CN114751982B

CN114751982B - Anti-human NGF antibodies, methods of making and uses thereof

Info

Publication number: CN114751982B
Application number: CN202210379185.6A
Authority: CN
Inventors: 王著; 高永娟; 陈思; 周若芸; 郑云程; 孙乃超; 李强
Original assignee: PHARMAB Inc; Anyuan Pharmaceutical Technology Shanghai Co ltd
Current assignee: PHARMAB Inc; Anyuan Pharmaceutical Technology Shanghai Co ltd
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2023-06-23
Anticipated expiration: 2037-12-15
Also published as: CN109929035B; CN109929035A; CN114751982A

Abstract

The present invention provides an antibody or antigen binding fragment thereof that binds NGF and lists the amino acid sequences of the heavy and light chain variable regions of the antibody. The NGF antibody or the antigen binding fragment thereof provided by the invention has higher affinity to NGF, and can effectively block the binding between NGF receptor and NGF. The antibody or antigen binding fragment thereof can inhibit the binding activity of NGF and a receptor in vitro, and is suitable for treating pain diseases associated with the hyperexpression and the rising level of NGF.

Description

Anti-human NGF antibodies, methods of making and uses thereof

Technical Field

The present invention relates to the field of therapeutic monoclonal antibodies, and more particularly to an anti-NGF antibody or antigen-binding fragment thereof and uses thereof.

Background

Nerve growth factor (Nerve Growth Factor, NGF) is the earliest neurotrophic factor discovered, has dual biological functions of neuronal nutrition and protrusion growth promotion, and has important regulatory effects on development, differentiation, growth, regeneration and expression of functional properties of central and peripheral neurons. NGF comprises three subunits, α, β, and γ, and β is the active subunit of NGF that exerts nerve regeneration and repair effects. Currently known NGF receptors include high affinity tyrosine kinase receptors (Tropomyosin receptor kinase A, trkA) and low affinity p75neurotrophin receptors (p 75Neurotrophin Receptor, p75 NTR).

Although initially the primary role of NGF is to promote neuronal survival and differentiation, more and more studies have been carried out over the last two decades to indicate that NGF is associated with persistent or chronic pain. In 1993, it was reported that administration of exogenous NGF at doses to rats induced pain (Lewis GR et al, J Neurosci,1993, 13:2136-2148), after which intravenous administration of NGF to humans was found to produce systemic muscle pain, and local administration also induced hyperalgesia and allodynia at the site of injection (Petty BG et al, ann Neurol,1994, 36:244-246). Also studies have shown that NGF upregulates neuropeptide expression in sensory neurons. Upon binding to TrkA and p75NTR receptors, NGF increases the pain response, upregulating a sensory neuron called nociceptors, a process that makes neurons more sensitive to potential pain stimuli (Holmes D, nat Rev Drug Discov,2012, 11:337-338). Current studies have demonstrated that NGF/TrkA expression is increased in articular cartilage of degenerative joint disease and NGF levels are increased in patients with rheumatoid arthritis and interstitial cystitis. If a monoclonal antibody that specifically binds to NGF and has a function of inhibiting the binding can be developed, it is expected that the monoclonal antibody will have a positive effect on prevention, diagnosis, and treatment of various diseases associated with NGF, such as pain.

Worldwide, tens of millions of patients suffer from chronic pain, and this figure is increasing as the population increases. Drugs currently used clinically for chronic pain treatment include non-steroidal anti-inflammatory drugs, anticonvulsants, opioids, etc., however, these drugs have many disadvantages, in which the efficacy of the non-steroidal anti-inflammatory drugs is limited and have side effects including gastrointestinal bleeding and renal toxicity; whereas opioids have side effects such as addiction. There is a need in the art for non-opioid analgesics that are pain-relieving, non-toxic, abuse-preventing, and therefore methods of treating chronic pain by inhibiting NGF are of great value. To date, there are many anti-human NGF antibodies in the development or clinical development phase, with the fastest growing ones including the anti-NGF monoclonal antibodies tanizumab from Pfizer/Lilly and Fasinumab from Regeneron/Sanofi. Tanizumab is the first anti-NGF antibody drug developed, and has been reported to show a strong and broad analgesic effect against pain such as joint pain associated with degenerative joint disease, chronic lumbago, and bladder pain associated with interstitial cystitis (Lane NE et al, N Engl J Med,2010, 363:1521-1531), and is currently undergoing phase III clinical trials for indications such as osteoarthritis, back pain, cancer pain, and the like. Phase II/III clinical study data for Fasinumab treatment of osteoarthritis pain showed that patients in the 4-dose Fasinumab-treated group achieved statistically significant improvement in pain relief. On the other hand, clinical experiments with multiple NGF inhibitors also indicate that NGF antibodies may face problems of limited use for severe patients, inability to long term use, and dose limitation, so that further safety validation is required for clinical application of NGF antibodies.

NGF is an extremely important factor in neuronal development, and in the development of drugs that inhibit NGF function, the effect of NGF dose on neurons needs to be considered. In one aspect, the effective dose of antibody drug depends on the neutralizing activity against the antigen and the amount of antigen present in the body, and an increase in neutralizing activity correlates with a decrease in the amount administered. anti-NGF antibody studies require access to CDR regions with different affinities for different epitopes or the same epitope. The immunogenicity of different CDRs is different, so that the tolerance speed and toxicity of the antibody are different, and the drug effect is directly influenced. On the other hand, the immune response of the subject to the antibody itself may form an immune complex, so that pharmacokinetics is changed, an allergic reaction is generated, etc., eliminating its therapeutic use. The human immune system has minimal antibody response against the humanized antibody relative to murine and chimeric antibodies, while the humanized antibody has a half-life similar to that of the natural human antibody, thus ensuring less frequent and lower dosage. Accordingly, development of an anti-NGF antibody that can maintain high neutralizing activity and is excellent in safety, and humanization of the anti-NGF antibody have been extremely important for treatment or prevention of various diseases associated with NGF.

Disclosure of Invention

The invention aims to provide a safe and reliable anti-human NGF antibody or antigen binding fragment thereof with remarkable functions. The antibodies or antigen binding fragments thereof are capable of antagonizing NGF binding to NGF receptors with high specificity. Therefore, the human NGF antibody or the antigen binding fragment thereof provided by the invention has higher specificity, is expected to improve the safety in clinical application, can achieve ideal drug effect with smaller dosage, and greatly reduces the treatment cost of patients.

In one aspect of the invention, there is disclosed an antibody or antigen-binding fragment thereof that binds human NGF, characterized in that the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences; and

a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 sequences, and selected from the group consisting of:

(1) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:1, and the CDR-H1 amino acid sequence shown in SEQ ID NO:3, and the CDR-H2 amino acid sequence shown in SEQ ID NO:5, a CDR-H3 amino acid sequence; and the light chain variable region thereof comprises the amino acid sequence as set forth in SEQ ID NO:7, and the CDR-L1 amino acid sequence shown in SEQ ID NO:9, and the CDR-L2 amino acid sequence shown in SEQ ID NO:11, a CDR-L3 amino acid sequence;

(2) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:2, and the CDR-H1 amino acid sequence shown in SEQ ID NO:4, and the CDR-H2 amino acid sequence shown in SEQ ID NO:6, a CDR-H3 amino acid sequence; and the light chain variable region thereof comprises the amino acid sequence as set forth in SEQ ID NO:8, and the CDR-L1 amino acid sequence shown in SEQ ID NO:10, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(3) The heavy chain variable region comprises SEQ ID NO:37, and the CDR-H1 amino acid sequence shown in SEQ ID NO:39, and the CDR-H2 amino acid sequence shown in SEQ ID NO:41, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:43, and the CDR-L1 amino acid sequence shown in SEQ ID NO:45, and the CDR-L2 amino acid sequence shown in SEQ ID NO:11, a CDR-L3 amino acid sequence;

(4) The heavy chain variable region comprises SEQ ID NO:47, and the CDR-H1 amino acid sequence shown in SEQ ID NO:48, and the CDR-H2 amino acid sequence shown in SEQ ID NO:41, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:49, and the CDR-L1 amino acid sequence set forth in SEQ ID NO:50, and the CDR-L2 amino acid sequence shown in SEQ ID NO:11, a CDR-L3 amino acid sequence;

(5) The heavy chain variable region comprises SEQ ID NO:47, and the CDR-H1 amino acid sequence shown in SEQ ID NO:48, and the CDR-H2 amino acid sequence shown in SEQ ID NO:41, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:49, and the CDR-L1 amino acid sequence set forth in SEQ ID NO:51, and the CDR-L2 amino acid sequence shown in SEQ ID NO:11, a CDR-L3 amino acid sequence;

(6) The heavy chain variable region comprises SEQ ID NO:38, and the CDR-H1 amino acid sequence shown in SEQ ID NO:40, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:44, and the CDR-L1 amino acid sequence shown in SEQ ID NO:46, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(7) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:53, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:56, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(8) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:53, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:57, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(9) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:54, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:57, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12, and a CDR-L3 amino acid sequence as set forth in seq id no.

Further, the antigen or antigen binding fragment thereof is murine, chimeric or humanized.

In some embodiments, the antibody is murine or chimeric, the heavy chain variable region of which further comprises the heavy chain FR region of murine IgG1, igG2a, igG2b, igG3, or a variant thereof; and a light chain FR region whose light chain variable region comprises a murine kappa, lambda chain or variant thereof.

Preferably, the murine or chimeric antibody comprises the amino acid sequence as set forth in SEQ ID NO:13 or 15, and a heavy chain variable region amino acid sequence shown in seq id no; and comprising the amino acid sequence as set forth in SEQ ID NO:14 or 16.

More preferably, murine antibody #56 and chimeric antibody AB5C1 of the present invention comprise the amino acid sequence as set forth in SEQ ID NO:13, a heavy chain variable region amino acid sequence shown in seq id no; and as set forth in SEQ ID NO:14, and a light chain variable region amino acid sequence shown in seq id no.

More preferably, the murine antibody #2 and chimeric antibody AB5D1 of the present invention comprise the amino acid sequence as set forth in SEQ ID NO:15, and a heavy chain variable region amino acid sequence shown in seq id no; and as set forth in SEQ ID NO:16, and a light chain variable region amino acid sequence shown in seq id no.

In some embodiments, the antibody is humanized. The preparation of humanized antibodies can be accomplished using CDR grafting techniques, surface remodeling techniques, computer modeling techniques, or other prior art techniques.

In some embodiments of the invention, the murine antibody #56 described above is humanized by CDR grafting. The humanized antibody thus produced, more preferably, has a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 17. 19, 21 or 23; and the light chain variable region thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 18. 20, 22 or 24. More preferably, the humanized antibodies AB5C2, AB5C3, AB5C4 and AB5C5 produced thereby comprise heavy chain variable regions comprising the amino acid sequence set forth in SEQ ID NO: 17. 19, 21 or 23; and light chain variable regions thereof comprise the amino acid sequence as set forth in SEQ ID NO: 18. 20, 22 or 24.

In some embodiments of the invention, the murine antibody #56 described above is humanized by a surface remodeling technique. More preferably, the humanized antibody AB5C6 thus produced comprises a heavy chain variable region as set forth in SEQ ID NO:25, an amino acid sequence shown in seq id no; and the light chain variable region thereof comprises the amino acid sequence as set forth in SEQ ID NO:26, and a polypeptide comprising the amino acid sequence shown in seq id no.

In some embodiments of the invention, the murine antibody #2 described above is humanized by CDR grafting. The humanized antibody thus produced, more preferably, has a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 27. 29, 31 or 33; and the light chain variable region thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 28. 30, 32 or 34. More preferably, the humanized antibodies AB5D2, AB5D3, AB5D4 and AB5D5 thus produced comprise heavy chain variable regions comprising the amino acid sequence set forth in SEQ ID NO: 27. 29, 31 or 33; and light chain variable regions thereof comprise the amino acid sequence as set forth in SEQ ID NO: 28. 30, 32 or 34.

In some embodiments of the invention, the murine antibody #2 described above is humanized by a surface remodeling technique. More preferably, the humanized antibody AB5D6 thus produced has a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:35, an amino acid sequence shown in seq id no; and the light chain variable region thereof comprises the amino acid sequence as set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no.

One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids from the sequences of the antibodies of the invention to obtain variants of the antibody sequences without substantially affecting the activity of the antibodies. They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The sequence of the variant of the invention may be at least 80% homologous to the sequence from which it was derived; more preferably, the sequence of the variant of the invention may be at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence from which it was derived.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are full length antibodies further comprising human or murine antibody constant regions; or only Fab, fab ', F (ab') ² Or an antigen binding fragment of ScFv.

In a preferred embodiment of this aspect, the heavy chain constant region sequence is selected from human IgGl, igG2, igG3 or IgG4.

In a preferred embodiment of this aspect, the light chain constant region sequence of the antibody or antigen binding fragment thereof is a human kappa antibody light chain constant sequence.

In one embodiment, the antibody binds human NGF. In particular, the antibodies are capable of blocking the interaction between human NGF and the corresponding human NGF receptor. K binding of said antibody or antigen binding fragment thereof to NGF _D The value is less than or equal to 5 multiplied by 10 ^-11 M is preferably 1X 10 ^-11 M or less K _D 。

In a second aspect of the invention there is provided a DNA molecule encoding an antibody or antigen binding fragment thereof as described above. Preferably, the DNA molecules encoding the heavy chain variable region of said antibody or antigen binding fragment thereof are set forth in SEQ ID NOs: 58. 60 and 62, and a DNA molecule encoding the light chain variable region of said antibody or antigen binding fragment thereof, as set forth in SEQ ID NO: 59. 61 and 63.

For example, a DNA molecule encoding the heavy chain variable region of a preferred chimeric antibody AB5C1 of the invention is set forth in SEQ ID NO:58, and a DNA molecule encoding the light chain variable region thereof as set forth in SEQ ID NO: 59.

As another example, a DNA molecule encoding the AB5C2 heavy chain variable region of a preferred humanized antibody of the invention is set forth in SEQ ID NO:60, and a DNA molecule encoding the light chain variable region thereof as set forth in SEQ ID NO: shown at 61.

For another example, a DNA molecule encoding the AB5C6 heavy chain variable region of a preferred humanized antibody of the invention is set forth in SEQ ID NO:62, and a DNA molecule encoding the light chain variable region thereof as set forth in SEQ ID NO: 63.

In a third aspect of the invention there is provided an expression vector comprising a DNA molecule as described above.

In a fourth aspect of the invention, there is provided a host cell transfected with an expression vector as described above. Preferably, the host cell is a CHO cell.

In a fifth aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, excipient or diluent, and an effective amount of an antibody or antigen-binding fragment thereof as described above.

In a sixth aspect the invention provides the use of the antibody or antigen binding fragment thereof or pharmaceutical composition, preferably for the treatment of any NGF-related disease, in particular NGF-related pain diseases. These diseases are usually associated with increased NGF expression and elevated levels. Such diseases include, but are not limited to, degenerative joint disease, rheumatoid arthritis, interstitial cystitis, osteonecrosis, lumbago, or diabetic peripheral neuropathy.

Preferably, chimeric, humanized anti-NGF antibodies, antigen-binding fragments thereof, may be used in the preparation of a medicament for the treatment of the disease; more preferably, a humanized anti-NGF antibody, antigen-binding fragment thereof, is used.

The inventors have found that the antibodies or antigen binding fragments thereof provided by the present invention have the following advantages:

1. the antibody provided by the invention has high affinity and affinity constant K _D The value is less than or equal to 5 multiplied by 10 ^-11 M, which is capable of effectively blocking the binding between NGF and its receptor, blocking the pain response;

2. The antibody provided by the invention has extremely strong specificity of binding with antigen, and the clinical administration dosage of the antibody can be expected to be reduced;

3. the antibody provided by the invention is expressed by CHO cells, and has the advantages of high yield, high activity, simple purification process and low production cost.

Detailed Description

Abbreviations and definitions

hNGF human nerve growth factor

Complementarity determining regions in immunoglobulin variable regions defined by the CDR using the IMGT numbering system

ELISA enzyme-linked immunosorbent assay

FR antibody framework region: immunoglobulin variable region excluding CDR regions

HRP horseradish peroxidase

IgG immunoglobulin G

Immunoglobulin alignment and numbering system advocated by Elvin a Kabat by Kabat

IMGT is an international immunogenetic information system proposed by LaFranc et al

mAb monoclonal antibodies

PCR polymerase chain reaction

The V region is an IgG chain segment of variable sequence between different antibodies. It extends to Kabat residue 109 of the light chain and residue 113 of the heavy chain.

VH immunoglobulin heavy chain variable regions

vK immunoglobulin kappa light chain variable region

K _D Equilibrium dissociation constant

kd dissociation rate constant

kon binding rate constant

Interpretation of the terms

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, encompass full-length antibodies (e.g., igG1 or IgG4 antibodies), various functional fragments thereof (e.g., may contain only antigen-binding portions, such as Fab, F (ab') ² Or ScFv fragments), and modified antibodies (e.g., humanized, glycosylated, etc.). Examples of antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, domain antibodies, single chain antibodies, fab ', F (ab') ² Fragments, etc. The invention also includes anti-NGF antibodies having glycosylation modifications. In some applications, modifications are made to remove undesired glycosylation sites, such as defragmentation on oligosaccharide chains to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function; in other applications, galactosylation modifications may be made to alter Complement Dependent Cytotoxicity (CDC).

The term "monoclonal antibody or mAb" refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen binding fragments may be obtained by recombinant techniques such as hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.

"antibody fragment" and "antigen-binding fragment" are intended to mean antigen-binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 85%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antibody fragments include, but are not limited to: fab, fab ', F (ab') ² And Fv fragments; a diabody; linear antibodies (linear antibodies); single chain antibody molecules, e.g., scFv, monoclonal antibodies (technologies from Genmab); nanobodies (technology from domanis); domain antibodies (technologies from Ablynx); and multispecific antibodies formed from antibody fragments. Engineered antibody variants are reviewed in Holliger et al, nat Biotechnol 2005, 23:1126-1136.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

"Fab ' fragments" contain a constant region portion between the VH and CH1 domains and CH1 and CH2 domains of one light and one heavy chain, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ') ² A molecule.

“F(ab′) ² The fragment "contains the constant region portion between the VH and CH1 domains and the CH1 and CH2 domains of two light chains and two heavy chains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, F (ab') ² Fragments consist of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody" or "ScFv antibody" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. For an overview of ScFv, see Pluckaphun, 1994,The Pharmacology of Monoclonal Antibodies (monoclonal antibody pharmacology), vol.113, rosenburg and Moore, inc., springer-Verlag, berlin, heidelberg, pages 269-315. See also International patent application publication No. WO88/01649 and U.S. Pat. No.4,946,778 and U.S. Pat. No.5,260,203.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

An "antigen binding fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable region or light chain variable region chains.

The term "hypervariable region" or "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. CDR region sequences may be defined by IMGT, kabat, chothia and AbM methods or amino acid residues within the variable region identified by any CDR region sequence determination method known in the art. Antibody CDRs can be identified as hypervariable regions initially defined by Kabat et al, e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain, see Kabat EA et al, 1991,Sequences of Proteins of Immunological Interest,Public Health Service,National Institutes of Health,Bethesda,Md; the positions of the CDRs can also be identified as originally defined by the "hypervariable loop" (HVL) structure described by Chothia et al. IMGT (ImMunoGeneTics) also provides numbering systems for immunoglobulin variable regions comprising CDRs, which are defined according to IMGT numbering, e.g., residues 27-32 (L1), 50-52 (L2) and 89-97 (L3) of the light chain variable domain and residues 26-35 (H1), 51-57 (H2) and 93-102 (H3) of the heavy chain variable domain, see, e.g., lefranc MP et al, dev Comp Immunol,2003,27:55-77, which are incorporated herein by reference. Other methods for CDR identification include "AbM definition", which is a compromise between Kabat and Chothia and is obtained using Oxford Molecular's AbM antibody model software; or "contact definition" of CDRs, which is based on observed antigen contact and is described in MacCallum RM et al, J.mol Biol,1996, 262:732-745. In the "configuration definition" method of CDRs, the positions of the CDRs can be identified as residues that contribute enthalpy to antigen binding, see, e.g., makabe K et al, J Biol Chem,2008,283:1156-1166. The methods used in the present invention may be defined using or according to any of these methods, including, but not limited to, any of Kabat definition, IMGT definition, chothia definition, abM definition, contact definition, and/or configuration definition.

The term "chimeric antibody (Chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The method comprises the steps of establishing chimeric antibody, selecting hybridoma secreting mouse-derived specific monoclonal antibody, cloning variable region genes from mouse hybridoma cells, cloning constant region genes of human antibody according to requirements, connecting the mouse variable region genes and the human constant region genes into chimeric genes, inserting the chimeric genes into a vector, and finally expressing chimeric antibody molecules in a eukaryotic expression system or a prokaryotic expression system. In a preferred embodiment of the invention, the antibody light chain variable region of said NGF chimeric antibody further comprises a light chain FR region of murine kappa, lambda or variant thereof. The antibody heavy chain variable region of the NGF chimeric antibody further comprises a heavy chain FR region of murine IgG1, igG2a, igG2b, or IgG3 or a variant thereof. The constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant region of human IgG 1.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences of non-human immunoglobulins. For most purposes, humanized antibodies are human immunoglobulins (recipient antibody) in which recipient hypervariable region residues are replaced by non-human (donor antibody) hypervariable region residues such as mouse, rat, rabbit or non-human primate antibodies of the desired specificity, affinity and activity. In some cases, framework Region (FR) residues of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may include residues that are present in neither the recipient antibody nor the donor antibody. These modifications can further enhance the performance of the antibody. Typically, a humanized antibody will comprise substantially all (at least one, and typically two) variable regions, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region (Fc). For more details, see citation Jones PT et al, nature,1986,321:522-525.

The terms "immunological binding" and "immunological binding properties" as used herein refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which immunization is intendedGlobulins are specific). The intensity or affinity of the immunological binding interactions can be determined by the equilibrium dissociation constant (K _D ) Representation, where K _D The smaller the value, the higher the affinity. The immunological binding properties of the selected polypeptides may be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kd or koff) can be calculated from the concentration and the actual rate of association and dissociation (Malmqvist M, nature,1993, 361:186-187). The kd/kon ratio is equal to the dissociation constant K _D (see generally Davies et al, annual Rev Biochem,1990, 59:439-473). K can be measured by any effective method _D Kon and kd values. In a preferred embodiment, the dissociation constant is measured using bioluminescence interferometry (e.g., the ForteBio oct method described in example 3). In other preferred embodiments, the dissociation constant may be measured using surface plasmon resonance techniques (e.g., biacore) or Kineca. When the equilibrium binding constant (K _D ) Is less than or equal to 5 multiplied by 10 ^-11 M is preferably ∈1×10 or less ^-11 M, the antibodies of the invention are believed to specifically bind to NGF epitopes.

The term "label" or "labeled" as used herein refers to the attachment of a biotin moiety to a polypeptide that incorporates a detectable marker, such as by incorporating a radiolabeled amino acid or by attaching a moiety that can be detected by labeling with an avidin (e.g., a fluorescent marker or enzymatically active streptavidin that can be detected by optical means or calorimetry). In some cases, the marker or markers may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of markers for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

Homologous antibodies

In a further aspect, the heavy and light chain variable regions comprised by the antibodies of the invention comprise amino acid sequences homologous to the amino acid sequences of preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-NGF antibodies of the invention.

For example, the invention provides a humanized NGF-binding antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region of (a) comprises a sequence selected from the group consisting of SEQ ID NOs: 17. 19, 21, 23, 25, 27, 29, 31, 33, or 35, an amino acid sequence that is at least 80% homologous to the amino acid sequence; more preferably, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 17. 19, 21, 23, 25, 27, 29, 31, 33, or 35, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequence; (b) said light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 18. 20, 22, 24, 26, 28, 30, 32, 34, or 36, an amino acid sequence that is at least 80% homologous to the amino acid sequence; more preferably, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 18. 20, 22, 24, 26, 28, 30, 32, 34, or 36, at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous amino acid sequence.

Antibodies with conservative modifications

The term "conservative modification" is intended to mean that an amino acid modification does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitutions refer to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been described in detail in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of the antibodies of the invention may be replaced with other amino acid residues from the same side chain family.

In certain embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 sequences, wherein one or more of these CDR sequences comprises a specific amino acid sequence or conservative modification thereof based on the preferred antibodies described herein, and wherein the antibody retains the desired functional properties of an anti-NGF antibody of the invention. Accordingly, the present invention provides an isolated binding NGF antibody or antigen binding portion thereof comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 sequences, wherein: (a) The heavy chain variable region CDR-H1 sequence comprises a sequence selected from the group consisting of SEQ ID NOs:1 and 2, and conservatively modified amino acid sequences thereof; and/or the heavy chain variable region CDR-H2 sequence comprises a sequence selected from SEQ ID NOs: 3 and 4, and conservatively modified amino acid sequences thereof; and/or the heavy chain variable region CDR-H3 sequence comprises a sequence selected from SEQ ID NOs: 5 and 6, and conservatively modified amino acid sequences thereof; and/or (b) the light chain variable region CDR-L1 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 7 and 8, and conservatively modified amino acid sequences thereof; and/or the light chain variable region CDR-L2 sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, and conservatively modified amino acid sequences thereof; and/or the light chain variable region CDR-L3 sequence comprises a sequence selected from the group consisting of SEQ ID NOs:11 and 12, and conservatively modified amino acid sequences thereof.

Monoclonal antibody preparation

Monoclonal antibodies of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody methodologies, such as standard somatic hybridization techniques described in Kohler G and Milstein C, nature, 1975:256:495. Although the somatic hybridization protocol is preferred, in principle other methods of preparing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, may also be used.

A preferred animal system for preparing hybridomas is the murine system. The preparation of hybridomas in mice is a very well established protocol. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known.

For expression of the antibody or antibody fragment thereof, the DNA encoding a portion or the full length light and heavy chain may be obtained by standard molecular biology techniques (e.g., PCR amplification or using cDNA clones of hybridomas expressing the antibody of interest), and the DNA may be inserted into an expression vector, such that the gene of interest is operably linked to transcriptional and translational regulatory sequences, and the transfected host cell expresses, preferably a eukaryotic expression vector, more preferably a mammalian cell, such as CHO and its derived cell lines.

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G. Subsequently or alternatively, the specific antigen or epitope thereof may be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Purification of immunoglobulins is for example discussed by Wilkinson D (The scientific, 2000,8:25-28,The Scientist,Inc., philadelphia Pa.).

The chimeric or humanized antibody of the present invention can be prepared according to the sequence of the murine monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the murine hybridomas of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create chimeric antibodies, the murine variable region can be linked to a human constant region using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to cabill et al). The isolated DNA encoding the VH region may be converted to a full length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (CH 1, CH2, and CH 3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., kabat EA et al, 1991,Sequences of Proteins of Immunological Interest,Public Health Service,National Institutes of Health,Bethesda,MD), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region.

To create humanized antibodies, murine CDR regions can be inserted into a human framework sequence using methods known in the art (see U.S. Pat. No.5,225,539 to Winter and U.S. Pat. Nos.5,530,101 to Queen et al; 5,585,089;5,693,762 and 6,180,370). Transgenic animals, such as HuMAb mice (Medarex, inc.) can also be utilized that contain human immunoglobulin gene miniloci (miniloci) encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, together with targeted mutations that inactivate endogenous μ and kappa chain loci (see, e.g., lonberg et al, nature,1994, 368:856-859); or "KM mice carrying human heavy chain transgenes and human light chain transchromosomes ^TM "(see patent WO 02/43478) for antibody humanization. Other methods of antibody humanization include surface remodeling techniques, phage display techniques, and the like.

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference.

Drawings

FIG. 1, indirect ELISA assay for the titers of different anti-human NGF murine monoclonal antibodies binding hNGF.

FIG. 2, competition ELISA assay for anti-human NGF murine monoclonal antibody #56 blocking the binding capacity of hNGF and its receptor TrkA.

FIG. 3-1, parallel comparison of amino acid sequences of anti-hNGF humanized antibodies AB5C2, AB5C3, AB5C4, AB5C5 and AB5C6 with murine antibody #56 heavy chain variable region. Wherein CDR region sequences (defined according to IMGT systems) are underlined.

FIG. 3-2, parallel comparison of the amino acid sequences of anti-hNGF humanized antibodies AB5C2, AB5C3, AB5C4, AB5C5 and AB5C6 with murine antibody #56 light chain variable region. Wherein CDR region sequences (defined according to IMGT systems) are underlined.

FIG. 4-1, parallel comparison of amino acid sequences of anti-hNGF humanized antibodies AB5D2, AB5D3, AB5D4, AB5D5 and AB5D6 with murine antibody #2 heavy chain variable region. Wherein CDR region sequences (defined according to IMGT systems) are underlined.

FIG. 4-2, parallel comparison of the amino acid sequences of anti-hNGF humanized antibodies AB5D2, AB5D3, AB5D4, AB5D5 and AB5D6 with murine antibody #2 light chain variable region. Wherein CDR region sequences (defined according to IMGT systems) are underlined.

FIG. 5, determination of NGF-dependent cell survival signals inhibited by anti-hNGF antibodies.

Fig. 6, AB5C1 effect on post-operative wound model. And (3) injection: p < 0.01 and P < 0.001 compared to model group.

Fig. 7, AB5C1 effect on partial mouse sciatic nerve ligation model. And (3) injection: p < 0.01 and P < 0.001 compared to model group.

Fig. 8, AB5C1 and AB5C2 effect on sodium urate-induced acute gouty arthritis model in mice. And (3) injection: p < 0.01 and P < 0.05 compared to model group.

Fig. 9, AB5C1 and AB5C6 effect on the complete freund's adjuvant induced inflammatory pain model. And (3) injection: p < 0.01 and P < 0.05 compared to model group.

Detailed Description

EXAMPLE 1 preparation of anti-hNGF murine monoclonal antibody

After 20. Mu.g of recombinant human NGF (hNGF, peproTech) was fully emulsified with complete Freund's adjuvant, four-week-old BALB/c mice were subjected to subcutaneous multipoint immunization with an immunization cycle of three weeks. Anti-human NGF antibody titers were tested by ELISA by orbital bleeding on day 10 after 3 rd immunization to monitor the effects of the mouse immune response. Mice producing the highest anti-human NGF antibody titers were boosted once 3 days prior to fusion. After 3 days, the mouse spleen was removed and fused with a mouse myeloma Sp2/0 cell line. Mixing 5×10 ⁸ Sp2/0 cells and 5X 10 ⁸ Mouse spleen cells were fused in 50% polyethylene glycol (PEG, molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) solution. Iscove's medium (containing 10% fetal bovine serum, 100U/ml penicillin, 100. Mu.g/ml streptomycin, 0.1mM hypoxanthine, 0.4. Mu.M aminopterin and 16. Mu.M thymidine) was used to adjust spleen cell count to 7.5X10 ⁵ Per ml, 0.2ml was added to the wells of a 96-well plate. Placed at 37 ℃ and 5% CO ₂ Is arranged in the incubator. After 10 days, the supernatants were individually tested for their ability to compete with Fc-tagged human TrkA receptor for binding to hNGF by high-throughput ELISA, and positive wells that compete with human TrkA receptor were screened (see example 3 for methods). And subcloning the hybridoma cells in the holes, and screening the hybridoma cells by a competition ELISA method to obtain 3 positive hybridoma monoclonal cell strains A2, A56 and A98.

Clones producing specific antibodies were cultured in RPMI 1640 medium supplemented with 10% fcs. When the cell density reaches about 5X 10 ⁵ At each cell/ml, the medium was replaced with serum-free medium. After 2 to 4 days, the cultured medium was centrifuged to collect a culture supernatant. Protein G columns were used to purify antibodies. The monoclonal antibody eluate was dialyzed against 150mM NaCl. The dialyzed solution was filter sterilized through a 0.2 μm filter to obtain purified murine monoclonal antibodies #2, #56 and #98 to be tested.

Example 2 determination of the titers of anti-hNGF murine antibodies

The titers of purified murine antibodies #2, #56 and #98 binding to hNGF were determined by indirect ELISA. Wherein each well was coated with 100. Mu.l of 0.2. Mu.g/ml hNGF, respectively, and the plate was left at 4℃for 16-20 hours. After the PBS buffer in the 96-well plate was aspirated, the plate was washed 1 time with PBST (pH 7.4, PBS containing 0.05% Tween 20), 200. Mu.l/well PBST/1% skimmed milk powder was added, and incubated at room temperature for 1h for blocking. After removing the blocking solution and washing the plate 3 times with PBST buffer, the anti-hNGF murine antibody to be tested diluted to a suitable concentration with PBST/1% nonfat milk powder was added, 100. Mu.l/well and incubated for 1.5h at room temperature. After removing the reaction system and washing the plate 3 times with PBST, the plate was incubated with 50. Mu.l/well of HRP-labeled polyclonal goat anti-mouse IgG (Jackson Laboratory) diluted with PBST/1% nonfat dry milk (dilution ratio 1:5000) as detection antibody for 1h at room temperature. After washing the plates 3 times with PBST, 100. Mu.l/well TMB was added and incubated at room temperature for 10-30min. The reaction was quenched by the addition of 50. Mu.l/well of 0.2M sulfuric acid. The absorbance was measured by a microplate reader at OD450 nm and the results are shown in FIG. 1.

As can be seen in FIG. 1, murine antibodies #2, #56 and #98 each bind hNGF, with the binding titers of #56 being optimal.

EXAMPLE 3 inhibition of binding of anti-hNGF murine antibodies to NGF receptor TrkA assay

The ELISA plate was coated with 100. Mu.l of 2.5. Mu.g/ml hNGF overnight at room temperature. The coating solution was discarded, each well was blocked with skimmed milk in Phosphate Buffered Saline (PBS) for 0.5h and washed with PBST. Then 50. Mu.l of a mixture of 2. Mu.g/ml human Fc tagged TrkA (a product of the Vermilion, beijing) and 50. Mu.l of different concentrations of #56 antibody (10-0.15. Mu.g/ml) were added to each well. As a negative control, polyclonal antibody (Jackson Laboratory) of goat anti-human IgG Fc labeled with HRP was used as a detection antibody without adding #56 antibody, and color development was performed by TMB and the absorbance at OD450/690nm was recorded. As can be seen from fig. 2, the #56 antibody is able to specifically block NGF binding to its receptor TrkA.

Example 4 affinity assay for anti-hNGF murine antibodies

Binding affinity constants of purified murine monoclonal antibodies #56, #2 to antigen were determined using the biological thin film interference technique (BLI) (ForteBio Octet RED)&QK system, PALL corporation). The multichannel parallel quantitative analysis concentration gradient was set as: 3.125, 6.25, 12.5, 25, 50 and 100nM, human NGF (His-tag) was affinity coupled to Ni-NTA sensors. After affinity analysis of the kinetic fitting curve, the above data were analyzed to calculate the affinity constant, the binding constant kon value of murine monoclonal antibody # 56=7.23×10 ⁵ Ms, dissociation constant kd value=8.89×10 ^-6 Equilibrium dissociation constant K _D Value = kd/kon = 1.23 x 10 ^-11 M (0.0123 nM); binding constant kon value for murine monoclonal antibody # 2=1.83×10 ⁶ Ms, dissociation constant kd value = 2.92 x 10 ^-5 Equilibrium dissociation constant K _D Value = kd/kon = 1.59 x 10 ^-11 M. The murine monoclonal antibodies #56 and #2 have extremely high binding affinity for hNGF, up to 10 ^-11 M order of magnitude.

Example 5 subtype identification and variable region amplification of anti-hNGF murine monoclonal antibodies

Identification of antibody subtypes: taking hybridoma cell culture supernatant, and adopting IsoStrip ^TM The mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology) identifies the antibody subtype. The mab #56 subtype was identified as IgG1 (Kappa) and the mab #2 subtype was identified as IgG1 (Kappa).

Antibody variable region amplification: candidate hybridoma cells a56 and A2 were cultured to a total number of 10 ⁷ The individual cells were collected by centrifugation at 1000rpm for 10min, total RNA was extracted using Trizol kit (Invitrogen), first strand cDNA was synthesized using reverse transcription kit SMART RACE, and antibody variable region DNA sequences corresponding to hybridoma cells were amplified using the first strand cDNA as a subsequent template. According to subtype identification results, heavy chain constant region sequences and light chain constant region sequences of the antibody subtype are obtained, specific nested PCR primers are designed, and primer sequences used in the amplification reaction are complementary with the first framework region and the constant region of the antibody variable region. Amplifying a target gene by adopting a conventional PCR method, and sequencing an amplified product to obtain a heavy chain variable region sequence SEQ ID NO of the hybridoma clone A56 secretion antibody # 56: 13 and light chain variable region sequences SEQ ID NO:14; the amino acid sequences of the heavy chain CDRs (CDR-H1, CDR-H2 and CDR-H3) of the antibody are respectively shown in SEQ ID NO:1,3 and 5, the amino acid sequences of the light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) are shown in SEQ ID NO:7,9 and 11. Hybridoma clone A2 secretes the heavy chain variable region sequence of antibody #2 SEQ ID NO:15 and light chain variable region sequences SEQ ID NO:16; the amino acid sequences of the heavy chain CDRs (CDR-H1, CDR-H2 and CDR-H3) of the antibody are respectively shown in SEQ ID NO:2,4 and 6, the amino acid sequences of the light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) are shown in SEQ ID NO:8, 10 and 12. The above CDR region sequences are defined using IMGT methods, and any other CDR region sequence determination methods known in the art may be used to identify amino acid residues in the CDR region within the variable region.

Example 6 humanization of anti-hNGF murine antibodies

6.1 determination of the variable region Gene sequence of the antibody and determination of the typing of the antibody

And performing humanization modification by adopting a CDR grafted antibody humanization modification method according to the variable region sequence of the antibody secreted by the obtained hybridoma cells. Briefly, the humanization engineering process involves the following steps: comparing the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryo antibody to find out a sequence with high homology; analyzing and researching HLA-DR affinity, and selecting a human embryo system framework sequence with low affinity; and then, analyzing the variable region and the frame amino acid sequence around the variable region by using a computer simulation technology and observing the spatial three-dimensional combination mode by using molecular docking. The amino acid sequence of the antibody secreted by each hybridoma cell is analyzed for key amino acids that interact with NGF and maintain the space framework by calculating electrostatic forces, van der waals forces, hydrophilicity and entropy values, and grafted back to the human embryonic antibody framework that has been selected.

Wherein, the murine antibody #56 takes a human IGHV4-38-2 x 02 heavy chain variable region and a human IGKV1-33 x 01 light chain variable region as template sequences, and totally constructs 4 different humanized antibodies, namely AB5C2, AB5C3, AB5C4 and AB5C5. Meanwhile, a human-mouse chimeric antibody AB5C1 is constructed, which is obtained by grafting a heavy chain variable region sequence of a mouse antibody to a human IgG1 heavy chain constant region and grafting a light chain variable region sequence of the mouse antibody to a human Kappa light chain constant region. The amino acid sequences of the variable regions of the humanized antibodies are shown in Table 1.

Wherein, the murine antibody #2 takes a human IGHV1-46 x 01 heavy chain variable region and a human IGKV1-NL1 x 01 light chain variable region as template sequences, and totally constructs 4 different humanized antibodies, namely AB5D2, AB5D3, AB5D4 and AB5D5. Meanwhile, a human-mouse chimeric antibody AB5D1 is constructed, which is obtained by grafting a heavy chain variable region sequence of a mouse antibody to a human IgG1 heavy chain constant region and grafting a light chain variable region sequence of the mouse antibody to a human Kappa light chain constant region. The amino acid sequences of the variable regions of the humanized antibodies are shown in Table 1.

TABLE 1 amino acid sequence of humanized antibody variable region

In addition, the surface remodelling method is also adopted to carry out humanized modification on the variable region sequence of the antibody secreted by the obtained hybridoma cells. The surface remodelling method is to humanize the amino acid residues on the surface of the heterologous antibody, and the method only replaces the region with obvious difference from the amino acid on the surface of the human antibody, and selects the amino acid substitution similar to the amino acid on the surface of the human antibody on the basis of maintaining the activity of the antibody and reducing the heterology. Specifically, the surface remodeling humanized modification process involves the following steps: firstly, comparing the amino acid sequence of the antibody secreted by each hybridoma cell with the amino acid sequence of the human embryo antibody to find out a sequence with high homology; the exposed surface amino acids were then substituted for adult embryonal antibody amino acids using in silico techniques when solvent accessibility was selected to be greater than 30%. Residues that affect the side chain size, charge, hydrophobicity, or the potential to form hydrogen bonds and thus affect the conformation of the complementarity determining regions of the antibody are as little replaced as possible. Wherein, the murine antibody #56 takes a human IGHV4-38-2 x 02 heavy chain variable region and a human IGKV1-33 x 01 light chain variable region as template sequences to construct a humanized antibody AB5C6; the murine antibody #2 was constructed as a humanized antibody AB5D6 using the human IGHV1-46 x 01 heavy chain variable region and the human IGKV1-NL1 x 01 light chain variable region as template sequences. The amino acid sequences of the variable regions of the humanized antibodies are shown in Table 1.

The affinity of the resulting humanized antibodies was measured by the method of example 4 and the results are shown in Table 2.

TABLE 2 affinity assay results for humanized antibodies

The amino acid sequences of the CDR regions of the murine monoclonal antibodies #56 and #2 and 10 humanized antibodies AB5C2, AB5C3, AB5C4, AB5C5, AB5C6, AB5D2, AB5D3, AB5D4, AB5D5 and AB5D6 prepared by the invention are shown in tables 3-1 to 3-2, wherein the amino acid sequences of the CDRs contained in the antibody variable regions are defined by Kabat and IMGT methods, respectively, and the amino acid sequences of the CDR region mutations in the humanized antibodies are underlined.

Table 3-1, CDR region sequences for exemplary anti-human NGF antibody #56 and humanized antibodies derived therefrom

TABLE 3-2 CDR region sequences for exemplary anti-human NGF antibody #2 and humanized antibodies derived therefrom

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FIG. 3-1 shows a parallel comparison of the amino acid sequences of the heavy chain variable regions of 5 anti-hNGF humanized antibodies and murine antibody # 56. FIG. 3-2 shows a side-by-side comparison of the amino acid sequences of the light chain variable regions of 5 humanized antibodies and murine antibody # 56. In the variable region, the complementarity determining regions and framework regions are as indicated, and the heavy and light chain variable region CDRs are defined using the IMGT method.

FIG. 4-1 shows a parallel comparison of the amino acid sequences of the heavy chain variable regions of 5 anti-hNGF humanized antibodies and murine antibody # 2. FIG. 4-2 shows a side-by-side comparison of the amino acid sequences of the light chain variable regions of 5 humanized antibodies and murine antibody # 2. In the variable region, the complementarity determining regions and framework regions are as indicated, and the heavy and light chain variable region CDRs are defined using the IMGT method.

6.2 construction of humanized antibody expression vectors and protein expression

The heavy and light chain coding cDNAs obtained in the above method were inserted into pCMAB2M eukaryotic expression vectors (constructed in this laboratory) to construct humanized expression vectors. The expression vector plasmid contains the cytomegalovirus early gene promoter-enhancer required for high level expression in mammalian cells. At the same time, the vector plasmid contains a selectable marker gene, which confers ampicillin resistance in bacteria and G418 resistance in mammalian cells. In addition, the vector plasmid contains a dihydrofolate reductase (DHFR) gene, and the antibody gene and DHFR gene can be co-amplified with Methotrexate (MTX) in a suitable host cell.

The recombinant expression vector plasmid constructed above is transfected into a mammalian host cell line to express the humanized antibody. For stable high levels of expression, a preferred host cell line is DHFR-deficient Chinese Hamster Ovary (CHO) cells (see U.S. patent No.4,818,679). The preferred transfection method is electroporation, other methods including calcium phosphate co-sedimentation, lipofection, and protoplast fusion can also be used. In electroporation, 2X 10 was added to the cuvette using a GenePulser (Bio-Rad Laboratories) set to 250V electric field and 960. Mu. Fd capacitance ⁷ Individual cells were suspended in 0.8ml of PBS and contained 10. Mu.g of the expression vector plasmid linearized with PvuI (Takara). 2 days after transfection, a solution containing 0.2mg/ml G418 and 200nM MTX (Sigma) was added. To achieve higher levels of expression, the transfected antibody gene was co-amplified with the DHFR gene inhibited by MTX drug. The secretion rate of each cell line was measured by limiting dilution subclone transfectants and ELISA method, and cell lines expressing high levels of the antibodies were selected. Conditioned medium of antibodies was collected for determination of their in vitro and in vivo biological activity.

Example 7 in vitro neutralization Activity assay of anti-hNGF antibodies

The biological activity of anti-NGF antibodies was determined by TF-1 cell (human leukemia cell) culture. TF-1 cell culture methods are based on the high dependence of TF-1 cell lines on granulocyte macrophage colony-stimulating factor (GM-CSF) and utilize NGF to bind to the NGF high affinity receptor TrkA on the surface of TF-1 cells and induce proliferation of TF-1 cells. TF-1 cells (ATCC) at 10 per ml ⁵ Cell density was resuspended and 50. Mu.l of RPMI 1640 medium (Life Technologies Corporation) containing 10% fetal bovine serum was added to each well of a 96-well plate followed by 50. Mu.l of different concentrations of antibody and 50 μl 800ng/ml hNGF. The blank control group was added with medium only, and the negative control group was not added with anti-hNGF antibody. Shaking for 30 minutes at room temperature, adding 100. Mu.l of 10 per well ⁵ TF-1 cells/ml, 5% CO at 37 ℃C ₂ Culturing in incubator for 5-6 days. After adding 20. Mu.l MTT (2.5 mg/ml) per well for a further 4h of incubation, 100. Mu.l 10% SDS was added for incubation overnight. Proliferation of cells was measured with an enzyme-labeled instrument at an excitation wavelength of 570nm and an emission wavelength of 620 nm. Three replicates were set for each treatment, and each replicate was assayed twice.

The results are shown in FIG. 5, from which it can be seen that the binding capacity of anti-hNGF antibodies to NGF is relatively low at antibody concentrations of 0.005-0.3. Mu.g/ml; at concentrations above 0.3 μg/ml, the added anti-human NGF monoclonal antibodies bind to NGF, reducing the effect of NGF on promoting TF-1 cell proliferation, indicating that NGF monoclonal antibodies are capable of antagonizing NGF function. Furthermore, it can be seen from the results that the anti-NGF humanized antibodies AB5C2, AB5C6 and murine monoclonal antibody #56 have better NGF binding capacity than the chimeric antibody AB5C1.

EXAMPLE 8 in vivo analgesic pharmacodynamic Studies of anti-hNGF antibodies

8.1 post-operative wound model pharmacodynamics study

The experiment leads to the injury stimulus of the mice by surgical knife injury at the flexor position of the rear sole of the mice, and finally leads to the thermal hyperalgesia of the mice; and the effect of the control and dosing anti-NGF antibodies on this pain type was observed.

SPF-grade C57BL/6J Male mice (Shanghai Laike laboratory animal Co., ltd.) weighing about 25g were randomly divided into 2 groups of 10 animals each according to body weight. The test animals were placed in a Hargreaves device (model 336, IITC Life Science) prior to measurement, the illumination intensity was set to 17% and the illumination cut-off time was 25s, for a period of time. Before administration, each mouse sequentially measures the basal thermal pain threshold of the sole, i.e. the time from the onset of intense light irradiation to the onset of the paw withdrawal response, i.e. the thermal pain response time, of the mouse. The average value of the total three times of 0.5-2h each time is taken as a base line (t 0 value). Prior to surgical treatment, the test groups were subcutaneously injected with AB5C1 at a dose of 10mg/kg, and the model groups were injected with an equivalent volume dose (10 ml/kg) of PBS buffer. After 1h, the right sole of the mouse was cut with a scalpel, the flexor was separated, and the two parts were cut vertically. The wound was sutured, and the recovery phase, the average of the thermal pain response time after the surgical sites 1, 24, 48, 72, 96h, i.e. the thermal pain threshold(s), was tested, and the percentage improvement of the thermal pain threshold was calculated according to the following formula: percentage improvement in thermal pain threshold = (post-dose mean thermal pain threshold-pre-dose mean thermal pain threshold)/pre-dose mean thermal pain threshold x 100%. Statistical analysis is carried out on the data, experimental data are expressed by mean ± standard deviation (Means ± SD), if the data conform to normal distribution, SPSS 18.0 software is adopted, single factor analysis of variance or T-TEST is adopted; the influence factors before and after treatment are different, and paired T-TEST is adopted; the scoring statistics adopt a nonparametric test, a Mann-whistney test or a Kruskal-wallis test, P is less than or equal to 0.05 and has a significant difference, and P is less than or equal to 0.01 and has a very significant difference.

From fig. 6, it can be seen that the mice in each group of 1h after the operation have lower thermal pain threshold due to pain, but the difference of the thermal pain threshold between the tested drug group and the model group is larger and larger along with the time, and the statistical significance of the time period from t24 h to 72h is obvious through the study's T-TEST, which shows that compared with the mice in the model group, the pretreatment of the tested drug AB5C1 obviously reduces the pain after the operation. At t72 h, the thermal pain threshold of the test drug group has been restored to the initial value, while the model group is still at a lower value; at t96 h, both groups recovered to the starting value.

8.2 pharmacodynamics study of ischial nerve ligation model

The effect of anti-NGF antibodies in a model of chronic pain induced by sciatic nerve ligation in mice was observed. SPF-class C57BL/6J female mice, weighing about 25g, were randomly divided into 3 groups of 5 animals each by body weight. The test animals were placed in a Hargreaves device (parameter set-up such as 8.1) for a period of time prior to measurement. Each mouse was sequentially measured for the basal thermal pain threshold of sole, 0.5-2h each time, three times total, and the average value thereof was taken as a base line (d 0 value). A scalpel is used for scratching the femur part on the right side of the mouse, fascia and muscle parts are separated, and sciatic nerves are found out; ligature the ischial nerve with 7-0 silk thread at 1/3-1/2 of ischial nerve. And sewing layer by layer, and recovering to the original state. The negative control group was subjected to a sham operation but did not bind the sciatic nerve. On the 10 th (d 10) and 17 th (d 17) post-surgery, AB5C1 was subcutaneously injected at a dose of 100mg/kg in the test group, an equal volume dose (20 ml/kg) of PBS buffer was injected in the negative control group and the model group, the thermal pain threshold values of d10, d17, d12, d14, d16, d18, d20, d21 and d23 before and after the administration were measured, and the thermal pain threshold improvement percentage (%) was calculated, as in 8.1. Statistical analysis of data was performed using software and analysis methods same as 8.1

As seen from the data in table 4, on day 10 after ligation, the thermal pain threshold values of each group showed a decreasing trend compared to the basic thermal pain threshold value measured before the experiment, as measured by the thermal pain sensitive experimental instrument; the percentage increase of the thermal pain threshold of the AB5C1 group, the model group and the negative control group are respectively as follows: -56.31% ± 6.98%, -50.99% ± 8.19% and-20.42% ± 5.35%, the first two groups being statistically significant, p=0.014 <0.05, as compared to the negative control group by T-TEST statistical analysis. After 48h (d 12) of subcutaneous administration on day 10, the thermal pain threshold of each group was measured, and the percentage increase of the AB5C1 thermal pain threshold of the administration group was found to be-23.65% + -5.17%, which is equivalent to (-18.70% + -5.98%) of the negative control group, the mean value was much higher than that of the model group (-51.44% + -1.28%), and the statistical significance was very significant, indicating that the tested drug had significantly better analgesic effect than the control group at 48 h; however, the thermal pain threshold showed a decreasing trend from day 4 to day 7 (d 14-d 17) after dosing, and the percentage increase in thermal pain threshold at day 7 was almost the same as the model group value, as shown in fig. 7, indicating that the drug was continuously metabolized and degraded in mice over time.

After one week of administration, the second administration was performed at d17, and it was observed that the percentage increase in the thermal pain threshold of the test drug AB5C1 was much higher than that of the model group at 24h (d 18) and 72h (d 20) after the second administration, the specific values are shown in Table 3, and the statistical differences were significant, and the effect of the drug in 48h was presumed to have a statistical difference, indirectly verifying the data of the first administration, indicating that the test drug AB5C1 had therapeutic effect on the sciatic nerve ligation model within 0-72h after the administration.

TABLE 4 thermal pain threshold of mice after dosing

8.3 pharmacodynamic Studies of sodium urate-induced gouty arthritis model in mice

The effect of anti-NGF antibodies on sodium urate-induced acute gouty arthritis models in mice was observed. SPF-grade C57BL/6J male mice, weighing about 25g, were randomly divided into 3 groups of 8 animals each according to body weight. The test animals were placed in a Hargreaves device (parameter set-up such as 8.1) for a period of time prior to measurement. Each mouse was sequentially measured for the basal thermal pain threshold of the sole at rest, 0.5-2h each time, three times total, and the average value thereof was taken as a base line (d 0 value). The test groups were subcutaneously injected with AB5C1 and AB5C2 at a dose of 10mg/kg, and the model groups were injected with an equal volume dose (10 ml/kg) of PBS buffer. 1h after administration, a 2.5% sodium urate (Sigma) solution was injected into the ankle region in 30. Mu.l; and measuring the thermal pain threshold after foot in 4, 24 and 48 hours after molding, taking the average value as an actual threshold three times each time for 0.5-2 hours, and calculating the improvement percentage (%) of the thermal pain threshold, wherein the formula is the same as 8.1. Statistical analysis was performed on the data using the same software and analysis method as 8.1.

From fig. 8, it can be seen that at 4-6 hours after administration, both AB5C1 and AB5C2 significantly prolonged the mice thermal pain threshold in the gout model, with the statistical differences of AB5C1 being very significant at 4-6 hours, 24 hours and 48 hours, whereas the statistical differences of AB5C2 were only significant on the day of administration; however, the mean value of the two groups of medicines is very high compared with that of the model group, and the thermal pain threshold of the mice is obviously prolonged.

8.4 pharmacodynamic Studies of complete Freund's adjuvant induced inflammatory pain model

To investigate whether anti-hNGF antibodies were able to reduce pain in a chronic peripheral inflammatory mouse model, the hind foot subcutaneous injection of complete freund's adjuvant in C57BL/6J mice caused thermal hyperalgesia, pain was measured. SPF-grade C57BL/6J male mice, weighing about 25g, were randomly divided into 3 groups of 8 animals each according to body weight. The test animals were placed in a Hargreaves device (parameter set-up such as 8.1) for a period of time prior to measurement. Each mouse was sequentially measured for the basal thermal pain threshold of the sole at rest, 0.5-2h each time, three times total, and the average value thereof was taken as a base line (d 0 value). The test groups were subcutaneously injected with AB5C1 and AB5C6 at a dose of 10mg/kg, and the model groups were injected with an equal volume dose (10 ml/kg) of PBS buffer. 1h after administration, 50. Mu.l of a 0.5% solution of complete Freund's adjuvant (BD company) was injected into the ankle region; and after modeling for 4, 24, 48, 72 hours and 96 hours, measuring the thermal pain threshold of the sole of the hind limb, taking an average value as an actual threshold three times each time for 0.5-2 hours, and calculating the thermal pain threshold improvement percent (%), wherein the formula is the same as 8.1. Statistical analysis was performed on the data using the same software and analysis method as 8.1.

FIG. 9 shows that the AB5C6 thermal pain threshold is higher than the model and test drug AB5C1 groups 4-72h after dosing and the statistical difference is significant; the drug effect of AB5C1 is slightly worse than that of AB5C6, and the statistical difference is obvious only at 48 hours. After 96h, there was no statistical difference between the two. In summary, the test agents all have a prophylactic effect on the treatment of the immunoinflammatory pain model, and AB5C6 is better than AB5C1.

While the preferred embodiments of the present invention have been illustrated and described, it will be appreciated by those skilled in the art that, based on the teachings herein, various changes may be made without departing from the scope of this invention.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents are intended to fall within the scope of the invention as defined in the claims appended hereto.

Sequence listing

<110> Anyuan medicine science and technology (Shanghai) Co., ltd

Xuhua (Shanghai) Biological Research and Development Center Co., Ltd.

<120> anti-human NGF antibodies, methods of making and uses thereof

<130> 2017

<160> 63

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Gly Phe Ser Leu Thr Gly Tyr Gly

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Gly Tyr Thr Phe Thr Asp Tyr Trp

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Ile Trp Ala Asp Gly Asp Thr

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Ala Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val

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Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr

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Gln Asp Ile Ser Asn Tyr

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Gln Asp Val Asn Thr Ala

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Tyr Thr Ser

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Trp Ala Ser

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Gln Gln Gly Asn Thr Leu Pro Arg Thr

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Gln Gln His Tyr Ser Ser Pro Trp Thr

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<213> #56 heavy chain variable region amino acid sequence ()

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Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

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Gly Met Ile Trp Ala Asp Gly Asp Thr Asp Tyr Asn Ser Ala Leu Lys

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Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

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Lys Val Asn Asn Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

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Gly Thr Thr Val Thr Val Ser Ser

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<213> #56 light chain variable region amino acid sequence ()

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Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Gln

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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<213> #2 heavy chain variable region amino acid sequence ()

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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

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50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

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Ser Val Thr Val Ser Ser

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<213> #2 light chain variable region amino acid sequence ()

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Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

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Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Tyr Ile Leu Thr Ile Ser Ser Val Gln Ala

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Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

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<212> PRT

<213> humanized antibody AB5C2 heavy chain variable region amino acid sequence ()

<400> 17

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Ala Asp Gly Asp Thr Asp Tyr Asn Ser Ala Leu Lys

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Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Arg Tyr Tyr Cys Ala

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Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

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<212> PRT

<213> humanized antibody AB5C2 light chain variable region amino acid sequence ()

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

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<213> humanized antibody AB5C3 heavy chain variable region amino acid sequence ()

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Trp Ala Asp Gly Asp Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 20

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<213> humanized antibody AB5C3 light chain variable region amino acid sequence ()

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Tyr Tyr Thr Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

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Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 120

<212> PRT

<213> humanized antibody AB5C4 heavy chain variable region amino acid sequence ()

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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

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Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

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Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 22

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<212> PRT

<213> humanized antibody AB5C4 light chain variable region amino acid sequence ()

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 120

<212> PRT

<213> humanized antibody AB5C5 heavy chain variable region amino acid sequence ()

<400> 23

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Ser Ile Trp Ala Asp Gly Asp Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 24

<211> 107

<212> PRT

<213> humanized antibody AB5C5 light chain variable region amino acid sequence ()

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 120

<212> PRT

<213> humanized antibody AB5C6 heavy chain variable region amino acid sequence ()

<400> 25

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Glu

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Ala Asp Gly Asp Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Val Asn Asn Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val Trp Gly Ala

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 26

<211> 107

<212> PRT

<213> humanized antibody AB5C6 light chain variable region amino acid sequence ()

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Glu Gly Thr Leu Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Gln Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 118

<212> PRT

<213> humanized antibody AB5D2 heavy chain variable region amino acid sequence ()

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Tyr Thr Arg Tyr Ile Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 28

<211> 107

<212> PRT

<213> humanized antibody AB5D2 light chain variable region amino acid sequence ()

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Thr

100 105

<210> 29

<211> 118

<212> PRT

<213> humanized antibody AB5D3 heavy chain variable region amino acid sequence ()

<400> 29

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Gly Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 30

<211> 107

<212> PRT

<213> humanized antibody AB5D3 light chain variable region amino acid sequence ()

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Trp Ala Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Thr

100 105

<210> 31

<211> 118

<212> PRT

<213> humanized antibody AB5D4 heavy chain variable region amino acid sequence ()

<400> 31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Gly Tyr Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 32

<211> 107

<212> PRT

<213> humanized antibody AB5D4 light chain variable region amino acid sequence ()

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Trp Ala Ser Arg Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Thr

100 105

<210> 33

<211> 118

<212> PRT

<213> humanized antibody AB5D5 heavy chain variable region amino acid sequence ()

<400> 33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Gly Tyr Thr Ser Tyr Ile Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 34

<211> 107

<212> PRT

<213> humanized antibody AB5D5 light chain variable region amino acid sequence ()

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

35 40 45

Tyr Trp Ala Ser Arg Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Thr

100 105

<210> 35

<211> 118

<212> PRT

<213> humanized antibody AB5D6 heavy chain variable region amino acid sequence ()

<400> 35

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Tyr Thr Arg Tyr Ile Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Ala Ala Tyr Tyr Thr Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 36

<211> 107

<212> PRT

<213> humanized antibody AB5D6 light chain variable region amino acid sequence ()

<400> 36

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ile Leu Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Ser Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 37

<211> 5

<212> PRT

<213> #56 CDR-H1

<400> 37

Gly Tyr Gly Val Asn

1 5

<210> 38

<211> 5

<212> PRT

<213> #2 CDR-H1

<400> 38

Asp Tyr Trp Met Gln

1 5

<210> 39

<211> 16

<212> PRT

<213> #56 CDR-H2

<400> 39

Met Ile Trp Ala Asp Gly Asp Thr Asp Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 40

<211> 17

<212> PRT

<213> #2 CDR-H2

<400> 40

Thr Ile Tyr Pro Gly Asp Gly Tyr Thr Arg Tyr Ile Gln Lys Phe Lys

1 5 10 15

Gly

<210> 41

<211> 12

<212> PRT

<213> #56 CDR-H3

<400> 41

Asp Ser Tyr Tyr Tyr Gly Tyr Asn Phe Phe Asp Val

1 5 10

<210> 42

<211> 9

<212> PRT

<213> #2 CDR-H3

<400> 42

Arg Ala Ala Tyr Tyr Thr Met Asp Tyr

1 5

<210> 43

<211> 11

<212> PRT

<213> #56 CDR-L1

<400> 43

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 44

<211> 11

<212> PRT

<213> #2 CDR-L1

<400> 44

Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala

1 5 10

<210> 45

<211> 7

<212> PRT

<213> #56 CDR-L2

<400> 45

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 46

<211> 7

<212> PRT

<213> #2 CDR-L2

<400> 46

Trp Ala Ser Thr Arg His Thr

1 5

<210> 47

<211> 5

<212> PRT

<213> #56 humanized antibody CDR-H1 ()

<400> 47

Gly Tyr Gly Trp Gly

1 5

<210> 48

<211> 16

<212> PRT

<213> #56 humanized antibody CDR-H2 ()

<400> 48

Ser Ile Trp Ala Asp Gly Asp Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 49

<211> 11

<212> PRT

<213> #56 humanized antibody CDR-L1 ()

<400> 49

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 50

<211> 7

<212> PRT

<213> #56 humanized antibody CDR-L2 ()

<400> 50

Tyr Thr Ser Asn Leu Glu Thr

1 5

<210> 51

<211> 7

<212> PRT

<213> #56 humanized antibody CDR-L2 ()

<400> 51

Tyr Thr Ser Asn Leu Glu Ser

1 5

<210> 52

<211> 5

<212> PRT

<213> #2 humanized antibody CDR-H1 ()

<400> 52

Asp Tyr Trp Met His

1 5

<210> 53

<211> 17

<212> PRT

<213> #2 humanized antibody CDR-H2 ()

<400> 53

Ile Ile Tyr Pro Gly Asp Gly Tyr Thr Ser Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 54

<211> 17

<212> PRT

<213> #2 humanized antibody CDR-H2 ()

<400> 54

Ile Ile Tyr Pro Gly Asp Gly Tyr Thr Ser Tyr Ile Gln Lys Phe Gln

1 5 10 15

Gly

<210> 55

<211> 11

<212> PRT

<213> #2 humanized antibody CDR-L1 ()

<400> 55

Arg Ala Ser Gln Asp Val Asn Thr Ala Leu Ala

1 5 10

<210> 56

<211> 7

<212> PRT

<213> #2 humanized antibody CDR-L2 ()

<400> 56

Trp Ala Ser Arg Leu Glu Ser

1 5

<210> 57

<211> 7

<212> PRT

<213> #2 humanized antibody CDR-L2 ()

<400> 57

Trp Ala Ser Arg Leu Glu Thr

1 5

<210> 58

<211> 360

<212> DNA

<213> chimeric antibody AB5C1 heavy chain variable region nucleotide sequence ()

<400> 58

caagtgcagc tgaaggaaag cggccccgga ctggtggccc cttcccagtc cctgagcatc 60

acctgtaccg tgtccggctt ctccctgaca ggctacggag tgaactgggt gaggcagccc 120

cctggaaaag gactggagtg gctcggaatg atttgggccg acggcgacac cgattataat 180

tccgccctga agtccaggct gtccatcagc aaggacaaca gcaagtccca agtcttcctc 240

aaggtgaaca acctgcagac cgatgataca gcccggtact actgcgcccg ggactcctac 300

tactacggct acaacttctt cgatgtgtgg ggcgccggaa ccaccgtcac agtgagcagc 360

<210> 59

<211> 321

<212> DNA

<213> chimeric antibody AB5C1 light chain variable region nucleotide sequence ()

<400> 59

gacatccaga tgacccagac cacatccagc ctcagcgcta gcctgggaga tagggtgaca 60

atctcctgta gggcctccca ggacattagc aactacctga actggtatca gcagaagccc 120

gagggcacac tcaagctgct gatctactac acctcccggc tccatagcgg cgtgccttcc 180

aggtttagcg gctccggctc cggcaccgac tactccctca ccatctcctc cctggaacag 240

gaggacatcg ccacctattt ttgccagcag ggcaacaccc tgcccaggac atttggcggc 300

ggcaccaagc tggagatcaa a 321

<210> 60

<211> 360

<212> DNA

<213> humanized antibody AB5C2 heavy chain variable region nucleotide sequence ()

<400> 60

caggtgcagc tgcaggagtc tggaccagga ctggtgaagc cttccgagac cctgagcctg 60

acctgcacag tgtctggctt ctccctgaca ggctacggag tgaactgggt gaggcagcca 120

cctggcaagg gactggagtg gctgggcatg atctgggctg acggcgatac cgactataac 180

tctgccctga agtcccgggt gaccatcagc aaggacacat ctaagaatca gttttccctg 240

aagctgtcca gcgtgaccgc cgctgacaca gctaggtact attgcgcccg ggatagctac 300

tattacggct acaatttctt tgacgtgtgg ggcgctggca ccacagtgac cgtgtcttcc 360

<210> 61

<211> 321

<212> DNA

<213> humanized antibody AB5C2 light chain variable region nucleotide sequence ()

<400> 61

gatatccaga tgacacagtc cccaagctct ctgtctgctt ccgtgggcga cagggtgacc 60

atcacatgtc gggcctccca ggatatcagc aactacctga attggtatca gcagaagcct 120

ggcaaggccc caaagctgct gatctattac acctctaggc tgcactccgg agtgccaagc 180

cggttcagcg gctctggctc cggcaccgac ttcaccttta caatctccag cctgcagcct 240

gaggatatcg ctacatactt ctgccagcag ggcaacaccc tgccaaggac atttggcggc 300

ggcaccaagg tggagatcaa g 321

<210> 62

<211> 360

<212> DNA

<213> humanized antibody AB5C6 heavy chain variable region nucleotide sequence ()

<400> 62

caggtgcagc tgaaggagag cggaccagga ctggtggctc catctgagac cctgtccatc 60

acctgtacag tgagcggctt ctctctgaca ggctacggcg tgaattgggt gagacagcca 120

ccaggcaagg gcctggaatg gctgggaatg atctgggctg atggcgacac cgattataac 180

agcgccctga agtctcgcct gacaatctct aaggacaata gcaagtctca ggtgtttctg 240

aaggtgaaca atctgcagac cgacgataca gctagatatt actgcgcccg cgactcctat 300

tactatggct acaacttctt tgacgtgtgg ggtgccggta ctaccgtcac cgtgtcttct 360

<210> 63

<211> 321

<212> DNA

<213> humanized antibody AB5C6 light chain variable region nucleotide sequence ()

<400> 63

gatattcaga tgacccagag ccctagctct ctgtccgcta gcgtgggcga cagagtgacc 60

atctcctgtc gcgccagcca ggatatctct aattacctga actggtatca gcagaagccc 120

gagggcaccc tgaagctgct gatctactat acatccagac tgcatagcgg cgtgccttct 180

cgcttttctg gctccggcag cggcaccgac tactctctga ccatttccag cctgcagcag 240

gaggatatcg ccacctattt ctgtcagcag ggcaataccc tgccaagaac atttggcggc 300

ggcacaaagc tggagatcaa g 321

Claims

1. An antibody or antigen-binding fragment thereof that binds to human nerve growth factor, comprising,

a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 sequences, wherein the amino acid sequences of the CDRs comprised by the heavy and light chain variable regions are defined using the Kabat or IMGT method and are selected from the group consisting of:

(1) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:2, and the CDR-H1 amino acid sequence shown in SEQ ID NO:4, and the CDR-H2 amino acid sequence shown in SEQ ID NO:6, a CDR-H3 amino acid sequence; and the light chain variable region thereof comprises the amino acid sequence as set forth in SEQ ID NO:8, and the CDR-L1 amino acid sequence shown in SEQ ID NO:10, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(2) The heavy chain variable region comprises SEQ ID NO:38, and the CDR-H1 amino acid sequence shown in SEQ ID NO:40, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:44, and the CDR-L1 amino acid sequence shown in SEQ ID NO:46, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(3) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:53, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:56, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(4) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:53, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:57, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12 to the CDR-L3 amino acid sequence shown in seq id no;

(5) The heavy chain variable region comprises SEQ ID NO:52, and the CDR-H1 amino acid sequence shown in SEQ ID NO:54, and the CDR-H2 amino acid sequence shown in SEQ ID NO:42, a CDR-H3 amino acid sequence shown in seq id no; and the light chain variable region comprises SEQ ID NO:55, and the CDR-L1 amino acid sequence shown in SEQ ID NO:57, and the CDR-L2 amino acid sequence shown in SEQ ID NO:12, and a CDR-L3 amino acid sequence as set forth in seq id no.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is murine, chimeric or humanized.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is murine or chimeric, and the heavy chain variable region thereof further comprises the heavy chain FR region of murine IgG1, igG2a, igG2b, igG3, or a variant thereof; and a light chain FR region whose light chain variable region comprises a murine kappa, lambda chain or variant thereof.

4. An antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:15, an amino acid sequence of seq id no; and the light chain variable region thereof comprises SEQ ID NO:16, and a sequence of amino acids.

5. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof is humanized and the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 27. 29, 31, 33 or 35; and the light chain variable region thereof comprises a sequence selected from the group consisting of SEQ ID NOs: 28. 30, 32, 34 or 36.

6. The antibody or antigen-binding fragment thereof of claim 5, comprising a heavy chain variable region and a light chain variable region, and selected from the group consisting of:

(a) The heavy chain variable region is as shown in SEQ ID NO:27 and the light chain variable region is set forth in SEQ ID NO: 28;

(b) The heavy chain variable region is as shown in SEQ ID NO:29 and the light chain variable region is set forth in SEQ ID NO: shown at 30;

(c) The heavy chain variable region is as shown in SEQ ID NO:31 and the light chain variable region is as set forth in SEQ ID NO: shown at 32;

(d) The heavy chain variable region is as shown in SEQ ID NO:33 and the light chain variable region is set forth in SEQ ID NO: shown at 34;

(e) The heavy chain variable region is as shown in SEQ ID NO:35 and the light chain variable region is as set forth in SEQ ID NO: shown at 36.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions, additions and/or deletions to obtain a variant of the antibody sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence from which it was derived.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof is a full-length antibody further comprising human or murine antibody constant regions; or only Fab, fab ', F (ab') ² Or an antigen binding fragment of ScFv.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof binds NGF to K _D The value is less than or equal to 5 multiplied by 10 ^-11 M。

10. A DNA molecule encoding the antibody or antigen binding fragment thereof of any one of claims 1-9.

11. An expression vector comprising the DNA molecule of claim 10.

12. A host cell comprising the expression vector of claim 11, preferably wherein the host cell is a CHO cell.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-9 and a pharmaceutically acceptable carrier, excipient or diluent.

14. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-9 or a pharmaceutical composition according to claim 13 in the manufacture of a medicament for the treatment of degenerative joint disease, rheumatoid arthritis, interstitial cystitis, osteonecrosis, lumbago or diabetic peripheral neuropathy.