CN108178798B - PH engineered NGF antibodies and medical uses thereof - Google Patents

Abstract

The invention provides an NGF antibody, an antigen-binding fragment thereof and medical application thereof, and also provides a chimeric antibody, a humanized antibody and a pH engineered antibody containing CDR regions of the antibody, a pharmaceutical composition containing the human NGF antibody and the antigen-binding fragment thereof, and application of the antibody in preparation of a medicament for treating and/or preventing pain, including postoperative pain, rheumatoid arthritis pain and osteoarthritis pain.

Description

PH engineered NGF antibodies and medical uses thereof

Technical Field

The present invention relates to an NGF antibody, antigen binding fragments, chimeric, humanized and pH engineered antibodies comprising the CDR regions of the NGF antibody, as well as pharmaceutical compositions comprising the NGF antibody and antigen binding fragments thereof, and uses thereof as a pain treatment and prevention medicament.

Background

Nerve Growth Factor (NGF) is the first recognized neurotrophin (neurotrophin) and has been fully established for its role in the development and survival of peripheral and central nerves. NGF has been shown to be a critical survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons and basal forebrain cholinergic neurons. Smeyne et al, Nature 368: 246249(1994) and Crowley et al, Cell 76: 10011011 (1994). NGF upregulates (upregulate) neuropeptide expression in sensory neurons (Lindsay and Harmer, Nature 337: 362364 (1989)) and regulates its activity via two distinct membrane-bound receptors, the TrkA tyrosine kinase receptor and the p75 common neurotrophin receptor (sometimes referred to as "high affinity" and "low affinity" NGF receptors, respectively). The p75 receptor is structurally related to other members of the tumor necrosis factor receptor family (Chao et al, Science 232: 518521 (1986)). For a general description of NGF, see Huang et al, Annu. Rev. Neurosci. 24: 677736 (2001), Bibel et al, GenesDev. 14: 29192937 (2000). The crystalline structure of NGF and of NGF in a complex with the trkA receptor have been defined. See Nature254:411(1991), Mature 401: 184188 (1996).

In addition to its role on the nervous system, NGF has become involved in processes outside the nervous system. For example, NGF has been shown to promote vascular permeability (Otten et al, EurJPharmacol. 106: 199201 (1984)), enhance T and B cell immune responses (Otten et al, Proc. Natl. Acadsi. USA 86: 1005910063(1989)), induce lymphocyte differentiation and mast cell proliferation, and cause the release of soluble biological signals from mast cells (Matsuda et al, Proc. Natl. Acad. Sci. USA85:65086512(1988); Pearce et al, J.Physiol. 372: 379393 (1986); Bischoff et al, Blood 79: 266269 (1992); Horigome et al, J.biol. chem. 268: 8114887 (1993)). Although exogenous addition of NGF has been shown to possess all of these effects, it is important to note that endogenous NGF is only rarely shown to be important in any of these processes in vivo (Torcia et al, cell. 85 (3): 34556 (1996)). Thus, it is unclear, if at all, what factors may inhibit the biological activity of endogenous NGF.

NGF is produced by a number of Cell types, including mast cells (Leon USA 91: 37393743 (1994)), B lymphocytes (Torcia et al, Cell 85: 345356 (1996)), keratinized cells (DiMarco et al, J.biol. chem. 268:2283822846)), smooth muscle cells (Ueyama et al, J.Hypertens. 11: 10611065 (1993)), fibroblasts (Lindholm et al, Eur.J.Neurosci. 2: 795801 (1990)), bronchial epithelial cells (Kassel et al, Clin.exp.Allergy 31: 143240: 2001), mesangial cells (Steiner et al, am.J.Physiol. 261: 792798 (1991)) and skeletal myotubes (Schwartz et al, Photochem. B200: 19566). NGF receptors have been found on a variety of cell types outside of the nervous system. For example, TrkA has been found on human monocytes, T and B lymphocytes, and mast cells.

A correlation between increased NGF content and inflammatory conditions has been observed in human patients and in several animal models. These include systemic lupus erythematosus (BracciLaudiero et al, Neuroreport4: 56565 (1993)), multiple sclerosis (BracciLaudiero et al, Neurosci.Lett. 147: 912 (1992)), psoriasis (Raychaudhuri et al, actaDerm.I' enerol. 78: 8486 (1998)), arthritis (Falcim et al, Ann.Rheum. Dis. 55: 745748 (1996)), interstitial cystitis (Okragly et al, J.Urology1 Θ 1: 438-441 (I "9)), and asthma (Braun et al, Eur.JImmunel.28: 32251 (403403403403403).

Similarly, the rise in the commercial NGF content in peripheral tissues is associated with hyperalgesia and inflammatory responses, and has been observed in many forms of arthritis. Synovium of patients affected by rheumatoid arthritis expresses high amounts of NGF, and it has been reported that NGF is not detectable in uninflammated synovium (Aloe et al, Arch. Rheum. 35: 351355 (1992)). Similar results were also seen in rats experimentally induced with rheumatoid arthritis (Aloe et al, Clin. Exp. Rheumatotol. 10: 203204 (1992)). Increased levels of NGF have been reported in transgenic arthritic mice, along with an increase in the number of mast cells (Aloe et al, int.J.tissue reactions Exp.Clin.Aspects15: 139143 (1993)). PCT publication No. WO02/096458 discloses the use of anti-NGF antibodies with certain properties to treat various NGF-related disorders, such as inflammatory conditions (e.g., rheumatoid arthritis). It has been reported that injection of purified anti-NGF antibody into arthritic transgenic mice bearing the human tumor necrosis factor a (TNFRi) gene results in a reduction in mast cell number and a reduction in histamine and substance P levels in the synovium of arthritic mice (Aloe et al, Rheumatol. int. 14: 249252 (1995)). Exogenous administration of NGF antibodies has been shown to reduce the elevated levels of TNF α that occur in arthritic mice (Manni et al, Rheumatotol. int. 18: 97102 (1998)).

Furthermore, increased expression of NGF and high affinity NGF receptor (TrkA) has been observed in human osteoarthritic chondrocytes (Iannone et al, Rheumatology 41: 14131418 (2002)).

Rodent anti-NGF antagonist antibodies have been reported. See, e.g., Hongo et al, Hybridoma (2000) 19 (3): 215227; Ruberti et al (1993) cell. molecular. neurobiol. 13(5): 559568. However, when rodent antibodies are used on humans for therapy, human anti-aging murine antibody responses develop in a large number of treated individuals. In addition, it has been found that the effector functions of mouse antibodies are less potent in human relation. Thus, there is a great need for anti-NGF antagonist antibodies, including humanized anti-NGF antagonist antibodies.

Several pharmaceutical companies are currently developing monoclonal antibodies against NGF, including blocking the activity of nerve growth factor with a neutralizing humanized monoclonal antibody (tanezumab) to alleviate pain from osteoarthritis. This finding suggests the possibility of using neutralizing antibodies, in theory, similar to those used in rheumatoid arthritis patients for Tumor Necrosis Factor (TNF).

The central pain response is usually accompanied by local inflammatory responses, such as osteoarticular pain. An acidic microenvironment (pH 4.0) is formed due to The local inflammatory response and The release of protons from activated osteoclasts and The like, whereas low pH not only activates TRPV1 and ASIC (acid-sensing ion channel) agents, but also severely affects The affinity of NGF antibodies (The Journal of Neuroscience, July 1995.15(7) 4919; pain. 2012 Aug 153(8): 1673-9.; Curr neuropharmacol. 2006 Jul; 4(3): 197. 206.). Therefore, the pH sensitivity screening or pH engineering modification of NGF therapeutic antibodies can significantly increase the clinical efficacy of NGF antibodies.

The present invention provides pH engineered, highly selective, humanized NGF antibodies.

Disclosure of Invention

The present invention provides an NGF antibody or antigen-binding fragment thereof, comprising:

an antibody heavy chain variable region selected from the group consisting of at least 1 HCDR as shown in seq id no:4, 5 or 6; and

an antibody light chain variable region, wherein at least 1 of said antibody light chain variable regions is selected from the group consisting of LCDRs as shown in SEQ ID NO:7, 8 or 9.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR1 as shown in SEQ ID NO. 4.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR2 as shown in SEQ ID NO. 5.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR3 as shown in SEQ ID NO 6.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 7.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises LCDR2 shown in SEQ ID NO. 8.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises the region as set forth in SEQ ID NO 9 LCDR 3.

In a preferred embodiment of the invention, there is provided an NGF antibody or antigen-binding fragment thereof according to the invention, wherein the antibody is a humanized antibody or fragment thereof.

In a preferred embodiment of the present invention, the humanized antibody or fragment thereof provided according to the present invention, the antibody light chain variable region thereof further comprises a light chain FR region of a humanized kappa, lambda chain or variant thereof.

In a preferred embodiment of the invention, there is provided according to the invention a humanized antibody or fragment thereof further comprising a light chain constant region of a humanized kappa, lambda chain or variant thereof.

In a preferred embodiment of the present invention, the humanized antibody or fragment thereof provided according to the present invention further comprises a heavy chain FR region of humanized IgG1, IgG2, IgG3, IgG4 or variants thereof in its antibody heavy chain variable region.

In a preferred embodiment of the invention, the humanized antibody or fragment thereof provided according to the invention further comprises a heavy chain constant region of humanized IgG1, IgG2, IgG3, IgG4 or variants thereof.

In a preferred embodiment of the present invention, there is provided an NGF antibody or antigen-binding fragment thereof according to the present invention, wherein the antibody is a chimeric antibody or fragment thereof.

In a preferred embodiment of the present invention, there is provided a chimeric NGF antibody or fragment thereof according to the present invention, wherein the variable heavy chain region sequence of the chimeric antibody is: 2, SEQ ID NO.

In a preferred embodiment of the present invention, there is provided a chimeric NGF antibody or fragment thereof according to the present invention, wherein the light chain variable region sequence of the chimeric antibody is: 3, SEQ ID NO.

In a preferred embodiment of the invention, there is provided an NGF chimeric antibody or fragment thereof according to the invention, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.

In a preferred embodiment of the present invention, there is provided a NGF chimeric antibody or a fragment thereof according to the present invention, further comprising a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a heavy chain constant region of human IgG2 or IgG4, or IgG1 which has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

In a preferred embodiment of the invention, there is provided an NGF antibody or antigen-binding fragment thereof according to the invention, wherein the antibody is a humanized antibody or fragment thereof.

In a preferred embodiment of the present invention, there is provided a NGF humanized antibody or fragment thereof, wherein the humanized antibody light chain variable region further comprises a light chain FR region of a human kappa, lambda chain or variant thereof.

In a preferred embodiment of the invention, a humanized NGF antibody or fragment thereof is provided according to the present invention, wherein light chain FR region sequences on the humanized antibody light chain variable region, such as combined sequences derived from human germline light chain IGKV1-39 and JK 4; it comprises the FR1, FR2, FR3 regions of human germline light chain IGKV1-39 and the FR4 region of JK 4.

In a preferred embodiment of the present invention, there is provided a NGF humanized antibody or fragment thereof according to the present invention, wherein the humanized antibody light chain variable region variant preferably has an amino acid change in the light chain variable region of 0-10; the amino acid change of the variable region sequence variant in the variable region of the humanized antibody light chain is preferably A43S.

In a preferred embodiment of the invention, there is provided a humanized NGF antibody or fragment thereof according to the present invention, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.

In a preferred embodiment of the present invention, the humanized antibody heavy chain variable region provided according to the present invention further comprises heavy chain FR regions of human IgG1, IgG2, IgG3, IgG4 or variants thereof.

In a preferred embodiment of the invention, there is provided a humanized NGF antibody or fragment thereof according to the present invention, wherein the heavy chain FR region sequence in the heavy chain variable region of said humanized antibody, such as the combined sequence derived from human germline heavy chain IgHV3-7 and JH6, comprises the FR1, FR2, FR3 regions of human germline heavy chain IgHV3-7 and the FR4 region of JH 6.

In a preferred embodiment of the invention, an NGF humanized antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprises a human IgG2 or IgG4 heavy chain constant region. Because IgG2 or IgG4 has no ADCC toxicity, or IgG1 which has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation is used. Said variant preferably comprises a heavy chain constant region mutation with reduced or absent ADCC effector function, more preferably N297A, L234A, L235A of IgG 1; IgG2/4chimera, F235E of IgG4, or L234A/E235A mutations.

In a preferred embodiment of the invention, an NGF antibody or antigen-binding fragment thereof is provided according to the invention, wherein the antigen-binding fragment is Fab, Fv, sFv, F (ab')₂。

The invention further provides a DNA molecule encoding an NGF antibody or antigen-binding fragment thereof as described above.

The invention further provides an expression vector comprising a DNA molecule as described above.

The present invention further provides a host cell transformed with the expression vector as described above.

In a preferred embodiment of the present invention, there is provided a host cell according to the present invention, wherein said host cell is a mammalian cell, preferably a CHO cell.

The invention further provides a pharmaceutical composition comprising an NGF antibody or antigen-binding fragment thereof according to the invention and a pharmaceutically acceptable excipient, diluent or carrier.

The invention further provides the use of an NGF antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, according to the invention in the manufacture of a medicament for the treatment of an NGF-mediated disease or disorder; wherein said disease is preferably hypercholesterolemia; more preferably NGF-induced pain of various types, including postoperative pain, rheumatoid arthritis pain, osteoarthritis pain, and the like.

Drawings

FIG. 1 SDS-PAGE analysis of purified human NGF protein

FIG. 2 SDS-PAGE analysis of purified murine human chimeric antibody N11 and humanized antibody G11-H1L1

FIG. 3 SDS-PAGE analysis of purified pH engineered, humanized antibody G11-H2L2

FIG. 4 binding curve of chimeric antibody N11 to human NGF protein at neutral pH (ForteBio).

FIG. 5 binding curve of chimeric antibody N11 to human NGF protein at slightly acidic pH (ForteBio). .

FIG. 6 binding curve of pH engineered, humanized antibody (G11-H2L2) to human NGF protein at neutral pH (ForteBio)

FIG. 7 binding curves of pH engineered, humanized antibodies (G11-H2L2) to human NGF protein at slightly acidic pH (ForteBio)

Detailed Description

Term of

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other 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.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j. diol. chem,243, p3558 (1968).

The antibody of the invention refers to immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes called immunoglobulins, i.e., IgM, IgD, IgG, IgA and IgE, with their corresponding heavy chains being the μ, δ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa or lambda chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1,2,3,4 or variants thereof.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, arranged sequentially from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDE2-3) or in accordance with Kabat and chothia numbering convention (HCDR 1).

The term "murine antibody" is in the present invention a monoclonal antibody to human NGF prepared according to the knowledge and skill in the art. Preparation is carried out by injecting a test subject with human NGF protein antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties. In a preferred embodiment of the present invention, the murine NGF antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or variant thereof.

The term "chimeric antibody" refers to an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody. Establishing a chimeric antibody, selecting and establishing a hybridoma secreting a mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to needs, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing a chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain variable region of the NGF chimeric antibody further comprises the light chain FR region of a murine kappa, lambda chain or variant thereof, and the antibody light chain variable region sequence is set forth in SEQ ID NO. 11. The variable region of the antibody heavy chain of the NGF chimeric antibody further comprises heavy chain FR regions of murine IgG1, IgG2, IgG3, IgG4 or variants thereof, and the sequence of the variable region of the antibody heavy chain is shown as SEQ ID NO. 9. The constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG2 or IgG4 heavy chain constant region, or IgG1 which has no ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the strong antibody variable antibody reaction induced by the chimeric antibody because of carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA sequences of, for example, human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (at the Internet)www.mrccpe.com.ac.uk/vbaseAvailable), and found in Kabat, e.a. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. In a preferred embodiment of the present invention, the mouse CDR sequences of the humanized NGF antibody are selected from SEQ ID NOs 3,4, 5, 6, 7 and 8. Human antibody variable region frameworks have been designed and selected in which light chain FR region sequences in the antibody light chain variable region, such as the combined sequences derived from human germline light chains IGKV1-39 and JK4, comprise the FR1, FR2, FR3 regions of human germline light chains IGKV1-39 and the FR4 region of JK 4; wherein the heavy chain FR region sequence in the heavy chain variable region of the antibody, such as the combined sequence derived from human germline heavy chain IgHV3-7 and JH6, comprises the FR1, FR2, FR3 region of human germline heavy chain IgHV3-7 and the FR4 region of JH 6. To avoid reduced immunogenicity and resulting reduced activity, the human antibody variable regions may be subjected to minimal back mutations to maintain activity.

"antigen-binding fragment" as used herein refers to Fab fragments, Fab 'fragments, F (ab') 2 fragments, and Fv fragments sFv fragments that bind to human NGF, which have antigen-binding activity; comprising one or more CDR regions of an antibody of the invention selected from SEQ ID NO 4 to SEQ ID NO 9. The Fv fragment contains the variable regions of the antibody heavy and light chains, but lacks the constant region, and has the smallest antibody fragment with the entire antigen-binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined together with different linkers into a single polypeptide chain, known as single chain antibodies (scFv) or single chain fv (sFv). The term "binds to NGF" in the context of the present invention refers to the ability to interact with human NGF. The term "antigen binding site" of the present invention refers to a three-dimensional spatial site that is not antigenically contiguous and is recognized by an antibody or antigen binding fragment of the present invention.

The term "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, as used herein refers to the direct killing of antibody-coated target cells by Fc fragments of cells expressing Fc receptors through recognition of the antibody. The ADCC effector function of an antibody may be reduced or eliminated by modification of the Fc-fragment of the IgG. The modification refers to mutation in the heavy chain constant region of an antibody, such as N297A, L234A, L235A selected from IgG 1; IgG2/4chimera, F235E of IgG4, or L234A/E235A mutations.

The fusion protein is a protein product obtained by coexpression of two genes through DNA recombination. Recombinant NGF extracellular region His fusion protein is a fusion protein co-expressed by NGF extracellular region and 6His segment through DNA recombination. The human NGF extracellular region refers to the part of NGF protein expressed outside a cell membrane, and the sequence is shown in the following SEQ ID NO:1 lineation region.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as the antibody test technical guide of cold spring harbor, chapters 5-8 and 15. For example, mice can be immunized with human NGF or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can likewise be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to incorporate one or more human FR regions in a CDR region of non-human origin. Human FR germline sequences can be obtained from the website http:// imgt. cities.fr of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN 012441351. In particular, the light chain FR germline used by the antibodies or antigen-binding fragments of the present invention includes A3 and O2. Specific germline heavy chain FRs for use with the antibodies or antigen-binding fragments of the invention include VH3-21 and VH 3-23.

The engineered antibodies or antigen binding fragments of the invention can be prepared and purified using conventional methods. For example, the CDNA sequences encoding the heavy chain (SEQ ID NO: 10) and light chain (SEQ ID NO: 11) can be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bind to human NGF. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, the column is run on an A or G Sepharose FF column containing the adjusted buffer. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting antibody fragments by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibodies or mabs of the present invention refer to antibodies derived from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques.

"administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, such as a composition comprising any of the binding compounds of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in each patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any of the statistical tests known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without altering the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.

The term "consisting essentially of … …" or variants thereof as used throughout the specification and claims is meant to encompass all such elements or groups of elements, and optionally other elements of similar or different nature than the elements, which other elements do not materially alter the basic or novel characteristics of a given dosing regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence mentioned may also comprise one or more amino acids, which do not significantly affect the properties of the binding compound.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

"exogenous" refers to a substance that is to be produced outside an organism, cell, or human body by context. "endogenous" refers to a substance produced in a cell, organism, or human body by background.

"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The present invention is further described below with reference to examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is usually performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1: preparation of rat monoclonal antibody

A murine monoclonal antibody against human NGF was generated by immunizing Balb/C mice with purified recombinant human NGF extracellular domain His fusion protein (huNGF-His) (SEQ ID NO: 1). Human NGF-His antigen is synthesized and expressed by taking P01138 in a Uniprot database as a template.

huNGF-His, recombinant human NGF protein (SEQ ID NO: 1):

MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTR NITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVG DKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAW RFIRIDTACVCVLSRKAVRRAHHHHHH。

for immunization with the human NGF extracellular domain His fusion protein, the purified antigens used were high (50ug) and low (10ug) doses, respectively. Immunization was performed 3 times on days 0,14, and 35, respectively, with complete Freund's adjuvant, and immune responses were monitored by postorbital bleeds. Plasma was screened by ELISA to obtain mice with titers of human anti-human NGF immunoglobulin. On day 56, the mice with the highest titers of anti-NGF human immunoglobulin were boosted. After 3 days, mice were sacrificed and spleens were removed for fusion with Sp20 mouse myeloma cells. And (4) screening the fused hybridoma cells to obtain a rat monoclonal antibody N11.

Example 2: molecular cloning of monoclonal antibody hybridoma cells

The heavy chain and light chain variable regions of rat monoclonal antibody N11 were subjected to clonal amplification PCR using high fidelity Phusion enzyme, and at least 28 different clones were picked for sequencing per amplification, and the common sequences obtained were as follows:

N11-VH (SEQ ID NO：2)

QVQLKESGPGLVQPSQTLSLTCTVSGFSLTNNNVNWVRQATGRGLEWMGGVWAGGATDYNSALKSRLTITRDTSKSQVFLKMHSLQSEDTATYYCARDGGYSSSTLYAMDAWGQGTSVTVSS

N11-VL (SEQID NO：3)

DIQMTQSPASLSASLGETVTIECRASEDIYNALAWYQQKPGKSPQLLIYNTDTLHTGVPSRFSGSGSGTQYSLKINSLQSEDVASYFCQHYFGYPRTFGGGTKLELK

it contains the following CDR sequences:

0

full-length heavy chain sequence of recombinant N11 chimeric antibody (SEQ ID NO: 10)

PDWTWRVFCLLAVAPGAHSQVQLKESGPGLVQPSQTLSLTCTVSGFSLTNNNVNWVRQATGRGLEWMGG VWAGGATDYNSALKSRLTITRDTSKSQVFLKMHSLQSEDTATYYCARDGGYSSSTLYAMDAWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Recombinant N11 chimeric antibody full-length light chain sequence (SEQ ID NO: 11) MDMRVPAQLLGLLLLWLPGTRCDIQMT QSPASLSASLGETVTIECRASEDIYNALAWYQQKPGKSPQLLIYNTDTLHTGVPSRFSGSGSGTQYSLKINSLQSED VASYFCQHYFGYPRTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The heavy-light chain plasmid vector of N11 is subjected to cotransfection of HEK-293 cells by Fectin293 liposome (Invitrogen), transient expression is carried out, the expression quantity can reach more than 50mg/L through electrophoresis observation, the obtained supernatant is subjected to preliminary purification through Protein-A affinity chromatography, further cation exchange purification and polymyxin B affinity column removal of endotoxin are adopted, and the purity of the product is more than 98% through SDS-PAGE (figure 2) and HPLC analysis.

Example 4: mouse antihumanization

The humanized G11-H1L1 antibody was obtained by conventional CDR grafting and back mutation of adjacent amino acids in the framework region, and the protein sequence of the heavy and light chains was as follows.

Heavy chain variable region sequence of recombinant G11-H1L1 humanized antibody (SEQ ID NO: 12)

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNNNWSWIRQPPGKGLEWIGYVWAGGATNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGGYSSSTLYAMDAWGQGTLVTVSS

Light chain variable region sequence (SEQ ID NO: 13) DIQMTQSPSSLSASVGDRVTITCRASEDIYNALAWYQQKPGKVPKLLIYNTDTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYFGYPRTFGQGTKLEIK of recombinant G11-H1L1 humanized antibody

Example 5: pH engineered G11 antibodies

Constructing a space structure model of NGF and Fab of G11, and finding that a plurality of Fab amino acid sites can avoid self protonation and generate ion repulsion with adjacent NGF basic amino acid under the condition of pH6.0 by point mutation; or to avoid self-positively charged NGF protonated amino acids that come into close contact with them to produce ion repulsion. From this, a G11-H2L2 antibody of a pH engineered antibody was obtained, whose heavy and light chain protein sequences are as follows.

Heavy chain variable region sequence of pH engineered antibody G11-H2L2 (SEQ ID NO: 14)

QVQLQESGPGLVKPSETLSLTCTVSGFSLTNNNVNWIRQPPGKGLEWIGGVWAGGATDYNSALKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDGGYSSSTLYAMDAWGQGTLVTVSS

Light chain variable region sequence (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITCRASEDIYNALAWYQQKPGKAPKLLIYNTDTLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHYFGYPRTFGQGTKLEIK of pH engineered antibody G11-H2L2

Example 6: fortebio dynamics determination of affinity of NGF antigen and monoclonal antibody at different pH

The biosensor (such as Biacore, ForteBio, etc.) method is a well-known method for objectively detecting the affinity and kinetics of proteins with each other. We analyzed the NGF antibodies to be tested according to the invention by ForteBio to characterize the affinity and binding kinetics.

Using a kit provided by ForteBio corporation, 2mg/ml of antibody solution was immobilized onto anti-human Fc chips by Loading step, and the appropriate concentration range for human and cynomolgus NGF antigen Loading was searched, i.e., antigen flow over (Association) immobilized human mab chips could generate 0.1-1 RU acceptable detection signal, and then bound antigen was dissociated with PBS, reaction signal was mechanically detected, and the binding constant ka and dissociation constant kd values of antigen-antibody binding were analyzed by ForteBio's own assay software in 1:1(Langmuir) Global binding model by 4-5 different concentration combinations. The results obtained in this way are shown in FIGS. 4-7, i.e.the AHC immobilized recombinant antibody and human NGF as mobile phase.

Fortebio analysis procedure:

Initial systemic Baseline Initial System equilibrium Baseline PBS, pH7.2 (or pH6.0), 30s

Loading, in which an antibody is fixed on an Anti-hFc chip and 2mg/ml monoclonal antibody is used for 90s

Baseline PBS, pH7.2 (or pH6.0), Baseline rebalancing

Association PBS-modulated NGF, different concentration gradients, antigen-antibody binding 60s

Dissociation Dissociation of antigen-antibody binding with PBS for 120s

TABLE 1 binding kinetics parameters of NGF antibodies to human NGF

Example 7: pharmacodynamics in macaque

The experiment content is that the humanized NGF antibody (G11-H2L2) with different single doses and engineered pH is intravenously instilled to macaque, the plasma concentration-time change (PK) and the Pharmacodynamics (PD) are observed, and theoretical and experimental basis is provided for the formulation of clinical test schemes. Kiwi 8, the Limited liability company of the biological resource research institute of Beijing Helercin (production permit SCXK (Jing) 2010-0007), male and female half, the weight of 3.68 + -0.14 kg. The animals were fed with standard monkey feed, water was freely available, and fresh fruits were given twice daily. Two dose groups of the test antibody were set for single administration, 1 and 3 mg. kg-1, respectively.

In the PK study, 50mL of the drug was prepared with physiological saline according to the body weight and the administration dose of the animal, the hind limb was subjected to constant-rate intravenous drip, 1mL of blood was collected in the contralateral vein after 1, 4, 8, 24, 48, 72, 96, 144, 192, 240 and 288 hours (both before the administration of the drug) before the intravenous drip and 15min and 30min (the drip was stopped) after the administration (both before the administration of the drug) after the completion of the drip within 30min, and serum was separated, and in the PD study, 1mL of blood was collected after the administration (both before the administration of the drug) and 1, 4, 8, 24, 48, 72, 96, 144 and 192 hours after the intravenous drip and 15min and 30min (the drip was stopped). All samples were centrifuged at room temperature, 5000rpm/min, 10min off. The supernatant was collected and stored at-80 ℃.

PK analysis method

In the PK study, the experimental antibody concentration levels in serum were studied using ELISA kit analysis.

The detection principle is that the method adopts a double-antibody sandwich ELISA method to quantify the experimental antibody. The goat anti-human IgG adsorbed by monkey serum is used as a primary antibody to be coated on a 96-well plate, a series of concentration standard substances and diluted blood samples are added, the anti-human NGF monoclonal chimeric antibody in the standard substance solution and the blood samples can be specifically combined with the primary antibody, then the goat anti-human IgG-HRP (secondary antibody) adsorbed by the diluted monkey serum is added, the secondary antibody can be specifically combined with the anti-human NGF monoclonal chimeric antibody combined with the primary antibody, then a substrate color developing agent is added, and finally, the reaction is stopped by sulfuric acid, and the reading is carried out under 450/560nm of an enzyme labeling instrument. The shade of the color is proportional to the concentration of the anti-human NGF monoclonal chimeric antibody in the sample.

Calculating the concentration of G11-H2L2 antibody in macaque serum sample, which is to draw a standard curve by using Microcal Origin software as concentration and OD value according to original data obtained by experiment and calculate the concentration of unknown sample. The correction curve equation is:

where a 1is an estimated value (OD value) of the upper asymptote of the curve, and a2 is an estimated value (OD value) of the lower asymptote of the curve. P is the slope of the calibration curve, X0 is IC₅₀The value is the concentration of the sample corresponding to the 50% light absorption value. The calibration curve is obtained by fitting the curve of the best, i.e., the curve with the smallest fitting error (the smallest χ 2 value). The concentration of the unknown sample (mMG/ml) was calculated from a calibration curve on the same batch of tests.

Pharmacokinetic parameter calculation and statistical analysis

Statistical moment method was used to calculate various pharmacokinetic parameters using Microsoft Excel 2007 software, and Student's were used between macaquest-The test was statistically judged. Experimental results Linear regression was performed using the Microcal origin7.0 regression equation and related statistical parameters and plotted. All individual serum drug concentration values less than LLOQ were not involved in PK calculations. All the above statistical calculations to obtain individual values were done by Excel 2007 (Microsoft Corporation).

Single intravenous drip of 1, 3 mg.kg for macaque^-1Pharmacokinetic parameters after G11-H2L 2. After single intravenous drip of the macaque antibody, the half-life t1/2 of the tail end phase is 95.6 +/-22.3 and 198.7 +/-58.8 hours after the macaque antibody is injected into the macaque in two doses (1 mg. kg < -1 >) which are lower and higher.

Claims (9)

1. A pH engineered NGF antibody or antigen-binding fragment thereof, wherein the heavy chain variable region sequence of the pH engineered NGF antibody is shown in SEQ ID NO. 14, and the light chain variable region sequence of the pH engineered NGF antibody is shown in SEQ ID NO. 15.

2. A DNA molecule encoding the NGF antibody or antigen-binding fragment thereof of claim 1.

3. An expression vector comprising the DNA molecule of claim 2.

4. A host cell transformed with the expression vector of claim 3.

5. The host cell of claim 4, wherein the host cell is a mammalian cell.

6. The host cell of claim 4, wherein the host cell is a CHO cell.

7. A pharmaceutical composition comprising an NGF antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable excipient, diluent or carrier.

8. Use of an NGF antibody or antigen-binding fragment thereof of claim 1, the pharmaceutical composition of claim 7, in the manufacture of a medicament for treating an NGF-mediated disease or disorder, wherein the disease or disorder is central pain.

9. Use of an NGF antibody or antigen-binding fragment thereof according to claim 1, a pharmaceutical composition according to claim 7, in the manufacture of a medicament for treating an NGF-mediated disease or disorder, wherein the disease or disorder is high-expression NGF-induced post-operative pain, rheumatoid arthritis pain, and osteoarthritis pain.

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