which can measure the effectiveness of a compound in inhibiting a biological function, such as the inhibition of NGF-induced TF-1 cell proliferation by a humanized anti-TrkA antibody.

[0088] The term "effector function" herein includes biochemical events generated by the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include FcγR-mediated effector functions such as ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis), and complement-mediated effector functions such as CDC (complement-dependent cytotoxicity). The effector function of an antibody can be changed by changing (i.e., enhancing or weakening, preferably enhancing) the affinity of the antibody to an effector molecule such as an Fc receptor or a complement component. Binding affinity can usually be changed by modifying the effector molecule binding site, and in this case it is suitable to determine the target site and modify at least part of the site in a suitable manner. It should also be understood that changing the binding site of an antibody to an effector molecule does not necessarily significantly change all binding affinities, but can change the geometric position of the interaction so that the effector mechanism is invalid as a non-productive binding. It should also be understood that the effector function can also be changed by modifying sites that are not directly involved in the binding of effector molecules but participate in the generation of effector functions. By altering the effector function of antibodies, many aspects of the immune response can be controlled, such as enhancing or suppressing various responses of the immune system, which may have beneficial effects in diagnosis and treatment.

[0089] The term "treatment" and its various parts of speech "treatment and / or treating and / or treat" herein means that there may be all processes that slow down, interrupt, block, control, prevent or reverse the progress of the disease described herein, but does not necessarily indicate the complete elimination of all symptoms of the disease. Treatment can also include the prevention of pain. Treatment includes the administration of the antibodies of the present application for the treatment of human diseases or disorders that will benefit from the reduction of TrkA activity, and includes: (a) inhibiting the further progression of the disease, that is, preventing its development; (b) alleviating the disease, that is, causing the regression of the disease or disorder or alleviating its symptoms or complications; (c) preventing the onset of symptomatic diseases.

[0090] As used herein, the term "reducing the incidence of pain" refers to any of lessening severity (which includes reducing the need for and / or amount of (e.g., exposure to) other drugs and / or therapies typically used for such a condition, including, for example, opiates), duration, and / or frequency (including, for example, delaying or increasing the time to onset of pain following surgery in an individual). As will be appreciated by those skilled in the art, individuals may vary in their response to treatment, and thus, for example, "a method of reducing the incidence of rheumatoid arthritis pain or osteoarthritis pain in an individual" reflects administration of an antibody based on the reasonable expectation that such administration may result in a reduced incidence in a particular individual.

[0091] The term "alleviating" pain or one or more symptoms of pain (such as rheumatoid pain or osteoarthritis pain) herein means a reduction or improvement of one or more symptoms of pain compared to when the antibody was not administered. "Alleviating" also includes shortening or reducing the duration of symptoms.

[0092] The term "alleviating" pain or one or more symptoms of pain (such as rheumatoid pain or osteoarthritis pain) herein refers to a reduction in the extent of one or more undesirable clinical presentations of postoperative pain in an individual or population of individuals treated with an antibody according to the present application.

[0093] As used herein, the term "delaying" the development of pain means slowing down, hindering, slowing down, delaying, stabilizing and / or postponing the progression of pain, such as postoperative pain, rheumatoid arthritis pain or osteoarthritis pain. Such delays can have different lengths of time, depending on the medical history and / or the individual to be treated. It will be apparent to one skilled in the art that a sufficient or significant delay may actually include prevention, as the individual will not experience pain. A method of "delaying" the development of symptoms is a method of reducing the likelihood of symptom development and / or reducing the extent of symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

[0094] The term "pain" herein refers to pain of any cause, including pain of nociceptive / inflammatory causes, pain of neuropathic causes, pain of hyperplastic causes, or pain of mixed causes. Pain particularly includes acute and chronic pain. Pain particularly includes acute or chronic pain of musculoskeletal or neuropathic origin. Pain also includes any pain with an inflammatory component. Pain also includes visceral pain (such as, for example, chronic prostatitis, interstitial cystitis (bladder pain) or chronic pelvic pain). Non-limiting examples of pain include postoperative pain, postoperative pain (including toothache), migraine, headache and trigeminal neuralgia, pain associated with burns, wounds or kidney stones, pain associated with trauma (including traumatic brain injury), neuropathic pain, pain associated with musculoskeletal diseases (such as rheumatoid arthritis, osteoarthritis), including, for example, non-radicular (non-neurological) and radicular CLBP (including LSR or sciatica), DNP, ankylosing spondylitis, seronegative (non-rheumatoid) arthritis, non-articular rheumatism and periarticular diseases, and pain associated with cancer (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy and post-herpetic neuralgia. Non-limiting examples of pain with inflammatory components (in addition to some of the above pain) include rheumatic pain, pain associated with mucositis and dysmenorrhea. Pain as defined herein particularly includes chronic pain of both musculoskeletal and neuropathic origins. Pain can also particularly include acute pain or sudden pain.

[0095] The term "post-operative pain" (interchangeably referred to as "post-incisional" or "post-traumatic pain") herein refers to pain caused or resulting from external trauma, such as a cut, puncture, incision, laceration, or wound into an individual's tissues, including pain caused by all surgical procedures, whether invasive or non-invasive.

[0096] The term "binding (or binding to)" herein refers to the interaction of an antibody with an epitope of human TrkA. The term "epitope" as used herein refers to a dispersed three-dimensional site of an antigen recognized by the antibody of the present application. The antibody of the present application can be incorporated into a pharmaceutical composition, which can be prepared by methods well known in the art and comprises the antibody of the present application and one or more pharmaceutically acceptable carriers and / or diluents (e.g., Remington, The Science and Practice of Pharmacy, 22nd edition, Loyd V., Ed., Pharmaceutical Press, 2012, which provides a compilation of formulation techniques generally known to practitioners. Carriers suitable for pharmaceutical compositions include any substance that retains molecular activity after binding to the antibody of the present application and is non-reactive to the patient's immune system.

[0097] In a first aspect of an embodiment of the present application, a TrkA-specific binding protein is provided, which has a TrkA binding domain, wherein the TrkA binding domain is selected from any one of the following TrkA binding domains 1 to 5:

[0098] In the TrkA binding domain 1, the sequence of CDR-H1 is GYSFT-Xaa1-YT, the sequence of CDR-H2 is Xaa2-NP-Xaa3-NGG-Xaa4, the sequence of CDR-H3 is A-Xaa5-SAYG-Xaa6-Xaa7-YWYFDV, the sequence of CDR-L1 is SSV-Xaa8-Y, the sequence of CDR-L2 is ATS, and the sequence of CDR-L The sequence of 3 is QQW-Xaa9-Xaa10-Xaa11-PPT; wherein Xaa1 is A or G; Xaa2 is I, F or L, Xaa3 is F or Y, Xaa4 is A or T, Xaa5 is I or G, Xaa6 is S or T, Xaa7 is D or N, Xaa8 is S or N, Xaa9 is S or T, Xaa10 is S or T, and Xaa11 is T or N;

[0099] In TrkA binding domain 2, the sequence of CDR-H1 is GYSFT-Xaa12-YT, the sequence of CDR-H2 is INP-Xaa13-NGG-Xaa14, the sequence of CDR-H3 is A-Xaa15-SAYG-Xaa16-Xaa17-YWYFDV, the sequence of CDR-L1 is QSLLNSRTRKN-Xaa18, the sequence of CDR-L2 is WAS, and the sequence of CDR-L3 is KQSYNLFT; wherein Xaa12 is A or G, Xaa13 is H, F or Y, Xaa14 is S or T, Xaa15 is I, G or H, Xaa16 is S or T, Xaa17 is S or N, and Xaa18 is S or Y;

[0100] In TrkA binding domain 3, the sequence of CDR-H1 is GYTFTSYT, the sequence of CDR-H2 is INPRS-Xaa19-YN, the sequence of CDR-H3 is ARLGGWGYYFDY, the sequence of CDR-L1 is KSVS-Xaa20-SGYSY, the sequence of CDR-L2 is LAS, and the sequence of CDR-L3 is QHSRELFT; among them, Xaa19 is N or D, and Xaa20 is S or T;

[0101] In TrkA binding domain 4, the sequence of CDR-H1 is shown in SEQ ID NO.124, the sequence of CDR-H2 is shown in SEQ ID NO.125, and the sequence of CDR-H3 is shown in SEQ ID NO.126; the sequence of CDR-L1 is shown in SEQ ID NO.149, the sequence of CDR-L2 is shown in SEQ ID NO.150, and the sequence of CDR-L3 is shown in SEQ ID NO.104;

[0102] In TrkA binding domain 5, the sequence of CDR-H1 is shown as SEQ ID NO.140, the sequence of CDR-H2 is shown as SEQ ID NO.141, the sequence of CDR-H3 is shown as SEQ ID NO.142, the sequence of CDR-L1 is shown as SEQ ID NO.157, the sequence of CDR-L2 is shown as SEQ ID NO.154, and the sequence of CDR-L3 is shown as SEQ ID NO.117.

[0103] The embodiments of the present application mainly provide a TrkA-specific binding protein having NGF binding to TrkA blocking activity.

[0104] Anti-TrkA antibodies have been reported, for example: Patent application WO 97 / 21732 (McGill University) describes a monoclonal antibody called 5C3, but the monoclonal antibody is a TrkA agonist and has no analgesic effect. Patent application WO00 / 73344 (Societa Italiana Per La Ricerca Scientifica) describes an anti-TrkA monoclonal antibody called MNAC13, also known as EP-B-118138 (Lay Line Genomics SpA's TrkA monoclonal antibody, a single-chain Fv (ScFv) variant, called MNAC 13ScFv. It was found that the variant binds to TrkA as effectively as MNAC 13 and has an analgesic effect. Patent WO 97 / 21732 (McGill University) describes an anti-TrkA monoclonal antibody called MNAC13ScFv. The 5C3 antibody recorded in the Journal of the American Medical Sciences does not have analgesic effect because it is a TrkA agonist. The disclosed antibody MNAC13 can bind to TrkA, and can further block NGF-induced pain stimulation by blocking TrkA from binding to NGF. However, the activity of the antibody MNAC13 in inhibiting TrkA binding to NGF is not high enough. Furthermore, the antibody has low activity in inhibiting pain associated with the NGF / TrkA signaling pathway and poor efficacy. Based on this, there is an urgent need for anti-TrkA antibodies with higher affinity, stronger specificity and blocking activity for pain treatment. In fact, compared with traditionally reported antibodies, the TrkA-specific binding protein provided in the embodiments of the present application has better effects.

[0105] In some embodiments, the TrkA-specific binding protein has any one of the following TrkA binding domains:

[0106] TrkA binding domain 1-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.107;

[0107] TrkA binding domain 1-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108;

[0108] TrkA binding domain 1-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.138, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.115;

[0109] TrkA binding domains 1-4 have CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.144, CDR-H3 shown in sequence SEQ ID NO.145, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.120;

[0110] TrkA binding domains 1-5 have CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.146, CDR-H3 shown in sequence SEQ ID NO.147, CDR-L1 shown in sequence SEQ ID NO.159, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108;

[0111] TrkA binding domain 2-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.130, CDR-H3 shown in sequence SEQ ID NO.131, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0112] TrkA binding domain 2-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.135, CDR-H3 shown in sequence SEQ ID NO.136, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0113] TrkA binding domain 2-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.143, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.158, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111;

[0114] TrkA binding domain 3-1 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.133, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.155, CDR-L2 shown in sequence SEQ ID NO.156, and CDR-L3 shown in sequence SEQ ID NO.114;

[0115] TrkA binding domain 3-2 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.148, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.160, CDR-L2 shown in sequence SEQ ID NO.156 and CDR-L3 shown in sequence SEQ ID NO.114.

[0116] The TrkA-specific binding protein of the present application may have the above-mentioned CDRs, or may be a derivative fragment having the above-mentioned CDRs. The derivative fragment is formed by amino acid substitution ("conservative modification" or "conservative substitution or replacement") at no more than 6 sites relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementary determining region. For example, the derivative fragment is replaced at 1, 2, 3, 4, 5 or 6 sites relative to its corresponding complementary determining region, and one amino acid may be replaced by another amino acid, or one amino acid may be replaced by multiple (for example, 2) amino acids.

[0117] In the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.

[0118] Table A

[0119]

[0120]

[0121] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

[0122] In the present application, the TrkA specific binding protein may be an antibody, an antigen binding fragment of an antibody, or a small modular immune drug. Further, the binding protein may be a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a biclonal antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an intrabody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immune drug.

[0123] The "antibody" mentioned in this application refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into κ chain or λ chain by the difference in the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0124] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conservative framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0125] The antibody of the present application may be a murine antibody (for example, M01, M02, M04, M05, M06, M07, M08, M10, M12, M13, M15 and M16 mentioned in step 1 of the present application), a chimeric antibody (for example, ch01, ch02, ch04, ch05, ch06, ch07, ch08, ch10, ch12, ch13, ch15 and ch16 mentioned in step 2 of the present application) or a humanized antibody.

[0126] The term "mouse antibody" in this application refers to a monoclonal antibody against human TrkA prepared according to the knowledge and skills in the art. During the preparation, TrkA or its epitope is used as an antigen to inject the test subject, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated. In some examples, the murine anti-TrkA antibody or its antigen-binding fragment may further contain a light chain constant region of a murine κ, λ chain or a variant thereof, or further contain a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.

[0127] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma that secretes mouse-specific monoclonal antibodies must first be established, and then the variable region genes must be cloned from the mouse hybridoma cells, and then the constant region genes of the human antibodies must be cloned as needed, and the mouse variable region genes and the human constant region genes must be connected into chimeric genes and inserted into an expression vector, and finally the chimeric antibody molecules are expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain, or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprising a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0128] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting the CDR sequence of a non-human species into the antibody variable region framework of a human, i.e., different types of human germline antibody framework sequences. The heterologous reaction induced by chimeric antibodies due to carrying a large amount of heterologous protein components can be overcome. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or public references. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk / vbase), and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid the decrease in immunogenicity and the decrease in activity caused by the decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity. The humanized antibodies disclosed herein also include humanized antibodies after further affinity maturation mutation of CDR by yeast display.

[0129] In the present application, the antibody or its antigen-binding fragment may further comprise a light chain constant region of a human or mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of a human or mouse IgG1, IgG2, IgG3, IgG4 or a variant thereof; preferably comprise a human IgG1, IgG2 or IgG4 heavy chain constant region, or an IgG1, IgG2 or IgG4 variant using amino acid mutations (e.g., L234A and / or L235A mutations, and / or S228P mutations).

[0130] The "variants" of the heavy chain constant region of the human antibody and the light chain constant region of the human antibody described in the present application refer to the variants of the heavy chain constant region or light chain constant region that are derived from humans and do not change the structure and function of the antibody variable region, which have been disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants that undergo site-directed modification and amino acid substitutions in the heavy chain constant region; specific substitutions such as YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain knob-into-hole structures (so that the antibody heavy chain has a combination of knob-Fc and hole-Fc), which have been shown to give antibodies new properties, but do not change the function of the antibody variable region.

[0131] "HuMAb", "humanized antibody", "fully human antibody", "fully human antibody" are used interchangeably and may be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic stimulation, and may be produced by any method known in the art. In certain techniques, human heavy and light chain loci elements are introduced into cell lines derived from embryonic stem cell lines in which endogenous heavy and light chain loci are targeted for disruption. The transgenic organism can synthesize human antibodies specific for human antigens, and the organism can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies may also be constructed by gene or chromosome transfection methods and phage display technology, or by in vitro activated B cells, all of which are known in the art.

[0132] The terms "full-length antibody", "intact antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, as distinguished from an antigen-binding fragment as defined below. The term specifically refers to an antibody that contains constant regions in both the light and heavy chains. The disclosure "antibody" encompasses "full-length antibodies" and antigen-binding fragments thereof.

[0133] The full-length antibody of the present application includes a full-length antibody formed by connecting the light chain variable region and the light chain constant region in the combination of the light and heavy chain variable regions in the following table, and connecting the heavy chain variable region and the heavy chain constant region. Those skilled in the art can select the light chain constant region and the heavy chain constant region of different antibody sources according to actual needs, such as the light chain constant region and the heavy chain constant region of human antibody sources.

[0134] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that retain the ability to specifically bind to an antigen (e.g., TrkA). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge on the hinge region, (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody; (v) dsFv, an antigen-binding fragment formed by VH and VL via an interchain disulfide bond; (vi) diabodies, bispecific antibodies and multispecific antibodies comprising fragments such as scFv, dsFv, Fab, etc. In addition, although the two domains of the Fv fragment, VL and VH, are connected by a synthetic linker, they can be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody can be an antibody of different isotypes, for example, an IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), an IgA1, an IgA2, an IgD, an IgE or an IgM antibody.

[0135] Fab is an antibody fragment having antigen-binding activity among fragments obtained by treating IgG antibody molecules with an enzyme having the same activity as papain.

[0136] F(ab')2 is an antibody fragment having antigen-binding activity obtained by digesting IgG with an enzyme having the same activity as pepsin.

[0137] Fab' is an antibody fragment having antigen-binding activity obtained by cleaving the above-mentioned F(ab')2.

[0138] Alternatively, the Fab' fragment can be produced by inserting a DNA encoding the Fab' fragment into an expression vector and introducing the vector into a host.

[0139] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other linkers useful in the present disclosure are described by, for example, but not limited to, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0140] Diabodies are antibody fragments in which scFv or Fab is dimerized, and are antibody fragments having bivalent antigen-binding activity. In the bivalent antigen-binding activity, the two antigens may be the same or different.

[0141] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants.

[0142] dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is substituted by a cysteine ​​residue via a disulfide bond between the cysteine ​​residues. The amino acid residue substituted by the cysteine ​​residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method (e.g., Protein Engineering, 7, 697 (1994)).

[0143] The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion or substitution of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0144] The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is a variable domain without CDRs.

[0145] The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948) and the ImmunoGenTics (IMGT) numbering convention (see Lecture 1, 1996). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003) etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL. Following the IMGT rule, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).Following the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0146] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody (e.g., a specific site on a TrkA molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0147] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-8M, such as about less than 10-9M, 10-10M, 10-11M, 10-12M or less.

[0148] The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies of the present disclosure bind to TrkA or its epitope with a dissociation equilibrium constant (KD) of less than about 10-7M, such as less than about 10-8M or 10-9M, for example, in the present disclosure, the affinity of the antibody to the cell surface antigen is determined by FACS to characterize the KD value.

[0149] Optionally, the species origin of the framework regions of the heavy chain variable region and the light chain variable region of the TrkA-specific binding protein is not particularly limited, and each independently can be selected from but not limited to: mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose.

[0150] In some embodiments, the TrkA-specific binding protein has a heavy chain variable region as shown in any one of SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29, SEQ ID NO.33, SEQ ID NO.37, SEQ ID NO.41 and SEQ ID NO.45.

[0151] In some embodiments, the light chain variable region of the TrkA-specific binding protein is as shown in any one of the sequences SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27, SEQ ID NO.31, SEQ ID NO.35, SEQ ID NO.39, SEQ ID NO.43 and SEQ ID NO.47.

[0152] In some embodiments, the TrkA-specific binding protein further comprises an antibody constant region;

[0153] Optionally, the sequence of the antibody constant region is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD;

[0154] Optionally, in the antibody constant region, the species of the light chain constant region and the heavy chain constant region are independently mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, alpaca, donkey, deer, mink, chicken, duck or goose;

[0155] Optionally, the type of the light chain constant region in the antibody constant region is κ type or λ type.

[0156] In some embodiments, the TrkA-specific binding protein has a heavy chain as shown in any one of the sequences SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69, SEQ ID NO.71, SEQ ID NO.73 and SEQ ID NO.75. Or its heavy chain sequence has no less than 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 990% or 100%) identity with these sequences.

[0157] In some embodiments, the TrkA-specific binding protein has a light chain as shown in any one of the sequences SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74 and SEQ ID NO.76. Or its light chain sequence has no less than 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 990% or 100%) identity with these sequences.

[0158] "Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, when the sequences are optimally aligned, if 6 of the 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous; if 95 of the 100 positions in the two sequences are matched or homologous, then the two sequences are 95% homologous. Typically, when two sequences are aligned, comparison is made to give the maximum percentage identity. For example, the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to BLAST algorithms frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.

[0159] The second aspect of the embodiments of the present application provides a nucleic acid molecule encoding the TrkA-specific binding protein.

[0160] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.

[0161] Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence when the amino acid sequences are aligned (introducing gaps if necessary) to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0162] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0163] In a third aspect of the embodiments of the present application, a vector is provided, which includes the nucleic acid molecule;

[0164] Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or a combination thereof.

[0165] The term "vector" is also "expression vector" and refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, the vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector disclosed herein can be autonomously replicated in a host cell introduced therein (e.g., a bacterial vector and an additional mammalian vector having a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., a non-additional mammalian vector). On the whole, the vector can be selected from, but is not limited to, a mammalian cell virus, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a combination thereof.

[0166] A fourth aspect of the embodiments of the present application provides a cell comprising the nucleic acid molecule or the vector.

[0167] The term "cell" or "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants or animal cells. Easily transformed bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0168] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content, due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, this is clear from the context.

[0169] In a fifth aspect of the embodiments of the present application, a method for preparing the TrkA specific binding protein is provided, which comprises: culturing the cells, and isolating the TrkA specific binding protein from the obtained culture.

[0170] The antibodies or antigen-binding fragments of the present application can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect host cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to TrkA. Positive clones are expanded and cultured in bioreactors to produce antibodies. The culture fluid that secretes antibodies can be purified by conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.

[0171] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold Spring Harbor Manual of Antibody Experimental Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human TrkA or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in the application are genetically engineered to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0172] The sixth aspect of the embodiments of the present application provides a pharmaceutical composition, which includes the TrkA-specific binding protein, the nucleic acid molecule, the expression vector, the cell or the antibody conjugate, and a pharmaceutically acceptable carrier.

[0173] In a seventh aspect of the embodiments of the present application, use of the TrkA-specific binding protein, the nucleic acid molecule, the vector, the cell, the antibody conjugate or the pharmaceutical composition in preparing a drug for treating a disease is provided;

[0174] The disease treated by the drug is pain, neuroma or neuronal disease;

[0175] Optionally, the pain is acute or chronic pain;

[0176] Optionally, the pain may be associated with any of the following: inflammatory pain, postoperative pain, neuropathic pain and cancer pain;

[0177] Optionally, the pain may be associated with any of the following: pancreatitis pain, kidney stone pain, endometriosis pain, IBD pain, postoperative adhesion pain, gallstone pain, headache, dysmenorrhea, musculoskeletal pain, sprain pain, visceral pain, ovarian cyst pain, prostatitis pain, cystitis pain, interstitial cystitis pain, postoperative pain, migraine, trigeminal neuralgia, burn and / or wound pain, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis pain, osteoarthritis pain, ankylosing spondylitis pain, periarticular lesion pain, tumor-related pain, chemotherapy-induced perineuropathy (CIPN) pain, pain from tumor bone metastasis and HIV infection pain.

[0178] In an eighth aspect of the embodiments of the present application, use of the TrkA specific binding protein, the nucleic acid molecule, the vector or the cell in preparing a TrkA detection product is provided.

[0179] A ninth aspect of the embodiments of the present application provides a TrkA detection product, which includes the TrkA specific binding protein;

[0180] Optionally, the TrkA detection product is a detection reagent, a diagnostic reagent, a test strip, a detection plate or a test kit.

[0181] The present application uses a TrkA-specific binding protein in a detection product to compete with TrkA in a test product.

[0182] When the term "competes" is used in the context of antigen binding proteins that compete for the same epitope, it means competition between antigen binding proteins, which is determined by an assay in which the antigen binding protein (e.g., antibody or functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., TrkA antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); using I-1 2 Solid phase direct labeling RIA with 5-label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically the assay involves the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Typically the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically interfere with each other for binding. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen binding protein to the common antigen.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0183] In a tenth aspect of the embodiments of the present application, a method for detecting TrkA is provided, which comprises using the above-mentioned TrkA-specific binding protein to capture TrkA in a test sample.

[0184] The present application relates to a method for immunodetection or determination of a target antigen (e.g., TrkA), a reagent for immunodetection or determination of a target antigen (e.g., TrkA), a method for immunodetection or determination of cells expressing a target antigen (e.g., TrkA), and a diagnostic agent for diagnosing a disease associated with target antigen (e.g., TrkA)-positive cells, which comprises the antibody or antibody fragment of the present application that specifically recognizes the target antigen (e.g., TrkA) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure as an active ingredient.

[0185] In the present application, the method for detecting or measuring the amount of target antigen (e.g., TrkA) can be any known method. For example, it includes immunoassay or assay. Immunoassay or assay is a method for detecting or measuring the amount of antibody or antigen using labeled antigen or antibody. Examples of immunoassay or assay include radioactive material labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods, etc.

[0186] In order to detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0187] In the application, there is no particular limitation on the sample to be tested for detecting or measuring the target antigen (e.g., TrkA), as long as it has the possibility of containing cells expressing the target antigen (e.g., TrkA), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.

[0188] According to the desired detection method, the detection product containing the monoclonal antibody or its antibody fragment of the present application may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a second antibody that recognizes a label of the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.

[0189] According to an eleventh aspect of the embodiments of the present application, a method for treating a disease is provided, which comprises administering an effective amount of the above-mentioned TrkA-specific binding protein or pharmaceutical composition to a subject.

[0190] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0191] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., treatment methods or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0192] "Effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity or delaying the onset of a condition, including the biochemical, histological and / or behavioral symptoms of the condition, its complications and the intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present application or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of the present disclosure in the patient.

[0193] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0194] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0195] Studies have shown that the NGF-TrkA signaling pathway is an effective target for the development of new analgesics. Compared with targeting NGF, targeting TrkA does not interfere with the p75 receptor-mediated function of NGF in physiological processes such as neuronal development, leading to a variety of neurotoxicity, nor does it produce unpredictable side effects such as osteoarticular necrosis caused by excessive neutralization of NGF by anti-NGF antibodies. TrkA antibodies are expected to have better safety.

[0196] The present application example screened out a series of high-affinity anti-TrkA monoclonal antibodies that block the binding of NGF and TrkA through hybridoma technology, and humanized the constant region of the mouse anti-TrkA monoclonal antibody, retaining the variable region of the mouse anti-TrkA monoclonal antibody, to obtain the corresponding anti-TrkA chimeric monoclonal antibody. The chimeric antibody candidate obtained in the present application can not only specifically target and bind to the TrkA receptor, blocking the binding of NGF and TrkA, but also compared with the prior art, the anti-TrkA antibody or its antigen-binding fragment developed in the present application has higher affinity, better specificity and stronger functional activity, and can inhibit pain.

[0197] Example 1

[0198] Step 1: Anti-human TrkA monoclonal antibody production

[0199] 1. Immunization

[0200] Six-week-old BALB / c female mice were selected, and recombinantly expressed human TrkA extracellular segment protein (Acro, TRA-H5259) containing a mouse Fc tag was mixed and emulsified with an equal amount of Freund's complete adjuvant (Sigma-Alderich, F5881) for initial immunization. Three weeks after the initial immunization, the immunogen was mixed and emulsified with Freund's incomplete adjuvant (Sigma-Alderich, F5506) for booster immunization. Blood was collected from immunized mice 7 days after the third immunization, and the serum titer of immunized mice was detected by ELISA. Mice with a serum titer of 1:128000 were selected for shock immunization, and 25 μg of recombinant protein was intraperitoneally inoculated into immunized mice.

[0201] 2. Spleen Cell Fusion

[0202] Three days after the shock immunization, the spleen and peripheral lymph nodes of the mice were taken to isolate B cells. An appropriate amount of B cells were fused with SP2 / 0 cells and screened in DMEM complete medium containing HAT (containing 20% ​​FBS, 1×HAT and 1×DMEM medium). The hybridoma cells were placed in 5% CO 2 , cultured at 37°C for 5-7 days until clones were formed, the supernatant was removed, and 200 μL / well of HT complete medium (containing 20% ​​FBS, 1×HT and 1×DMEM medium) was added, and incubated at 37°C, 5% CO 2 ELISA was performed after 3 days of culture.

[0203] 3. Hybridoma Screening

[0204] 1μg / mL human TrkA extracellular segment recombinant protein (Sino, 11073-H08H) was coated, and the hybridoma culture supernatant was tested by ELISA. The positive clones with strong binding ability to human TrkA protein were selected for further competitive ELISA screening. The clones with high activity blocking the binding of human TrkA and NGF were selected for amplification and cryopreservation, and subcloning was performed at the same time. After 2-3 rounds of subcloning, a single clone was obtained. Each round of subcloning cells needs to be tested by ELISA binding and competitive ELISA.

[0205] Finally, 12 positive hybridoma monoclonal cell lines were screened and obtained, namely 1H2H8B5, 28G11G12B8, 1B1A1H8, 15G1H6H10, 9C7C3B8, 20C11A1G10, 24F10E11B1, 15E10D11H12, 26F3F11B4, 20A3H7F3, 15A4A8F2 and 26F10C2C1.

[0206] The hybridoma monoclonal cell lines were expanded and cultured in serum-free medium, and the culture medium was collected and purified from it using a protein G column to obtain mouse anti-human TrkA monoclonal antibodies MM01 (1H2H8B5), MM02 (28G11G12B8), MM04 (1B1A1H8), MM05 (15G1H6H10), MM06 (9C7C3B8), MM07 (20C11A1G10), MM08 (24F10E11B1), MM10 (15E10D11H12), MM12 (26F3F11B4), MM13 (20A3H7F3), MM15 (15A4A8F2) and MM16 (26F10C2C1), and the purified antibody proteins were stored at -80°C for activity detection.

[0207] Step 2: Sequencing of mouse antibody variable regions and recombinant expression of chimeric antibodies

[0208] The antibody variable regions of the 12 hybridoma monoclonal cells obtained by screening were cloned, and then connected with the human IgG4 heavy chain constant region and the human Kappa light chain constant region to construct an expression vector. The CHO cells were transiently transfected for recombinant expression of chimeric antibodies for activity evaluation.

[0209] 1. The process of cloning the variable region from hybridoma is as follows: collect hybridoma cells in logarithmic growth phase, extract RNA with Trizol (Invitrogen, 15596-018) (according to the steps in the kit instructions) and perform reverse transcription (PrimeScriptTM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription is amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing. The obtained antibody variable region DNA sequence and the corresponding amino acid sequence are shown in SEQ ID NO.1-SEQ ID NO.48.

[0210] 2. The above murine heavy chain variable region and light chain variable region are respectively connected to the heavy chain constant region of the following human IgG4 antibody and the human κ light chain constant region to form chimeric antibodies ch01, ch02, ch04, ch05, ch06, ch07, ch08, ch10, ch12, ch13, ch15 and ch16. The constant region is selected from the sequence SEQ ID NO.49-SEQ ID NO.52.

[0211] 3. The corresponding heavy chain variable region is connected to the human IgG4 heavy chain constant region shown in SEQ ID NO.49 to form the heavy chain of the full-length antibody, and the light chain variable region is connected to the human κ light chain constant region shown in SEQ ID NO.51 to form the light chain of the full-length antibody. Exemplarily, the full-length sequence of the chimeric antibody is shown in SEQ ID NO.53-SEQ ID NO.76.

[0212] Table 1. Summary of anti-TrkA chimeric antibody sequences

[0213]

[0214] 4. The chimeric antibody is fully connected for gene synthesis, and then connected to the corresponding plasmid after enzyme digestion. The constructed plasmid is transiently transfected into CHO cells for expression. After 7-10 days of expression, the cell culture supernatant is purified by MabSelect column (GE Healthcare) balanced with corresponding buffer (such as phosphate buffer, pH7.4), and then eluted with sodium citrate (or other buffer). The antibody obtained in this step can be identified by SDS-PAGE (or SEC-HPLC) for purity and other subsequent characterization studies.

[0215] 5. The positive control antibody used in the present example is YZ-40-13 (from patent WO2009098238A1), and the amino acid sequences of the heavy chain and light chain of the full-length antibody of YZ-40-13 are shown in SEQ ID NO.77-SEQ ID NO.78, respectively. The positive control antibody gene was synthesized by the same method as described in the above steps of this example, transfected into CHO cells for recombinant expression, and purified to obtain the positive control antibody for subsequent activity evaluation and comparison.

[0216] Step 3: Antibody sequence characterization and analysis

[0217] According to the sequencing results of step 2, the antibody sequences were analyzed to obtain CDRs defined by the Kabat and IMGT systems. The following tables (Table 2, Table 3) list the CDRs of 12 anti-TrkA antibodies based on the definitions of the Kabat and IMGT systems.

[0218] Table 2. CDRs of anti-TrkA antibodies defined based on the Kabat system

[0219]

[0220] Table 3. CDRs of anti-TrkA antibodies defined based on the IMGT system

[0221]

[0222]

[0223] Step 4: ELISA binding experiment of chimeric antibody with human, cynomolgus monkey, rat and mouse TrkA recombinant protein

[0224] The recombinantly expressed human TrkA (Sino, 11073-H08H), cynomolgus monkey TrkA (Sino, 91011-C08H), rat TrkA (Sino, 80404-R08H) and mouse TrkA (Sino, 51103-M08H) extracellular domain proteins were diluted to 1 μg / mL, and 100 μL / well of the 96-well ELISA plate was coated with the antigen protein. After washing and blocking, 3× gradient dilutions of the chimeric antibody to be tested, the positive control antibody and the human IgG4 isotype control antibody (isotype control) were added and incubated at room temperature for 1 hour; after washing 3 times, horseradish peroxidase-conjugated goat anti-human Fc antibody (Abcam, ab6858) was added and incubated at room temperature for 1 hour. After washing 3 times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, 1M sulfuric acid was used to stop the color development, and the absorbance value at 450nm was read by an ELISA reader.

[0225] The 12 chimeric anti-TrkA antibodies and positive control antibodies all bound to the extracellular domain proteins of human and cynomolgus monkey TrkA in a dose-dependent manner. Antibodies ch02, ch04, ch05, ch06, ch10, and ch13 could weakly bind to the extracellular domain proteins of rat TrkA; antibodies ch02, ch04, ch05, ch10, and ch13 could weakly bind to the extracellular domain proteins of mouse TrkA. ELISA binding curves and EC50 are shown in Figure 1 And Table 4.

[0226] Table 4. EC50 Summary (nM)

[0227] Antibody ID Human TrkA Cynomolgus monkey TrkA Rat TrkA Mouse TrkA ch01 0.01239 0.02259 >6.667 >6.667 ch02 0.01252 0.02148 >6.667 >6.667 ch04 0.01150 0.01735 >6.667 >6.667 ch05 0.01386 0.02912 >6.667 >6.667 ch06 0.02233 0.02564 >6.667 >6.667 ch07 0.02247 0.04127 >6.667 >6.667 ch08 0.01689 0.02069 >6.667 >6.667 ch10 0.02195 0.03341 >6.667 >6.667 ch12 0.01721 0.02697 4.137 >6.667 ch13 0.01588 0.03037 1.534 >6.667 ch15 0.02155 0.04009 >6.667 >6.667 ch16 0.02270 0.02591 >6.667 >6.667 YZ-40-13 0.02474 0.00357 0.1115 4.747 isotype >6.667 >6.667 >6.667 >6.667

[0228] Step 5: Binding experiment of chimeric antibody and TrkA homologous family protein

[0229] Recombinant human TrkA extracellular domain protein (Sino, 11073-H08H), TrkB (Sino, 10047-H08H), TrkC (Sino, 10048-H08H) and P75 (Acro, TN2-H82E3) were diluted to 1 μg / mL, and 100 μL / well of 96-well ELISA plate was coated with antigen protein. After washing and blocking, 3× gradient dilutions of chimeric antibodies to be tested, positive control antibodies and Human IgG4 isotype control antibodies (isotype control) were added and incubated at room temperature for 1 hour; after washing 3 times, horseradish peroxidase-conjugated goat anti-human Fc antibody (Abcam, ab6858) was added and incubated at room temperature for 1 hour. After washing 3 times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, 1M sulfuric acid was used to stop the color development, and the absorbance value at 450nm was read by an ELISA reader.

[0230] All 12 chimeric anti-TrkA antibodies and positive control antibodies can specifically bind to the recombinant human TrkA extracellular domain protein, but not to TrkB, TrkC and P75 proteins. The test results are shown in Figure 2 .

[0231] Step 6: Anti-TrkA chimeric antibody cell level flow cytometry binding experiment (TF-1 cell)

[0232] 1. Flow cytometry (FACS) was used to detect the binding of anti-TrkA antibodies to TF-1 cells expressing full-length TrkA.

[0233] TrkA / CHO cells were cultured in RPMI1640 medium (Gibco, C11875500BT) containing 10% FBS (Gibco, 10099141) and 5 ng / mL GM-CSF (Sino, 10015-HNAH); negative control cells 293T were cultured in DMEM medium (Gibco, C11995500BT) containing 10% FBS (Gibco, 10099141).

[0234] 2. Collect cells in logarithmic growth phase, resuspend the cells in PBS containing 2% FBS, and add 1×10 5 100 μL of cells were added, and 50 μL of 5 μg / mL anti-TrkA antibody or human IgG control was added and reacted at room temperature for 1 hour. After centrifugation and washing 3 times with FACS buffer at 400 g, 1 μg / mL PE-labeled goat anti-human IgG antibody (Invitrogen, PA1-86078) was added and incubated at room temperature for 30 minutes.

[0235] 3. After washing three times by centrifugation at 300 g with FACS buffer, the geometric mean fluorescence intensity was measured on a BD FACS CantoII flow cytometer to analyze the binding of TrkA antibody to TF-1 cells and 293T cells.

[0236] Results Figure 3 , 12 candidate chimeric antibodies and positive control antibodies could specifically bind to TF-1 cells expressing TrkA, but not to negative control cells 293T.

[0237] Test Example 1: Anti-TrkA chimeric antibody blocks the binding of human TrkA and ligand NGF

[0238] The TrkA extracellular region (33-417) protein fused to human Fc (Acro, TRA-H5259) was diluted to 1 μg / mL, and 100 μL / well of the 96-well ELISA plate was coated with the antigen protein, washed and blocked. The anti-TrkA antibody was diluted to 10000 ng / mL (66.67 nM) as the starting concentration, and then 10 times diluted 3 times to 0.17 ng / mL (0.0011 nM), and 100 μL of the diluted antibody was added to each well. Biotin-labeled human NGF (Novoprotein, CI51) was diluted to 1 μg / mL, 100 μL was added to each well, and incubated at room temperature for 1 hour. After washing 3 times, streptavidin-labeled horseradish peroxidase (Streptavidin-HRP, Biolegend, 405210) was added and incubated at room temperature for 1 hour. After washing five times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, and 1 M sulfuric acid was used to terminate the color development. The absorbance at 450 nm was read by a microplate reader.

[0239] The results are as follows Figure 4 As shown, the anti-TrkA antibodies of the examples of the present application can block the binding of human TrkA to the ligand NGF, and the blocking effect is significantly enhanced with the increase of the antibody concentration. When the antibody concentration is 20nM, the 12 anti-TrkA antibodies of the examples of the present application can almost completely block the binding of TrkA to NGF. However, the control antibody YZ-40-13 has no significant blocking effect on the binding of TrkA to NGF within the measured concentration (0.03nM-66.67nM). The IC50 of the 12 anti-TrkA antibodies and YZ-40-13 of the examples of the present application in blocking the binding of human TrkA to the ligand NGF is shown in Table 5.

[0240] Table 5. Summary of IC50 of antibodies blocking the binding of human TrkA to ligand NGF

[0241] Antibody ch01 ch02 ch04 ch05 ch06 ch07 ch08 ch10 ch12 ch13 ch15 ch16 YZ-40-13 isotype IC50(nM) 9.794 20.89 22.24 19.74 8.602 39.71 29.46 16.61 13.57 20.55 39.21 18.77 >66.67 >66.67

[0242] Test Example 2: In vitro neutralization activity detection of anti-TrkA chimeric antibody (NGF-induced TF-1 cell proliferation assay)

[0243] The growth of TF-1 cells (human blood leukemia cells, ATCC, CRL-2003) is highly dependent on GM-CSF (granulocyte macrophage colony stimulating factor). NGF binds to TrkA on the surface of TF-1 cells and activates its downstream signaling pathway, which can also induce the growth of TF-1, thus eliminating the need to rely on GM-CSF.

[0244] 1. TF-1 cells were cultured in RPMI1640 medium (Gibco, C11875500BT) containing 10% FBS (Gibco, 10099141) and 5 ng / mL GM-CSF (Sino, 10015-HNAH). Cells were collected in the logarithmic growth phase, thoroughly washed to remove GM-CSF in the original growth medium, and resuspended in medium without GM-CSF. The cells were counted and diluted to a concentration of 6.25×10 4 5000 cells / mL were diluted in 80 μL culture medium and inoculated into 96-well cell culture plates (Thermo, 167008).

[0245] 2. Anti-TrkA antibody was diluted to 300 μg / mL (2000 nM) as the starting concentration, and 9 concentrations were prepared by 3-fold serial dilution. 10 μL was added to each well of cells. After incubation at room temperature for 0.5 h, 10 μL of 50 ng / mL human NGF (Novoprotein, CI51) was added to each well of cells. The cells were incubated at 37°C and 5% CO. 2 The cells were cultured in an incubator for 72 h.

[0246] 3. Add 10 μL of CCK-8 colorimetric reagent to each well, detect the absorbance at 450 nm with a microplate reader, calculate the cell viability, and use Graphpad Prism 9.0 to perform nonlinear fitting on the data to calculate the IC50 of each antibody in inhibiting NGF-induced TF-1 cell proliferation.

[0247] The results are as follows Figure 5As shown, the anti-TrkA antibodies ch01, ch02, ch04, ch06, ch10, ch13, ch15 and ch16 of the examples of the present application can significantly inhibit the proliferation of TF-1 cells induced by human NGF, and the neutralization activity EC50 is shown in Table 6. The inhibitory effect of the positive control antibody YZ-40-13 on the proliferation of TF-1 cells is much weaker than that of the antibody of the present application. When the antibody concentration is 30 μg / mL (200 nM), the anti-TrkA antibody of the present application can completely inhibit the proliferation of TF-1 cells induced by NGF, and the inhibition rate of the positive control antibody YZ-40-13 on the proliferation of TF-1 cells induced by NGF at this concentration is lower than that of the chimeric antibodies ch01, ch02, ch04, ch06, ch10, ch13, ch15 and ch16 of the present application.

[0248] Table 6. Summary of EC50 of neutralizing activity of antibodies in inhibiting NGF-induced TF-1 cell proliferation

[0249] Antibody ch01 ch02 ch04 ch06 ch10 ch13 ch15 ch16 YZ-40-13 isotype EC50(nM) 0.9259 0.3259 0.2409 0.3299 1.670 0.3932 0.9266 1.423 >200 >200

[0250] Test Example 3: Effect of anti-TrkA chimeric antibody on CFA-induced inflammatory pain in mice

[0251] B-hNTrk1 / hNGF C57BL / 6 mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., Cat. No. 110820), male, SPF grade, 6-8 weeks old, adapted for 5 days. After the adaptation period, the animals were divided into 3 groups, namely, blank control group, normal saline group (i.e., model group) and anti-TrkA antibody ch02 group, with 10 animals in each group. Before the mice were injected with the modeling agent CFA (complete Freund's adjuvant) in the soles of their feet, the pain threshold was measured by VonFrey, which was the basic pain threshold.

[0252] The next day, except for the blank control group, 10 μL CFA was injected subcutaneously into the right sole of the mouse to establish an inflammatory pain model. The blank control group was given an equal volume of normal saline. After the toes were obviously swollen (about 24 hours after modeling), the pain threshold of the mouse sole was tested in the same way, which was the pain threshold before drug administration.

[0253] Subsequently, each group was subcutaneously injected with normal saline and anti-TrkA antibody ch02 at a dose of 10 mg / kg, and the pain threshold was measured 6h, 24h, 48h and 72h after administration to evaluate the effect of the test substance on the pain threshold.

[0254] The results are as follows Figure 6As shown, 6h after administration, the PWT of the ch02 group (0.130±0.076) increased compared with the PWT of the model group (0.111±0.116). One day after administration, the PWT of the ch02 group (0.161±0.105) increased compared with the PWT of the model group (0.149±0.070). Two days after administration, the PWT of the ch02 group (0.457±0.256) increased significantly compared with the PWT of the model group (0.132±0.079) (P<0.01). Three days after administration, the PWT of the ch02 group (0.525±0.144) increased compared with the PWT of the model group (0.174±0.101). Five days after administration, the PWT of G3-ch02 (0.408±0.183) increased compared with the PWT of the model group (0.319±0.111).

[0255] The above results indicate that anti-TrkA antibody ch02 can improve the decrease in pain threshold caused by CFA from 6 hours to 5 days after administration.

[0256] Test Example 4: Humanization of anti-TrkA antibody

[0257] 1. Humanization of antibodies: Humanization is performed according to the sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the antibodies secreted by the hybridoma cells obtained in step 2. The amino acid sequences of VL and VH of the mouse antibodies are compared and searched in the human embryonic antibody amino acid sequence database to find human IGHV and IGKV sequences with high homology as humanization templates. Through computer simulation technology, the possible steric hindrance and mutual influence of amino acids in the variable region and framework region are analyzed to determine the framework region amino acids that are critical to maintaining the activity of humanized antibodies, and these amino acids are retained during the humanization process. The humanization of the light chain and heavy chain variable regions is completed by CDR transplantation technology. After that, the following multiple humanized antibodies are obtained through the selected antibody constant region template.

[0258] 2. The anti-TrkA antibody ch02 was humanized to obtain a series of exemplary antibodies.

[0259] 3. The obtained humanized antibody heavy chain variable region sequence is grafted to the human IgG4 heavy chain constant region, and the light chain variable region sequence is grafted to the human Kappa light chain constant region, and gene synthesis is performed, and the corresponding plasmid is connected after enzyme digestion. The constructed plasmid is transiently transfected into CHO cells for expression. After 7-10 days of expression, the cell culture supernatant is purified with a MabSelect column (GE Healthcare) balanced with a corresponding buffer (such as phosphate buffered saline, pH7.4), and then eluted with sodium citrate or other buffers. The antibodies obtained in this step can be identified by SDS-PAGE or SEC-HPLC for purity, etc., and used for subsequent other characterization studies.

[0260] 4. The variable region sequences of the ch02 humanized antibodies (MM02H-8, MM02H-20, MM02H-24, MM02H-25, MM02H-30, MM02H-53, MM02H-56, MM02H-59) that can bind to Human TrkA with high activity as detected by ELISA are as follows (SEQ ID NOs. 161 to 180).

[0261] Test Example 5: Humanized anti-TrkA antibody binds to human, cynomolgus monkey, rat and mouse TrkA recombinant proteins

[0262] The recombinantly expressed human TrkA (Sino, 11073-H08H), cynomolgus monkey TrkA (Sino, 91011-C08H), rat TrkA (Sino, 80404-R08H) and mouse TrkA (Sino, 51103-M08H) extracellular domain proteins were diluted to 1 μg / mL, and 100 μL / well of the 96-well ELISA plate was coated with the antigen protein. After washing and blocking, 3× gradient dilutions of the chimeric antibody to be tested, humanized antibody and Human IgG4 isotype control antibody (isotype control) were added and incubated at room temperature for 1 hour; after washing 3 times, horseradish peroxidase-conjugated goat anti-human Fc antibody (Abcam, ab6858) was added and incubated at room temperature for 1 hour. After washing 3 times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, 1M sulfuric acid was used to stop the color development, and the absorbance value at 450nm was read by an ELISA reader.

[0263] The chimeric antibody and 8 humanized anti-TrkA antibodies all bound to human and cynomolgus monkey TrkA extracellular domain proteins in a dose-dependent manner and with high affinity, and could weakly bind to rat and mouse TrkA extracellular domain proteins. ELISA binding curves and EC50 are shown in Figure 7 And Table 7.

[0264] Table 7. Summary of EC50 of humanized antibodies binding to TrkA of different species (nM)

[0265]

[0266]

[0267] Test Example 6: Humanized anti-TrkA antibody binds to human TrkA homologous family proteins

[0268] Recombinant human TrkA extracellular domain protein (Sino, 11073-H08H), TrkB (Sino, 10047-H08H), TrkC (Sino, 10048-H08H) and P75 (Acro, TN2-H82E3) were diluted to 1 μg / mL, and 100 μL / well of 96-well ELISA plate was coated with antigen protein. After washing and blocking, 3× gradient dilutions of humanized antibodies to be tested, positive control antibodies and Human IgG4 isotype control antibodies (isotype control) were added and incubated at room temperature for 1 hour; after washing 3 times, horseradish peroxidase-conjugated goat anti-human Fc antibody (Abcam, ab6858) was added and incubated at room temperature for 1 hour. After washing 3 times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, 1M sulfuric acid was used to stop the color development, and the absorbance value at 450nm was read by an ELISA reader.

[0269] The chimeric antibody, 8 humanized anti-TrkA antibodies and positive control antibody can specifically bind to the recombinant human TrkA extracellular domain protein, but not to TrkB, TrkC and P75 proteins. The test results are shown in Figure 8 .

[0270] Test Example 7: Humanized anti-TrkA antibody blocks the binding of human TrkA and ligand NGF

[0271] The TrkA extracellular region (33-417) protein fused to human Fc (Acro, TRA-H5259) was diluted to 1 μg / mL, and 100 μL / well of the 96-well ELISA plate was coated with the antigen protein, washed and blocked. The anti-TrkA antibody was diluted to 0.823 nM as the starting concentration, and then 6 concentrations were diluted 3 times to 0.0034 nM, and 90 μL of the diluted antibody was added to each well. Biotinylated human NGF (Novoprotein, CI51) was diluted to 1 μg / mL, 10 μL was added to each well, and incubated at room temperature for 1 hour. After washing 3 times, streptavidin-labeled horseradish peroxidase (Streptavidin-HRP, Biolegend, 405210) was added and incubated at room temperature for 1 hour. After washing 5 times, tetramethylbenzidine (TMB, 4A Biotech, 4AMB200) was added for color development, 1M sulfuric acid was used to stop the color development, and the absorbance value at 450nm was read by an ELISA reader.

[0272] The results are as follows Fig. 9As shown, the chimeric antibodies and humanized anti-TrkA antibodies in the examples of the present application can block the binding of human TrkA to the ligand NGF, and the blocking effect is significantly enhanced with the increase of antibody concentration. When the antibody concentration is higher than 0.27nM, the 8 strains of humanized anti-TrkA antibodies and chimeric antibodies in the test examples of the present application can completely block the binding of TrkA to NGF. However, the control antibody YZ-40-13 has no significant blocking effect on the binding of TrkA to NGF within the measured concentration (0.0034nM-0.823nM). The IC50 of the 8 strains of humanized anti-TrkA antibodies and YZ-40-13 in the examples of the present application to block the binding of human TrkA to the ligand NGF is shown in Table 8.

[0273] Table 8. Summary of IC50 of antibodies blocking the binding of human TrkA to ligand NGF

[0274] Antibody ch02 MM02H-8 MM02H-20 MM02H-24 MM02H-25 MM02H-30 MM02H-53 MM02H-56 MM02H-59 YZ-40-13 isotype IC50(nM) 0.4704 0.02739 0.04084 0.05907 0.04723 0.04266 0.06411 0.05908 0.04544 >66.67 66.67

[0275] Test Example 8: In vitro neutralization activity detection of humanized anti-TrkA antibodies (NGF-induced TF-1 cell proliferation assay)

[0276] 1. TF-1 cells were cultured in RPMI1640 medium (Gibco, C11875500BT) containing 10% FBS (Gibco, 10099141) and 5 ng / mL GM-CSF (Sino, 10015-HNAH). Cells were collected in the logarithmic growth phase, thoroughly washed to remove GM-CSF in the original growth medium, and resuspended in medium without GM-CSF. The cells were counted and diluted to a concentration of 6.25×104 cells / mL. 5000 cells per well were diluted in 80 μL medium and inoculated into 96-well cell culture plates (Thermo, 167008).

[0277] 2. Dilute the humanized anti-TrkA antibody to 300 μg / mL (2000 nM) as the starting concentration, perform a 3-fold gradient dilution to prepare 9 concentrations, add 10 μL to each well of cells, incubate at room temperature for 0.5 h, then add 10 μL of 50 ng / mL human NGF (Novoprotein, CI51) to each well of cells, incubate at 37°C, 5% CO 2 The cells were cultured in an incubator for 72 h.

[0278] 3. Add 100 μL CellTiter lumi TM The optical luminescence signal value was read by a multifunctional microplate reader, and the cell viability was calculated. The data were nonlinearly fitted using Graphpad Prism 10.0 to calculate the EC50 of each antibody in inhibiting NGF-induced TF-1 cell proliferation.

[0279] The results are as follows Fig.10 As shown, the humanized anti-TrkA antibodies of the embodiments of the present application can significantly inhibit the proliferation of TF-1 cells induced by human NGF, and the neutralization activity EC50 is shown in Table 9. The inhibitory effect of the positive control antibody YZ-40-13 on the proliferation of TF-1 cells is significantly weaker than that of the antibody of the present application. When the antibody concentration reaches 10 μg / mL (66.67 nM), the anti-TrkA antibody of the present application can completely inhibit the proliferation of TF-1 cells induced by NGF, and the inhibition rate of the positive control antibody YZ-40-13 on the proliferation of TF-1 cells induced by NGF at this concentration is lower than that of the humanized antibodies MM02H-8, MM02H-20, MM02H-24, MM02H-25, MM02H-30, MM02H-53, MM02H-56 and MM02H-59 of the present application.

[0280] Table 9. Summary of EC50 of humanized anti-TrkA antibodies inhibiting TF-1 cell proliferation

[0281] Antibody ch02 MM02H-8 MM02H-20 MM02H-24 MM02H-25 MM02H-30 MM02H-53 MM02H-56 MM02H-59 YZ-40-13 isotype EC50(nM) 3.356 3.512 3.340 1.161 0.997 2.520 2.475 3.378 2.098 >200 >200

[0282] Test Example 9: Effect of humanized anti-TrkA antibody MM02H-24 on CFA-induced inflammatory pain in mice

[0283] B-hNTrk1 / hNGF C57BL / 6 mice (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., Cat. No. 110820), male, SPF grade, 6-8 weeks old, adaptive feeding for 5 days. After the adaptation period, the animals were divided into 4 groups, namely blank control group, normal saline group (i.e. model group), humanized antibody MM02H-24 (10mpk) group and MM02H-24 (30mpk) group, with 10 animals in each group. Before the modeling agent CFA was injected into the sole of the mouse (Day-1), the pain threshold was measured by VonFrey, which was the basic pain threshold.

[0284] The next day, except for the blank control group, 10 μL CFA was injected subcutaneously into the right sole of the mouse to establish an inflammatory pain model (Day 0). The blank control group was given an equal volume of saline. After the toes were obviously swollen (about 24 hours after modeling, Day 1), the pain threshold of the mouse sole was tested in the same way, which was the pain threshold before drug administration.

[0285] Subsequently, each group was subcutaneously injected with normal saline and humanized anti-TrkA antibody MM02H-24, and the pain threshold was measured 1 day (Day 2), 2 days (Day 3), 3 days (Day 4) and 5 days (Day 6) after administration to evaluate the effect of the test substance on the pain threshold.

[0286] The results are as follows Fig.11As shown, 1 day after administration, the drug administration group could improve the decrease in pain threshold caused by CFA compared with the G2-Model (normal saline) group. 2 days after administration, the MM02H-24 (30 mpk) group significantly improved the decrease in pain threshold caused by CFA compared with the G2-Model (normal saline) group (P < 0.05). 3 days after administration, compared with the G2-Model (normal saline) group, both the MM02H-24 (10 mpk) group and the MM02H-24 (30 mpk) group could significantly improve the decrease in pain threshold caused by CFA (P < 0.01). 5 days after administration, compared with the G2-Model (normal saline) group, both the MM02H-24 (10 mpk) group and the MM02H-24 (30 mpk) group could significantly improve the decrease in pain threshold caused by CFA (P < 0.01).

[0287] The above results indicate that the anti-humanized TrkA antibody MM02H-24 can significantly improve the decrease in pain threshold caused by CFA from 1 day to 5 days after administration, with a significant statistical difference compared with the model group.

[0288] The present application example screened and obtained high-affinity antibodies against the extracellular domain of TrkA. Compared with traditional technologies, the screened antibodies have higher specificity and functional activity. The anti-TrkA antibodies of the present application example have higher cellular functional activity and pharmacodynamic activity than existing TrkA antibody drugs, and can be used to treat pain associated with the NGF / TrkA signaling pathway, including nociceptive / inflammatory pain, postoperative pain, neuropathic pain, hyperplastic pain, cancer pain, osteoarthritis, acute or chronic pain of mixed etiology, and other tumors that express positively.

[0289] Sequence Description:

[0290] (1) mAb MM01 mouse heavy chain variable region amino acid sequence (SEQ ID NO.1) (121aa)

[0291] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYYWHWIRQFPGNKLEWMGYIYYSGNSNYNPSLKSRISIT RDTSKNQFFLQLNSVTTEDTATYYCAREDLVWLRRGFASWGQGTLVTVSA.

[0292] (2) mAb MM01 murine heavy chain variable region nucleotide sequence (SEQ ID NO.2) (363 bp)

[0293] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCA

[0294] CTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGCACTGGATCCGGCAGTTTCCAGGAAACAAA

[0295] CTGGAATGGATGGGCTACATATATTACAGTGGTAACTCTAACTACAACCCATCTCTCAAAAGTCGAATC

[0296] TCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAATTGAATTCTGTGACTACTGAGGACAC

[0297] AGCCACATATTACTGTGCAAGAGAGGATCTAGTATGGTTACGACGGGGATTTGCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA。

[0298] (3) Amino acid sequence of the murine light chain variable region of mAb MM01 (SEQ ID NO.3) (111aa)

[0299] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPRFLIYAASNQGSGVPARFSGSGSG TDFSLNIHPMEEDDSAMYFCQQSKEVPYTFGGGTKLEIK。

[0300] (4) Nucleotide sequence of the murine light chain variable region of mAb MM01 (SEQ ID NO.4) (333bp)

[0301] GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTG

[0302] CAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAGAAACCAGGAC

[0303] AGCCACCCAGATTCCTCATCTATGCTGCATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGC

[0304] AGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATTCTGCAATGTATTTCTGTCAGCAAAGTAAAGAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA.

[0305] (5) mAb MM02 mouse heavy chain variable region amino acid sequence (SEQ ID NO.5) (121aa)

[0306] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGENLEWIGLINPFNGGTRYNQKFKGKATLTVDKSSSTAYMELFSLTSDDSAVYYCAGSAYGSDYWYFDVWGAGTTVTVSS.

[0307] (6) mAb MM02 murine heavy chain variable region nucleotide sequence (SEQ ID NO.6) (363 bp)

[0308] GAGGTCCAGCTCCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCA

[0309] AGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAGAGAACCT

[0310] TGAGTGGATTGGACTTATTAATCCTTTCAATGGGTGGTACTAGGTACAACCAGAAGTTCAAGGGCAAGG

[0311] CCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCTTCAGTCTGACATCTGACGAC

[0312] TCTGCAGTCTATTACTGTGCAGGATCTGCCTACGGTAGTGACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0313] (7) mAb MM02 mouse light chain variable region amino acid sequence (SEQ ID NO.7) (106aa)

[0314] QIVLSQSPAILSASPGETVTMTCRASSSVSYMHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYYCQQWTSTPPTFGAGTKLELK.

[0315] (8) mAb MM02 mouse light chain variable region nucleotide sequence (SEQ ID NO.8) (318 bp)

[0316] CAAATTGTTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGACGGTCACAATGACTTG

[0317] CAGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAAACC

[0318] TGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC

[0319] CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAATGGACTAGTACCCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0320] (9) mAb MM04 mouse heavy chain variable region amino acid sequence (SEQ ID NO.9) (121aa)

[0321] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGTRYNQKFKGKATLTVDKSSSTAYMELFSLTSEDSAVYYCAGSAYGSDYWYFDVWGAGTTVTVSS.

[0322] (10) mAb MM04 murine heavy chain variable region nucleotide sequence (SEQ ID NO.10) (363 bp)

[0323] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCA

[0324] AGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCT

[0325] TGAGTGGATTGGACTTATTAATCCTTTCAATGGTGGTACTAGGTACAACCAGAAGTTCAAGGGCAAGG

[0326] CCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCTTCAGTCTGACATCTGAGGAC

[0327] TCTGCAGTCTATTACTGTGCAGGATCTGCCTACGGTAGTGACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。

[0328] (11) Amino acid sequence of the murine light chain variable region of mAb MM04 (SEQ ID NO.11) (106aa)

[0329] QIVLSQSPAILSASPGETVTMTCRASSSVSYMHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYYCQQWSSTPPTFGAGTKLELK。

[0330] (12) Nucleotide sequence of the murine light chain variable region of mAb MM04 (SEQ ID NO.12) (318bp)

[0331] CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGACGGTCACAATGACTTG

[0332] CAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAAACC

[0333] TGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC

[0334] CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAATGGAGTAGTACCCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0335] (13) mAb MM05 mouse heavy chain variable region amino acid sequence (SEQ ID NO.13) (121aa)

[0336] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGTSLEWIGLINPHNGGTNYNQKFKGKATLTLDKSSSSAYMELLSLTSEDSAVYYCAHSAYGSSYWYFDVWGAGTTVTVSS.

[0337] (14) mAb MM05 murine heavy chain variable region nucleotide sequence (SEQ ID NO.14) (363 bp)

[0338] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCA

[0339] AGGCTTCTGGTTACTCATTCACTGGCTACACCATGAATTGGGTGAAACAGAGCCATGGAACGAGCCTT

[0340] GAGTGGATTGGACTTATTAATCCTCACAATGGTGGTACTAACTACAACCAGAAGTTCAAGGGCAAGGC

[0341] CACATTAACTTTAGACAAGTCATCCAGCTCAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACT

[0342] CTGCAGTCTATTACTGTGCACACTCTGCCTACGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0343] (15) mAb MM05 mouse light chain variable region amino acid sequence (SEQ ID NO.15) (112aa)

[0344] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

[0345] (16) mAb MM05 mouse light chain variable region nucleotide sequence (SEQ ID NO.16) (336 bp)

[0346] GACATTTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTG

[0347] CAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAA

[0348] CCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTT

[0349] CACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGCAAGCAATCTTATAATCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0350] (17) mAb MM06 mouse heavy chain variable region amino acid sequence (SEQ ID NO.17) (119aa)

[0351] QVQLQQSGAELARPGASVKMSCKASGYTFTSYTIHWAKQRPGQGLEWIGKINPRSNYNNYNQKFKEKATLSADKSSSTAYMQLSSLTSEDSAVYYCARLGGWGYYFDYWGQGTTLTVSS.

[0352] (18) mAb MM06 murine heavy chain variable region nucleotide sequence (SEQ ID NO.18) (357 bp)

[0353] CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGTA

[0354] AGGCTTCTGGCTACACCTTTACTAGCTACACGATACACTGGGCAAAACAGAGGCCTGGACAGGGTCT

[0355] GGAATGGATTGGAAAAATAAATCCTAGAAGTAATTATAATAATTACAATCAGAAGTTCAAGGAGAAGG

[0356] CCACATTGAGTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGA

[0357] CTCTGCAGTCTATTACTGTGCAAGATTAGGGGGCTGGGGTTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0358] (19) Amino acid sequence of the murine light chain variable region of mAb MM06 (SEQ ID NO.19) (110aa)

[0359] DIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSG TDFTLNIHPVEEEDAATYYCQHSRELFTFGSGTKLEIK。

[0360] (20) Nucleotide sequence of the murine light chain variable region of mAb MM06 (SEQ ID NO.20) (330bp)

[0361] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATG

[0362] CAGGGCCAGCAAAAGTGTCAGTTCATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGAC

[0363] AGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC

[0364] AGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。

[0365] (21) Amino acid sequence of the murine heavy chain variable region of mAb MM07 (SEQ ID NO.21) (121aa)

[0366] EVQLQQSGPELVRPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGSNYNQKFKGKATLTVDKSSSTAYMDLLSLTSEDSAVYYCAGSAYGSSYWYFDVWGAGTTVTVSS。

[0367] (22) Nucleotide sequence of the murine heavy chain variable region of mAb MM07 (SEQ ID NO.22) (363bp)

[0368] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAGGCCTGGAGCTTCAATGAAGATATCCTGCA

[0369] AGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCT

[0370] TGAGTGGATTGGACTTATTAATCCTTTCAATGGTGGTTCTAACTACAACCAGAAGTTCAAGGGCAAGG

[0371] CCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGACCTCCTCAGTCTGACATCTGAGGAC

[0372] TCTGCAGTCTATTACTGTGCAGGGTCTGCCTACGGTAGTAGCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0373] (23) mAb MM07 mouse light chain variable region amino acid sequence (SEQ ID NO.23) (112aa)

[0374] DIMMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYFCKQSYNLFTFGSGTKLEIK.

[0375] (24) mAb MM07 mouse light chain variable region nucleotide sequence (SEQ ID NO.24) (336 bp)

[0376] GACATATTATGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTG

[0377] CAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAA

[0378] CCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTT

[0379] CACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTTCTGCAAGCAATCTTATAATCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0380] (25) mAb MM08 mouse heavy chain variable region amino acid sequence (SEQ ID NO.25) (121aa)

[0381] EVQLQQSGPELVKPGASMKISCKASGYSFTAYTMTWVKQSHGKNLEYIGLFNPYNGGTSYNQKFKGKAT

[0382] LTVDKSSSTAYMELLSLLTSEDSAVYYCAISAYGTNYWYFDVWGAGTTVTVSS

[0383] (26) mAb MM08 murine heavy chain variable region nucleotide sequence (SEQ ID NO.26) (363 bp)

[0384] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCA

[0385] AGGCTTCTGGTTACTCATTCACTGCCTACACCATGACCTGGGTGAAGCAGAGCCATGGAAAGAACCTT

[0386] GAGTATATTGGACTTTTTAATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGC

[0387] CACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACT

[0388] CTGCAGTCTATTACTGTGCAATCTCTGCCTACGGTACTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0389] (27) mAb MM08 mouse light chain variable region amino acid sequence (SEQ ID NO.27) (106aa)

[0390] QVVLSQSPAILSASPGEMVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSY SLTISRVEAEDAATYYCQQWSTNPPTFGAGTKLDLK.

[0391] (28) mAb MM08 murine light chain variable region nucleotide sequence (SEQ ID NO.28) (318 bp)

[0392] CAAGTTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGATGGTCACAATGACTTG

[0393] CAGGGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCC

[0394] TGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC

[0395] CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTACTAACCCACCCACGTTCGGTGCTGGGACCAAGCTGGACCTGAAA。

[0396] (29) Amino acid sequence of the murine heavy chain variable region of mAb MM10 (SEQ ID NO.29) (124aa)

[0397] KVQLQQSGAELVKPGASVKLSCKASGYTFTEYIVHWVKQRSGQGLEWIGWFYPGSGTIKYNEKFKDKATLTADKSSNTVYMELSRLTSEDSAVYFCARHEEPGGYYVGRVFAYWGQGTLVTVSA。

[0398] (30) Nucleotide sequence of the murine heavy chain variable region of mAb MM10 (SEQ ID NO.30) (372bp)

[0399] AAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTGAAACCCGGGGCATCAGTGAAACTGTCCTGC

[0400] AAGGCTTCTGGCTACACCTTCACTGAATATATTGTACACTGGGTAAAGCAGAGGTCTGGACAGGGTCT

[0401] TGAGTGGATTGGGTGGTTTTACCCTGGAAGTGGTACTATAAAGTACAATGAGAAATTCAAGGACAAGG

[0402] CCACATTGACTGCGGACAAATCCTCCAACACAGTCTATATGGAGCTTAGTAGATTGACATCTGAAGAC

[0403] TCTGCGGTCTATTTCTGTGCAAGACACGAAGAACCCGGGGGTTACTACGTAGGGAGGGTTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0404] (31) mAb MM10 mouse light chain variable region amino acid sequence (SEQ ID NO.31) (113aa)

[0405] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNDQKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTVSSVKAEDLAVYYCHQYYYYPWTFGGGTKLEIK.

[0406] (32) mAb MM10 mouse light chain variable region nucleotide sequence (SEQ ID NO.32) (339 bp)

[0407] GACATTTGTGATGTCGCAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTG

[0408] CAAGTCCAGTCAGAGCCTTTTATATAGTAACGATCAAAAGAGCTACTTGGCCTGGTACCAGCAGAAAC

[0409] CAGGACAGTCTCCTAAATTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTC

[0410] ACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCGTCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCACCAATATTATTACTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA. (33) mAbMM12 mouse heavy chain variable region amino acid sequence (SEQ ID NO.33) (121aa)

[0411] EVQLQQSGPELVKPGASMKISCKASGYSFTAYTMTWVKQSHGKNLEYIGLINPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAISAYGTNYWYFDVWGAGTTVTVSS.

[0412] (34) mAb MM12 murine heavy chain variable region nucleotide sequence (SEQ ID NO.34) (363 bp)

[0413] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCA

[0414] AGGCTTCTGGTTACTCATTCACTGCCTACACCATGACCTGGGTGAAGCAGAGCCATGGAAAGAACCTT

[0415] GAGTATATTGGACTTATTAATCCTTACAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGC

[0416] CACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGACT

[0417] CTGCAGTCTATTACTGTGCAATCTCTGCCTACGGTACTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0418] (35) mAb MM12 mouse light chain variable region amino acid sequence (SEQ ID NO.35) (112aa)

[0419] DIVMSQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGS GSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIK.

[0420] (36) mAb MM12 mouse light chain variable region nucleotide sequence (SEQ ID NO.36) (336 bp)

[0421] GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTTTGAGCTG

[0422] CAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTCCTTGGCTTGGTACCAGCAGAAA

[0423] CCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTT

[0424] CACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA。

[0425] (37) Amino acid sequence of the murine heavy chain variable region of mAb MM13 (SEQ ID NO.37) (121aa)

[0426] EVQLQQSGPELVKPGTSMKISCRASGYSFTGYTMNWVKQSHGKNLEWIGLLNPFNGGASYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVYYCAGSAYGTNYWYFDVWGAGTTVTVSS。

[0427] (38) Nucleotide sequence of the murine heavy chain variable region of mAb MM13 (SEQ ID NO.38) (363bp)

[0428] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAACTTCAATGAAGATATCCTGCA

[0429] GGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCT

[0430] TGAGTGGATTGGACTTCTTAATCCTTTCAATGGTGGTGCTAGCTACAACCAGAAGTTCAAGGGCAAGG

[0431] CCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGAC

[0432] TCTGCAGTCTATTATTGTGCAGGATCTGCCTACGGAACTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0433] (39) mAb MM13 mouse light chain variable region amino acid sequence (SEQ ID NO.39) (106aa)

[0434] QIVLSQSPAILSASPGDKVTMTCRASSSVSYIHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYSL TISRVEAEDAATYYCQQWTSNPPTFGAGTKLELK.

[0435] (40) mAb MM13 murine light chain variable region nucleotide sequence (SEQ ID NO.40) (318 bp)

[0436] CAAATTGTTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGACAAGGTCACAATGACTTG

[0437] CAGGCCAGCTCAAGTGTAAGTTACATTCACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAAACC

[0438] TGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC

[0439] CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0440] (41) mAb MM15 mouse heavy chain variable region amino acid sequence (SEQ ID NO.41) (121aa)

[0441] EVQLQQSGPELVKPRASMKISCRASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGATYNQKFKAKATLTVDKSSSTAYMELLSLTSEDSAVYYCAGSAYGSNYWYFDVWGAGTTVTVSS.

[0442] (42) mAb MM15 murine heavy chain variable region nucleotide sequence (SEQ ID NO.42) (363 bp)

[0443] GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTAGAGCTTCAATGAAGATATCCTGCA

[0444] GGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCT

[0445] TGAGTGGATTGGACTTATTAATCCTTTCAATGGTGGTGCTACCTACAACCAGAAGTTCAAGGCCAAGG

[0446] CCACATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCTCAGTCTGACATCTGAGGAC

[0447] TCTGCAGTCTATTACTGTGCAGGATCTGCCTACGGTTCTAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA.

[0448] (43) mAb MM15 mouse light chain variable region amino acid sequence (SEQ ID NO.43) (106aa)

[0449] QIVLSQSPAILSASPGDKVTMTCRASSSVNYMHWYQQKSGSSPKTWIYATSNLASGVPARFSGSGSGTSYS LTISRVEAEDAATYYCQQWSSTPPTFGAGTKLELK.

[0450] (44) mAb MM15 mouse light chain variable region nucleotide sequence (SEQ ID NO.44) (318 bp)

[0451] CAAATTGTTCTCTCCCAGTCTCCAGCTATCCTGTCTGCATCTCCAGGGGACAAGGTCACAATGACTTG

[0452] CAGGGCCAGTTCAAGTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGATCCTCCCCCAAAACC

[0453] TGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC

[0454] CTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTACCCCACCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

[0455] (45) Amino acid sequence of the murine heavy chain variable region of mAb MM16 (SEQ ID NO.45) (119aa)

[0456] QVQLQQSGAELARPGASVKMSCKASGYTFTSYTIHWVKQRPGQGLEWIGKINPRSDYNYYNQKFKDKATLSADKSSSTAYMQLSSLTSEDSAVYYCARLGGWGYYFDYWGQGTTLTVSS。

[0457] (46) Nucleotide sequence of the murine heavy chain variable region of mAb MM16 (SEQ ID NO.46) (357bp)

[0458] CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGC

[0459] AAGGCTTCTGGCTACACCTTTACTAGCTACACGATACACTGGGTAAAACAGAGGCCTGGACAGGGTC

[0460] TGGAATGGATTGGAAAAATTAATCCTAGAAGTGATTATAATTATTACAATCAGAAGTTCAAGGACAAG

[0461] GCCACATTGAGTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGG

[0462] ATTCTGCAGTCTATTACTGTGCAAGATTAGGGGGCTGGGGTTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0463] (47) mAb MM16 mouse light chain variable region amino acid sequence (SEQ ID NO.47) (110aa)

[0464] DIVLTQSPASLAVSLGQRATMSCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLDSGVPARFSGSGS GTDFTLNIHPVEEEDAATYYCQHSRELFTFGSGTKLEIK.

[0465] (48) mAb MM16 murine light chain variable region nucleotide sequence (SEQ ID NO.48) (330 bp)

[0466] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATGTCATG

[0467] CAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGAC

[0468] AGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGATTCTGGGGTCCCTGCCAGGTTCAGTGGC

[0469] AGTGGGTCTGGGACAGATTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAACACAGTAGGGAGCTATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0470] (49) Amino acid sequence of the heavy chain constant region of human IgG4 antibody (SEQ ID NO.49) (327aa)

[0471] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP

[0472] SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVV

[0473] VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS

[0474] SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0475] (50) Nucleotide sequence of the heavy chain constant region of human IgG4 antibody (SEQ ID NO.50) (981bp)

[0476] GCTAGCACCAAGGGCCCATCGGTCTTCCCGCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAG

[0477] CCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGC

[0478] CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC

[0479] GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCA

[0480] GCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACC

[0481] TGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCC

[0482] GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACT

[0483] GGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCA

[0484] CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTG

[0485] CAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC

[0486] CGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG

[0487] ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGG

[0488] AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCT

[0489] AACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA。

[0490] (51) Amino acid sequence of human κ light chain constant region (SEQ ID NO.51) (107aa)

[0491] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0492] (52) Human κ light chain constant region nucleotide sequence (SEQ ID NO.52) (321bp)

[0493] CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC

[0494] CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG

[0495] CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCC

[0496] TCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT。

[0497] (53) Amino acid sequence of the heavy chain of chimeric antibody ch01 (SEQ ID NO.53) (448aa)

[0498] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYYWHWIRQFPGNKLEWMGYIYYSGNSNYNPSLKSRISIT

[0499] RDTSKNQFFLQLNSVTTEDTATYYCAREDLVWLRRGFASWGQGTLVTVSAASTKGPSVFPLAPCSRSTSE

[0500] STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN

[0501] TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG

[0502] VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

[0503] LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0504] (54) Amino acid sequence of the light chain of chimeric antibody ch01 (SEQ ID NO.54) (218aa)

[0505] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPRFLIYAASNQGSGVPARFSGSGSG

[0506] TDFSLNIHPMEEDDSAMYFCQQSKEVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF

[0507] YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0508] (55) Amino acid sequence of the heavy chain of chimeric antibody ch02 (SEQ ID NO.55) (448aa)

[0509] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGENLEWIGLINPFNGGTRYNQKFKGKAT

[0510] LTVDKSSSTAYMELFSLTSDDSAVYYCAGSAYGSDYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRST

[0511] SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0512] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0513] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0514] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0515] (56) Amino acid sequence of the light chain of chimeric antibody ch02 (SEQ ID NO.56) (213aa)

[0516] QIVLSQSPAILSASPGETVTMTCRASSSVSYMHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYS

[0517] LTISRVEAEDAATYYCQQWTSTPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

[0518] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0519] (57) Amino acid sequence of the heavy chain of chimeric antibody ch04 (SEQ ID NO.57) (448aa)

[0520] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGTRYNQKFKGKAT

[0521] LTVDKSSSTAYMELFSLTSEDSAVYYCAGSAYGSDYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRST

[0522] SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0523] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0524] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0525] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0526] (58) Amino acid sequence of the light chain of chimeric antibody ch04 (SEQ ID NO.58) (213aa)

[0527] QIVLSQSPAILSASPGETVTMTCRASSSVSYMHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYS

[0528] LTISRVEAEDAATYYCQQWSSTPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

[0529] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0530] (59) Amino acid sequence of the heavy chain of chimeric antibody ch05 (SEQ ID NO.59) (448aa)

[0531] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGTSLEWIGLINPHNGGTNYNQKFKGKAT

[0532] LTLDKSSSSAYMELLSLTSEDSAVYYCAHSAYGSSYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRST

[0533] SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0534] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0535] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0536] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0537] (60) Amino acid sequence of the light chain of chimeric antibody ch05 (SEQ ID NO.60) (219aa)

[0538] DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG

[0539] SGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN

[0540] NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0541] (61) Amino acid sequence of the heavy chain of chimeric antibody ch06 (SEQ ID NO.61) (446aa)

[0542] QVQLQQSGAELARPGASVKMSCKASGYTFTSYTIHWAKQRPGQGLEWIGKINPRSNYNNYNQKFKEKA

[0543] TLSADKSSSTAYMQLSSLTSEDSAVYYCARLGGWGYYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTS

[0544] ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

[0545] NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

[0546] GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY

[0547] TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0548] (62) Amino acid sequence of the light chain of chimeric antibody ch06 (SEQ ID NO.62) (217aa)

[0549] DIVLTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSG

[0550] TDFTLNIHPVEEEDAATYYCQHSRELFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP

[0551] REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0552] (63) Amino acid sequence of the heavy chain of chimeric antibody ch07 (SEQ ID NO.63) (448aa)

[0553] EVQLQQSGPELVRPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGSNYNQKFKGKAT

[0554] LTVDKSSSTAYMDLLSLTSEDSAVYYCAGSAYGSSYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRST

[0555] SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0556] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0557] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0558] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0559] (64) Amino acid sequence of the light chain of chimeric antibody ch07 (SEQ ID NO.64) (219aa)

[0560] DIMMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG

[0561] SGSGTDFTLTISSVQAEDLAVYFCKQSYNLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN

[0562] FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0563] (65) Amino acid sequence of the heavy chain of chimeric antibody ch08 (SEQ ID NO.65) (448aa)

[0564] EVQLQQSGPELVKPGASMKISCKASGYSFTAYTMTWVKQSHGKNLEYIGLFNPYNGGTSYNQKFKGKAT

[0565] LTVDKSSSTAYMELLSLTSEDSAVYYCAISAYGTNYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTS

[0566] ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

[0567] NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

[0568] GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY

[0569] TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0570] (66) Amino acid sequence of the light chain of chimeric antibody ch08 (SEQ ID NO.66) (213aa)

[0571] QVVLSQSPAILSASPGEMVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSY

[0572] SLTISRVEAEDAATYYCQQWSTNPPTFGAGTKLDLKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

[0573] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0574] (67) Amino acid sequence of the ch10 heavy chain of the chimeric antibody (SEQ ID NO.67) (451aa)

[0575] KVQLQQSGAELVKPGASVKLSCKASGYTFTEYIVHWVKQRSGQGLEWIGWFYPGSGTIKYNEKFKDKAT

[0576] LTADKSSNTVYMELSRLTSEDSAVYFCARHEEPGGYYVGRVFAYWGQGTLVTVSAASTKGPSVFPLAPCS

[0577] RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD

[0578] HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW

[0579] YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP

[0580] QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0581] (68) Amino acid sequence of the ch10 light chain of the chimeric antibody (SEQ ID NO.68) (220aa)

[0582] DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSNDQKSYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTG

[0583] SGSGTDFTLTVSSVKAEDLAVYYCHQYYYYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL

[0584] LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0585] (69) Amino acid sequence of the ch12 heavy chain of the chimeric antibody (SEQ ID NO.69) (448aa)

[0586] EVQLQQSGPELVKPGASMKISCKASGYSFTAYTMTWVKQSHGKNLEYIGLINPYNGGTSYNQKFKGKAT

[0587] LTVDKSSSTAYMELLSLTSEDSAVYYCAISAYGTNYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRSTS

[0588] ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

[0589] NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

[0590] GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY

[0591] TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0592] (70) Amino acid sequence of the light chain of chimeric antibody ch12 (SEQ ID NO.70) (219aa)

[0593] DIVMSQSPSSLAVSAGEKVTLSCKSSQSLLNSRTRKNSLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGS

[0594] GSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN

[0595] FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0596] (71) Amino acid sequence of the heavy chain of chimeric antibody ch13 (SEQ ID NO.71) (448aa)

[0597] EVQLQQSGPELVKPGTSMKISCRASGYSFTGYTMNWVKQSHGKNLEWIGLLNPFNGGASYNQKFKGKA

[0598] TLTVDKSSSTAYMELLSLTSEDSAVYYCAGSAYGTNYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRS

[0599] TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK

[0600] PSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0601] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0602] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0603] (72) Amino acid sequence of the chimeric antibody ch13 light chain (SEQ ID NO.72) (213aa)

[0604] QIVLSQSPAILSASPGDKVTMTCRASSSVSYIHWYQQKPGSSPKTWIYATSNLASGVPARFSGSGSGTSYSL

[0605] TISRVEAEDAATYYCQQWTSNPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

[0606] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0607] (73) Amino acid sequence of the chimeric antibody ch15 heavy chain (SEQ ID NO.73) (448aa)

[0608] EVQLQQSGPELVKPRASMKISCRASGYSFTGYTMNWVKQSHGKNLEWIGLINPFNGGATYNQKFKAKAT

[0609] LTVDKSSSTAYMELLSLTSEDSAVYYCAGSAYGSNYWYFDVWGAGTTVTVSSASTKGPSVFPLAPCSRST

[0610] SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0611] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV

[0612] DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV

[0613] YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0614] (74) Amino acid sequence of the light chain of chimeric antibody ch15 (SEQ ID NO.74) (213aa)

[0615] QIVLSQSPAILSASPGDKVTMTCRASSSVNYMHWYQQKSGSSPKTWIYATSNLASGVPARFSGSGSGTSYS

[0616] LTISRVEAEDAATYYCQQWSSTPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

[0617] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0618] (75) Amino acid sequence of the heavy chain of chimeric antibody ch16 (SEQ ID NO.75) (446aa)

[0619] QVQLQQSGAELARPGASVKMSCKASGYTFTSYTIHWVKQRPGQGLEWIGKINPRSDYNYYNQKFKDKA

[0620] TLSADKSSSTAYMQLSSLTSEDSAVYYCARLGGWGYYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTS

[0621] ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

[0622] NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

[0623] GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY

[0624] TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0625] (76) Amino acid sequence of the ch16 light chain of the chimeric antibody (SEQ ID NO.76) (217aa)

[0626] DIVLTQSPASLAVSLGQRATMSCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLDSGVPARFSGSGS

[0627] GTDFTLNIHPVEEEDAATYYCQHSRELFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY

[0628] PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0629] (77) Amino acid sequence of the heavy chain of the positive control antibody YZ-40-13 (SEQ ID NO.77) (450aa)

[0630] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYTMSWARQAPGKGLEWVAYISKGGGSTYYPDTVKGRFTI

[0631] SRDNSKNTLYLQMNSLRAEDSAVYYCARGAMFGNDFFFPMDRWGQGTLVTVSSASTKGPSVFPLAPCSR

[0632] STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0633] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY

[0634] VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ

[0635] VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0636] (78) Amino acid sequence of the light chain of the positive control antibody YZ-40-13 (SEQ ID NO.78) (213aa)

[0637] EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYTTSNLASGIPARFSGSGSGTDFTLT

[0638] ISSLEPEDFAVYYCHQWSSYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. (79) Amino acid sequence of the heavy chain variable region of MM02H-8 (SEQ ID NO.161) (121aa):

[0639] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAHGENLEWIGLINPFNGGTRYNQKFKGRATLTVDKSTSTAYMELRSLRSDDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0640] (80) MM02H-8 heavy chain variable region nucleotide sequence (SEQ ID NO.162) (363 bp):

[0641] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCTCTGTGAAGGTGTCCTGTA

[0642] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAGACAGGCTCATGGAGAGAACTT

[0643] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0644] GCTACCCTGACAGTGGACAAGAGCACCAGCACAGCCTATATGGAACTGAGGTCCCTGAGGTCTGATG

[0645] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0646] (81) MM02H-8 light chain variable region amino acid sequence (SEQ ID NO.163) (106aa):

[0647] DIQLTQSPSSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKTWIYATSNLASGVPSRFSGSGSGTDYT LTISSLQPEDFATYYCQQWTSTPPTFGQGTKLEIK.

[0648] (82) MM02H-8 light chain variable region nucleotide sequence (SEQ ID NO.164) (318 bp):

[0649] GACATCCAACTTACCCAGAGCCCATCCTCCCTGTCTGCCTCTGTGGGAGACAGGGTGACCATCACTTG

[0650] TAGGGCATCCTCCTCTGTGTCCTATATGCACTGGTATCAACAGAAGCCTGGCAAGGCTCCAAAGACCT

[0651] GGATTTATGCCACCAGCAACCTGGCATCTGGAGTGCCAAGCAGGTTCTCTGGCTCTGGCTCTGGCACA

[0652] GACTACACCCTGACCATCTCCTCCCTCCAACCTGAGGACTTTGCCACCTACTACTGTCAACAGTGGACCAGCACACCTCCAACCTTTGGACAAGGCACCAAATTGGAGATTAAG。

[0653] (83) Amino acid sequence of the variable region of the heavy chain of MM02H-20 (SEQ ID NO.165) (121aa):

[0654] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSSSTAYMELFRLRSDDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS。

[0655] (84) Nucleotide sequence of the variable region of the heavy chain of MM02H-20 (SEQ ID NO.166) (363bp):

[0656] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCTCTGTGAAGGTGTCCTGTA

[0657] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAGACAGGCTCCTGGACAAGGATT

[0658] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0659] GCTACCCTGACAGTGGACAAGTCCTCCAGCACAGCCTATATGGAACTGTTCAGACTGAGGTCTGATGA

[0660] CACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0661] (85) MM02H-20 light chain variable region amino acid sequence: same as MM02H-8 (SEQ ID NO.163) (106aa).

[0662] (86) MM02H-20 light chain variable region nucleotide sequence: same as MM02H-8 (SEQ ID NO.164) (318 bp).

[0663] (87) MM02H-24 heavy chain variable region amino acid sequence (SEQ ID NO.167) (121aa):

[0664] QVQLVQSGAEVKKPGASMKVSCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0665] (88) MM02H-24 heavy chain variable region nucleotide sequence (SEQ ID NO.168) (363 bp):

[0666] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGGTGTCCTGTA

[0667] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0668] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0669] GCTACCCTGACAGTGGACAAGAGCATCAGCACAGCCTATATGGAACTGAGCAGACTGAGGTCTGATG

[0670] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0671] (89) MM02H-24 light chain variable region amino acid sequence: same as MM02H-8 (SEQ ID NO.163) (106aa).

[0672] (90) MM02H-24 light chain variable region nucleotide sequence: same as MM02H-8 (SEQ ID NO.164) (318 bp).

[0673] (91) MM02H-25 heavy chain variable region amino acid sequence (SEQ ID NO.169) (121aa):

[0674] QVQLVQSGAEVKKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSISTAYMELSRLRSDDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0675] (92) MM02H-25 heavy chain variable region nucleotide sequence (SEQ ID NO.170) (363 bp):

[0676] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGATTTCCTGTA

[0677] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0678] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0679] GCTACCCTGACAGTGGACAAGAGCATCAGCACAGCCTATATGGAACTGAGCAGACTGAGGTCTGATG

[0680] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0681] (93) MM02H-25 light chain variable region amino acid sequence: same as MM02H-8 (SEQ ID NO.163) (106aa).

[0682] (94) MM02H-25 light chain variable region nucleotide sequence: same as MM02H-8 (SEQ ID NO.164) (318 bp).

[0683] (95) MM02H-30 heavy chain variable region amino acid sequence (SEQ ID NO.171) (121aa):

[0684] QVQLVQSGAEVKKPGASMKVSCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSISTAYMELFRLRSDDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0685] (96) MM02H-30 heavy chain variable region nucleotide sequence (SEQ ID NO.172) (363 bp):

[0686] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGGTGTCCTGTA

[0687] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0688] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0689] GCTACCCTGACAGTGGACAAGAGCATCAGCACAGCCTATATGGAACTGTTCAGACTGAGGTCTGATG

[0690] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0691] (97) MM02H-30 light chain variable region amino acid sequence: same as MM02H-8 (SEQ ID NO.163) (106aa).

[0692] (98) MM02H-30 light chain variable region nucleotide sequence: same as MM02H-8 (SEQ ID NO.164) (318 bp).

[0693] (99) MM02H-53 heavy chain variable region amino acid sequence (SEQ ID NO.173) (121aa):

[0694] QVQLVQSGAEVKKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSSSTAYMELSSLRSEDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0695] (100) MM02H-53 heavy chain variable region nucleotide sequence (SEQ ID NO.174) (363 bp):

[0696] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGATTTCCTGTA

[0697] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0698] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0699] GCTACCCTGACAGTGGACAAGTCCTCCAGCACAGCCTATATGGAACTGTCCTCCCTGAGGTCTGAGG

[0700] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0701] (101) MM02H-53 light chain variable region amino acid sequence (SEQ ID NO.175) (106aa):

[0702] DIQLTQSPSSSLSASVGDRVTMTCRASSSVSYMHWYQQKPGKAPKTWIYATSNLASGVPSRFSGSGSGTEY TLTISSLQPEDFATYYCQQWTSTPPTFGQGTKVEIK.

[0703] (102) MM02H-53 light chain variable region nucleotide sequence (SEQ ID NO.176) (318 bp):

[0704] GACATCCAACTTACCCAGAGCCCATCCTCCCTGTCTGCCTCTGTGGGAGACAGGGTGACAATGACTTG

[0705] TAGGGCATCCTCCTCTGTGTCCTATATGCACTGGTATCAACAGAAGCCTGGCAAGGCTCCAAAGACCT

[0706] GGATTTATGCCACCAGCAACCTGGCATCTGGAGTGCCAAGCAGGTTCTCTGGCTCTGGCTCTGGCACA

[0707] GAATACACCCTGACCATCTCCTCCCTCCAACCTGAGGACTTTGCCACCTACTACTGTCAACAGTGGACCAGCACACCTCCAACCTTTGGACAAGGCACCAAGGTGGAGATTAAG.

[0708] (103) MM02H-56 heavy chain variable region amino acid sequence (SEQ ID NO.177) (121aa):

[0709] QVQLVQSGAEVKKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0710] (104) MM02H-56 heavy chain variable region nucleotide sequence (SEQ ID NO.178) (363 bp):

[0711] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGATTTCCTGTA

[0712] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0713] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0714] GCTACCCTGACAGTGGACAAGAGCACCAGCACAGCCTATATGGAACTGTCCTCCCTGAGGTCTGAGG

[0715] ACACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0716] (105) MM02H-56 light chain variable region amino acid sequence: same as MM02H-53 (SEQ ID NO.175) (106aa).

[0717] (106) MM02H-56 light chain variable region nucleotide sequence: same as MM02H-53 (SEQ ID NO.176) (318 bp).

[0718] (107) MM02H-59 heavy chain variable region amino acid sequence (SEQ ID NO.179) (121aa):

[0719] QVQLVQSGAEVKKPGASMKISCKASGYSFTGYTMNWVKQAPGQGLEWIGLINPFNGGTRYNQKFKGRATLTVDKSSSTAYMELFSLRSEDTAVYYCAGSAYGSDYWYFDVWGQGTMVTVSS.

[0720] (108) MM02H-59 heavy chain variable region nucleotide sequence (SEQ ID NO.180) (363 bp):

[0721] CAGGTCCAACTTGTCCAGTCTGGAGCAGAGGTGAAGAAGCCTGGAGCCAGTATGAAGATTTCCTGTA

[0722] AGGCATCTGGCTACTCCTTCACAGGCTACACAATGAACTGGGTGAAGCAGGCTCCTGGACAAGGATT

[0723] GGAGTGGATTGGACTGATAAACCCATTCAATGGAGGCACCAGATACAACCAGAAGTTCAAGGGCAGG

[0724] GCTACCCTGACAGTGGACAAGTCCTCCAGCACAGCCTATATGGAACTGTTCTCCCTGAGGTCTGAGGA

[0725] CACAGCAGTCTACTACTGTGCTGGCTCTGCCTATGGCTCTGACTACTGGTATTTTGATGTGTGGGGACAAGGCACAATGGTGACAGTGTCCAGT.

[0726] (109) MM02H-59 light chain variable region amino acid sequence: same as MM02H-53 (SEQ ID NO.175) (106aa).

[0727] (110) MM02H-59 light chain variable region nucleotide sequence: same as MM02H-53 (SEQ ID NO.176) (318 bp).

[0728] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0729] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A TrkA-specific binding protein having a TrkA binding domain, wherein the TrkA binding domain is selected from any one of the following TrkA binding domains 1 to 5: In the TrkA binding domain 1, the sequence of CDR-H1 is GYSFT-Xaa1-YT, the sequence of CDR-H2 is Xaa2-NP-Xaa3-NGG-Xaa4, the sequence of CDR-H3 is A-Xaa5-SAYG-Xaa6-Xaa7-YWYFDV, the sequence of CDR-L1 is SSV-Xaa8-Y, the sequence of CDR-L2 is ATS, and the sequence of CDR-L3 is QQW-Xaa9-Xaa10-Xaa11-PPT; Xaa1 is A or G; Xaa2 is I, F or L, Xaa3 is F or Y, Xaa4 is A or T, Xaa5 is I or G, Xaa6 is S or T, Xaa7 is D or N, Xaa8 is S or N, Xaa9 is S or T, Xaa10 is S or T, Xaa11 is T or N; In TrkA binding domain 2, the sequence of CDR-H1 is GYSFT-Xaa12-YT, the sequence of CDR-H2 is INP-Xaa13-NGG-Xaa14, the sequence of CDR-H3 is A-Xaa15-SAYG-Xaa16-Xaa17-YWYFDV, the sequence of CDR-L1 is QSLLNSRTRKN-Xaa18, the sequence of CDR-L2 is WAS, and the sequence of CDR-L3 is KQSYNLFT; wherein Xaa12 is A or G, Xaa13 is H, F or Y, Xaa14 is S or T, Xaa15 is I, G or H, Xaa16 is S or T, Xaa17 is S or N, and Xaa18 is S or Y; In TrkA binding domain 3, the sequence of CDR-H1 is GYTFTSYT, the sequence of CDR-H2 is INPRS-Xaa19-YN, the sequence of CDR-H3 is ARLGGWGYYFDY, the sequence of CDR-L1 is KSVS-Xaa20-SGYSY, the sequence of CDR-L2 is LAS, and the sequence of CDR-L3 is QHSRELFT; among them, Xaa19 is N or D, and Xaa20 is S or T; In TrkA binding domain 4, the sequence of CDR-H1 is shown in SEQ ID NO.124, the sequence of CDR-H2 is shown in SEQ ID NO.125, and the sequence of CDR-H3 is shown in SEQ ID NO.126; the sequence of CDR-L1 is shown in SEQ ID NO.149, the sequence of CDR-L2 is shown in SEQ ID NO.150, and the sequence of CDR-L3 is shown in SEQ ID NO.104; In TrkA binding domain 5, the sequence of CDR-H1 is shown in SEQ ID NO.140, the sequence of CDR-H2 is shown in SEQ ID NO.141, the sequence of CDR-H3 is shown in SEQ ID NO.142, the sequence of CDR-L1 is shown in SEQ ID NO.157, the sequence of CDR-L2 is shown in SEQ ID NO.154, and the sequence of CDR-L3 is shown in SEQ ID NO.

117.

2. The TrkA-specific binding protein according to claim 1, which has any one of the following TrkA binding domains: TrkA binding domain 1-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.107; TrkA binding domain 1-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.128, CDR-H3 shown in sequence SEQ ID NO.129, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108; TrkA binding domain 1-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.138, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.115; TrkA binding domain 1-4 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.144, CDR-H3 shown in sequence SEQ ID NO.145, CDR-L1 shown in sequence SEQ ID NO.151, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.120; TrkA binding domains 1-5 have CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.146, CDR-H3 shown in sequence SEQ ID NO.147, CDR-L1 shown in sequence SEQ ID NO.159, CDR-L2 shown in sequence SEQ ID NO.152, and CDR-L3 shown in sequence SEQ ID NO.108; TrkA binding domain 2-1 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.130, CDR-H3 shown in sequence SEQ ID NO.131, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111; TrkA binding domain 2-2 has CDR-H1 shown in sequence SEQ ID NO.127, CDR-H2 shown in sequence SEQ ID NO.135, CDR-H3 shown in sequence SEQ ID NO.136, CDR-L1 shown in sequence SEQ ID NO.153, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111; TrkA binding domain 2-3 has CDR-H1 shown in sequence SEQ ID NO.137, CDR-H2 shown in sequence SEQ ID NO.143, CDR-H3 shown in sequence SEQ ID NO.139, CDR-L1 shown in sequence SEQ ID NO.158, CDR-L2 shown in sequence SEQ ID NO.154, and CDR-L3 shown in sequence SEQ ID NO.111; TrkA binding domain 3-1 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.133, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.155, CDR-L2 shown in sequence SEQ ID NO.156, and CDR-L3 shown in sequence SEQ ID NO.114; TrkA binding domain 3-2 has CDR-H1 shown in sequence SEQ ID NO.132, CDR-H2 shown in sequence SEQ ID NO.148, CDR-H3 shown in sequence SEQ ID NO.134, CDR-L1 shown in sequence SEQ ID NO.160, CDR-L2 shown in sequence SEQ ID NO.156 and CDR-L3 shown in sequence SEQ ID NO.

114.

3. The TrkA-specific binding protein according to claim 1 or 2, which is an antibody, an antigen-binding fragment of an antibody, or a small modular immune drug; Optionally, the binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a scFv fragment, a biclonal antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an intrabody, a nanobody, a binding domain immunoglobulin fusion protein or a small module immune drug.

4. The TrkA-specific binding protein according to claim 1 or 2, wherein the species of the framework regions of the heavy chain variable region and the light chain variable region are independently mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, alpaca, donkey, deer, mink, chicken, duck or goose.

5. The TrkA-specific binding protein according to claim 4, wherein the heavy chain variable region is shown in any one of the sequences SEQ ID NO.1, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.13, SEQ ID NO.17, SEQ ID NO.21, SEQ ID NO.25, SEQ ID NO.29, SEQ ID NO.33, SEQ ID NO.37, SEQ ID NO.41 and SEQ ID NO.

45.

6. The TrkA-specific binding protein according to claim 4, wherein the light chain variable region is as shown in any one of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.11, SEQ ID NO.15, SEQ ID NO.19, SEQ ID NO.23, SEQ ID NO.27, SEQ ID NO.31, SEQ ID NO.35, SEQ ID NO.39, SEQ ID NO.43 and SEQ ID NO.

47.

7. The TrkA-specific binding protein according to any one of claims 1 to 2 and 5 to 6, further comprising an antibody constant region; Optionally, the sequence of the antibody constant region is selected from the sequence of any one of the constant regions of IgG, IgA, IgM, IgE and IgD; Optionally, in the antibody constant region, the species of the light chain constant region and the heavy chain constant region are independently mouse, human, cow, horse, pig, sheep, dog, cat, rabbit, camel, alpaca, donkey, deer, mink, chicken, duck or goose; Optionally, the type of the light chain constant region in the antibody constant region is κ type or λ type.

8. The TrkA-specific binding protein according to claim 7, whose heavy chain is shown in any one of the sequences SEQ ID NO.53, SEQ ID NO.55, SEQ ID NO.57, SEQ ID NO.59, SEQ ID NO.61, SEQ ID NO.63, SEQ ID NO.65, SEQ ID NO.67, SEQ ID NO.69, SEQ ID NO.71, SEQ ID NO.73 and SEQ ID NO.

75.

9. The TrkA-specific binding protein according to claim 7, wherein the light chain is represented by any one of SEQ ID NO.54, SEQ ID NO.56, SEQ ID NO.58, SEQ ID NO.60, SEQ ID NO.62, SEQ ID NO.64, SEQ ID NO.66, SEQ ID NO.68, SEQ ID NO.70, SEQ ID NO.72, SEQ ID NO.74 and SEQ ID NO.

76.

10. A nucleic acid molecule encoding the TrkA-specific binding protein according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid molecule of claim 10; Optionally, the vector is selected from mammalian cell virus, bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or a combination thereof.

12. A cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11; Optionally, the cell is a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

13. A method for preparing a TrkA-specific binding protein according to any one of claims 1 to 9, comprising: Cultivating the cell according to claim 12, and isolating the TrkA-specific binding protein from the obtained culture.

14. A pharmaceutical composition comprising the TrkA-specific binding protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the expression vector according to claim 11 or the cell according to claim 12, and a pharmaceutically acceptable carrier.

15. Use of the TrkA-specific binding protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11 or the cell according to claim 12 in the preparation of a drug for treating a disease; The disease treated by the drug is pain, neuroma or neuronal disease; Optionally, the pain is acute pain; Optionally, the pain is chronic pain; Optionally, the pain is related to any one of: inflammatory pain, postoperative pain, neuropathic pain and cancer pain; Optionally, the pain may be associated with any of the following: pancreatitis pain, kidney stone pain, endometriosis pain, IBD pain, postoperative adhesion pain, gallstone pain, headache, dysmenorrhea, musculoskeletal pain, sprain pain, visceral pain, ovarian cyst pain, prostatitis pain, cystitis pain, interstitial cystitis pain, postoperative pain, migraine, trigeminal neuralgia, burn and / or wound pain, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis pain, osteoarthritis pain, ankylosing spondylitis pain, periarticular lesion pain, tumor-related pain, chemotherapy-induced perineurial lesion pain, pain from tumor bone metastasis and HIV infection pain.

16. Use of the TrkA specific binding protein according to any one of claims 1 to 9, the nucleic acid molecule according to claim 10, the vector according to claim 11 or the cell according to claim 12 in preparing a TrkA detection product.

17. A TrkA detection product comprising the TrkA specific binding protein according to any one of claims 1 to 9; Optionally, the TrkA product is a detection reagent, a diagnostic reagent, a test strip, a detection plate or a test kit.

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