Anti-trka antibody or antigen-binding fragment thereof, preparation method thereof, and application thereof

The present invention provides an anti-TrkA antibody or an antigen-binding fragment thereof, a preparation method, and an application thereof. The present invention also provides an isolated polynucleotide encoding the anti-TrkA antibody or the antigen-binding fragments thereof, and a vector comprising the isolated polynucleotide. The present invention also provides a use of the antibody or the antigen-binding fragment thereof in the preparation of a drug for treating pain.

1. 11. An antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region VH and a light chain variable region VL: the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, and VH CDR3 as shown in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 25, VL CDR2 as shown in SEQ ID NO: 26, and VL CDR3 as shown in SEQ ID NO: 27; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 4, VH CDR2 as shown in SEQ ID NO: 5, and VH CDR3 as shown in SEQ ID NO: 6; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 28, VL CDR2 as shown in SEQ ID NO: 29, and VL CDR3 as shown in SEQ ID NO: 30; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 7, VH CDR2 as shown in SEQ ID NO: 8, and VH CDR3 as shown in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 31, VL CDR2 as shown in SEQ ID NO: 32, and VL CDR3 as shown in SEQ ID NO: 33; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 10, VH CDR2 as shown in SEQ ID NO: 11, and VH CDR3 as shown in SEQ ID NO: 12; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 34, VL CDR2 as shown in SEQ ID NO: 35, and VL CDR3 as shown in SEQ ID NO: 36; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:13, VH CDR2 as shown in SEQ ID NO:14 and VH CDR3 as shown in SEQ ID NO: 15, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 37, VL CDR2 as shown in SEQ ID NO: 38, and VL CDR3 as shown in SEQ ID NO: 39; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:16, VH CDR2 as shown in SEQ ID NO:17 and VH CDR3 as shown in SEQ ID NO: 18, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 40, VL CDR2 as shown in SEQ ID NO: 41, and VL CDR3 as shown in SEQ ID NO: 42; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:19, VH CDR2 as shown in SEQ ID NO:20 and VH CDR3 as shown in SEQ ID NO: 21, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 43, VL CDR2 as shown in SEQ ID NO: 44, and VL CDR3 as shown in SEQ ID NO: 45; or the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 22, VH CDR2 as shown in SEQ ID NO: 23, and VH CDR3 as shown in SEQ ID NO: 24, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 46, VL CDR2 as shown in SEQ ID NO: 47, and VL CDR3 as shown in SEQ ID NO: 48.

RELATED APPLICATIONS
The present application is a U.S. National Phase of International Application Number PCT/CN2022/077414, filed Feb. 23, 2022, and claims priority to Chinese Application Number 202110222357.4, filed Feb. 28, 2021.
INCORPORATION BY REFERENCE
The sequence listing provided in the file entitled S220019_IPUS_Sequence_Listing_Rev1.txt, which is an ASCII text file that was created on Jan. 5, 2023, and which comprises 52,809 bytes, is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention belongs to the technical field of biological immunity, and in particular relates to anti-TrkA antibodies or antigen-binding fragments thereof capable of specifically binding to human Tropomyosin Receptor Kinase A. The present invention also relates to preparation methods and uses of the antibodies or antigen-binding fragments thereof.
BACKGROUND OF THE INVENTION
Tropomyosin Receptor Kinase (TrkA, also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, or TRK1-transforming tyrosine kinase protein) is a member of Tropomyosin Receptor Kinase (Trk) family, which is encoded by NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1) gene. The Trk family includes three receptors, namely TrkA, TrkB and TrkC, each exerting different biological roles by binding to specific neurotrophin ligands. Trk receptors are transmembrane receptors, consisting of an extracellular region containing a ligand-binding domain, a transmembrane domain (TM), and an intracellular domain containing a tyrosine kinase domain. TrkA is predominantly expressed in neural crest neurons, sympathetic neurons, and cholinergic neurons within the basal forebrain and striatum (Holtzman D M et al. (1992) Neuron 9(3): 465-78; Verge V M et al. (1992) J. Neurosci. 12 (10): 4011-22), and it is also expressed in some non-neuronal tissues and cells including B lymphocytes (Torcia M et al. (1996) Cell 85(3): 345-56). TrkA selectively binds to Nerve Growth Factor (NGF) and is a high-affinity receptor for NGF. NGF binding to TrkA activates TrkA kinase activity, resulting in activation of multiple signaling pathways, including Ras/MAPK, PI3K/Akt, and PLCγ pathway. In addition to TrkA, NGF also binds to the p75 common neurotrophin receptor (p75 neurotrophin receptor, p75NTR, also known as the “low-affinity” NGF receptor).
The NGF/TrkA signaling pathway is involved in cell differentiation, proliferation, survival and pain. Among them, NGF/TrkA is related to the occurrence of pain and peripheral nociceptive sensitization. The activation of NGF/TrkA pathway can increase the phosphorylation of Transient Receptor Potential Vanilloid subtype 1 (TRPV1), thereby making it sensitive. Further, NGF/TrkA can increase the expression of multiple proteins, including TRPV1, Na/Ca/K ion channels, CGRP, substance P, and Brain-Derived Neurotrophic Factor (BDNF), etc., these proteins subsequently sensitize nociceptive neurons, increase nociception signals, which are transmitted from the dorsal root ganglia to the center to promote the activation of secondary neurons in the central nervous system (Denk F et al. (2017) Annu. Rev. Neurosci. 40: 307-325). In the injured and inflammatory tissues, NGF is highly expressed, and the activation of nociceptive neurons by TrkA is triggered by multiple mechanisms, resulting in pain signals. Association between the NGF/TrkA signaling pathway and pain has been demonstrated in animal models: after injected with NGF, thermal stimulus-induced paw withdrawal latency in rats was significantly shortened (Lewin et al. (1994) Eur J Neurosci, 6: 1903-1912); while the pain was suppressed by blocking the NGF/TrkA signal by administering the rats anti-NGF antibodies, TrkA-IgG, small molecule TrkA inhibitors, etc. (Woolf C J et al. (1994) Neuroscience 62: 327-331; McMahon S B et al. (1995) Net. Med. 1: 774-780; Koltzenburg M et al. (1999) Eur. J. Neurosci. 11: 1698-1704; Bagal S K et al. (2019) J Med Chem. 62(1): 247-265). In humans, NGF levels were elevated in patients with pain such as rheumatoid arthritis, interstitial cystitis, pancreatitis, prostatitis, diabetic neuropathy, and cancer pain (Aloe et al. (1992) Arthritis and Rheumatism 35: 351-355; Mantyh P W et al. (2011) Anesthesiology 115(1): 189-204). After injected with NGF, healthy persons would suffer from hyperalgesia and local pain (Petty et al. (1994) Ann Neurol. 36: 244-246), suggesting that the increased level of NGF is required for hyperalgesia. Homozygous missense mutations occurring in the NGF R gene can cause HSANS symptoms in humans, such patients were insensitive to pain, cold, and heat (Larsson et al. (2009) Neurobiol Dis, 33: 221-228). A genetic study on patients with Congenital Insensitivity to Pain with Anhidrosis (CIPA) shows that mutations within the extracellular or intracellular domain of TrkA gene (NTRK1) can cause CIPA, in such patients TrkA can not be activated by NGF, resulting in loss of pain perception in the patients (Mardy S et al. (2001) Human mutation 18: 462-471).
Globally, tens of millions of patients are suffering from chronic pain, and this number continues to increase as the population increases. Currently, agents clinically used for the treatment of chronic pain include non-steroidal anti-inflammatory agents, anticonvulsants, opioids, etc. However, these agents have many disadvantages. Among them, the non-steroidal anti-inflammatory agents have limited efficacy, and have side effects including gastrointestinal bleeding and kidney toxicity; while opioids have side effects such as addiction. Only less than 30% of chronic pain patients benefit from existing treatments (Kalso E et al. (2004) Pain 112(3): 372-80). There is an urgent need for non-toxic and abuse-free non-opioid analgesics that can relieve pain in the field. As a new analgesic mechanism, NGF/TrkA provides a possibility to solve this problem. So far, several anti-human NGF antibodies are in the R&D or clinical development stage. Clinical trials have shown that NGF antibodies have a strong and wide-ranging analgesic effect on joint pain associated with degenerative joint disease, on chronic low back pain, and on bladder pain associated with interstitial cystitis (Lane N E et al. (2010) N Engl J Med 363: 1521-1531. On the other hand, clinical trials of multiple NGF antibodies have also shown that NGF antibodies increase the risk of accelerated progression of osteoarthritis in patients (Thomas J S et al. (2019) JAMA 322: 37-48), and these NGF antibodies may face problems such as being restricted to severely ill people, not for a long-term use, and dose limitations, making the clinical application of NGF antibodies need further safety verification.
TrkA-targeting antibodies are theoretically better options for treatment than NGF inhibitors, since TrkA antibodies do not affect the binding of NGF to p75 receptor, the function of which is related to neuron development, osteoblast differentiation, proliferation, myoblast differentiation, muscle repair, etc. (Akiyama Y et al. (2014) Differentiation, 87: 111-118; Deponti et al. (2009) Molecular Biology of the Cell, 20: 3620-3627; Mikami et al. (2012) Differentiation, 84: 392-399). TrkA antibodies have superior performance in terms of safety.
Based on this, there is currently a need for TrkA antibodies with higher affinity and stronger specificity.
SUMMARY OF THE INVENTION
Based on the deficiency of the prior art, the main purpose of the present invention is to provide an anti-TrkA antibody with higher affinity and stronger specificity. The invention also provides methods for preparing the antibodies and uses thereof. The anti-TrkA antibodies of the invention are useful for the treatment of pain, including inflammatory pain, postoperative pain, neuropathic pain, cancer pain, osteoarthritis, etc. Compared with the prior art, the anti-TrkA antibodies or antigen binding fragments thereof developed in the invention have higher affinities, better specificities and stronger activities.
In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:25, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:27, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 1, VH CDR2 as shown in SEQ ID NO: 2, and VH CDR3 as shown in SEQ ID NO: 3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 25, VL CDR2 as shown in SEQ ID NO: 26, and VL CDR3 as shown in SEQ ID NO: 27.
    
    


In one aspect, the present invention provides an anti-TrkA antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:28, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:30, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 4, VH CDR2 as shown in SEQ ID NO: 5, and VH CDR3 as shown in SEQ ID NO: 6; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 28, VL CDR2 as shown in SEQ ID NO: 29, and VL CDR3 as shown in SEQ ID NO: 30.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:8, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:31, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:32, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:33, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 7, VH CDR2 as shown in SEQ ID NO: 8, and VH CDR3 as shown in SEQ ID NO: 9; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 31, VL CDR2 as shown in SEQ ID NO: 32, and VL CDR3 as shown in SEQ ID NO: 33.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:11, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:34, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:35, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:36, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 10, VH CDR2 as shown in SEQ ID NO: 11, and VH CDR3 as shown in SEQ ID NO: 12; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 34, VL CDR2 as shown in SEQ ID NO: 35, and VL CDR3 as shown in SEQ ID NO: 36.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:15, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO: 37, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:38, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:39, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:13, VH CDR2 as shown in SEQ ID NO:14 and VH CDR3 as shown in SEQ ID NO:15, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 37, VL CDR2 as shown in SEQ ID NO: 38, and VL CDR3 as shown in SEQ ID NO: 39.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:16, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:17, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:18, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:40, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:41, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:42, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:16, VH CDR2 as shown in SEQ ID NO:17 and VH CDR3 as shown in SEQ ID NO:18, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 40, VL CDR2 as shown in SEQ ID NO: 41, and VL CDR3 as shown in SEQ ID NO: 42.
    
    


In one aspect, the present invention provides an anti-TrkA antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:20, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:21, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:43, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:44, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO:19, VH CDR2 as shown in SEQ ID NO:20 and VH CDR3 as shown in SEQ ID NO:21, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 43, VL CDR2 as shown in SEQ ID NO: 44, and VL CDR3 as shown in SEQ ID NO: 45.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising:

    
    
        (a) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs):
        (i) VH CDR1 consisting of the following sequence: SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (ii) VH CDR2 consisting of the following sequence: SEQ ID NO:23, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (iii) VH CDR3 consisting of the following sequence: SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        and/or
        (b) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs):
        (iv) VL CDR1 consisting of the following sequence: SEQ ID NO:46, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same,
        (v) VL CDR2 consisting of the following sequence: SEQ ID NO:47, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same, and
        (vi) VL CDR3 consisting of the following sequence: SEQ ID NO:48, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the same;
        preferably, the substitution(s) described in any one from (i) to (vi) is (are) conservative substitution(s);
        preferably, the VH of the antibody or antigen-binding fragment thereof comprises: VH CDR1 as shown in SEQ ID NO: 22, VH CDR2 as shown in SEQ ID NO: 23, and VH CDR3 as shown in SEQ ID NO: 24, and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 46, VL CDR2 as shown in SEQ ID NO: 47, and VL CDR3 as shown in SEQ ID NO: 48.
    
    


In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to TrkA, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:

    
    
        the heavy chain variable region comprises three CDRs comprised in the heavy chain variable region as shown in any one of SEQ ID NOs: 49-56; and the light chain variable region comprises three CDRs comprised in the light chain variable region as shown in any one of SEQ ID NOs: 57-64;
        preferably, the three CDRs comprised in the heavy chain variable region and/or the three CDRs comprised in the light chain variable region are defined by the Kabat or IMGT numbering system.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 49;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:49; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:49;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:57;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:57; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:57;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: VH having sequence as shown in SEQ ID NO:49 and VL having sequence as shown in SEQ ID NO:57.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 50;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:50; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:50;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:58;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:58; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:58;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 50 and a VL having sequence as shown in SEQ ID NO: 58.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 51;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:51; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:51;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:59;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:59; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:59;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO:51 and a VL having sequence as shown in SEQ ID NO:59.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 52;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:52; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:52;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:60;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:60; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:60;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 52 and a VL having sequence as shown in SEQ ID NO: 60.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 53;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:53; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:53;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:61;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:61; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:61;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 53 and a VL having sequence as shown in SEQ ID NO: 61.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 54;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:54; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:54;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:62;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:62; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:62;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 54 and a VL having sequence as shown in SEQ ID NO: 62.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 55;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:55; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:55;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:63;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:63; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:63;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 55 and a VL having sequence as shown in SEQ ID NO: 63.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in SEQ ID NO: 56;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:56; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:56;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in SEQ ID NO:64;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to SEQ ID NO:64; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO:64;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in SEQ ID NO: 56 and a VL having sequence as shown in SEQ ID NO: 64.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof further comprises:

    
    
        (a) a heavy chain constant region (CH) of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; and
        (b) a light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having up to 20 conservative amino acid substitutions (e.g. up to 15, up to 10, or up to 5 amino acid conservative substitutions; e.g. 1, 2, 3, 4 or 5 conservative amino acid substitutions) compared to the sequence from which it is derived;
        preferably, the heavy chain constant region is IgG heavy chain constant region, such as IgG1, IgG2, IgG3 or IgG4 heavy chain constant region;
        preferably, the light chain constant region is kappa light chain constant region.
    
    


The antibody or antigen-binding fragment thereof according to the present invention, wherein the antigen-binding fragment is selected from Fab, Fab′, (Fab′)2, Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb); and/or, the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.
The antibody or antigen-binding fragment thereof according to the present invention, wherein the humanized antibody comprises:

    
    
        (a) a heavy chain variable region (VH) comprising amino acid sequence selected from:
        (i) sequence as shown in any one of SEQ ID NOs: 81, 85, 89, 97, 99 and 101;
        (ii) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence as shown in any one of SEQ ID NOs: 81, 85, 89, 97, 99 and 101; or
        (iii) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in any one of SEQ ID NOs: 81, 85, 89, 97, 99 and 101;
        and/or,
        (b) a light chain variable region (VL) comprising amino acid sequence selected from:
        (iv) sequence as shown in any one of SEQ ID NOs: 83, 87, 91, 93 and 95;
        (v) sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence as shown in any one of SEQ ID NOs: 83, 87, 91, 93 and 95; or
        (vi) sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence as shown in any one of SEQ ID NOs: 83, 87, 91, 93 and 95;
        preferably, the substitution(s) described in (ii) or (v) is (are) conservative substitution(s);
        preferably, the antibody or antigen-binding fragment thereof comprises: a VH having sequence as shown in any one of SEQ ID NOs: 81, 85, 89, 97, 99 and 101, and a VL having sequence as shown in any one of SEQ ID NOs: 83, 87, 91, 93 and 95.
    
    


More preferably, the humanized antibody is selected from 42F5-01, 42F5-03, 42F5-04, 42F5-05, 42F5-08, 42F5-11, 42F5-13, 42F5-14 or 42F5-15.
The antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof is labeled with a detectable marker, such as enzyme (e.g. horseradish peroxidase), radioisotope, fluorescent dye, luminescent substance (such as chemiluminescent substance), or biotin.
In another aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, or the heavy chain variable region and/or light chain variable region thereof;

    
    
        preferably, the polynucleotide comprises nucleotide coding sequence as shown in any one of SEQ ID NOs: 65-80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 and 102.
    
    


The present invention also provides a vector comprising the isolated nucleic acid molecule; preferably, the vector is a cloning vector or an expression vector.
The present invention also provides a host cell comprising the isolated nucleic acid molecule or the vector.
The present invention also provides a method for preparing the antibody or antigen-binding fragment thereof, comprising, culturing the host cell under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture;

    
    
        preferably, the host cell is a mammalian cell, more preferably a human, murine, sheep, horse, dog or cat cell, further preferably a Chinese hamster ovary cell.
    
    


The present invention also provides a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof;

    
    
        preferably, the bispecific or multispecific molecule specifically binds to TrkA, and additionally specifically binds to one or more other targets;
        preferably, the bispecific or multispecific molecule at least further comprises a second specifically binding molecule (e.g., a second antibody) for a second target.
    
    


The present invention also provides a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule, and a pharmaceutically acceptable carrier and/or excipient. The present invention further provides the use of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the pharmaceutical composition or the host cell in the preparation of a medicament for the treatment of various conditions or diseases.
The present invention also provides a method for treating various conditions or diseases, including administering to a subject in need (suitably a mammalian subject, particularly a human subject) a therapeutically effective amount of the antibody or antigen-binding fragment thereof, or the bispecific or multispecific molecule, or the pharmaceutical composition or the host cell. The condition or disease is pain, preferably chronic pain or acute pain, more preferably chronic pain; further the pain may be associated with any of the following: inflammatory pain, postoperative pain, neuropathic pain, cancer pain, etc.; more preferably, the pain may be associated with any of the following: pain in pancreatitis, pain in nephrolithiasis, pain in endometriosis, pain in IBD, pain in postoperative adhesion, pain in gallstone, headache, dysmenorrhea, musculoskeletal pain, pain in sprain, visceral pain, pain in ovarian cyst, pain in prostatitis, pain in cystitis, pain in interstitial cystitis, postoperative pain, migraine, trigeminal neuralgia, burn and/or wound pain, trauma-related pain, neuropathic pain, pain associated with musculoskeletal disease, pain in rheumatoid arthritis, pain in osteoarthritis, pain in ankylosing spondylitis, periarticular pathological pain, tumor pain, pain due to bone metastases, pain in HIV infection. The condition or disease is neuroma or neuronal disorder. The antibodies provided by the present invention for use as medicaments have stronger binding ability and specificity compared with the existing antibodies.



DESCRIPTION OF THE DRAWINGS
Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:
FIG. 1 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to human TrkA protein.
FIG. 2 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to the extracellular region of TrkA protein (192-402).
FIG. 3 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to monkey TrkA protein.
FIG. 4 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to mouse TrkA protein.
FIG. 5 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to rat TrkA protein.
FIG. 6 shows the FACS results of the binding of the anti-TrkA antibodies of the present invention to CHO cells expressing human TrkA protein.
FIG. 7 is a schematic diagram showing efficiencies of the anti-TrkA antibodies of the present invention and of the prior art to block the binding of human TrkA to ligand NGF.
FIG. 8 is a schematic diagram showing efficiencies of the anti-TrkA antibodies of the present invention and of the prior art to inhibit NGF-induced TF-1 cell proliferation.
FIG. 9 is a schematic diagram showing efficiencies of the anti-TrkA antibodies of the present invention and of the prior art to inhibit NGF-induced TrkA/Ba/F3 cell proliferation.
FIG. 10 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to human TrkA.
FIG. 11 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to human TrkB.
FIG. 12 shows the ELISA results of the binding of the anti-TrkA antibodies of the present invention to human TrkC.
FIG. 13 shows the basal pain threshold of the CFA-induced inflammatory mouse model, which was measured by VonFrey before the injection of the modeling agent CFA into the planta.
FIG. 14 shows the pain threshold (i.e., pre-dose pain threshold) of the CFA-induced inflammatory mouse model about 24 hours after the injection of the modeling agent CFA into the planta.
FIG. 15 shows the pain threshold of the CFA-induced inflammatory mouse model 24 hours after being treated with the anti-TrkA antibodies 33H5 and 42F5 of the present invention, wherein *P<0.05 v.s. normal saline; ***P<0.001 v.s. normal saline.
FIG. 16 shows the pain threshold of the CFA-induced inflammatory mouse model 48 hours after being treated with the anti-TrkA antibodies 33H5 and 42F5 of the present invention, wherein *P<0.05 v.s. normal saline; ***P<0.001 v.s. normal saline.
FIG. 17 shows the ELISA results of the binding of the humanized anti-TrkA antibodies of the present invention to human TrkA.
FIG. 18 shows the FACS results of the binding of the humanized anti-TrkA antibodies of the present invention to CHO cells expressing human TrkA, TrkB or TrkC protein.
FIG. 19 shows the results of the binding of the humanized anti-TrkA antibodies of the present invention to CHO cells expressing human TrkA protein.
FIG. 20 shows efficiencies of the humanized anti-TrkA antibodies of the present invention to inhibit NGF-induced TF-1 cell proliferation.
FIG. 21 shows efficiencies of the humanized anti-TrkA antibodies of the present invention to inhibit NGF-induced TrkA/Ba/F3 cell proliferation.



DETAILED DESCRIPTION OF THE INVENTION
Example 1: Preparation of Murine Anti-TrkA Antibodies
As an immunogen, recombinant human TrkA protein containing murine Fc tag (Beijing ACROBiosystems Co., Ltd. TRA-H5254) was mixed and emulsified with an equal volume of Freund's complete adjuvant (Sigma-Alderich, F5881) for initial immunization. Ten of each of 6-week-old BALB/c and C57 mice (Jiangsu Huafukang) were prepared, and each animal was injected subcutaneously with 50 μg Immunogen (excluding the weight of the adjuvant, similarly hereafter). The immunogen was mixed and emulsified with incomplete Freund's adjuvant (Sigma-Alderich, F5506) for subsequent booster immunizations. Two weeks after the initial immunization, each animal was injected intraperitoneally with 25 μg immunogen for the first booster immunization; 2 weeks later, each animal was injected subcutaneously with 25 μg immunogen for the second booster immunization. 4-5 weeks later, 25 μg immunogen was injected intraperitoneally for the last immunization shock.
Mouse B cells were separated after the last immunization, mixed with SP2/0 cells (Cell Bank of Chinese Academy of Sciences, TCM18), and fused according to the operation manual available from Electroporator, BTX. The fusion cells were cultured, and then screened for hybridoma cells capable of binding to TrkA and of inhibiting human TrkA to bind to ligand NGF by Enzyme Linked Immunosorbent Assay (ELISA). Subcloning was performed by limiting dilution method, and eight positive hybridoma monoclonal cell lines were obtained by screening with the same ELISA method, named 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42A11 respectively.
The hybridoma monoclonal cell lines were expanded and cultured with serum-free medium, and the medium was pooled and purified by protein G column to obtain murine anti-human TrkA monoclonal antibodies 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42A11.
Example 2: ELISA Detection of the Binding of Murine Anti-TrkA Antibodies to Human TrkA
The binding abilities of the anti-TrkA antibodies were detected by using a TrkA extracellular region (33-417) protein comprising human Fc (Beijing ACROBiosystems Co., Ltd., TRA-H5259). Each well of a 96-well microtiter plate was coated with 50 ng of human TrkA, washed and blocked, and then added with serially diluted antibodies, and incubated at room temperature for 1 hour. After washing three times, horseradish peroxidase-conjugated goat anti-mouse Fc antibody (Biolegend, 405306) was added and incubated at room temperature for 1 hour. After washing three time, tetramethylbenzidine (TMB, Biolegend, 421101) was added for color development. 1M HCl was used to terminate the color development, and the absorbance values were read at 450 nm with a microplate reader.
All of the anti-TrkA antibodies secreted by the eight hybridomas bound to human TrkA extracellular region in a dose-dependent manner (FIG. 1), and the EC50 values of the eight antibodies binding to human TrkA are shown in Table 1.
Next, the binding abilities of the anti-TrkA antibodies were detected with a fusion protein (fused to human Fc) containing only the portion of human TrkA extracellular region that binds to the ligand NGF (TrkA 192-402) (Beijing ACROBiosystems Co., Ltd., TRA-H5258). ELISA results show that the anti-TrkA antibodies bind to the amino acid region 192-402 of the TrkA extracellular region, as shown in FIG. 2, and the EC50 is shown in Table 2.
In addition, the species cross-reactivities of anti-TrkA antibodies were detected with monkey TrkA (Hangzhou Haoyang Biotechnology Co., Ltd., HSP037-05), rat TrkA (Beijing Yiqiao Shenzhou Technology Co., Ltd., 80243-R03H), mouse TrkA (Beijing Yiqiao Shenzhou Technology Co., Ltd., 51103-M02H) extracellular region fusion protein (fused to human Fc). The ELISA results show that all of the eight anti-TrkA antibodies can strongly bind to TrkA proteins of the three other species. The results are shown in FIGS. 3-5.







TABLE 1







EC50 values of the eight anti-TrkA antibodies binding to human TrkA (33-417)















Antibodies
5A3
11G8
26E9
33H5
40D6
42F5
42H6
42A11





EC50 (nM)
0.242
0.114
0.099
0.204
0.202
0.128
0.135
0.187
















TABLE 2







EC50 values of the eight anti-TrkA antibodies binding to human TrkA (192-402)















Antibodies
5A3
11G8
26E9
33H5
40D6
42F5
42H6
42A11





EC50 (nM)
0.154
0.071
0.068
0.173
0.195
0.134
0.175
0.234









Example 3: Affinity Assay of Murine Anti-TrkA Antibodies Binding to Human TrkA
The affinities of the antibodies were detected with Biomolecular Interaction Detection Platform (ForteBio Octet Red96 (PALL)).
Human TrkA extracellular region (33-417) protein containing human Fc tag was fixed onto a chip by using an anti-human Fc capture sensor (Fortebio, 18-5088), and then bound to gradient concentrations of anti-TrkA antibody. Buffer (1×Kinetics Buffer: PBS+0.1% BSA+0.05% Tween20) was added for dissociation, and finally the affinity kinetic constants of antigen-antibody binding were calculated by instrument algorithm. The results are shown in Table 3.







TABLE 3







Affinities of anti-TrkA antibodies binding to human TrkA












Antibodies
KD (M)
kon (1/Ms)
kdis (1/s)







5A3
1.55E−11
1.21E+06
1.88E−05



11G8
<1.0E−12
9.92E+05
<1.0E−07



26E9
2.67E−11
9.23E+05
2.46E−05



33H5
1.62E−11
1.29E+06
2.09E−05



40D6
2.71E−11
9.18E+05
2.49E−05



42F5
2.18E−12
9.47E+05
2.07E−06



42H6
6.77E−12
1.47E+06
9.91E−06



42A11
1.42E−12
8.89E+05
1.26E−06










Example 4: Detection of Murine Anti-TrkA Antibodies Binding to TrkA/CHO Cells by Flow Cytometry
The binding of anti-TrkA antibodies to CHO-K1 cells expressing full-length TrkA (TrkA/CHO) was detected by flow cytometry (FACS). TrkA/CHO cells were cultured in F12K medium (Hyclone, SH30026) containing 10% FBS and 5 μg/mL puromycin, and the negative control cells CHO-K1 were cultured in F12K medium containing 10% FBS. Cells were collected during the logarithmic growth phase, resuspended in PBS containing 2% FBS, and added to each well of a 96-well plate at 105 cells (50 μl). 50 μl of 20 μg/mL anti-TrkA antibody, or mouse IgG control was added, and reacted at room temperature for 1 hour. 1 μg/mL Alexa Flour 647-labeled goat anti-mouse IgG antibody (Jackson, 115-605-062) was added and incubated at room temperature for 30 minutes. The binding of antibodies was detected by flow cytometry (BioRad, ZE5). As shown in FIG. 6, all of the eight anti-TrkA antibodies bind to TrkA/CHO cells, but not to CHO-K1 cells; the control antibody mouse IgG neither binds to CHO-K1 nor to TrkA/CHO.
Example 5: Murine Anti-TrkA Antibodies Block the Binding of Human TrkA to the Ligand NGF
A control antibody BXhVH5VL1 was synthesized based on the sequence information provided by Chinese patent application CN101939337A, with reference to the optimal antibody BXhVH5VL1 therein, having the heavy chain variable region of (SEQ ID NO: 5) and the light chain variable region of (SEQ ID NO: 7).
Each well of a 96-well microtiter plate was coated with 50 ng of TrkA extracellular region (33-417) protein fused to human Fc, washed three times and blocked with 3% BSA for 1 hour. 10,000 ng/mL (66.67 nM) anti-TrkA antibody was 3-fold serially diluted up to 10 concentrations, until to 0.17 ng/mL (0.0011 nM). Each well was added with 100 μl and incubated at room temperature for 30 minutes. 100 μl of 1 μg/mL biotin-labeled human NGF was added, incubated at room temperature for 1 hours, and washed for three times. Streptavidin-labeled horseradish peroxidase (Streptavidin-HRP, Biolegend, 405210) was added and incubated for 0.5 hours. After washing three times, TMB was added to the plate, and the absorbance at 450 nm was read by a microplate reader after the color development was terminated.
The results are shown in FIG. 7. The anti-TrkA antibodies of the present invention 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 reaches 20 nM, the eight anti-TrkA antibodies can almost completely block the binding of TrkA to NGF. While the control antibody BXhVH5VL1 has no significant effect on blocking TrkA binding to NGF at all of the tested concentrations (0.001-100 nM). The IC50 values of the eight anti-TrkA antibodies of the present invention and BXhVH5VL1 for blocking the binding of human TrkA to the ligand NGF are shown in Table 4.







TABLE 4







IC50 of anti-TrkA antibodies for blocking TrkA-NGF binding
















Antibodies
5A3
11G8
26E9
33H5
40D6
42F5
42H6
42A11
BXhVH5VL1





IC50 (nM)
1.763
1.900
2.040
1.498
2.284
2.352
2.185
2.054
>100









Example 6: Detection of the In Vitro Neutralization Activities of Murine Anti-TrkA Antibodies
A. NGF-Induced TF-1 Cell Proliferation
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 can also induce the growth of TF-1 cells after binding to TrkA on the surface of TF-1 cells to activate the downstream signaling pathway, thereby eliminating the need of GM-CSF. TF-1 cells were cultured in RPMI 1640 culture medium (HyClone, SH30027) containing 10% FBS (Gibco, 10091148) and 2 μg/mL GM-CSF (R&D, 215-GM-010), cells were collected during logarithmic growth phase, washed thoroughly to remove the GM-CSF comprised in the original medium, and resuspended with GM-CSF-free medium. 5000 cells per well were diluted in 80 μl medium and plated into a white 96-well cell culture plate with transparent bottom (Corning, 3610). Anti-TrkA antibody was 4-fold serially diluted, starting from 400 μg/mL (2666.67 nM) to prepare 10 concentrations, 10 μL was added into each well, and incubated at room temperature for 0.5 hours. 10 μL of 50 ng/mL human NGF was added to each well. After incubation at 37° C., 5% CO2 for 72 hours, 100 μL of CellTiter-Glo® Cell Viability Detection Reagent (Promega, G7573) was added, and the optical luminescence signals were read by using a Multimode Reader (SpectraMax). As shown in FIG. 8, the anti-TrkA antibodies of the present invention are capable of inhibiting the proliferation of TF-1 cells induced by human NGF, and the IC50 values are shown in Table 5. The inhibitory effect of antibody BXhVH5VL1 on the proliferation of TF-1 cells is much weaker than those of the antibodies of the present invention. When the antibody concentration is 40 μg/mL (266.67 nM), the anti-TrkA antibodies of the present invention can completely inhibit the NGF-induced TF-1 cell proliferation, whereas the inhibition rate of the antibody BXhVH5VL1 on the NGF-induced TF-1 cell proliferation at this concentration is less than 40%.







TABLE 5







IC50 values of the anti-TrkA antibodies inhibiting NGF-induced TF-1 cell proliferation
















Antibodies
5A3
11G8
26E9
33H5
40D6
42F5
42H6
42A11
BXhVH5VL1





IC50 (nM)
6.487
1.092
1.393
2.136
8.532
1.302
3.576
1.462
>200









B. Assay of NGF-Induced TrkA/Ba/F3 Cell Proliferation

The growth of Ba/F3 cells involves two pathways: IL3-dependent and IL3-independent. The IL3-independent growth requires Ba/F3 cells to stably express active kinases. Ba/F3 cells expressing full-length human TrkA (TrkA/Ba/F3) were constructed, and the proliferation of such cells requires the addition of NGF for induction.
TrkA/Ba/F3 cells were cultured in RPMI 1640 culture medium containing 10% FBS and 100 ng/mL NGF. The cells were collected and washed thoroughly to remove NGF comprised in the original growth medium, 3000 cells per well were resuspended in 80 μL of NGF-free medium and plated in a white 96-well cell culture plate with transparent bottom (Corning, 3610). 10 μL of serially diluted anti-TrkA antibody was added to the cells in each well, incubated at room temperature for 0.5 hours, and 10 μL of 50 ng/mL human NGF was added to each well at the final concentration of 5 ng/mL. The highest concentration of serially diluted anti-NGF antibody was 40 μg/mL (266.67 nM). After incubation at 37° C., 5% CO2 for 48 hours, 100 μL of CellTiter-Glo® Cell Viability Detection Reagent (Promega, G7573) was added. Optical luminescence signals were read by using a Multimode Reader (SpectraMax).
During the observation of the growth of TrkA/Ba/F3 cells, the proliferation of the cells added with human NGF is normal; the eight anti-TrkA antibodies of the present invention can all inhibit the NGF-induced TrkA/Ba/F3 cell proliferation, and the inhibitory effect is increased with the increase of the concentration. When the antibody concentration is increased to 2.5 μg/mL (16.67 nM) or higher, the anti-TrkA antibodies inhibit the TrkA/Ba/F3 cell proliferation by 100%; while the inhibition rate of antibody BXhVH5VL1 at the highest concentration of 40 μg/mL (266.67 nM) for TrkA/Ba/F3 cell proliferation is less than 50% (FIG. 9). IC50 values are as shown in Table 6.







TABLE 6







IC50 values of the anti-TrkA antibodies inhibiting NGF-induced TrkA/Ba/F3 cell proliferation
















Antibodies
5A3
11G8
26E9
33H5
40D6
42F5
42H6
42A11
BXhVH5VL1





IC50 (nM)
0.422
1.646
1.539
0.413
1.805
1.252
0.782
0.738
>200









Example 7: Detection of the Binding of Murine Anti-TrkA Antibodies to TrkA Proteins of the Same Family
Each well of a 96-well microtiter plate was coated with 50 ng of human TrkA extracellular region protein, human TrkB extracellular region protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10047-H03H), human TrkC extracellular region protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 10048-H03H). The bindings of the anti-TrkA antibodies to human TrkA and the two proteins of the same family were detected according to the method described in Example 2.
Eight murine anti-TrkA antibodies can bind to TrkA with high affinity (FIG. 10), but substantially do not bind to TrkB (FIG. 11) and TrkC (FIG. 12).
Example 8: The Effects of Anti-TrkA Antibodies on CFA-Induced Inflammatory Pain in Mice
C57BL/6 mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.), male, SPF grade, 6-8 weeks old, were kept for 5-day adaptive feeding. After the adaptation, the animals were divided into 3 groups, namely the saline group, the anti-TrkA antibody 33H5 group, and the anti-TrkA antibody 42F5 group, 10 animals in each group. The pain threshold (i.e., basal pain threshold) was measured by VonFrey before the injection of the modeling agent CFA into the planta.
On the next day, mice were injected with 25 μl CFA to the planta to induce inflammation. After the toes were significantly swollen (about 24 hours after modeling), the pain threshold was measured in mouse planta in a similar way, which was taken as the pre-dose pain threshold (FIG. 14).
Subsequently, each group were subcutaneously injected with normal saline, anti-TrkA antibody 33H5, or anti-TrkA antibody 42F5 at a dose of 10 mg/kg, and the pain thresholds were measured at 24 hours and 48 hours post administration (FIGS. 15-16). The effects of the test substances on pain threshold were evaluated.
The results are shown in FIGS. 13-16. Both anti-TrkA antibodies 33H5 and 42F5 can significantly improve the CFA-induced decrease in pain threshold at 24 h and 48 h post administration, and there are statistical differences compared to the normal saline group.
Example 9: Sequencing and Sequence Analysis of Murine Anti-TrkA Antibody Variable Regions
Total RNA of hybridoma cells was extracted by using TRIzol kit (Ambion, 15596-026), and was used as a template to synthesize the first-strand cDNA (Takara). The antibody light chain and heavy chain fragments were obtained by rapid amplification of cDNA ends (RACE), and the amplified fragments were cloned into standard vectors respectively. After sequencing, the sequences of the heavy chain and light chain variable regions of the anti-TrkA antibodies 5A3, 11G8, 26E9, 33H5, 40D6, 42F5, 42H6 and 42A11 obtained are as follows:








Anti-TrkA Antibody 5A3:



Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 49)



QVQLQQSGAELMKPGASVKISCKAIGYTFSRYWIEWVKQRPGHGLEWIGEILPGRGVTN



 


YNENFKGKATFTVDISSTTTYIQFSSLTSEDSAVYYCARSNYGDYDFWGQGTSLTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 65)



CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAA



 


GATATCCTGCAAGGCTATTGGGTACACATTCAGTAGGTACTGGATAGAGTGGGTAAAGCAG


 


AGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGAGGTGTTACTAAC


 


TACAATGAGAACTTCAAGGGCAAGGCCACATTCACTGTAGATATATCCTCCACCACAACCT


 


ACATTCAATTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGATCGAA


 


TTATGGTGACTACGACTTCTGGGGCCAAGGCACCTCTCTCACAGTCTCCTCA


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 57)



QTVVTQESALTTSPGETVTLTCRSSSGAVTTSNHANWVQEKPDHLFTSLMGGTNNRAPG



 


VPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 66)



CAGACTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACA



 


CTCACTTGTCGCTCAAGTTCTGGGGCTGTTACAACTAGTAACCATGCCAACTGGGTCCAA


 


GAAAAACCTGATCATTTATTCACTAGTCTAATGGGTGGTACCAATAACCGAGCTCCAGGTG


 


TTCCTGCCAGATTCTCAGGCTCCCTGATTGGCGACAAGGCTGCCCTCACCATCACAGGGG


 


CGCAGACTGAGGATGAGGCAATATATTTCTGTGCTCTCTGGTACAGCAACCATTGGGTGTT


 


CGGTGGAGGAACTAAACTGACTGTCCTA


 


Anti-TrkA Antibody 11G8:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 50)



QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPNNGL



 


TNYDEKFKTKATLTIDKSSRTAYIQLSSLTSEDSAVYYCAKYGNYVAFAFWGQGTLVTVSA


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 67)



CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAA



 


GCTGTCCTGCAAGGCTTCCGGCTACACCTTTACCAGCTACTGGATGCACTGGGTGAAGCA


 


GAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCTTACTAA


 


CTACGATGAGAAATTCAAGACCAAGGCCACACTGACCATAGACAAATCCTCCAGAACAGC


 


CTACATACAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAAATAT


 


GGTAACTACGTCGCGTTTGCTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 58)



DIQMTQSPASLSATVGETVTITCRASENIYSYVAWYQQKQGKSPQLLVHNAKTLAEGVPS



 


RFSGSGSGTQFSLKINGLHPEDFGSYYCQHHYGIPLTFGAGTKLELK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 68)



GACATTCAGATGACTCAGTCTCCAGCCTCCCTATCTGCAACTGTGGGAGAAACTGTC



 


ACCATCACATGTCGAGCAAGTGAAAATATTTACAGTTATGTAGCATGGTATCAGCAGAAAC


 


AGGGAAAATCTCCTCAACTCCTGGTCCATAATGCAAAAACCTTAGCAGAAGGTGTACCAT


 


CAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACGGCCTGCACC


 


CTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTATTCCGCTCACGTTCGGCGCT


 


GGGACCAAGCTGGAGCTGAAA


 


Anti-TrkA Antibody 26E9:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 51)



QVQLQQPGAELVKPGASVKLSCKSSGYTFTNYWMHWVKQRPGQGLEWIGEIYPSNGRT



 


NYNEKFKNRATLTVDISSSTAYMQLSSLTSEDSAVYYCARSRYDPMEDWGQGTSVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 69)



CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCTGTGAA



 


GCTGTCCTGCAAGTCTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAAGCA


 


GCGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAGCAACGGTCGTACTAA


 


CTACAATGAGAAGTTCAAAAACAGGGCCACACTGACTGTAGACATTTCCTCCAGCACAGC


 


CTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAG


 


TAGGTACGACCCTATGGAAGACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCT


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 59)



QIVLTQSPAIMSASPGEKVTMTCSASSSVGYMHWYQQKSGTSPKRWIYDTSKLASGVPT



 


RFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSIPLTFGSGTKLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 70)



CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTC



 


ACCATGACCTGCAGTGCCAGCTCAAGTGTGGGTTACATGCACTGGTACCAGCAGAAGTCA


 


GGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAACT


 


CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCT


 


GAGGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTATCCCACTCACGTTCGGCTCGG


 


GGACAAAGTTGGAAATAAAG


 


Anti-TrkA Antibody 33H5:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 52)



QVQLQQPGAELVKPGASVQLSCKASGYTFTSYWIHWVKQRPGQGLEWIGEINPNNGLT



 


NYIEKFKNKATLTIDKSSNTAYMQLSGLTPEDSAVYYCAKYGNYVAFAYWGQGTLVTVSA


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 71)



CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGCA



 


GCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGTTACTGGATACACTGGGTGAAACA


 


GAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAACGGTCTTACTAA


 


CTACATTGAGAAATTCAAGAACAAGGCCACACTGACTATTGACAAATCCTCCAACACAGC


 


CTACATGCAACTCAGCGGCCTGACACCTGAGGACTCTGCGGTCTATTACTGTGCAAAATAT


 


GGTAACTACGTCGCGTTTGCTTACTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCA


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 60)



DIQMTQSPASLSASVGDTVTITCRASENIYTYLAWYQQKQGKSPQLLVHNTKTLAEGVP



 


SRFSGSGSGTQFSLKISSLQPEDFGTYYCQHHYGVPLTFGAGTKLELK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 72)



GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGACACTGTCA



 


CCATCACATGTCGAGCAAGTGAAAATATCTACACTTATTTAGCTTGGTATCAGCAGAAACA


 


GGGAAAATCTCCTCAACTCCTGGTCCATAATACAAAAACCTTAGCAGAAGGTGTGCCCTC


 


AAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAGCAGCCTGCAGCC


 


TGAAGATTTTGGGACTTATTACTGTCAACATCATTATGGTGTTCCGCTCACGTTCGGTGCTG


 


GGACCAAGCTGGAGCTGAAA


 


Anti-TrkA Antibody 40D6:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 53)



QVQLQQSGTELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGRSSTN



 


YNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCTRVSQLHIYFDYWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 73)



CAGGTTCAGCTGCAGCAGTCTGGAACTGAACTGATGAAGCCTGGGGCCTCAGTGAA



 


GATATCCTGCAAGGCTACTGGCTACACATTCAGTAGATACTGGATAGAGTGGGTAAAACAG


 


AGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGGAGTAGTACTAAC


 


TACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCCGATACATCCTCCAACACAGCC


 


TACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAGTTT


 


CCCAACTGCACATTTACTTTGACTACTGGGGCCAAGGGACCACTGTCACAGTCTCCTCC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 61)



DIVMTQVVPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFQQRPGQSPQLLIYRMSNLA



 


SGVPDRFSGSGSGTAFTLRISRVEAEDVAFYYCMQHLEFPLTFGAGTKLELK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 74)



GATATTGTGATGACTCAGGTTGTACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTAT



 


CCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTATATTGG


 


TTCCAGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCT


 


CAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACATTGAGAATC


 


AGTAGAGTGGAGGCTGAGGATGTGGCTTTTTATTACTGTATGCAACATCTAGAATTTCCGC


 


TCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAA


 


Anti-TrkA Antibody 42F5:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 54)



QVQLQQPGAELVKPGASVKLSCKSSGYTFTNYWMHWVRQRPGQGLEWIGEIYPNNGR



 


VNYNEKFKNRATLTVDISSSTAYMQLSSLTSEDSAVYYCARSRYDPMEDWGQGTSVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 75)



CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAACCTGGGGCTTCAGTGAA



 


GCTGTCCTGCAAGTCTTCTGGCTATACCTTCACCAACTACTGGATGCACTGGGTGAGGCA


 


GAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATCTATCCTAACAACGGTCGTGTTAA


 


CTACAATGAGAAGTTCAAGAACAGGGCCACACTGACTGTAGACATATCCTCCAGCACAGC


 


CTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGGAG


 


TAGGTACGACCCTATGGAAGACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCT


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 62)



QVVLTQSPAIMSASPGEKVTMTCSASSSVGYMHWYQQKSGTSPKRWIYDTSKLASGVP



 


TRFSGSGSGTSYSLTISSMEAEDAATYFCQQWSSIPLTFGSGTRLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 76)



CAAGTTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTC



 


ACCATGACCTGCAGTGCCAGCTCAAGTGTAGGTTACATGCACTGGTACCAGCAGAAGTCA


 


GGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCAACT


 


CGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCT


 


GAGGATGCTGCCACTTATTTCTGCCAGCAGTGGAGTAGTATCCCACTCACGTTCGGCTCGG


 


GGACAAGGTTGGAAATAAAG


 


Anti-TrkA Antibody 42H6:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 55)



QVQLQQPGVELVKPGASVKLSCKTSGYTFTSYWMHWVKQRPGQGLEWIGEIYSSNGLT



 


NYNEKFKNKATLTVDKSSSTAYMQLTSLTSEDSAIYYCARHWYVFLDHWGQGTTLTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 77)



CAGGTCCAACTGCAGCAGCCTGGGGTTGAACTGGTGAAGCCTGGGGCTTCAGTGAA



 


GCTGTCCTGCAAGACTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCA


 


GAGGCCTGGACAAGGCCTTGAATGGATTGGAGAGATTTATTCCAGTAACGGTCTTACTAA


 


CTACAATGAGAAGTTCAAGAATAAGGCCACACTGACTGTAGATAAATCCTCCAGCACAGC


 


CTACATGCAACTCACCAGCCTGACATCTGAAGACTCTGCGATCTATTACTGTGCAAGACAT


 


TGGTACGTCTTCCTTGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 63)



QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNHANWVQEKPDHLFTGLIGGINNRAPGV



 


PARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTRLTVL


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 78)



CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACA



 


CTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACCATGCCAACTGGGTCCAA


 


GAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTATCAACAACCGAGCTCCAGGTG


 


TTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGG


 


CACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAATCATTGGGTGTT


 


CGGTGGAGGAACCAGACTGACTGTCCTA


 


Anti-TrkA Antibody 42A11:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO: 56)



QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEIYPSNGR



 


TNYNEKFKTKATLTVDKSSSTAYMHLSSLTSEDSAVYYCAGSRYDAMDFWGQGTSVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO: 79)



CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGAAGCCTGGGGCTTCAGTGAA



 


GCTGTCCTGTAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCATTGGGTGAAACA


 


GAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAGCAACGGTCGTACTAA


 


CTACAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAG


 


CCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAGGATC


 


GAGATACGATGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO: 64)



QIVLTQSPAIMSASPGEKVTMTCSASSIISYMHWYQQKSGTSPKRWIYDTSKLASGVPAR



 


FSGSGSGTSYSLTISGMEAEDAATYYCHQWTSNPLTFGGGTKLELK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO: 80)



CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTC



 


ACCATGACCTGCAGTGCCAGCTCAATTATAAGTTACATGCACTGGTACCAGCAGAAGTCA


 


GGCACCTCCCCCAAAAGATGGATTTATGACACTTCCAAACTGGCTTCTGGAGTCCCTGCTC


 


GCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGTGGCATGGAGGCTG


 


AAGATGCTGCCACTTATTACTGCCACCAGTGGACTAGTAACCCGCTCACGTTCGGTGGTG


 


GGACCAAGCTGGAACTGAAA






The CDRs defined by the Kabat and IMGT systems were obtained after sequence analysis. The following table (Table 7) lists the CDRs of the eight anti-TrkA antibodies defined on the basis of the Kabat and IMGT systems.







TABLE 7





Anti-TrkA antibody CDRs defined according to Kabat and


IMGT systems







Monoclonal antibody heavy chain CDRs: CDRs defined


according to Kabat system are shown in black and bold;


CDRs defined by IMGT are underlined.













Clone

SEQ ID

SEQ ID

SEQ ID


No.
CDR-H1
No.
CDR-H2
No.
CDR-H3
No.





5A3
GYTFSRYWIE
 1
EILPGRGVTNY
 2
ARSNYGDYDF
 3





NENFKG





 


11G8
GYTFTSYWMH
 4
EINPNNGLTNY
 5
AKYGNYVAFAF
 6





DEKFKT





 


26E9
GYTFTNYWMH
 7
EIYPSNGRTNY
 8
ARSRYDPMED
 9





NEKFKN





 


33H5
GYTFTSYWIH
10
EINPNNGLTNYI
11
AKYGNYVAFAY
12





EKFKN





 


40D6
GYTFSRYWIE
13
EILPGRSSTNY
14
TRVSQLHIYFDY
15





NEKFKG





 


42F5
GYTFTNYWMH
16
EIYPNNGRVNY
17
ARSRYDPMED
18





NEKFKN





 


42H6
GYTFTSYWMH
19
EIYSSNGLTNY
20
ARHWYVFLDH
21





NEKFKN





 


42A11
GYTFTNYWMH
22
EIYPSNGRTNY
23
AGSRYDAMDF
24





NEKFKT










Monoclonal antibody light chain CDRs: CDRs defined


according to Kabat system are shown in black and bold;


CDRs defined by IMGT are underlined.













Clone

SEQ ID

SEQ ID

SEQ ID


No.
CDR-L1
No.
CDR-L2
No.
CDR-L3
No.





5A3
RSSSGAVTTSN
25
GTNNRAP
26
ALWYSNHWV
27



HAN







 


11G8
RASENIYSYVA
28
NAKTLAE
29
QHHYGIPLT
30


 


26E9
SASSSVGYMH
31
DTSKLAS
32
QQWSSIPLT
33


 


33H5
RASENIYTYLA
34
NTKTLAE
35
QHHYGVPLT
36


 


40D6
RSSKSLLHSNG
37
RMSNLAS
38
MQHLEFPLT
39



NTYLY







 


42F5
SASSSVGYMH
40
DTSKLAS
41
QQWSSIPLT
42


 


42H6
RSSTGAVTTSN
43
GINNRAP
44
ALWYSNHWV
45



HAN







 


42A11
SASSIISYMH
46
DTSKLAS
47
HQWTSNPLT
48









Example 10: Humanization and Characterization of Murine Anti-TrkA Antibodies Humanization of Antibodies
The light chain variable region (VL) and heavy chain variable region (VH) sequences of the antibodies secreted by the hybridoma cells obtained above were humanized. The amino acid sequences of murine antibody VL and VH were aligned and searched in the human embryonic antibody amino acid sequence database, to find human IGHV and IGKV sequences with high homology as humanization templates. The potential steric hindrance and interaction between the amino acids of the variable regions and the framework regions were analyzed by computer simulation technology, to determine the amino acids in the framework regions that are critical for maintaining the activity of the humanized antibodies. Such amino acids were retained during the humanization process. Humanization of the light and heavy chain variable regions was accomplished by CDR grafting technology. Then, the following humanized antibodies were obtained by using the selected antibody constant region templates.
The following exemplary antibodies were obtained after the humanization of the anti-TrkA antibody 42F5: 42F5-01, 42F5-03, 42F5-04, 42F5-05, 43F5-08 and 42F5-11. The humanized antibodies 42F5-01 and 42F5-03 were obtained with human IGHV1-2 as the heavy chain variable region template and human IGKV1-39 as the light chain variable region template; the humanized antibody 42F5-05 was obtained with human IGHV1-2 as the heavy chain variable region template and human IGKV1-6 as the light chain variable region template; the humanized antibody 42F5-11 was obtained with human IGHV1-2 as the heavy chain variable region and human IGKV3-11 as the light chain variable region template; the humanized antibody 42F5-04 was obtained with human IGHV1-69 as the heavy chain variable region template and human IGKV1-39 as the light chain variable region template; the humanized antibody 42F5-08 was obtained with human IGHV1-69 as the heavy chain variable region template and human IGKV1-6 as the light chain variable region template. Among the obtained humanized antibodies, the heavy chain variable region sequence of 42F5-01 is identical to that of 42F5-05, the heavy chain variable region sequence of the humanized antibody 42F5-03 is identical to that of 42F5-11, and the heavy chain variable region sequence of the humanized antibody 42F5-04 is identical to that of 42F5-08; the light chain variable region sequence of the humanized antibody 42F5-03 is identical to that of 42F5-04.
IGHV1-2, IGHV1-69, IGKV1-39, IGKV1-6 and IGKV3-11 are human germline IgG gene based on the human immunoglobulin gene database IMGT (The International ImMunoGeneTics Information System) and NCBI (The National Center for Biotechnology Information). In addition, since the antibody 42F5 comprises deamidation site NG in the heavy chain complementarity determining region sequences, the heavy chain variable regions of the humanized antibody 43F5-01 were further engineered in order to remove the potential impact of the site on the antibody. 42F5-01 was used as a template, the NG55-56 in the heavy chain variable region was mutated to SG55-56 or QG55-56 or NA55-56, respectively, resulting in humanized antibodies 42F5-13, 42F5-14 and 42F5-15.
For gene synthesis, the obtained humanized antibody heavy chain variable region sequence was grafted to the human IgG4 heavy chain constant region, and the light chain variable region sequence was grafted to the human Kappa light chain constant region. After enzymatic digestion, the fragments were ligated into the corresponding plasmids. The constructed plasmid was transiently transfected into CHO cells for expression. After 7-10 days of expression, the cell culture supernatant was purified with Mab Select column (GE Healthcare) pre-equilibrated with a corresponding buffer (such as phosphate buffered saline (pH 7.4)), and then eluted with sodium citrate or other buffers. The resulting antibodies were identified by SDS-PAGE or SEC-HPLC to determine purity and for subsequent characterizations.
Variable Region Sequences of the Humanized Antibodies of Anti-TrkA Antibody 42F5
The variable region sequences of the humanized anti-TrkA antibody 42F5: 42F5-01, 42F5-03, 42F5-04, 42F5-05, 42F5-08, 42F5-11, 42F5-13, 42F5-14 and 42F5-15 are as follows:








42F5-01:



Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 81)



QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNNG



 


RVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 82)



CAGGTGCAGCTGGTGCAGAGCGGAGCTGAGGTGAAGAAGCCAGGAGCTTCCGTGA



 


AGGTGAGCTGCAAGTCCAGCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGC


 


AGGCTCCAGGACAGGGACTGGAGTGGATGGGCGAGATCTACCCTAACAATGGCAGGGTG


 


AACTACAACGAGAAGTTTAAGAACAGAGTGACCATGACAGTGGACATCAGCATCTCTACC


 


GCTTACATGGAGCTGTCTAGGCTGCGGTCCGACGATACAGCCGTGTACTATTGTGCTAGAT


 


CTCGCTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACCGTGTCTTCC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 83)



DVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRLIYDTSKLASGVPS



 


RFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 84)



GACGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGATAGAGTG



 


ACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCG


 


GCAAGGCCCCTAAGAGGCTGATCTACGACACATCTAAGCTGGCTTCCGGAGTGCCAAGCC


 


GGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAG


 


AGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGG


 


CACACGCCTGGAGATCAAG


 


42F5-03:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 85)



QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWIGEIYPNNGR



 


VNYNEKFKNRATLTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 86)



CAGGTGCAGCTGGTGCAGAGCGGAGCTGAGGTGAAGAAGCCAGGAGCTTCCGTGA



 


AGGTGAGCTGCAAGTCCAGCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGC


 


AGGCTCCAGGACAGGGACTGGAGTGGATCGGCGAGATCTACCCTAACAATGGCAGGGTG


 


AACTACAACGAGAAGTTTAAGAACAGAGCCACCCTGACAGTGGACATCAGCATCTCTACC


 


GCTTACATGGAGCTGTCTAGGCTGCGGTCCGACGATACAGCCGTGTACTATTGTGCTAGAT


 


CTCGCTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACCGTGTCTTCC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 87)



DVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRWIYDTSKLASGVPS



 


RFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 88)



GACGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGATAGAGTG



 


ACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCG


 


GCAAGGCCCCTAAGAGGTGGATCTACGACACATCTAAGCTGGCTTCCGGAGTGCCAAGCC


 


GGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCAG


 


AGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGGG


 


CACACGCCTGGAGATCAAG


 


42F5-04:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 89)



QVQLVQSGAEVKKPGSSVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWIGEIYPNNGR



 


VNYNEKFKNRATLTVDISTSTAYMELSSLRSEDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 90)



CAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGCTCCAGCGTGAA



 


GGTGAGCTGCAAGTCTTCCGGCTACACCTTCACAAACTATTGGATGCACTGGGTGAGGCA


 


GGCTCCAGGACAGGGACTGGAGTGGATCGGCGAGATCTACCCTAACAATGGCAGAGTGA


 


ACTACAACGAGAAGTTTAAGAACCGCGCCACCCTGACAGTGGACATCTCTACCTCCACAG


 


CTTACATGGAGCTGAGCTCTCTGAGAAGCGAGGATACCGCCGTGTACTATTGTGCTAGGTC


 


TCGGTATGACCCCATGGAGGATTGGGGCCAGGGCACCACAGTGACAGTGTCCAGC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 87)



Identical to that of 42F5-03



 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 88)



Identical to that of 42F5-03



 


42F5-05:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 81)



Identical to that of 42F5-01



 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 82)



Identical to that of 42F5-01



 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 91)



QVQLTQSPSSLSASVGDRVTITCSASSSVGYMHWYQQKPGKAPKRLIYDTSKLASGVPS



 


RFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 92)



CAGGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGACAGAGT



 


GACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCC


 


GGCAAGGCCCCTAAGAGGCTGATCTACGATACATCTAAGCTGGCTTCCGGAGTGCCAAGC


 


CGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCA


 


GAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGG


 


GCACACGCCTGGAGATCAAG


 


42F5-08


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 89)



Identical to that of 42F5-04



 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 90)



Identical to that of 42F5-04



 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 93)



RFSGSGSGTDYTLTISSLQPEDFATYFCQQWSSIPLTFGQGTRLEIK



 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 94)



CAGGTGCAGCTGACCCAGTCTCCTTCCAGCCTGTCCGCCAGCGTGGGCGACAGAGT



 


GACCATCACATGCTCCGCTTCTTCCAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCC


 


GGCAAGGCCCCTAAGAGGTGGATCTACGATACATCTAAGCTGGCTTCCGGAGTGCCAAGC


 


CGGTTCTCTGGCTCCGGCAGCGGAACCGACTACACCCTGACAATCTCTTCCCTGCAGCCA


 


GAGGATTTCGCCACATATTTTTGTCAGCAGTGGAGCTCTATCCCCCTGACCTTTGGCCAGG


 


GCACACGCCTGGAGATCAAG


 


42F5-11:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 85)



Identical to that of 42F5-03



 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 86)



Identical to that of 42F5-03



 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 95)



EVVLTQSPATLSLSPGERATLSCSASSSVGYMHWYQQKPGQAPRRWIYDTSKLASGVPA



 


RFSGSGSGTDYTLTISSLEPEDAAVYFCQQWSSIPLTFGQGTRLEIK


 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 96)



GAGGTGGTGCTGACCCAGTCCCCAGCCACACTGAGCCTGTCTCCAGGAGAGAGAGC



 


CACCCTGTCCTGCTCCGCCTCCAGCTCTGTGGGCTACATGCACTGGTATCAGCAGAAGCC


 


AGGACAGGCTCCTAGGCGGTGGATCTACGACACCTCTAAGCTGGCTTCCGGAGTGCCAGC


 


TCGCTTCTCTGGCTCCGGCAGCGGCACAGACTACACCCTGACAATCTCCAGCCTGGAGCC


 


TGAGGATGCCGCCGTGTACTTCTGTCAGCAGTGGTCTTCCATCCCACTGACCTTTGGCCAG


 


GGCACAAGGCTGGAGATCAAG


 


42F5-13:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 97)



QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNSG



 


RVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 98)



CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCTGTGAA



 


GGTGAGCTGCAAGAGCTCCGGCTACACCTTTACCAATTATTGGATGCACTGGGTGAGGCA


 


GGCTCCCGGCCAGGGACTGGAGTGGATGGGCGAGATATATCCCAATAGCGGCCGGGTGAA


 


TTATAATGAGAAGTTTAAGAATCGGGTGACCATGACCGTGGATATCAGCATCTCCACCGCC


 


TACATGGAGCTGAGCAGGCTGAGGAGCGATGACACCGCTGTGTACTACTGCGCTAGGTCC


 


AGGTATGACCCCATGGAGGATTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 83)



Identical to that of 42F5-01



 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 84)



Identical to that of 42F5-01



 


42F5-14:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 99)



QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNQG



 


RVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 100)



CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAA



 


GGTGTCCTGTAAGTCCAGCGGCTATACCTTCACCAACTATTGGATGCACTGGGTGAGGCA


 


GGCCCCTGGCCAGGGACTGGAGTGGATGGGCGAGATATATCCTAACCAGGGCCGGGTGA


 


ATTATAACGAGAAGTTCAAGAATAGGGTGACCATGACCGTGGACATCTCCATCAGCACCG


 


CTTACATGGAGCTGTCCAGGCTGCGGAGCGACGATACCGCCGTGTACTACTGTGCCAGGT


 


CCCGGTATGATCCCATGGAGGACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 83)



Identical to that of 42F5-01



 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 84)



Identical to that of 42F5-01



 


42F5-15:


Amino acid sequence of the heavy chain variable region:


(SEQ ID NO. 101)



QVQLVQSGAEVKKPGASVKVSCKSSGYTFTNYWMHWVRQAPGQGLEWMGEIYPNNA



 


RVNYNEKFKNRVTMTVDISISTAYMELSRLRSDDTAVYYCARSRYDPMEDWGQGTTVTVSS


 


Nucleic acid sequence of the heavy chain variable region:


(SEQ ID NO. 102)



CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCTGTGAA



 


GGTGTCCTGTAAGAGCAGCGGCTACACCTTTACCAATTATTGGATGCACTGGGTGAGGCA


 


GGCCCCCGGCCAGGGATTGGAGTGGATGGGCGAGATATATCCTAACAATGCCAGGGTGAA


 


CTATAATGAGAAGTTTAAGAACCGGGTGACCATGACCGTGGACATCAGCATCTCCACCGC


 


CTATATGGAGCTGAGCCGGCTGCGGTCCGACGACACCGCTGTGTACTACTGCGCCCGGTC


 


CCGGTATGATCCTATGGAGGACTGGGGCCAGGGCACCACCGTGACCGTGAGCTCC


 


Amino acid sequence of the light chain variable region:


(SEQ ID NO. 83)



Identical to that of 42F5-01



 


Nucleic acid sequence of the light chain variable region:


(SEQ ID NO. 84)



Identical to that of 42F5-01














TABLE 8







Variable region sequences of the humanized antibodies of anti-TrkA antibody 42F5










Heavy chain
Light chain











Antibody
Amino acid sequence
Nucleic acid sequence
Amino acid sequence
Nucleic acid sequence


number
of the heavy chain:
of the heavy chain:
of the light chain:
of the light chain:





42F5-01
SEQ ID NO. 81
SEQ ID NO. 82
SEQ ID NO. 83
SEQ ID NO. 84


42F5-03
SEQ ID NO. 85
SEQ ID NO. 86
SEQ ID NO. 87
SEQ ID NO. 88


42F5-04
SEQ ID NO. 89
SEQ ID NO. 90
SEQ ID NO. 87
SEQ ID NO. 88


42F5-05
SEQ ID NO. 81
SEQ ID NO. 82
SEQ ID NO. 91
SEQ ID NO. 92


42F5-08
SEQ ID NO. 89
SEQ ID NO. 90
SEQ ID NO. 93
SEQ ID NO. 94


42F5-11
SEQ ID NO. 85
SEQ ID NO. 86
SEQ ID NO. 95
SEQ ID NO. 96


42F5-13
SEQ ID NO. 97
SEQ ID NO. 98
SEQ ID NO. 83
SEQ ID NO. 84


42F5-14
SEQ ID NO. 99
SEQ ID NO. 100
SEQ ID NO. 83
SEQ ID NO. 84


42F5-15
SEQ ID NO. 101
SEQ ID NO. 102
SEQ ID NO. 83
SEQ ID NO. 84









B. Characterization of Humanized Anti-TrkA Antibodies

1) The Binding of Humanized Antibodies to Human TrkA

The affinities of humanized antibodies of anti-TrkA antibody 42F5 were studied with human TrkA extracellular region (33-417) protein containing mouse Fc. Each well of a 96-well microtiter plate was coated with 50 ng of human TrkA, washed and blocked, then added with serially diluted humanized antibodies, and incubated for 1 hour at room temperature. After washing three times, horseradish peroxidase-conjugated goat anti-human IgG antibody (ThermoFisher, A18817) was added and reacted at room temperature for 1 hour. After washing three time, tetramethylbenzidine (TMB, Biolegend) was added for color development. 1M HCl was used to terminate the color development, and the absorbance values were read at 450 nm with a microplate reader.
The binding results are shown in Table 9 and FIG. 17. All the humanized antibodies of anti-TrkA antibody 42F5 with different sequences have similar binding to human TrkA, and the level of binding is comparable to that of human-mouse chimeric antibody 42F5-Chi (which contains variable regions of murine anti-TrkA antibody 42F5 and constant regions of human antibody).







TABLE 9







EC50 values of humanized antibodies of anti-TrkA antibody 42F5 binding to TrkA

















Antibodies
42F5-Chi
42F5-01
42F5-03
42F5-04
42F5-05
42F5-08
42F5-11
42F5-13
42F5-14
42F5-15





EC50 (nM)
0.062
0.061
0.056
0.070
0.072
0.055
0.050
0.087
0.071
0.054









2) The Binding of Humanized Antibodies to Cells Expressing TrkA/TrkB/TrkC

The specificities of the humanized antibodies of anti-TrkA antibody 42F5 were studied by using cell lines TrkA/CHO (ThermoFisher, K1516), TrkB/CHO (ThermoFisher, K1491) and TrkC/CHO (ThermoFisher, K1515) expressing TrkA, TrkB, and TrkC respectively. CHO-K1 was used as a negative control. The binding of the humanized antibodies to the four cell lines was detected by flow cytometry.
The cells were cultured according to the manufacturer's instruction. The cells were harvested in the logarithmic growth phase, re-suspended in flow buffer (PBS+2% FBS, pH 7.4), and added into 96-well plate, 105 cells per well (50 μl). 50 μl of 100 μg/mL humanized anti-TrkA antibody was added, incubated at room temperature for 60 min, and centrifuged at 1200 rpm. The supernatant was discarded and the cells were washed 3 times. FITC-labeled goat anti-human Fc antibody (Abeam, ab97224) was diluted to 5 μg/mL with flow buffer and used to re-suspend the cells, incubated at room temperature for 30 min in the dark, centrifuged, and the supernatant was discarded. After washing 3 times, the cells were re-suspended with 7AAD solution (Biolegend, 420403), incubated for 10 min at room temperature, and the binding of the antibody was detected with a flow cytometer (BioRad, ZE5).
The results are shown in FIG. 18. Similar to the human-mouse chimeric antibody 42F5-Chi, the humanized antibodies of anti-TrkA antibody 42F5 bind to TrkA/CHO cells, but do not bind to TrkB/CHO, TrkC/CHO or CHO-K1 cells, indicating that the humanized anti-TrkA antibodies have favorable specificities.
The binding curve of the humanized anti-TrkA antibodies and TrkA/CHO cells was detected with the same flow cytometry. The antibodies were diluted 3-fold from a concentration of 66.67 μg/mL for a total of 10 concentrations. The results are shown in FIG. 19. The humanized antibodies of anti-TrkA antibody 42F5 bind to TrkA/CHO cells at a level comparable to the human-mouse chimeric antibody 42F5-Chi. The EC50 values are summarized in Table 10.







TABLE 10







EC50 values of the humanized antibodies of anti-TrkA antibody 42F5 binding to TrkA (nM)















Antibodies
42F5-chi
42F5-01
42F5-05
42F5-08
42F5-11
42F5-13
42F5-14
42F5-15





EC50 (nM)
0.490
0.605
0.680
0.535
0.513
0.518
0.547
0.609









3) Assay of TF-1 Cell Proliferation Induced by NGF

The effects of the humanized antibodies of anti-TrkA antibody 42F5 on inhibiting the NGF/TrkA pathway were detected by TF-1 cell proliferation as described in Example 6A. The results are shown in FIG. 20, all the humanized antibodies of anti-TrkA antibody 42F5 can inhibit the proliferation of TF-1 cells induced by NGF, and inhibitory levels are comparable to that of the human-mouse chimeric antibody 42F5-Chi. The IC50 values are shown in Table 11.







TABLE 11







IC50 values of the humanized antibodies of anti-TrkA antibody 42F5 inhibiting NGF-


induced TF-1 cell proliferation

















Antibodies
42F5-chi
42F5-01
42F5-03
42F5-04
42F5-05
42F5-08
42F5-11
42F5-13
42F5-14
42F5-15





IC50 (nM)
1.547
1.566
1.431
1.691
1.483
1.649
1.404
0.867
1.940
0.850









4) Assay of NGF-Induced TrkA/Ba/F3 Cell Proliferation

The effects of the humanized antibodies of anti-TrkA antibody 42F5 on inhibiting the NGF/TrkA pathway were detected by TrkA/Ba/F3 proliferation as described in Example 6B. The results are shown in FIG. 21, all the humanized anti-TrkA antibodies can inhibit the proliferation of TrkA/Ba/F3 cells induced by NGF, and inhibitory levels are comparable to that of the human-mouse chimeric antibody 42F5-Chi. The IC50 values are shown in Table 12.







TABLE 12







IC50 values of the humanized antibodies of anti-TrkA antibody 42F5 inhibiting NGF-


induced TrkA/Ba/F3 cell proliferation

















Antibodies
42F5-chi
42F5-01
42F5-03
42F5-04
42F5-05
42F5-08
42F5-11
42F5-13
42F5-14
42F5-15





IC50
2.916
2.966
2.91
3.695
4.417
3.541
2.814
3.309
3.273
2.71


(nM)



















While specific embodiments of the present invention have been illustrated and described in detail, it should be appreciated that the present invention is not intended to be limited to the specific embodiments described.