CN117050175A - Antibodies against human nerve growth factor - Google Patents

Abstract

The present application relates to neutralizing antibodies or antigen binding fragments thereof that specifically bind NGF, and methods of making such antibodies. The application also relates to the use of said antibodies in the treatment of diseases or disorders associated with NGF, in particular in the alleviation or amelioration of pain in an individual.

Description

Antibodies against human nerve growth factor

The application is a divisional application of application 202010313548.7 with the name of 'anti-human nerve growth factor antibody' on the application day of 2020, 4 and 17.

Technical Field

The application relates to the field of antibodies, in particular to an antibody specifically binding NGF, a preparation method and application thereof.

Background

Nerve Growth Factor (NGF) is the earliest neurotrophin (neurotrophic factor) found and is widely distributed in brain, ganglion, iris, heart, spleen, placenta and other tissues, fibroblasts, smooth muscle, skeletal muscle, glial cells, schwann cells and the like. NGF is a member of the group of Neurotrophins (NTs) that are a group of structurally related proteins that further include brain-derived neurotrophic factors (BDNF), NT-3, NT-4, NT-5. (Wyman et al, gene Therapy (1999), 6:1648-1660). The activity of NGF is mediated through two different membrane-bound receptors, the tyrosine kinase A (TrkA) receptor and the p75 receptor, which p75 receptor is structurally related to other members of the tumor necrosis factor receptor family (Chao, et al, science 232:518-521,1986).

NGF mediates a variety of diseases or conditions such as pain (RICHARDS, N.Br J Anaesth (2013), 111 (1): 46-51), arthritis, collagen vascular diseases, demyelinating diseases, airway inflammatory diseases (Hoyle, cytokine Growth Factor Rev (2003), 14:551-8.; lommatzsch, M.Ann NY Acad Sci (2003), 992:241-9.), neurological diseases, metabolic diseases (such as diabetes) (Yasuda, H.prog Neurobiol (2003), 69 (4): 229-85.), viral infection related diseases (Garaci, E.PNAS (2003), 100 (15) 8927-8932), cardiac arrhythmias (W004/032852), psoriasis and cancer (Nakagawara, A.cancer Lett (2001), 169 (2): 107-14.).

Inflammation or nerve injury of peripheral tissues is a cause of pathophysiological pain. NGF is capable of functioning to modulate peripheral and central neurons, and it has been demonstrated that NGF can interact with pain signaling systems in vivo and can cause hyperalgesia when administered locally or systemically to many species (sarcoelli, expert rev. Neurotherapeutics (2004), 4 (1): 115-127).

Pain is a perception based on signals received from the surrounding environment and transmitted and interpreted by the nervous system (Millan, prog. Neurobiol (1999) 57:1-164), severe pain affecting the quality of life of humans, creating a huge economic burden to society. Pain treatment is one of the most common complaints in clinic and society. However, the treatment of pain is only partially effective and many of these treatments are themselves associated with a number of adverse effects. For example, opioids have a better analgesic effect, but they are also renal toxins, and at high doses tend to cause gastrointestinal irritation, ulcers, bleeding and confusion, and long-term use of opioids with concomitant drug tolerance and dependence.

Due to the tremendous clinical demands and market space in the pain area. The development of new drugs for pain pathway targets is important and urgent for the treatment of pain. In recent years, due to the increasing awareness of NGF's role in acute and chronic pain and hyperalgesia, anti-NGF antagonists have become a new focus of pain treatment research. A number of murine and humanized anti-NGF antibodies have been reported and some of the anti-NGF antibody drug clinical trial data showed positive results, however, the murine anti-NGF antibodies, humanized anti-NGF antibodies have problems with immunogenicity, etc.

Accordingly, there remains a need in the art for compositions or methods that can be used to prevent or treat diseases associated with NGF, such as those associated with pain, and the present application meets that need.

Disclosure of Invention

The present application provides a novel antibody or antigen-binding fragment thereof capable of specifically binding NGF with high affinity, particularly an antibody or antigen-binding fragment thereof that specifically binds human NGF. In a preferred embodiment, the anti-NGF antibodies provided herein are human antibodies that cross-react with non-human NGF (including non-human primate NGF and/or mouse NGF). In another embodiment, a human antibody or antigen-binding fragment thereof that specifically binds human NGF described herein is a neutralizing antibody that neutralizes NGF.

In one aspect, the invention provides a human antibody or antigen-binding fragment thereof that specifically binds human NGF and cross-reacts with neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), brain-derived neurotrophic factor (BDNF). These antibodies are characterized by binding NGF with high affinity and high specificity and being capable of neutralizing NGF activity.

In a specific embodiment, the anti-NGF antibody and antigen fragment thereof comprises one, two or three CDRs (preferably three CDRs) selected from the VH region sequences of the antibodies shown in table I. In other embodiments, the antibodies of the invention comprise one, two or three CDRs (preferably three CDRs) selected from the VL region sequences of the antibodies shown in table I. In some embodiments, an antibody of the invention comprises the 6 CDR region sequences of an antibody shown in table I. In a preferred embodiment, the CDR sequences of the antibodies are those shown in table II.

In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises a) heavy chain Complementarity Determining Regions (CDRs): (i) a CDR1 comprising the amino acid sequence of SEQ ID NO. 1, or a sequence comprising one or more and not more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequence, (ii) a CDR2 comprising the amino acid sequence of SEQ ID NO. 2, or a sequence comprising one or more and not more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequence, (iii) a CDR3 comprising the amino acid sequence of SEQ ID NO. 3, or said sequence comprising one or more and not more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequence, and B) a light chain complementarity determining region (i) a CDR1 comprising the amino acid sequence of SEQ ID NO. 4, or a sequence comprising one or more and not more than 5 amino acid substitutions (e.g., conservative substitutions) deletions or insertions relative to said sequence, (ii) a CDR2 comprising one or more amino acid substitutions (e.g., conservative substitutions) of SEQ ID NO. 5, or amino acid substitutions comprising one or more than one amino acid substitutions (e.g., conservative substitutions) of said sequence of SEQ ID NO. 3, and a light chain complementarity determining region (e.g., CDR 1) comprising the amino acid sequence of SEQ ID NO. 4, the anti-NGF antibodies comprising the modified CDRs therein still have the ability to bind NGF.

In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises a) heavy chain Complementarity Determining Regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID No. 1, (ii) CDR2 consisting of the amino acid sequence of SEQ ID No. 2, (iii) CDR3 consisting of the amino acid sequence of SEQ ID No. 3, and B) light chain Complementarity Determining Regions (CDRs): (i) CDR1 consisting of the amino acid sequence of SEQ ID NO. 4, (ii) CDR2 consisting of the amino acid sequence of SEQ ID NO. 5, (iii) CDR3 consisting of the amino acid sequence shown in SEQ ID NO. 6.

In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises or consists of a heavy chain variable region VH comprising or having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 7, an anti-NGF antibody comprising said VH having the ability to bind NGF. In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region VH comprising the 3 heavy chain variable region CDRs of an antibody shown in Table II and having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 7, an anti-NGF antibody comprising said VH has the ability to bind NGF. In some embodiments, the heavy chain variable region VH of an anti-NGF antibody comprises an amino acid sequence having one or more substitutions (e.g. conservative substitutions), insertions or deletions compared to the amino acid sequence of SEQ ID NO. 7, and an anti-NGF antibody comprising said VH has the ability to bind NGF.

In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises or consists of a light chain variable region VL comprising or consisting of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 8, an anti-NGF antibody comprising said VL having the ability to bind NGF. In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region VL comprising 3 CDRs of the light chain variable region of an antibody set forth in Table II and having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 8, an anti-NGF antibody comprising said VL having the ability to bind NGF. In some embodiments, the light chain variable region VL of an anti-NGF antibody comprises an amino acid sequence having one or more substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of SEQ ID NO. 8, and an anti-NGF antibody comprising said VH has the ability to bind NGF.

In some embodiments, an anti-NGF antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

1) The heavy chain variable region VH comprises or consists of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or more to the amino acid sequence of SEQ ID No. 7; the light chain variable region VL comprises or consists of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or more to the amino acid sequence shown in SEQ ID NO. 8, or

2) The heavy chain variable region VH comprises the heavy chain variable region CDRs shown in table II and has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 7; the light chain variable region VL comprises the CDRs of the light chain variable region shown in Table II and has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 8, and an anti-NGF antibody comprising said VH and VL has the ability to bind NGF, or

3) The heavy chain variable region VH comprises an amino acid sequence having one or more substitutions (e.g. conservative substitutions), insertions or deletions compared to the amino acid sequence of SEQ ID No. 7; the light chain variable region VL comprises an amino acid sequence having one or more substitutions (e.g., conservative substitutions), insertions or deletions compared to the amino acid sequence of SEQ ID NO. 8, and an anti-NGF antibody comprising said VH and VL has the ability to bind NGF.

In a preferred embodiment, the invention provides an anti-NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region VH comprises or consists of the amino acid sequence shown in SEQ ID NO. 7; the light chain variable region VL comprises or consists of the amino acid sequence shown in SEQ ID NO. 8.

In some embodiments, the anti-NGF antibody or antigen-binding fragment thereof further comprises heavy and/or light chain constant region sequences from human antibody germline consensus sequences. The light chain constant region is preferably a kappa (CK) or lambda (C lambda) chain constant region of human origin. The heavy chain constant region may be a gamma, mu, alpha, delta, or epsilon chain, and in some embodiments is of human IgG1, igG2, igG3, igG4, igA1, igA2, igM, igD, and IgE isotype. Each heavy chain, light chain type is characterized by a specific constant region having sequences well known in the art.

In some embodiments, the constant region is preferably a human IgG constant region, such as a human IgG1, igG2, igG3, or IgG4 isotype constant region. In some embodiments, the heavy and/or light chain constant regions are described, for example, in Sequences of Proteins of Immunological Interest, NIH Publication No.91-3242, any of which may be used in the present invention.

In a preferred embodiment, the invention provides an anti-NGF antibody or antigen-binding fragment thereof, wherein the heavy chain constant region sequence is a human IgG2 constant region, the heavy chain constant region sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO. 9; in another embodiment, the heavy chain constant region sequence of the antibody may also be a human IgG4 constant region, the heavy chain constant region sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO. 10; in one embodiment, the light chain constant region of the antibody comprises or consists of the amino acid sequence shown in SEQ ID NO. 11.

It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein the amino acid positions are identified by the (1991) EU index system of Kabat et al.

In some embodiments, to prevent glycosylation of the antibody, e.g., a modification in the human IgG constant region, such modification may be N297A or N297Q (Sazinsky, PNAS (2008), 105 (51): 20167-20172).

In some embodiments, to alter Fc receptor interactions, such as modifications in the human IgG constant region, such modifications may be L234A and/or L235E or L235A.

In some embodiments, to extend half-life, for example, a modification is made in the human IgG constant region, which modification may be R435H.

In some embodiments, to prevent or reduce strand exchange, modifications are made, for example, in the human IgG constant region, which may be S228P (Angal, S.mol Immunol (1993), 30:105-108)

In some embodiments, to enhance FcRn binding, for example, modifications are made to the human IgG constant region, such modifications may be M252Y, S254T, T E (DallAcqua et al, J.biol. Chem (2006), 281 (33): 23514-23524; zalevsky et al, nature Biotech (2010), 28 (2): 157-159), M428L or N434S.

In some embodiments, to alter antibody-dependent cellular cytotoxicity (ADCC), and/or complement-dependent cytotoxicity (CDC), such as in human IgG constant regions, such modifications are known (including, but not limited to, natsume et al, cancer Res (2008), 68 (10): 3863-72; idusogene et al, J.Immunol (2001), 166 (4): 2571-5; moore et al mAbs (2010,2): 181-189; lazar et al, PNAS (2006), 103 (11): 4005-4010; shields et al J.biol. Chem. (2001), 276 (9): 6591-6604;Stavenhagen Cancer Res (2007), 67 (18): 8882-8890; alegre et al, J.Immunol (1992) 148: 3461-3468; kaneko, niwa et al Biodrugs (2011), 25 (1): 1-11.).

In some embodiments, heterodimerization may also be induced by T366W modification, and optionally further by introducing disulfide bonds via S354C and Y349C modifications on the opposite CH3 domains (Carter, journal of Immunological Methods (2001), 248:7-15.).

In some embodiments, the antibodies of the invention also encompass antibodies that compete for binding to NGF with any of the antibodies described above, as well as antibodies that bind to the same epitope of NGF as any of the antibodies described above.

In some embodiments, at least a portion of the framework sequence of an anti-NGF antibody is a human consensus framework sequence.

In one embodiment, the anti-NGF antibodies of the invention are intact antibodies, such as IgG1, igG2, igG3, igG4, igM, igA, and IgE antibodies. In another embodiment, the anti-NGF antibodies of the invention encompass only antigen-binding portions thereof, such as: fab, fab '-SH, fv, scFv or (Fab') ₂ Fragments. In one embodiment, an anti-NGF antibody of the invention may be modified to achieve a function, such as elimination of residual effector function (Glu may eliminate residual effector function (Reddy et al, 2000)).

In some embodiments, the anti-NGF antibodies of the invention are neutralizing antibodies for neutralizing NGF.

In a second aspect, the invention provides a nucleic acid encoding any of the above anti-NGF antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector.

In a third aspect, the invention provides a host cell comprising said vector, or comprising said nucleic acid or antibody. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from mammalian cells, such as CHO cells, HEK293 cells, or COS cells, or other cells suitable for the production of antibodies or antigen binding fragments thereof. In another embodiment, the host cell is a prokaryotic cell, e.g., an E.coli cell, a yeast cell.

In a fourth aspect, the invention provides a method of preparing an anti-NGF antibody or antigen-binding fragment thereof, wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody or antigen-binding fragment thereof, and optionally isolating the antibody or antigen-binding fragment thereof. In a certain embodiment, the method further comprises recovering the anti-NGF antibody or antigen-binding fragment thereof from the host cell.

In one embodiment, the invention provides an anti-NGF antibody or antigen-binding fragment thereof prepared by a method of the invention.

In a fifth aspect, the invention provides a composition comprising any anti-NGF antibody or antigen-binding fragment thereof described herein, preferably the composition is a pharmaceutical composition.

In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, an anti-NGF antibody and antigen-binding fragments thereof comprised in a composition are conjugated to a conjugated moiety. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

In a sixth aspect, the invention provides a method for treating a disease or disorder associated with NGF comprising administering to a subject a therapeutically effective amount of an anti-NGF antibody of the invention, or administering a pharmaceutical composition of the invention.

The invention also relates to a method for alleviating or ameliorating a symptom associated with an NGF-mediated disease or condition, comprising administering to a subject a therapeutically effective amount of an anti-NGF antibody of the invention, or administering a pharmaceutical composition of the invention.

In a specific embodiment, the NGF-mediated disease or condition encompasses any medical disease or condition associated with increased NGF levels or increased sensitivity to NGF, including, but not limited to, pain, arthritis, collagen vascular diseases, demyelinating diseases, airway inflammatory diseases, neurological diseases, metabolic diseases (such as diabetes), viral infection-related diseases, cardiac arrhythmias, psoriasis, and cancer.

In one embodiment, the NGF-mediated disease or condition is a pain-related condition, inflammatory pain, post-operative incision pain, neuropathic pain, fracture pain, gout joint pain, burn pain, cancer pain, complex regional pain syndrome, post-herpetic neuralgia, or pain associated with sickle cell crises. In one embodiment, the pain is pathophysiologic pain.

Further, the methods improve, reduce, inhibit or prevent any NGF-related disease or disorder, or reduce symptoms associated with such disease, such as pain, by eliminating, inhibiting or reducing the activity of NGF. In one embodiment, the invention treats the NGF-related disease or disorder by administering an anti-NGF antagonist, such as an antibody or antigen-binding fragment thereof, alone. In another embodiment, the presently disclosed methods of treatment combine an anti-NGF antibody with a second therapeutic agent to treat a NGF-related disease or disorder. The second therapeutic agent is selected from the group consisting of other magic agents, analgesics, anti-inflammatory agents, chemotherapeutic agents, or immunotherapeutic agents.

In one embodiment, the invention provides a method of alleviating or ameliorating NGF-mediated pain comprising administering to a subject a therapeutically effective amount of an anti-NGF antibody of the invention, or a therapeutically effective amount of a pharmaceutical composition.

In a seventh aspect, the invention provides a method of detecting NGF in a subject or sample, the method comprising: (a) Contacting a subject or sample with any anti-NGF antibody or fragment thereof described herein; and (b) detecting the formation of a complex between the anti-NGF antibody or fragment thereof and NGF. In a preferred embodiment, the anti-NGF antibodies and antigen-binding fragments thereof of the invention further comprise a detectable label.

In one embodiment, the antibodies provided herein are useful for detecting, diagnosing, and monitoring diseases or disorders associated with altered or aberrant NGF expression.

In an eighth aspect, the invention relates to the use of any anti-NGF antibody or fragment thereof described herein in the manufacture of a medicament or kit for treating a NGF-related disease or disorder in a subject.

In one embodiment, the invention provides the use of a composition comprising said anti-NGF antibody in the manufacture of a medicament or kit for treating a disease or disorder associated with NGF in a subject.

In another embodiment, the invention also relates to the use of any of the anti-NGF antibodies or fragments thereof described herein, or a composition comprising the anti-NGF antibodies, in the manufacture of a medicament for alleviating or ameliorating NGF-mediated pain.

In another embodiment, any of the anti-NGF antibodies or fragments thereof herein is for use as a medicament.

In a ninth aspect, the invention provides a kit, e.g., a diagnostic kit, a detection kit, a therapeutic kit, etc., comprising an antibody or composition of the invention.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, is suitable for use with any and all anti-NGF antibodies, or fragments, methods and uses thereof, of the invention described herein.

Advantageous effects of the invention

The present invention provides human antibodies and antigen binding fragments thereof capable of specifically recognizing/binding NGF, which have neutralizing effect against NGF and cross-reacting NT-3, NT-4, BDNF. Since the anti-NGF antibodies of the invention are fully human antibodies, they have low immunogenicity to a subject while treating NGF-related diseases or conditions, or alleviating NGF-related pain, and thus can effectively avoid a corresponding immune response by a subject against them, thus reducing side effects associated with conventional anti-NGF antibodies.

Drawings

Fig. 1: sample (Ns 006) anti-NGF antibody titer assay;

Fig. 2: antibody binding activity assay results;

fig. 3: antibody TRN1028 affinity curve;

fig. 4: antibody TRN1028 inhibits TF-1 cell proliferation;

fig. 5: effect of single subcutaneous administration on CFA induced inflammatory pain MWT in mice (n=9).

Detailed Description

1.1 definition

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For purposes of explaining the present specification, the following definitions will be used, and terms used in the singular form may also include the plural, and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and/or" is understood to mean either one of the selectable items or both of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to include the stated elements, integers or steps but do not exclude any other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

The term "nerve growth factor" or "NGF" refers to the native sequence NGF of all mammalian species (including humans), and in addition encompasses any form thereof that retains at least part of the biological activity of NGF, such as NGF mutants, NGF orthologs, NGF paralogs, and the like.

The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An intact antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, e.g. an antibody naturally occurring in a camel may comprise only heavy chains.

As used herein, "monoclonal antibody" or "mAb" refers to an antibody derived from, for example, a single copy or clone of a eukaryotic, prokaryotic, or phage clone, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope except for possible variant antibodies that are typically present in very small amounts (e.g., variant antibodies that contain natural mutations or are produced during production of a monoclonal antibody preparation). The modifier "monoclonal" refers to the characteristic of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.

"Natural antibody" refers to naturally occurring immunoglobulin molecules having different structures. "native sequence Fc domain" comprises an amino acid sequence identical to the amino acid sequence of an Fc domain found in nature. Natural sequence human Fc domains include, for example, natural sequence human IgG1 Fc domains (non-a and a allotypes); a native sequence human IgG2 Fc domain; a native sequence human IgG3 Fc domain; and a native sequence human IgG4 Fc domain; and naturally occurring variants thereof.

"human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues.

The term "neutralizing antibody" is an antibody or antibody fragment that reduces or inhibits the biological activity of NGF, such as reduces or inhibits the binding of NGF to one or more of its receptors, preferably TrkA. The reduction in biological activity may be partial or complete. The extent to which an antibody neutralizes NGF is referred to as the neutralizing potency of the antibody. Neutralizing efficacy of an antibody may be determined or measured using one or more assays known to the ordinarily skilled artisan and/or described or referenced herein, including, but not limited to, competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays, and enzyme-linked immunosorbent assays (ELISA).

An "anti-NGF antibody" refers to an antibody that binds NGF and inhibits NGF biological activity and/or downstream pathways mediated by NGF signaling. anti-NGF antibodies comprise antibodies that block, antagonize, inhibit, or reduce (including significantly reduce) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding, and/or elicit a response to NGF cells.

In some embodiments, the invention also encompasses fragments of anti-NGF antibodies. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

"biological activity" of NGF generally refers to the ability to bind to NGF receptors and/or activate NGF receptor signaling pathways. For example, biological activity includes any one or more of the ability to bind NGF receptors (e.g., p75 and/or TrkA); the ability to promote dimerization of TrkA receptors and/or autophosphorylation of TrkA; the ability to activate NGF receptor signaling pathways; promoting cell differentiation, proliferation, survival, growth, and other changes in cell physiology; the ability to promote survival of mouse E13.5 trigeminal neurons and the ability to mediate pain, including post-operative pain.

Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of one light chain variable region (LCVR or VL) and one light chain constant region. The light chain constant region consists of one domain CL. VH and VL regions can be further divided into hypervariable regions known as Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

"complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are the amino acid regions of an antibody variable region that are primarily responsible for binding to an epitope. CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus.

Various schemes for determining the CDR sequence of a given VH or VL amino acid sequence are known in the art: kabat Complementarity Determining Regions (CDRs) are determined based on sequence variability and are most commonly used (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, md. (1991)), while Chothia refers to the position of the structural loop (Chothia et al, (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:877-883), abM CDRs are a compromise between Kabat CDRs and Chothia structural loops, and "Contact" (Contact) CDRs are based on analysis of available complex crystal structures by AbM antibody modeling software of Oxford Molecular. Residues of each of these CDRs are as follows, according to different CDR determination schemes.

In one embodiment, the CDRs of the antibodies of the invention are CDR sequences according to the Kabat numbering system at the following Kabat residue positions:

Positions 24-34 (CDR 1), positions 50-56 (CDR 2), and positions 89-97 (CDR 3) in the VL, and positions 27-35 (CDR 1), positions 50-65 (CDR 2), and positions 93-102 (CDR 3) in the VH.

CDRs may also be determined based on having the same Kabat numbering positions as the reference CDR sequences (e.g., any of the exemplary CDRs of the invention).

The term "variant" in connection with an antibody is intended herein to encompass an antibody that has had an amino acid change in a region of the antibody of interest (e.g., a heavy chain variable region or a light chain variable region or a heavy chain CDR region or a light chain CDR region) by at least 1, e.g., 1-30, or 1-20 or 1-10, e.g., 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions, wherein the variant substantially retains the biological properties of the antibody molecule prior to the change. In one aspect, the invention encompasses variants of any of the antibodies described herein. In one embodiment, the antibody variant retains at least 60%,70%,80%,90%, or 100% of the biological activity (e.g., antigen binding capacity) of the pre-altered antibody. It will be appreciated that the heavy chain variable region or the light chain variable region, or the individual CDR regions, of an antibody may be altered individually or in combination. In some embodiments, the amino acids in one or more or all three heavy chain CDRs change by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the amino acid changes are amino acid substitutions, preferably conservative substitutions.

The term "conservative substitution" refers to the substitution of one amino acid with another within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table:

original residue	Exemplary substitution	Preferably conservative substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp,Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine (N-leucine)	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine (N-leucine)	Leu

In some embodiments, the antibody variant has at least 80%, 90% or 95% or 99% or more amino acid identity over the region of the antibody sequence of interest to the parent antibody.

An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237,5,789,199 and 5,840,523, also Charlton, methods in Molecular Biology, volume 248 (b.k.c.lo, editions, humana Press, totowa, NJ, 2003), pages 245-254, which describe expression of antibody fragments in e.coli. After expression, the antibodies may be isolated from the bacterial cell paste in the soluble fraction and may be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with a partially or fully human glycosylation pattern. See gerngros, nat. Biotech.22:1409-1414 (2004), and Li et al, nat. Biotech.24:210-215 (2006). Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney (293 or 293 cells, as described, for example, in Graham et al, J.Gen. Virol.36:59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA 77:216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., yazaki and Wu, methods in Molecular Biology, volume 248 (B.K.C.Lo, ed., humana Press, totowa, NJ), pages 255-268 (2003).

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the reference polypeptide sequence after aligning the sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

When referring to percentages of sequence identity in the present application, these percentages are calculated relative to the full length of the longer sequence unless otherwise specifically indicated. Full length calculations relative to longer sequences are applicable to both nucleic acid sequences and polypeptide sequences.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be determined by equilibrium dissociation constants (K _D ) Expressed by the following formula. The equilibrium dissociation constant is the dissociation rate constant and the association rate constant (k, respectively _dis And k _on ) Is a ratio of (2). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

In one embodiment of the present invention, the anti-NGF antibodies of the present invention have an equilibrium dissociation constant (KD) for NGF of 1X 10 or less ^-7 M is 1×10 or less ^-8 M is 1×10 or less ^-9 M is 1×10 or less ^-10 M is 1×10 or less ^{- 11} M is 1×10 or less ^-12 M is 1×10 or less ^-13 M, etc.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to a carrier.

The term "pharmaceutical composition" refers to a formulation that exists in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the formulation is administered.

In another aspect, the invention provides a pharmaceutical composition comprising one or more monoclonal antibodies that bind NGF or an immunologically active fragment thereof. It will be appreciated that the anti-NGF antibodies or pharmaceutical compositions provided herein may be incorporated into a formulation with suitable carriers, excipients and other agents for administration in combination, thereby providing improved transfer, delivery, tolerability, etc.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered.

Pharmaceutically acceptable carriers suitable for use in the present invention may be conventional, see "Handbook of PharmaceuticalExcipients", seventh edition, R.C.Rowe, P.J.Seskey and S.C.Owen, pharmaceuticalPress, london, chicago; and "Remington' sphaerobical sciences", e.w. martin, mack Publishing Co, easton, PA publications, 21 st edition, 2012, describing compositions and formulations suitable for drug delivery of the disclosed antibodies.

The term "effective amount" refers to an amount or dose sufficient to obtain, or at least partially obtain, a desired effect after administration in a single or multiple doses, and "therapeutically effective amount" refers to an amount that produces the desired effect in the subject being treated, including an improvement in the condition of the subject (e.g., an improvement in one or more symptoms), a delay in the progression of symptoms, and/or the like. An effective disease-preventing amount refers to an amount sufficient to prevent, arrest or delay the onset of a disease. Determination of an effective amount is well within the ability of those skilled in the art, e.g., a therapeutically effective amount depends on the particular disease involved; the extent or severity of the disease; response of individual patients; specific antibodies administered; mode of administration; the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy, etc.

As used herein, "treating" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

The term "subject" or "individual" is primate (e.g., human and non-human primates such as monkeys). In certain embodiments, the individual or subject is a human.

By "alleviating" pain or one or more symptoms of pain is meant alleviating or ameliorating one or more symptoms of pain as compared to the absence of administration of the anti-NGF antibody. "alleviating" also includes shortening or reducing the duration of symptoms.

The term "pain" refers to any etiology, including acute and chronic pain, as well as any pain with an inflammatory component. As used herein, "pain" includes nociception and sensation of pain, and pain can be objectively and subjectively assessed using pain scores and other methods known in the art.

The term "hyperalgesia" refers to an enhanced pain response of the body to normal nociceptive or unpleasant stimuli.

1.2 sequences of exemplary anti-NGF antibodies of the invention TRN1028

TABLE I antibodiesTRN1028Heavy chain variable region sequence and light chain variable region sequence:

TABLE II antibodiesTRN1028Is a CDR sequence of (a):

SEQ ID NO:9：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC

VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS

NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

SEQ ID NO:10：

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG

PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF

NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

SEQ ID NO:11：

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA

GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP

TECS

Examples

The following examples further illustrate the invention, however, it is to be understood that the examples are presented by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art. A description of these methods can be found, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons, updated 7 in 2008); short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology Greene Pub.associates and Wiley-Interscience; glover, DNA Cloning: A Practical Approach, vol.I & II (IRL Press, oxford, 1985); anand, techniques for the Analysis of Complex Genomes, (Academic Press, new York, 1992); transcription and Translation (b.hames & s.higgins, eds., 1984); perbal, A Practical Guide to Molecular Cloning (1984); harlow and Lane, antibodies, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1998) Current Protocols in Immunology Q.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach and w.strober, eds., 1991); annual Review of Immunology; and journal monographs such as Advances in Immunology.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compositions of the present invention may be made and utilized and are not intended to limit the scope of what is claimed.

EXAMPLE 1 preparation of human anti-NGF antibodies

1) PBMC isolation

After a patient suffering from various diseases is injected with the murine nerve growth factor (Jin Loujie) and treated for 30 days (according to the order), 10mL of peripheral blood is collected in an anticoagulant tube containing heparin, and plasma and PBMC cells are separated by a density centrifugation method. The specific operation is as follows: taking 10mL of venous blood, and centrifuging at 400g and 22 ℃ for 15min; sucking the supernatant transparent plasma layer after centrifugation, and sub-packaging at-80deg.C for freezing preservation; fully and uniformly mixing the rest part with an equal amount of RPMI1640 culture medium, slowly adding the mixture into a sterile centrifuge tube containing lymphocyte separation liquid Ficoll, and keeping the layering of the liquid level complete; centrifuging at 2000rpm for 20min, sucking PBMC cells in cloud fog layer with capillary tube, placing into another sterile centrifuge tube, adding RPMI1640 culture medium with volume of 5 times or more, centrifuging at 1500rpm for 10min, washing twice repeatedly, and counting cells at 1×10 ⁷ And freezing at-80 deg.C.

2) Sample anti-NGF antibody titer detection

hNGF (Sino Biological) was diluted to 2. Mu.g/mL with phosphate coating buffer at pH 9.6, coated overnight at 4℃in 0.1 mL/Kong Jiazhi 96-well plates, and blocked with blocking solution at 37℃for 2h. 100. Mu.L of serial diluted plasma samples were added as primary antibody, incubated at 37℃for 1h, then added with goat anti-human IgG (diluted 1:10000) secondary antibody labeled with HRP, incubated at 37℃for 1h, then added with 100. Mu.L/well of substrate color development solution TMB, left at 37℃for 5min in the absence of light, the reaction was stopped with 2M sulfuric acid, and OD450 values were read. The results (as in figure 1) show that sample Ns006 has a higher absorbance, indicating that the anti-NGF antibody in the Ns006 sample is higher in titer and can bind hNGF. PBMCs labeled as Ns006 samples were then flow sorted.

3) Flow cell sorter for sorting single plasma cells

From the results of the serological experiments described above, single plasma cells were sorted from PBMCs in correspondingly numbered samples by flow cytometry (BD, facs Aria Sorp); specific cell populations were selected from PBMC by setting up the gating CD3/CD14/CD16/CD235a-CD19+CD20+/-CD38hi CD27 hi. And selecting a memory plasma cell group specific to hNGF protein (> 3%), and placing single plasma cells in a 96-well PCR plate (20 mu L of single cell lysate per well) so that each well contains one plasma cell, and storing at-80 ℃ in a refrigerator for later use.

4) Isolation of antibody variable region genes from single plasma cells

cDNA first strand was synthesized by reverse transcription from the memory plasma cells obtained in step 3) of example 1 using constant region primers (see primer information disclosed in CN 107760690B) and Superscript III reverse transcriptase (Invitrogen, carlsbad, calif.). The antibody gene was isolated by the following PCR procedure.

First round PCR: mu.L of the system was added with 5. Mu.L of the reverse transcription reaction product, 25. Mu.L of Taq enzyme Mix, 25. Mu.M of each subtype heavy and light chain antibody constant region primer. Reaction conditions:

pre-denaturation at 95 ℃ for 5min;

circulation conditions: denaturation at 94℃for 30s, annealing at 55℃for 60s, extension at 72℃for 90s; repeating the cycle for 35 times;

finally, the reaction product was used for a second round of PCR reactions by extension at 72℃for 7min.

Second round PCR: mu.L of the system was added with 3. Mu.L of the first round PCR reaction product, 25. Mu.L of Taq enzyme Mix, 20. Mu.M of each subtype of heavy and light chain antibody variable region primer. Reaction conditions:

pre-denaturation at 95 ℃ for 5min;

circulation conditions: denaturation at 94℃for 30s, annealing at 58℃for 60s (or annealing of Lambda strand at 64℃for 60 s), extension at 72℃for 90s; repeating the cycle for 35 times;

finally, the mixture was extended at 72℃for 7min. The obtained PCR product is identified by agarose gel electrophoresis with 1.2 percent, and the variable region gene is obtained, wherein the amino acid sequence VH of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence VL of the light chain variable region is shown as SEQ ID NO. 8.

5) Construction, expression and purification of recombinant antibody expression vectors

In one embodiment, the PCR product of the antibody variable region gene identified above is cloned onto pcDNA3.1+/C- (K) -DYK plasmid containing human IgG constant region by conventional method to construct expression vector of human anti-NGF antibody, and then the expression vector is transformed into E.coli DH 5. Alpha. Competent bacteria for vector amplification to extract vector (recombinant plasmid). The obtained recombinant plasmid was co-transfected into HEK293 cells, and then HEK293 cells were cultured and the antibodies were expressed according to a conventional method. The culture supernatant was collected, centrifuged at 4000rpm for 1 hour, and the supernatant was purified by Protein A affinity chromatography. Purified antibodies were checked for expression and purification by SDS-PAGE. SDS-PAGE showed successful expression of the antibody, designated TRN1028, which had a relative molecular weight of about 180KD, a heavy chain of about 55KD and a light chain of about 30KD.

Example 2 detection of antibody binding Activity

The binding activity of the antibody TRN1028 to hNGF was detected by ELISA assay. Specifically, 2. Mu.g/mL of hNGF was used as an antigen to be coated in 96-well plates at 4℃overnight with a carbonate buffer pH of 9.6; the blocking solution was blocked at 37℃for 2h. Antibody TRN1028 was diluted 3-fold with 12 gradients starting with a blocking solution-fold ratio of 10 μg/mL; antibody TRN1028 was then added to the 96-well plate for each gradient, followed by incubation at 37 ℃ for 1h. PBST buffer was washed, 100. Mu.l/well LHRP-labeled goat anti-human IgG (1:10000) was added and incubated for 1h at 37 ℃. Washing with PBST buffer solution, adding TMB color development solution 100 μl/well, standing at 37deg.C for 5min, and H ₂ SO ₄ The reaction was terminated. And detecting, calculating and analyzing the result at the wavelength of 450 nm. In this experiment, an anti-NGF antibody, tanizumab (CAS number 880266-57-9, CN 102746399B, also referred to as E3), disclosed in the prior art with good efficacy was used as a positive control.

Results show (see fig. 2): the EC50 of the anti-NGF antibody E3 binding hNGF is 2.96ng/mL; whereas the EC50 of human anti-NGF antibody TRN1028 of the application binding to hNGF is 1.86ng/mL; it was shown that human anti-NGF antibody TRN1028 has strong binding activity.

Example 3 binding affinity of antibodies

The binding force between an antibody and an epitope is referred to as antibody affinity and represents the ability of an antibody molecule to bind to the epitope. In this example, surface plasmon resonance (BIACORE 3000) was used to detect the antigen binding affinity of antibody TRN 1028. Immobilizing an anti-human IgG (Fc) antibody on the surface of a CM5 sensing chip (GE) by an amino coupling method, and capturing the anti-NGF antibody as a ligand to enable the capturing level to reach about 400 RU; different concentrations of hNGF protein were passed through the detection channel as analyte, and binding of antigen to antibody and dissociation of bound complex were monitored in real time. Kinetic analysis (calculation of dissociation constant (KD)) was performed with Biacore X100 Evaluation Software (2.0.1).

The results are shown in fig. 3, with the antigen dissociation constant of TRN1028 as low as 2.52×10 ^-11 M, dissociation rate of 2.63×10 ^-04 And/s, the TRN1028 antibody has very strong binding affinity with the hNGF antigen.

Example 4 neutralizing Activity of antibodies

In this example, TF-1 cells (human erythroid leukemia cells) were used to examine the ability of anti-NGF antibodies to block NGF-dependent TrkA receptor-mediated cell proliferation activity (Cheva 1ier et al, blood (1994), 83 (6): 1479-1485). NGF is capable of binding to TrkA, which acts as a high affinity receptor for NGF, forming a dimer upon binding to NGF, activating an intracellular domain tyrosine kinase region, causing corresponding tyrosine phosphorylation, thereby activating a downstream signal transduction system, performing a function of modulating target cells, and thereby inducing TF-1 cell proliferation.

TF-1 cells in log phase were grown (ATCC, CRL-2003) ^TM ) Placing at 37deg.C and 5% CO ₂ Medium starvation culture for 24h. A32 ng/mL concentration of hNGF solution was prepared in minimal medium and added to 96-well cell culture plates at 25uL per well. Preparing a solution of the antibody of the present invention at a concentration of 6ug/mL with minimal medium, and taking the solution as a starting pointInitial concentration followed by 3-fold dilution yielded 8 concentration gradients. 25uL of serial dilutions of human anti-NGF antibodies were added to each 96-well plate and incubated at 37℃for 30min. 50uL density of 6X 10 after starvation culture for 24h was added to each well of 96-well plates ⁵ TF-1 cells/mL, hNGF final concentration of 8ng/mL, cell final density of 3X 10 ⁵ /mL. After further incubation for 72h MTS reagent was added and incubation was carried out for 2h at 37℃and detection was then carried out. EC50 of antibody TRN1028 was calculated by absorbance and signal to antibody concentration lazy was analyzed and E3 antibody served as control.

The results show (see fig. 4) that the dose response curves for antibody TRN1028 and the control group are typical S-type dose-response curves. The calculations indicate that the EC50 of blocking NGF signal by antibody TRN1028 and the control are 12.41ng/mL, 14.35ng/mL, respectively. The antibody disclosed by the invention can inhibit proliferation of TF-1 cells induced by NGF and has better neutralization activity.

Example 5 Cross-reactivity of antibodies with BDNF, NT-3, NT-4

The present invention determines the cross-reactivity of the antibody TRN1028 to other neurotrophic factors by ELISA. Coating 96-well plates with hBDNF, hNT-3 and hNT-4 (Absin) as antigens at 200 ug/well respectively, and standing overnight at 4deg.C; then sealing the mixture for 2 hours at normal temperature by using sealing liquid. Antibody TRN1028 (ranging from 10ug/ml to 0 ug/ml) was incubated at a concentration of decreasing concentration at 100 ul/Kong Jiaru for 1h at 37 ℃. Antibody TRN1028 was detected by incubating TMB with goat-anti-human IgG-HRP (1:10000) and substrate chromogenic solution. OD values were measured at double wavelengths of 450-630 nm.

The results showed that antibody TRN1028 reacted with hBDNF, hNT-3 and hNT-4, and the binding EC50 was calculated to be 1.377ng/mL, 3.35ng/mL and 1053ng/mL, respectively. Namely, the human anti-NGF antibody of the invention can cross react with BDNF, NT-3, NT-4 and other neurotrophic factors, and has extremely strong broad spectrum. NGF and BDNF, NT-3, NT-4 belong to the neurotrophic family, which have a certain homology in amino acid sequence. However, there are differences in their biological functions: BDNF has important protection and treatment effects on diseases such as AD, cerebral ischemia injury, PD, depression anxiety and the like, and is possibly more closely related to pain related to NGF and internal heterology than NGF, and the BDNF is also found to play a role in the occurrence and development of tumors; the NT-3 gene can cause the obvious deficiency of skin touch-pressure receptors, and NT-3 also has an effect on congenital nervous system diseases; NTs are involved in the onset of inflammatory skin diseases characterized by cell death (stress alopecia) or hyper-proliferation (atopic dermatitis, psoriasis, etc.) through neurogenic inflammatory reactions or induction of cytokine imbalance and autoimmune responses. The property of TRN1028 to bind BDNF, NT-3 and NT-4 may provide unexpected effects in the treatment of multiple, recurrent disease or cancer pain.

Example 6 anti-hyperalgesic Activity of antibodies

To evaluate the role of antibody TRN1028 in modulating pain, the following experiments were performed.

Mice (C57 BL/6N, male) were selected, and the mechanical paw withdrawal threshold (Mechanical Withdrawal Threshold, MWT) of the left hind paw sole of the mice was measured 3 times with VonFrey (manufacturer: IITC) fiber, and the average of 3 times was taken as the basal pain threshold. The next day (day 1), except for the blank control group, each group of mice was inflamed by injecting 30 μl CFA (3 mg/ml) into the sole of the left hind foot intradermally. After 24h (day 2), mice were tested for left hind paw MWT 3 times in the same way, and the average value was taken as the pain threshold before dosing. Animals with too large or too small MWT were knocked out, 45 animals were selected and equally divided into 5 groups (n=9) according to MWT values, followed by subcutaneous injection of PBS or test subjects: group 1 is blank control group, PBS is given, group 2 is model control group, PBS is given, groups 3, 4 and 5 are respectively 10mg/kg of test substance TRN1028, E3 and negative control antibody; and the pain threshold was measured 24 hours (day 3), 48 hours (day 4) and 72 hours (day 5) after the administration, and the pain threshold improvement rate after the administration was calculated.

Pain threshold improvement% = (MWT dosing group-MWT model group)/MWT model group x 100%.

There was no significant difference in MWT values for the TRN1028, E3 and negative control mice 24h after single subcutaneous injection. After 48h and 72h of dosing, the MWT of the mice in the E3 group and the TRN1028 group still have statistically significant differences (P < 0.01-0.001) compared with the MWT of the mice in the model group, and the pain threshold improvement rate is more than 20%. While the MWT value was slightly higher in the negative control group than in the model group, the difference was not statistically significant (P > 0.05) (FIG. 5, table 1). Thus, it was shown that the antibody TRN1028 has a remarkable pain relieving and treating effect on CFA-induced inflammatory response, i.e. the human anti-NGF antibody of the invention has an anti-hyperalgesic activity.

TABLE 1 pain threshold for CFA-induced inflammatory response by a single subcutaneous injection

Claims (15)

1. A human antibody or antigen-binding fragment thereof that specifically binds human Nerve Growth Factor (NGF) comprising 3 complementarity determining region CDRs of a heavy chain variable region as set forth in SEQ ID No. 7 and 3 complementarity determining region CDRs of a light chain variable region as set forth in SEQ ID No. 8.

2. The human antibody or antigen-binding fragment thereof that specifically binds to human nerve growth factor of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7 or a sequence having at least 90% identity to SEQ ID No. 7, and the light chain variable region comprises SEQ ID NO:8 or a sequence comprising at least 90% identity to SEQ ID No. 8.

3. The human antibody or antigen-binding fragment thereof that specifically binds to human nerve growth factor of claim 1 or 2, wherein the antibody is a human monoclonal antibody.

4. The human antibody or antigen-binding fragment thereof that specifically binds to human nerve growth factor of claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, or (Fab') ₂ Fragments.

5. The human antibody or antigen-binding fragment thereof that specifically binds to a human nerve growth factor of claim 1 or 2, comprising a constant region sequence, wherein at least a portion of the constant region sequence is a human consensus constant region sequence.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID No. 9 or SEQ ID No. 10 and the light chain constant region comprises the amino acid sequence shown in SEQ ID No. 11.

7. A nucleic acid molecule encoding a human antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 that specifically binds human nerve growth factor.

8. A vector comprising the nucleic acid molecule of claim 7, preferably said vector is an expression vector.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8, preferably the host cell is a prokaryotic cell or a eukaryotic cell; more preferably, the host cell is selected from the group consisting of E.coli cells, yeast cells, mammalian cells, such as CHO cells, HEK293 cells or COS cells, or other cells suitable for the production of antibodies or antigen binding fragments thereof.

10. A composition comprising the human antibody or antigen-binding fragment thereof of any one of claims 1-6 that specifically binds NGF and a pharmaceutically acceptable carrier.

11. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the composition of claim 10, in the manufacture of a medicament for reducing, alleviating or inhibiting pain or arthritis.

12. The use of claim 11, wherein the pain is pathophysiologic pain.

13. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the composition of claim 10, in the manufacture of a medicament for co-therapeutic use with a second therapeutic agent to reduce or inhibit pain or arthritis.

14. The use of claim 13, wherein the second therapeutic agent is selected from the group consisting of other neurotrophins, analgesics, anti-inflammatory agents, chemotherapeutic agents, or immunotherapeutic agents.

15. The use of claim 13 or 14, wherein the pain is pathophysiological pain.