Monoclonal antibody targeting Nav1.7 protein

Abstract

The invention relates to a monoclonal antibody of a targeted Nav1.7 protein. Specifically, the invention provides the Nav1.7-targeting antibody or the antigen binding fragment thereof, the antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprise complementary determining regions CDR selected from the following groups: the heavy chain variable region comprises the following complementary determining regions CDR: VHCDR1-3 shown as SEQ ID NO: 2-4; and the light chain variable region comprises the following complementarity determining regions CDR: VLCDR1 to VLCDR3 as shown in SEQ ID NO: 6 to SEQ ID NO: 8. The antibody provided by the invention can specifically recognize and bind to Nav1.7 protein and reduce pain response.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a monoclonal antibody targeting Nav1.7 protein. Background Art

[0002] Sodium channels (Nav), as the core members of the voltage-gated sodium channel family (VGSCs), are the molecular basis for the generation of action potentials in all excitable cells. Such channels are multi-subunit transmembrane glycoproteins composed of α and β subunits, and there are 9 subtypes (Nav1.1-1.9) of α subunits in the human body, which regulate electrical signal conduction through tissue-specific expression. Abnormalities in their functions (such as abnormal activation or inactivation) can lead to major diseases such as neurological diseases (epilepsy, pain), cardiovascular diseases (arrhythmia), and muscle dysfunction. Nav channels are key targets for anticonvulsants, local anesthetics, antiarrhythmic drugs, and analgesics, but these small molecule drugs usually act by binding to the open or inactivated states of the channels. Due to the high sequence homology among Nav family members, traditional drugs are prone to cause off-target effects, resulting in serious side effects (such as cardiotoxicity).

[0003] Therefore, there is an urgent need in the art to develop a Nav1.7 antibody drug with high affinity and high specificity to provide a more precise treatment plan for pain and Nav1.7-related diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide a Nav1.7 antibody drug with high affinity and high specificity.

[0005] In the first aspect of the present invention, there is provided an antibody targeting Nav1.7 or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region comprising the following complementarity-determining regions CDR:

[0006] The heavy chain variable region comprises the following complementarity-determining regions CDR:

[0007] VHCDR1 shown in SEQ ID NO:2,

[0008] VHCDR2 shown in SEQ ID NO:3, and

[0009] VHCDR3 shown in SEQ ID NO:4;

[0010] And the light chain variable region comprises the following complementarity-determining regions CDR:

[0011] VLCDR1 shown in SEQ ID NO:6,

[0012] VLCDR2 as shown in SEQ ID NO:7, and

[0013] VLCDR3 as shown in SEQ ID NO:8.

[0014] In another preferred example, the CDR sequences of SEQ ID NO:2-4 and SEQ ID NO:6-8 are as follows:

[0015] SEQ ID NO.2: DTYMH;

[0016] SEQ ID NO.3: RIDPANGNTKYDPKFQG;

[0017] SEQ ID NO.4: TTALRDYWYFDV;

[0018] SEQ ID NO.6: RSSKSLLHSNGNTYLY;

[0019] SEQ ID NO.7: RMSNLAS;

[0020] SEQ ID NO.8: MQHLEYPFT.

[0021] In another preferred example, the variable region of the heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it; and / or

[0022] the variable region of the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it.

[0023] In another preferred example, the antibody has a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:5.

[0024] In another preferred example, the SEQ ID NO.1:

[0025] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWI GRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARTT ALRDYWYFDVWGAGPTVTVSS;

[0026] In another preferred embodiment, the SEQ ID NO.5:

[0027] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIKR。

[0028] In another preferred embodiment, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:11 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it; and / or

[0029] the amino acid sequence of the light chain is as shown in SEQ ID NO:12 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with it.

[0030] In another preferred embodiment, the antibody has a heavy chain as shown in SEQ ID NO:11 and a light chain as shown in SEQ ID NO:12.

[0031] In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the three heavy chain CDRs and the heavy chain framework regions for connecting the heavy chain CDRs; the light chain variable region comprises the three light chain CDRs and the light chain framework regions for connecting the light chain CDRs.

[0032] In another preferred embodiment, the antibody targeting Nav1.7 or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein ("dsFv").

[0033] In another preferred embodiment, the antibody specifically binds to Nav1.7 or its derivative protein.

[0034] In another preferred embodiment, the antibody can specifically bind to Nav1.7 derived from human, mouse and cynomolgus monkey.

[0035] In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

[0036] In another preferred example, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.

[0037] In another preferred example, the heavy chain of the antibody further comprises a heavy chain constant region.

[0038] In another preferred example, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.

[0039] In another preferred example, the antibody is a diabody or a single-chain antibody.

[0040] In another preferred example, the antibody is a monoclonal antibody.

[0041] In another preferred example, the antibody comprises a monospecific, bispecific, trispecific or multispecific antibody.

[0042] In a second aspect of the present invention, there is provided a recombinant protein having:

[0043] (i) an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0044] (ii) optionally, a tag sequence for assisting expression and / or purification.

[0045] In another preferred example, the tag sequence includes a 6His tag, a GGGS sequence, a FLAG tag. In another preferred example, the recombinant protein (or polypeptide) includes a fusion protein.

[0046] In another preferred example, the recombinant protein is a fusion protein.

[0047] In another preferred example, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against Nav1.7), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0048] In another preferred example, the bispecific or multispecific antibody not only binds to Nav1.7, but also specifically binds to an additional target antigen (such as other tumor antigens, such as other antigens of gastric cancer or antigens of other tumors).

[0049] In another preferred example, the recombinant protein is a monomer, a dimer, or a multimer.

[0050] In another preferred example, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

[0051] In a third aspect of the present invention, there is provided a chimeric antigen receptor (CAR), wherein the antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence (scFv) of an antibody targeting Nav1.7, and the heavy-chain variable region and light-chain variable region of the scFv comprise the following complementarity-determining regions (CDRs):

[0052] The heavy-chain variable region comprises the following CDRs:

[0053] VHCDR1 shown in SEQ ID NO:2,

[0054] VHCDR2 shown in SEQ ID NO:3, and

[0055] VHCDR3 shown in SEQ ID NO:4;

[0056] And the light-chain variable region comprises the following CDRs:

[0057] VLCDR1 shown in SEQ ID NO:6,

[0058] VLCDR2 shown in SEQ ID NO:7, and

[0059] VLCDR3 shown in SEQ ID NO:8.

[0060] In a fourth aspect of the present invention, there is provided a polynucleotide encoding the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, or the chimeric antigen receptor (CAR) as described in the third aspect of the present invention.

[0061] In a fifth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the fourth aspect of the present invention.

[0062] In another preferred embodiment, the vector is selected from the group consisting of: DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0063] In another preferred embodiment, the vector is a retroviral vector.

[0064] In a sixth aspect of the present invention, there is provided a host cell containing the vector as described in the fifth aspect of the present invention or having the polynucleotide as described in the fourth aspect of the present invention integrated into its chromosome exogenously.

[0065] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0066] In another preferred embodiment, the cell is a mammalian cell.

[0067] In another preferred example, the cell is a NK cell or a T cell.

[0068] In another preferred example, the host cell is an engineered immune cell.

[0069] In the seventh aspect of the present invention, there is provided a method for preparing a CAR-NK cell or a CAR-T cell, wherein the CAR-NK cell or the CAR-T cell expresses the chimeric antigen receptor described in the third aspect of the present invention, comprising the following steps:

[0070] Transfecting the polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention into a NK cell or a T cell, thereby obtaining the CAR-NK cell or the CAR-T cell.

[0071] In the eighth aspect of the present invention, there is provided a pharmaceutical composition, which contains the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein such as described in the second aspect of the present invention, the chimeric antigen receptor described in the third aspect of the present invention, the polynucleotide such as described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the host cell described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0072] In another preferred example, the pharmaceutical composition is a preparation, preferably a liquid preparation.

[0073] In another preferred example, the dosage form of the pharmaceutical composition is an injection.

[0074] In another preferred example, the pharmaceutical composition comprises 0.01-99.99% of the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein such as described in the second aspect of the present invention, the host cell such as described in the sixth aspect of the present invention, or a combination thereof and 0.01-99.99% of a pharmaceutical carrier, and the percentages are by mass of the pharmaceutical composition.

[0075] In another preferred example, the pharmaceutical composition is used for preventing and / or treating Nav1.7-related diseases or pain associated with Nav1.7-related diseases.

[0076] In another preferred embodiment, the Nav1.7-related disease is selected from the group consisting of: acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, causalgia, general neuralgia, neurodegenerative disorders, movement disorders, neuroendocrine disorders, ataxia, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head or neck pain, severe or intractable pain, breakthrough pain, postoperative pain, hereditary erythermalgia, dental pain, rhinitis, cancer pain or bladder disorders, or a combination thereof.

[0077] In another preferred embodiment, the cancer pain is associated with a cancer selected from the group consisting of prostate cancer, breast cancer, and cervical cancer.

[0078] In another preferred embodiment, the pharmaceutical composition further comprises other pain therapeutics.

[0079] In another preferred embodiment, the other pain therapeutics are selected from the group consisting of: opioids, COX-2 inhibitors, local anesthetics, NMDA modulators, cannabinoid receptor agonists, P2X family modulators, VR1 antagonists, substance P antagonists, second Nav1.7 antagonists, cytokine or cytokine receptor antagonists, antiepileptic drugs, nerve growth factor (NGF) inhibitors, low-dose colchicine, aspirin, NSAIDs, steroids, low-dose cyclosporine A, tumor necrosis factor (TNF) or TNF receptor inhibitors, uric acid synthesis inhibitors, uric acid secretion promoters, other inflammatory inhibitors such as caspase 1 inhibitors, p38, IKK1 / 2, CTLA-4Ig, and corticosteroids.

[0080] In a ninth aspect of the present invention, there is provided an immunoconjugate comprising:

[0081] (a) an antibody moiety selected from the group consisting of: an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, or a combination thereof; and

[0082] (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0083] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, etc.

[0084] In a tenth aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the vector as described in the fifth aspect of the present invention, or the host cell as described in the sixth aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention,

[0085] (a) for preparing a detection reagent or kit; and / or

[0086] (b) for preparing a drug or preparation for preventing and / or treating Nav1.7-related diseases.

[0087] In another preferred embodiment, the Nav1.7-related diseases are selected from the group consisting of: acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, causalgia, general neuralgia, neurodegenerative disorders, movement disorders, neuroendocrine disorders, ataxia, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head or neck pain, severe or intractable pain, breakthrough pain, postoperative pain, hereditary erythermalgia, toothache, rhinitis, cancer pain, or bladder disorders, or a combination thereof.

[0088] In an eleventh aspect of the present invention, there is provided a method for in vitro detecting (including diagnostic or non-diagnostic) Nav1.7 protein in a sample, the method comprising the steps of:

[0089] (1) contacting the sample with the antibody or its antigen-binding fragment as described in the first aspect of the present invention or the recombinant protein as described in the second aspect of the present invention;

[0090] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of Nav1.7 protein in the sample.

[0091] In another preferred embodiment, the detection is for non-therapeutic and non-diagnostic purposes in vitro.

[0092] In another preferred embodiment, the method is an Immunocytochemistry staning (ICC) detection method, an Immunohistochemistry (IHC) detection method, a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0093] In a twelfth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0094] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for expression;

[0095] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody or its antigen-binding fragment according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

[0096] In a thirteenth aspect of the present invention, a test plate is provided, the test plate comprising: a substrate (support plate) and a test strip, wherein the test strip contains the antibody or its antigen-binding fragment according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof.

[0097] In a fourteenth aspect of the present invention, a kit is provided, the kit comprising:

[0098] (1) a first container containing the antibody or its antigen-binding fragment according to the first aspect of the present invention; and / or

[0099] (2) a second container containing a secondary antibody against the antibody according to the first aspect of the present invention;

[0100] Alternatively, the kit contains the test plate according to the thirteenth aspect of the present invention.

[0101] In a fifteenth aspect of the present invention, a method for treating a disease associated with abnormal Nav1.7 expression or function is provided, comprising administering an effective amount of the antibody or its antigen-binding fragment according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the host cell according to the sixth aspect of the present invention, or the pharmaceutical composition according to the eighth aspect of the present invention to a subject in need of treatment.

[0102] In another preferred embodiment, the disease associated with abnormal Nav1.7 expression or function is a pain-related disease.

[0103] In another preferred embodiment, the pain is pain with high expression of IL-1β.

[0104] In another preferred embodiment, the pain is selected from the group consisting of: acute pain, chronic pain, neuropathic pain, inflammatory pain, arthritis, osteoarthritis, migraine, cluster headache, trigeminal neuralgia, causalgia, general neuralgia, neurodegenerative disorders, movement disorders, neuroendocrine disorders, ataxia, visceral pain, acute gout, postherpetic neuralgia, diabetic neuropathy, sciatica, back pain, head or neck pain, severe or intractable pain, breakthrough pain, postoperative pain, hereditary erythermalgia, dental pain, rhinitis, cancer pain or bladder disorders, or a combination thereof.

[0105] In the sixteenth aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the host cell as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention in the preparation of a medicament for treating a disease associated with abnormal Nav1.7 expression or function.

[0106] In another preferred embodiment, the abnormal Nav1.7 expression refers to overexpression of Nav1.7.

[0107] In another preferred embodiment, the overexpression refers to the ratio of the expression level of Nav1.7 (F1) to the expression level under physiological conditions (F0) (i.e., F1 / F0) ≥ 1.5, preferably ≥ 2, more preferably ≥ 2.5.

[0108] In another preferred embodiment, the medicament is used for preventing and / or treating Nav1.7-related diseases or pain associated with Nav1.7-related diseases.

[0109] In another preferred embodiment, the medicament is used for treating and / or alleviating pain with up-regulated IL-1β expression.

[0110] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 Shows the binding curve of the Nav1.7 antibody to A549 cells in the examples of the present invention.

[0112] Figure 2 Shows the binding curve of the Nav1.7 antibody to MCF-7 cells in the examples of the present invention.

[0113] Figure 3 Shows the binding curve of the Nav1.7 antibody to chondrocytes C28 / I2 in the examples of the present invention.

[0114] Figure 4 The figure shows the scatter plot of the flow cytometry detection results corresponding to the addition amount of 20 μg / mL of the present invention S5AD08.

[0115] Figure 5 The figure shows the relevant results of the inhibition of the ADAMTS1 gene expression up-regulated by IL-1β by the Nav1.7 antibody of the present invention.

[0116] Figure 6 The figure shows the binding curve of the Nav1.7 antibody and CHOK1-mSCN9A in the examples of the present invention.

[0117] Figure 7 The figure shows the results of the inhibition of the pain response time of mice by the Nav1.7 antibody in the examples of the present invention. Among them, the vertical coordinate is cumulative nociceptive behaviors, which refers to the total time (seconds) of the behavior of licking or biting the injected foot of the mouse within every 5 minutes. Detailed implementation mode

[0118] Through extensive and in-depth research, the inventor of the present invention unexpectedly discovered a class of monoclonal antibodies against Nav1.7 for the first time. The antibodies provided by the present invention can specifically recognize and bind to the Nav1.7 protein and effectively inhibit the pain response. The antibodies of the present invention have excellent binding activity to Nav1.7 from various species. In addition, the antibodies of the present invention have a strong ability to inhibit the expression of the ADAMTS1 gene up-regulated by IL-1β. On this basis, the present invention was completed.

[0119] Terms

[0120] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0121] As used herein, the term "comprising" or "containing" includes "including", "consisting essentially of", "substantially consisting of", and "consisting of"; "consisting essentially of", "substantially consisting of", and "consisting of" are subordinate concepts of "containing", "having" or "including".

[0122] Voltage-gated sodium channel 1.7 (Nav1.7)

[0123] The Nav1.7 subtype is encoded by the SCN9A gene located on chromosome 2. Its α subunit contains 27 exons and encodes 1977 amino acids. This protein is composed of four homologous domains (DI - DIV) connected in series by intracellular linker loops and the N / C termini. Each domain contains 6 transmembrane helices (S1 - S6). Among them, the pore region is formed by the S5 - S6 transmembrane segments and the intervening P - loop to form a sodium - selective filter; the S4 helix of the voltage - sensing module is rich in arginine residues and cooperates with the carboxyl terminus of S3 to form a voltage - sensing domain (VSD), mediating membrane potential - dependent channel activation.

[0124] Nav1.7 and Pain

[0125] Nav1.7 is mainly enriched in the dorsal root ganglia (DRG) of the peripheral nervous system, especially highly expressed in small - diameter unmyelinated DRG and large - diameter DRG neurons. By enhancing DRG neuron firing, it promotes synaptic transmission in the spinal dorsal horn (such as the wind - up phenomenon). Inflammation or injury leads to up - regulation of Nav1.7 expression or enhanced function, reducing the pain threshold (such as heat pain and mechanical pain). Gain - of - function and loss - of - function mutations of Nav1.7 are closely related to hereditary erythermalgia and congenital insensitivity to pain respectively, making it a star target for pain management. Nav1.7 is the "master switch" for peripheral pain signal conduction. Abnormalities in its function lead to various pain diseases. Drugs targeting Nav1.7 are expected to achieve highly effective and low - side - effect analgesia. Currently, the drugs under investigation for this target are mainly small molecules and polypeptides, and no antibody drugs have entered the clinical stage. Due to the lack of specificity of small molecules and polypeptides, antibody drugs are a good alternative.

[0126] Antibody

[0127] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, which is composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy chain and light chain also have regularly spaced intra - chain disulfide bonds. One end of each heavy chain has a variable region (VH), followed by multiple constant regions. One end of each light chain has a variable region (VL), and the other end has a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.

[0128] As used herein, the term "variable" refers to the fact that certain portions of the variable regions in antibodies differ in sequence and that these form the binding and specificity of various particular antibodies for their particular antigens. However, the variability is not evenly distributed throughout the antibody variable regions. It is concentrated in three segments in the light and heavy chain variable regions called complementarity determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions which generally assume a β-sheet conformation, connected by three CDRs which form loops and which in some instances form part of the β-sheet structure. The CDRs in each chain are held in close proximity by the FR regions and together with the CDRs of the other chain form the antibody's antigen-binding site (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions do not directly participate in binding of the antibody to an antigen, but they exhibit various effector functions, such as participation of antibody-dependent cell cytotoxicity.

[0129] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes (designated kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant regions. There are five principal classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are designated alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known to those skilled in the art.

[0130] In general, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity determining regions (CDRs), which divide these segments into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form loop structures which are brought into proximity in the spatial structure by the β-sheets formed by the intervening FRs, and the CDRs on the heavy chain and the corresponding CDRs on the light chain form the antigen-binding site of the antibody. Which amino acids form the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.

[0131] In the present invention, the term "antigen-binding fragment" refers to a fragment having antigen-binding ability and includes Fab, F(ab′), F(ab′)2, Fv, etc. Among antibody fragments, Fab (antigen-binding fragment) has a structure containing a light-chain variable region, a heavy-chain variable region, a light-chain constant region, and a first heavy-chain constant region (CH1), and has one antigen-binding site. The difference between Fab′ and Fab is that it has a hinge region including at least one cysteine residue at the C-terminus of the heavy-chain CH1 domain. In the F(ab′)2 antibody, the cysteine residues in the hinge region of Fab′ form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments are known in the prior art, where Fv only has a heavy-chain variable region and a light-chain variable region. The diabody variable fragment (dcFv) is non-covalently linked to the heavy-chain variable region and the light-chain variable region, and the single-chain variable fragment (scFv) is usually covalently linked to the heavy-chain variable region or the C-terminus via a peptide linker to form a dimer, such as a diabody Fv. These antibody fragments can be obtained using proteases (for example, Fab can be obtained by cleaving intact antibodies with papain, while F(ab′)2 fragments can be obtained by cleaving with pepsin), or can be prepared using genetic recombination techniques.

[0132] In the present invention, antibodies include murine, chimeric, humanized, or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, which include human and non-human parts, can be obtained by standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, for example, a chimeric antibody having the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin (see, for example, U.S. Patent Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species and having one or more complementarity-determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0133] Unless otherwise indicated, the amino acid positions within the antibody molecules according to the present invention are numbered according to Kabat.

[0134] As used herein, "antibody variant" or "monoclonal antibody variant" includes an antibody having a modified amino acid sequence compared to a parental antibody but having the same or altered binding affinity for a target antigen. Antibody variants differ from the parental antibody by the substitution, deletion, or addition of one or more amino acid residues at specific positions within the variable domains (including CDR domains) and / or constant regions of the antibody in order to modify certain properties of the antibody, such as binding affinity and / or receptor function, e.g., ADCC, FcRn binding, etc. The histidine-mutated antibodies of the present invention without further modification are not referred to as "antibody variants" according to the present invention. Antibody variants according to the present invention show 80-99% sequence homology compared to the parental antibody, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence homology, depending on the specific positions of the amino acid residues to be substituted, deleted, or added.

[0135] In the present invention, the antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0136] In the present invention, the antibodies of the present invention also include their conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids in the amino acid sequence of the antibody of the present invention with amino acids having similar or close properties. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0137] Table A

[0138] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0139] Pharmaceutical composition

[0140] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or the corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substances to be formulated and the condition to be treated.

[0141] The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the preferred route of administration of the pharmaceutical composition of the present invention is injection or oral administration. The injection administration preferably includes routes such as intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is various conventional dosage forms in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions, etc.

[0142] The antibody of the present invention can also be expressed intracellularly from a nucleotide sequence for cell therapy. For example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0143] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the monoclonal antibody (or its conjugate) described above of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight - about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used together with other therapeutic agents.

[0144] In the present invention, preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiological or pharmaceutically acceptable drug excipient. The drug excipient is a conventional drug excipient in the art, and preferably includes pharmaceutically acceptable excipients, fillers, or diluents, etc. More preferably, the pharmaceutical composition includes 0.01 - 99.99% of the above protein and 0.01 - 99.99% of the pharmaceutical carrier, and the percentages are by mass percentage of the pharmaceutical composition.

[0145] In the present invention, preferably, the dosage of the pharmaceutical composition is an effective amount, and the effective amount is an amount capable of alleviating or delaying the progression of a disease, degenerative, or traumatic condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using no more than conventional experiments.

[0146] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the route of administration and the patient's health status, which are within the scope of the skills of a skilled physician.

[0147] The technical solution of the present invention has the following main advantages:

[0148] (1) The antibody obtained in the present invention has high affinity and specifically targets Nav1.7.

[0149] (2) The antibody obtained in the present invention is an antibody that can treat nerve and related pain diseases. The antibody exerts a therapeutic effect on nerve and related pain diseases by specifically binding to Nav1.7.

[0150] (3) Compared with using polypeptides for ELISA experiments, the present invention focuses on cell screening. The spatial conformation of the antibody of the present invention is more accurate, and it treats pain by causing changes in voltage-gated sodium channels.

[0151] (4) The antibody obtained in the present invention can be used alone to treat nerve and related pain diseases; it can also be conjugated with small molecules and polypeptides to form a conjugate with the efficacy of treating nerve and related pain diseases; it can also be used to construct bispecific antibodies, and the obtained bispecific antibodies can be used to treat nerve and related pain diseases.

[0152] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels without special instructions.

[0153] Example 1 Preparation of Antibody Against Human Nav1.7

[0154] To prepare antibodies against human Nav1.7, Balb / c female mice aged 6 - 8 weeks were selected as the immunization targets. A method of mixed immunization with plasmids encoding human Nav1.7 and cells was adopted. 50 μg of nucleic acid was injected via the tail vein at the 1st, 3rd, 5th, 7th, and 9th weeks, for a total of 5 immunizations. The titer of the immune serum was detected by the indirect ELISA method, and the mouse with the highest titer was screened at the 10th week. At the 11th week, the HEK293 - Nav1.7 cell line was taken, and the cell seeding density was 1*10E6 cells for booster immunization. Three days later, the spleen tissue of the mice was taken out, and the spleen cells were fused with SP2 / 0 cells using the BTX electrofusion system to generate hybridoma cells, and 4000 clones were plated. Subsequently, candidate clones were obtained through primary screening by cell ELISA and secondary screening by flow cytometry. Finally, the antibody sequence S5AD08 corresponding to the candidate clone was obtained through hybridoma sequencing. Among them,

[0155] The VH amino acid sequence of S5AD08 (SEQ ID NO:1, the underlined sequences are CDR1 - 3 sequences (KABAT definition))

[0156] EVQLQQSGAELVKPGASVKLSCTASGFNIK DTYMH WVKQRPEQGLEWIG RIDPANGNTKYDPKFQG KATITADTSSNTAYLQLSSLTSEDTAVYYCAR TTALRDYWYFDV WGAGPTVTVSS

[0157] The VL amino acid sequence of S5AD08 (SEQ ID NO:5, the underlined sequences are CDR1 - 3 sequences (KABAT definition))

[0158] DIVMTQAAPSVPVTPGESVSISC RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPFT FGSGTKLEIKR

[0159] Example 2 Construction and expression of the full - length S5AD08 antibody

[0160] Based on the above Example 1, the VH and VL of the antibody sequence S5AD08 retrieved were respectively constructed into eukaryotic expression vectors containing the hIgG1 kappa constant region. The VH and VL of S5AD08 were co - transfected into freestyle293F cells. After culturing for 3 - 7 days, the supernatant was collected and purified using a protein A column to obtain the antibody protein corresponding to S5AD08.

[0161] The CDR sequences of the S5AD08 antibody are shown in Table 1 below:

[0162] Table 1

[0163]

[0164] Among them, the amino acid sequences of the hIgG1 constant region and the kappa chain constant region are shown as follows respectively:

[0165] Amino acid sequence of hIgG1 constant region (SEQ ID NO:9)

[0166] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0167] Amino acid sequence of kappa chain constant region (SEQ ID NO:10)

[0168] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0169] Amino acid sequence of the heavy chain (H) of S5AD08 (SEQ ID NO: 11, the CDR1-3 sequences (KABAT definition) are underlined)

[0170] EVQLQQSGAELVKPGASVKLSCTASGFNIK DTYMH WVKQRPEQGLEWIG RIDPANGNTKYDPKFQG KATITADTSSNTAYLQLSSLTSEDTAVYYCAR TTALRDYWYFDVWGAGPTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] Amino acid sequence of the light chain (L) of S5AD08 (SEQ ID NO: 12, CDR1-3 sequences (KABAT definition) underlined)

[0172] DIVMTQAAPSVPVTPGESVSISC RSSKSLLHSNGNTYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC MQHLEYPFT FGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0173] Amino acid sequence of the heavy chain (H) of SVmab-1 (SEQ ID NO: 13)

[0174] QGQMQQSGAELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGGTSYNQKFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARQDGNYRYWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0175] Amino acid sequence of the light chain (L) of SVmab-1 (SEQ ID NO: 14)

[0176] ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKLEIKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0177] Example 3: Detection of cell affinity and non-specific binding of Nav1.7 antibody

[0178] It was found from querying the The Human Protein Atlas database that A549 (human non-small cell lung cancer cells) expresses SCN5A, SCN7A, SCN8A and SCN9A, and MCF-7 (human breast cancer cells) only expresses SCN5A, SCN7A, SCN8A and does not express SCN9A.

[0179] In this example, the binding ability of the Nav1.7 antibody (S5AD08) to the Nav1.7-positive cell line A549 was detected by flow cytometry. Among them, the positive control SVmab-1 is a Nav1.7 antibody disclosed by Duke University (the heavy and light chain sequences of SVmab-1 are shown in SEQ ID NO: 13 and 14, respectively). The S curve is plotted as shown in Figure 1 , and the EC50 results are shown in Table 2:

[0180] Table 2

[0181] Anti-Nav1.7 S5AD08 SVmab-1 EC50 (ug / mL) 0.5848 113.0

[0182] Figure 1 The results in and Table 2 show that compared with SVmab-1 (EC50 = 113 ug / mL), the EC50 of the Nav1.7 antibody S5AD08 of the present invention binding to the Nav1.7-positive cell line A549 is only 0.5848 ug / mL, indicating that the Nav1.7 antibody S5AD08 of the present invention has excellent binding activity.

[0183] In addition, this example verified whether the Nav1.7 antibody has a non-specific binding reaction, and the Nav1.7-negative cell line MCF-7 was used for cell non-specific binding detection. The specific experimental procedure is as follows:

[0184] 3×10 5 cells were taken respectively, gradient-diluted Nav1.7 antibody protein was added, after incubation for 1 h, washed once with PBS, Anti-hFc-APC (purchased from Jackson immunology) was added, and after washing once with PBS, it was detected by flow cytometry.

[0185] The results showed that the binding ability of S5AD08 to A549 cells was higher than that of SVmab-1 ( Figure 1 ), and there was no non-specific binding to MCF-7 cells ( Figure 2 ), indicating that the Nav1.7 antibody S5AD08 of the present invention has excellent specificity.

[0186] Example 4 Detection of Nav1.7 Antibody Function

[0187] The chondrocyte cell line C28 / I2 is a human cell model commonly used to study osteoarthritis (OA), cartilage degradation and inflammatory responses. It has been found that Nav1.7 (encoded by the SCN9A gene) is expressed in chondrocytes and may be involved in pain signal transduction and cartilage metabolism regulation.

[0188] Among them, IL-1β significantly upregulates the expression of ADAMTS1 in C28 / I2 cells through the NF-κB / MAPK pathway, promotes cartilage matrix degradation and inflammation amplification, and is a potential intervention node for the treatment of osteoarthritis.

[0189] This example mainly demonstrates the binding ability of the Nav1.7 antibody to chondrocytes C28 / I2 expressing SCN9A and its inhibitory ability on the IL-1β-induced upregulated ADAMTS1 gene. The specific process is as follows:

[0190] To detect the binding ability of the Nav1.7 antibody to chondrocytes C28 / I2 expressing SCN9A, 3×10 5 cells were taken respectively, and gradient-diluted Nav1.7 antibody protein was added. After incubation for 1 h, it was washed once with PBS, Anti-hFc-APC (purchased from Jackson immunology) was added, and after washing once with PBS, it was detected by flow cytometry. The flow scatter plot corresponding to the addition amount of 20 μg / mL of S5AD08 is as Figure 4 shown, showing that its positive rate with chondrocytes C28 / I2 is as high as 99.13%. The EC50 of the flow cytometry detection of the binding of the Nav1.7 antibody to chondrocytes C28 / I2 is shown in Table 3:

[0191] Table 3

[0192] Anti-Nav1.7 S5AD08 SVmab-1 EC50 (ug / mL) 1.722 3.838

[0193] The results show that: the binding ability of the Nav1.7 antibody to chondrocytes C28 / I2 expressing SCN9A is higher than that of SVmab-1 ( Figure 3 ), and the EC50 value of the binding of the Nav1.7 antibody to chondrocytes C28 / I2 is about 1 / 2 of that of SVmab-1.

[0194] To verify the inhibitory ability of the Nav1.7 antibody on the IL-1β-induced upregulated ADAMTS1 gene. IL-1β was fixed at 10 ng / mL, 20 μg / mL of S5AD08, SVmab-1 and the Nav1.7 inhibitor PF-04856264 (purchased from MCE) were added to each well, and human chondrocytes were treated for 48 h. The RNA of chondrocytes was extracted for reverse transcription, and qPCR was performed using cDNA as a template to detect the expression of the ADAMTS1 gene.

[0195] The results show that: compared with the IgG1 group, S5AD08, SVmab-1 and the Nav1.7 inhibitor PF-04856264 can all reduce the expression level of the ADAMTS1 gene, but compared with SVmab-1 and the Nav1.7 inhibitor PF-04856264, S5AD08 significantly reduces the expression level of the ADAMTS1 gene (Figure 5 ) indicates that the Nav1.7 antibody S5AD08 of the present invention has a strong ability to inhibit the expression of the ADAMTS1 gene upregulated by IL-1β.

[0196] Example 5 Effect of Nav1.7 Antibody on Mouse Pain Model

[0197] This example mainly demonstrates the binding ability of the Nav1.7 antibody to cells overexpressing murine Nav1.7, and verifies the effect of S5AD08 on the pain response of mice in a mouse pain model. The specific process is as follows:

[0198] Construct a full-length plasmid expressing murine Nav1.7 (UniProtKB, Q62205) and transiently transfect it into CHO-K1 cells. Flow cytometry is used to detect its binding to the Nav1.7 antibody. The specific steps are as follows:

[0199] Take 3×10 5 CHO-K1-mSCN9A cells, add gradient-diluted Nav1.7 antibody, after incubating for 1 h, wash 3 times with PBS, add Anti-hFc-APC (purchased from Jackson immunology), and perform flow cytometry analysis.

[0200] The results show that compared with SVmab-1, the Nav1.7 antibody S5AD08 of the present invention has a higher binding activity to CHO-K1-mSCN9A cells. ( Figure 6 )

[0201] Formalin test: In the mouse formalin model (i.e., an inflammatory pain model), pain in the first and second phases is inhibited after deletion of Nav1.7 in DRG neurons. Examine the formalin model to determine whether S5AD08 can relieve pain by inhibiting Nav1.7. The specific process is as follows:

[0202] Thirty minutes after 6 CD1 mice were intravenously injected (i.v.) with an antibody (50 mg / kg), 20 μL of 5% formalin was injected subplantarly into the left hind paw of the mice to induce acute pain, and the Isotype was the 2019-nCoV NP antibody (Suzhou Novoprotein Science & Technology Co., Ltd., Cat. No.: DA027) of the same hIgG1 subtype. After formalin injection, each mouse was immediately returned to the laboratory and video-recorded individually for one hour. The video was played back to visually score the pain responses (licking, flicking, lifting the injected paw) of each affected animal. The pain behavior was scored using the licking / biting time recording method, that is, the total time of licking, biting, flicking or lifting the injected foot of the mouse within a specific time period (every 5 minutes) was directly recorded, and the cumulative time of the first phase (0-10 minutes) and the second phase (15-45 minutes) was focused on. The statistical results of the reaction time of the mice to pain are shown in Figure 7 .

[0203] The results showed that S5AD08 could effectively inhibit the pain response of mice. Formalin injection induced a typical biphasic pain response. The first phase was the acute phase (0-10 minutes), which was acute pain caused by direct chemical stimulation. The second phase was the inflammatory phase (15-45 minutes), which was persistent pain mediated by peripheral inflammation and central sensitization. The sodium channel Nav1.7 was mainly involved in acute physiological pain (the first phase), so S5AD08 might be ineffective against inflammatory pain in the second phase 30 min later.

[0204] All documents mentioned in this invention are cited in this application as references, as if each document was cited individually as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An antibody targeting Nav1.7 or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region, and the heavy-chain variable region and the light-chain variable region comprise the following complementarity-determining regions CDR: The heavy-chain variable region comprises the following complementarity-determining regions CDR: VHCDR1 shown in SEQ ID NO:2, VHCDR2 shown in SEQ ID NO:3, and VHCDR3 shown in SEQ ID NO:4; And the light-chain variable region comprises the following complementarity-determining regions CDR: VLCDR1 shown in SEQ ID NO:6, VLCDR2 shown in SEQ ID NO:7, and VLCDR3 shown in SEQ ID NO:

8.

2. A recombinant protein, characterized in that, The recombinant protein has: (i) the antibody or its antigen-binding fragment as described in claim 1; and (ii) an optional tag sequence for assisting expression and / or purification.

3. A chimeric antigen receptor CAR, characterized in that, The antigen-binding domain of the chimeric antigen receptor contains the single-chain variable region sequence scFv of an antibody targeting Nav1.7, and the heavy-chain variable region and the light-chain variable region of the scFv comprise the following complementarity-determining regions CDR: The heavy-chain variable region comprises the following complementarity-determining regions CDR: VHCDR1 shown in SEQ ID NO:2, VHCDR2 shown in SEQ ID NO:3, and VHCDR3 shown in SEQ ID NO:4; And the light-chain variable region comprises the following complementarity-determining regions CDR: VLCDR1 shown in SEQ ID NO:6, VLCDR2 shown in SEQ ID NO:7, and VLCDR3 shown in SEQ ID NO:

8.

4. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, or the chimeric antigen receptor CAR as described in claim 3.

5. A carrier, characterized in that, The vector contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, The host cell contains the vector as described in claim 5 or the exogenous polynucleotide as described in claim 4 is integrated into the chromosome.

7. A method for preparing CAR-NK cells or CAR-T cells, characterized in that, The step of the CAR-NK cell or CAR-T cell expressing the chimeric antigen receptor as described in claim 3 comprises: Transducing the polynucleotide as described in claim 4 or the vector as described in claim 5 into NK cells or T cells, thereby obtaining the CAR-NK cell or CAR-T cell.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, the chimeric antigen receptor as described in claim 3, the polynucleotide as described in claim 4, the vector as described in claim 5, or the host cell as described in claim 6, and a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of the antibody or its antigen-binding fragment as described in claim 1, or the recombinant protein as described in claim 2 in the preparation of a drug for treating a disease associated with abnormal Nav1.7 expression or function.

10. The use according to claim 9, wherein The drug is used for treating and / or alleviating pain with up-regulated IL-1β expression.

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