HK1166998B - Human anti-ngf neutralizing antibodies as selective ngf pathway inhibitors - Google Patents

Description

Human anti-NGF neutralizing antibodies as selective NGF pathway inhibitors

The present application is a divisional application of the invention patent application having application number "200480026242.2," entitled "human anti-NGF neutralizing antibody as a selective NGF pathway inhibitor.

This application is related to and claims priority from U.S. provisional application serial No. 60/487,431 filed on 15/7/2003, the disclosure of which is incorporated herein by reference.

Technical Field

The present invention relates to human monoclonal antibodies that bind Nerve Growth Factor (NGF). Compositions and methods for treating pain and pain-related disorders are also described.

Background

More than two hundred thousand people in the united states are disabled daily due to chronic Pain (Jessell and Kelly, 1991, "Pain and danalingesia", PRINCIPLES OF NEURAL SCIENCE, 3 rd edition (edited by Kandel, Schwartz and Jessell), Elsevier, New York). Unfortunately, current pain treatments are only partially effective, and many of these treatments themselves can lead to debilitation or cause dangerous side effects. For example, while nonsteroidal anti-inflammatory drugs ("NSAIDs"), such as aspirin, ibuprofen, and indomethacin, have moderate effects on inflammatory pain, they are also renal toxins, and at high doses, they tend to cause gastrointestinal irritation, ulceration, bleeding, and confusion. Patients treated with opioids also often suffer from confusion, and prolonged opioid use is accompanied by concomitant drug tolerance and dependence. Local anesthetics such as lidocaine and mexiletine cause loss of normal sensation while suppressing pain.

Pain is a perception based on signals received from the surrounding environment and transmitted and interpreted by the nervous system (for a review see Millan, 1999, prog.57: 1-164). Noxious stimuli such as heat and touch can cause specialized receptors in the skin to transmit signals to the central nervous system ("CNS"). This process is called nociception, and the peripheral sensory neurons that mediate this process are nociceptors. Depending on the signal strength from nociceptors and the extraction and interpretation of this signal by the CNS, humans may or may not experience noxious stimuli as painful stimuli. When a person's perception of pain is properly aligned with the intensity of the stimulus, the pain can perform its intended protective function. However, certain types of tissue damage can lead to a phenomenon known as hyperalgesia or early pain perception (pronociception), in which a relatively innocuous stimulus is perceived as intense pain because the pain threshold of humans has been reduced. Hyperalgesia is caused by both inflammation and nerve damage. Persons affected by inflammatory diseases such as sunburn, osteoarthritis, colitis, myocarditis, dermatitis, myositis, neuritis, collagen vascular disease (which includes rheumatoid arthritis and lupus), and the like, often experience increased perception of pain. Similarly, trauma, surgery, amputation, abscesses, causalgia, collagen vascular disease, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, stroke, thalamic pain syndrome, diabetes, herpes infections, acquired immunodeficiency syndrome ("AIDS"), toxins and chemotherapy can cause nerve damage resulting in excessive pain.

As the understanding of the mechanisms by which nociceptors transduce external signals under normal and hyperalgesic conditions increases, one can target processes involving hyperalgesia to inhibit the drop in pain threshold, thereby reducing the amount of pain experienced.

Neurotrophic factors have been shown to play important roles in the transmission of physiological and pathological pain. Nerve Growth Factor (NGF) appears to be of particular importance (for a review see McMahon, 1996, phil.351: 431 to 40; and Apfel 2000, The clinical journal of Pain16: S7-S11). Local and systemic administration of NGF has been shown to cause hyperalgesia and allodynia (Lewis et al, 1994, Eur. JNeurosci).6: 1903-1912). Intravenous infusion of NGF produces systemic myalgia in humans, and topical administration causes, in addition to systemic effects, hyperalgesia and allodynia at the site of injection (Apfel et al, 1998, Neurology)51: 695-702). There is also substantial evidence suggesting that endogenous NGF is involved in diseases characterized primarily by pain. For example, NGF is upregulated in Dorsal Root Ganglion (DRG) neurolemmal cells for at least 2 months following peripheral nerve injury, and increased NGF levels have been reported in joints of animals with various models of arthritis (e.g., Aloe et al, 1993, Growth Factors)9: 149-155). In humans, NGF levels are elevated in synovial fluid of patients with rheumatoid Arthritis or other types of Arthritis (e.g., Aloe et al, 1992, Arthritis and Rheumatism)35: 351-355). Furthermore, antagonism of NGF function has also been shown to prevent hyperalgesia and allodynia in models of neuropathic and chronic inflammatory pain. For example, systemic injection of anti-NGF neutralizing antibodies can prevent allodynia and hyperalgesia in animal models of neuropathic pain (e.g. nerve trunk or spinal nerve ligation) (Ramer et al, 1999 eur.j.11: 837-846; and Ro et al, 1999, Pain79: 265-274). Examples of anti-NGF antibodies known in the art include, for example, PCT publications WO 01/78698, WO 01/64247, WO02/096458 and WO 2004/032870; U.S. patent nos. 5,844,092, 5,877,016 and 6,153,189; hongo et al, 2000, Hybridoma19: 215-227; hongo et al, 1993, cell. mol.biol.13: 559-568; and GenBank accession numbers U39608, U39609, L17078 or L17077.

Clearly, there is a need for new pain therapies that are safe and effective, particularly those targeting small molecule pain mediators or aggravators (exaterbators) such as NGF.

Summary of The Invention

The present invention provides novel human monoclonal antibodies that are therapeutically useful for managing pain. In particular, the invention provides monoclonal antibodies that bind Nerve Growth Factor (NGF). Preferably, the monoclonal antibody is a human monoclonal antibody that neutralizes the biological activity of NGF and is useful in ameliorating the effects of NGF-mediated pain responses. The invention also provides cells that produce the monoclonal antibodies of the invention, most preferably also secrete the monoclonal antibodies into the cell culture medium. In addition to their use in the treatment and management of pain, the antibodies of the invention are also useful in the treatment of neuropathic and inflammatory pain-related responses.

The invention further provides fusion proteins comprising an antibody Fc region sequence and one or more sequences identified as: SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22 and SEQ ID NO: 79-130. Such molecules may be prepared using, for example, the methods described in International patent application publication WO 00/24782, which is incorporated herein by reference. Such molecules may be expressed, for example, in mammalian cells (e.g., chinese hamster ovary cells) or bacterial cells (e.g., e.

In certain aspects, the invention provides antibodies, preferably monoclonal antibodies, most preferably human antibodies and human monoclonal antibodies, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: 6, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, the variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 10, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof. Preferably, the heavy chain comprises SEQ id no: 4, or a pharmaceutically acceptable salt thereof.

In certain aspects, the invention provides an antibody, preferably a Human antibody, more preferably a monoclonal antibody, most preferably a Human monoclonal antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE heavy chain constant regions, or any allelic variation thereof (as discussed in Kabat et al, 1991, Sequences of Proteins of immunological Interest, fifth edition, u.s.department of Health and Human Services, NIH publication No. 91-3242, which is incorporated herein by reference), the variable region of the heavy chain comprising the amino acid sequence of SEQ ID NO: 10, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof. Preferably, the antibody of the invention comprises SEQ id no: 4, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides an antibody, preferably a human antibody, more preferably a monoclonal antibody, most preferably a human monoclonal antibody, comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 8, or an antigen-binding or immunologically functional immunoglobulin fragment thereof, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the antibodies of the invention comprise a heavy chain and a light chain, wherein the heavy chain variable region comprises SEQ ID NO: 10, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof. In other aspects, the light chain variable region comprises SEQ ID NO: 12, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof. In further aspects, the heavy chain comprises any of SEQ ID NOs: 14. SEQ ID NO: 18 or SEQ id no: 20, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof. In still other aspects, the light chain comprises any of SEQ ID NOs: 16. 20, 24, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

The invention also provides an antibody that specifically binds NGF, wherein the heavy chain comprises a variable region comprising the amino acid sequence of SEQ ID NO: 10, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, and a light chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 12, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

The invention further provides an isolated human antibody that specifically binds NGF, wherein the antibody comprises:

(a) a heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 79, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, and a light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 80, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof;

(b) a heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, and a light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof;

(c) a heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, and a light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 84, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof; or

(d) A heavy chain having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 86, or an antigen-binding or immunologically functional immunoglobulin fragment thereof, and a light chain having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention also provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and wherein the heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO: 10, wherein the light chain comprises a light chain variable region, and wherein the light chain variable region comprises a sequence that is at least 75%, preferably 80%, more preferably at least 85%, even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% identical to the amino acid sequence represented by SEQ ID NO: 12, preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% identical, wherein the antibody specifically binds NGF.

The invention also provides an antibody that specifically binds NGF, wherein the heavy chain comprises SEQ ID NO: 14, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, and a light chain comprising the amino acid sequence of SEQ ID NO: 16, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, and wherein the heavy chain variable region comprises an amino acid sequence identical to SEQ ID NO: 14. SEQ ID NO: 18 or SEQ ID NO: 22, wherein the light chain comprises a light chain variable region, and wherein the light chain variable region comprises a sequence at least 75%, preferably 80%, more preferably at least 85%, even more preferably at least 9O%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% identical to the amino acid sequence represented by SEQ ID NO: 16, preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99%, wherein the antibody specifically binds NGF.

The present invention also provides single-chain antibodies, single-chain Fv antibodies, F (ab) 'antibodies, and F (ab')₂An antibody.

In a particular aspect, the invention provides a polypeptide comprising SEQ ID NO: 16 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In addition, the present invention provides a polypeptide comprising any of SEQ ID NOs: 14. SEQ ID NO: 18 or SEQ ID NO: 22 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

The invention also relates to an isolated human antibody that specifically binds NGF, wherein the antibody comprises (a) a human heavy chain framework region, a human heavy chain CDR1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region; and (b) a human light chain framework region, a human light chain CDR1 region, a human light chain CDR2 region, and a human light chain CDR3 region. In certain aspects, the human heavy chain CDR1 region can be SEQ ID NO: 22, the heavy chain CDR1 region, the human light chain CDR1 region of the monoclonal antibody (mAb) designated 4D4 can be SEQ ID NO: 24, the light chain CDR1 region of mAb 4D4. In other aspects, the human heavy chain CDR2 region can be SEQ ID NO: 18, the heavy chain CDR2 region and the human light chain CDR2 region of mAb 4D4 can be SEQ ID NO: 20, the light chain CDR2 region of mAb 4D4. In still other aspects, the human heavy chain CDR3 region is SEQ ID NO: the heavy chain CDR3 region and the human light chain CDR3 region of mAb 4D4 shown in SEQ ID NO: 16, the light chain CDR3 region of mAb 4D4.

The invention also provides an isolated human antibody that specifically binds nerve growth factor, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 79. SEQ ID NO: 81. SEQ ID NO: 83. SEQ ID NO: 85 or SEQ ID NO: 87, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

The invention also provides an isolated human antibody that specifically binds NGF, comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. SEQ ID NO: 80. SEQ ID NO: 82. SEQ ID NO: 84. SEQ ID NO: 86. SEQ ID NO: 88. SEQ ID NO: 89. SEQ ID NO: 90. SEQ ID NO: 91 or SEQ ID NO: 131, or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof.

The inventionAn antibody is characterized by the ability to antagonize at least one in vitro and/or in vivo activity associated with an NGF polypeptide. Preferably, the invention provides an isolated human antibody against human NGF capable of high affinity binding to NGF polypeptide, wherein the antibody binds to human NGF polypeptide and binds to human NGF polypeptide at about 50x 10^-12Dissociation constant (K) of M or less_D) (measured with KinExA) disassociation from human NGF polypeptide, or in an in vitro neutralization assay at about 1X10^-8IC of M or less₅₀Inhibits NGF-induced survival.

In a preferred embodiment, the present invention provides an isolated human antibody against human NGF having the following characteristics:

a) at about 1X10 in an in vitro neutralization assay^-9IC of M or less₅₀Inhibition of NGF-induced survival;

b) has a sequence comprising SEQ ID NO: 14 amino acid sequence of heavy chain CDR 3;

c) has a sequence comprising SEQ ID NO: 16 amino acid sequence light chain CDR 3.

The invention also provides an isolated human antibody or antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment that specifically binds NGF with high affinity, wherein the antibody or fragment binds at about 1x10^-9Or lower K_DDisassociation from human NGF polypeptide at about 1X10 in standard in vitro assays^-8IC of M or less₅₀Neutralizing human NGF biological activity, and wherein the antibody or fragment comprises a heavy chain variable region comprising:

a) a CDR1 region comprising the amino acid sequence of the formula:

a¹a²a³a⁴a⁵

wherein:

a¹is a polar hydrophilic amino acid residue; a is²Is an aromatic amino acid residue; a is³Is an aliphatic, polar hydrophobic, aromatic amino acid residue; a is⁴Is a neutral hydrophobic or aliphatic amino acid residue; a is⁵Is aliphatic or polarAn aqueous amino acid residue;

b) a CDR2 region comprising the amino acid sequence of the formula:

b¹b²b³b⁴b⁵b⁶b⁷b⁸b⁹b¹⁰b¹¹b¹²b¹³b¹⁴b¹⁵b¹⁶b¹⁷

wherein:

b¹is an aliphatic, polar hydrophobic or aromatic amino acid residue; b²Is an aliphatic hydrophobic amino acid residue; b³Is a polar hydrophilic or aromatic amino acid residue; b⁴Is a polar hydrophilic, hydrophobic or aromatic amino acid residue; b⁵-b⁹Independently is a polar hydrophilic or aliphatic amino acid residue; b¹⁰Is a polar hydrophilic, aromatic or aliphatic amino acid residue; b¹¹Is an aromatic or hydrophobic amino acid residue; b¹²Is an aliphatic hydrophobic or polar hydrophilic amino acid residue; b¹³Is an aliphatic, hydrophobic or polar hydrophilic amino acid residue; b¹⁴And b¹⁶Independently is a polar hydrophilic amino acid residue; b¹⁵Is an aliphatic or aromatic hydrophobic amino acid residue; b¹⁷Is an aliphatic acidic amino acid residue;

c) a CDR3 region comprising the amino acid sequence of the formula:

c¹c²c³c⁴c⁵c⁶c⁷c⁸c⁹c¹⁰c¹¹c¹²c¹³c¹⁴c¹⁵c¹⁶c¹⁷

wherein:

c¹is absent or is an aliphatic amino acid residue; c. C²Either absent or polar hydrophilic or aromatic hydrophobic amino acid residues; c. C³And c⁴Independently absent or polar hydrophilic, aromatic hydrophobic or aliphatic amino acid residues；c⁵Absent or polar hydrophilic, aliphatic or aromatic amino acid residues; c. C⁶Absent or polar hydrophilic or aliphatic amino acid residues; c. C⁷Is a polar hydrophilic or aliphatic amino acid residue; c. C⁸Is a polar hydrophilic, hydrophobic or aromatic amino acid residue; c. C⁹Is a polar hydrophilic, aliphatic or aromatic hydrophobic amino acid residue; c. C¹⁰Is a polar hydrophilic, aromatic hydrophobic or aliphatic hydrophobic amino acid residue; c. C¹¹-c¹³Independently a polar hydrophilic or aromatic hydrophobic amino acid residue; c. C¹⁴Is an aliphatic or aromatic hydrophobic amino acid residue; c. C¹⁵Is a polar hydrophilic or neutral hydrophobic amino acid residue; c. C¹⁶Absent or polar hydrophilic amino acid residues; c. C¹⁷Are aromatic hydrophobic or aliphatic hydrophobic amino acid residues.

In one aspect, a¹Is a polar hydrophilic amino acid residue; a is²Is an aromatic hydrophobic amino acid residue; a is³Is an aliphatic hydrophobic amino acid residue; a is⁴Is neutral hydrophobic; a is⁵Is a polar hydrophilic amino acid residue; b¹Is an aliphatic or aromatic amino acid residue; b²Is Ile; b³Is a polar hydrophilic amino acid residue; b⁴Is a polar hydrophilic or aromatic amino acid residue; b⁵-b⁹Independently is a polar hydrophilic or aliphatic amino acid residue; b¹⁰Is an aliphatic amino acid residue; b¹¹Is Tyr; b¹²Is an aliphatic hydrophobic amino acid residue; b¹³Is an aliphatic or polar hydrophilic amino acid residue; b¹⁴And b¹⁶Independently is a polar hydrophilic amino acid residue; b¹⁵Is an aliphatic hydrophobic amino acid residue; b¹⁷Is an aliphatic acidic amino acid residue; c. C¹Is absent or is an aliphatic amino acid residue; c. C²Either absent or polar hydrophilic or aromatic hydrophobic amino acid residues; c. C³And c⁴Independently absent or polar hydrophilic, aromatic hydrophobic or aliphatic amino acid residues; c. C⁵Absent or polar hydrophilic amino acid residues; c. C⁶Is absent or polarHydrophilic or aliphatic amino acid residues; c. C⁷Is a polar hydrophilic or aliphatic amino acid residue; c. C⁸Is a polar hydrophilic, hydrophobic or aromatic amino acid residue; c. C⁹Is a polar hydrophilic, aliphatic or aromatic hydrophobic amino acid residue; c. C¹⁰Is a polar hydrophilic, aromatic hydrophobic or aliphatic hydrophobic amino acid residue; c. C¹¹-c¹³Independently a polar hydrophilic or aromatic hydrophobic amino acid residue; c. C¹⁴Is an aliphatic or aromatic hydrophobic amino acid residue; c. C¹⁵Is a polar hydrophilic or neutral hydrophobic amino acid residue; c. C¹⁶Absent or polar hydrophilic amino acid residues; c. C¹⁷Are aromatic hydrophobic or aliphatic hydrophobic amino acid residues.

In a particular aspect, a¹Is Ser, Asp or Thr; a is²Is Tyr; a is³Is Ala, Ser, Trp or Gly; a is⁴Is Met or Ile; a is⁵Is His, Gly or Asn; b¹Is Tyr, Gly, Ile or Asp; b²Is Ile; b³Is Ser, Thr, Tyr or Asn; b⁴Is Trp, Arg or Pro; b⁵Is Ser, Asn or Gly; b⁶Is Ser, Arg, Asp or Gly; b⁷Is Ser, His or Gly; b⁸Is Ser, Ile, Asp or Thr; b⁹Is Leu, Ile or Thr; b¹⁰Is Gly, Lys or Phe; b¹¹Is Tyr; b¹²Is Ala or Ser; b¹³Asp, Gly or Pro; b¹⁴Is Ser; b¹⁵Is Val or Phe; b¹⁶Is Lys or Gln; b¹⁷Is Gly; c. C¹Absent or an aliphatic amino acid residue; c. C²Is absent or is Tyr; c. C³And c⁴Independently is absent, Tyr, Asn, Val or Glu; c. C⁵Is absent, Ser, Gly or Trp; c. C⁶Is absent, Ser, Gly, Glu or Leu; c. C⁷Is Gly, Arg or Asp; c. C⁸Is Trp, Pro, Ser or Thr; c. C⁹Is His, Gly or Tyr; c. C¹⁰Is Val, Tyr or Arg; c. C¹¹-c¹³Independently Ser, Phe, Tyr, Asp, or Asn; c. C¹⁴Is Phe, Val or Gly; c. C¹⁵Is Met or Asp; c. C¹⁶Is absent, Asp or Asn; c. C¹⁷Is Tyr or Val.

In another specific aspect, a¹Is Ser or Asp; a is²Is Tyr; a is³Is Ala or Ser; a is⁴Is Met or Ile; a is⁵Is His or Asn; b¹Is Tyr or Gly; b²Is Ile; b³Is Ser, Thr, Tyr or Asn; b⁴Is Trp, Arg or Pro; b⁵Is Ser or Ash; b⁶Is Ser or Arg; b⁷Is His or Gly; b⁸Is Ile or Thr; b⁹Is Leu, Ile or Thr; b¹⁰Is Gly or Phe; b¹¹Is Tyr; b¹²Is Ala or Ser; b¹³Is Asp or Gly; b¹⁴Is Ser; b¹⁵Is Val or Phe; b¹⁶Is Lys or Gln; b¹⁷Is Gly; c. C¹Is absent or Gly; c. C²Is absent or is Tyr; c. C³And c⁴Independently is absent, Tyr, Gly, or Val; c. C⁵Is absent or is Ser; c. C⁶Is Ser or Gly; c. C⁷Is Gly or Arg; c. C⁸Is Trp or Pro; c. C⁹Is His, Gly or Tyr; c. C¹⁰Is Val or Tyr; c. C¹¹-c¹³Independently Ser, Tyr, Phe, or Asp; c. C¹⁴Is Phe or Val; c. C¹⁵Is Met or Asp; c. C¹⁶Absent or Asp; c. C¹⁷Is Tyr or Val.

In other particular aspects:

a) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 22, heavy chain CDR2 has the amino acid sequence represented by SEQ ID NO: 18, heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof;

b) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 92, heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 93, and heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO: 94, or a pharmaceutically acceptable salt thereof;

c) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 98, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 99, heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO: 100, or a pharmaceutically acceptable salt thereof;

d) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 104, and heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 105, and heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 106;

e) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 110, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 111, heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence represented by seq id no;

f) heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 116, and heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 117 and heavy chain CDR3 has the amino acid sequence shown in SEQ ID NO: 118, or a pharmaceutically acceptable salt thereof.

The invention also provides an isolated human antibody or antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment that specifically binds NGF, wherein the antibody or fragment thereof comprises a light chain variable region comprising:

a) a CDR1 region comprising the amino acid sequence of the formula:

a¹a²a³a⁴a⁵a⁶a⁷a⁸a⁹a¹⁰a¹¹a¹²

wherein:

a¹is a polar hydrophilic amino acid residue; a is²、a¹¹And a¹²Independently is an aliphatic or hydrophobic amino acid residue; a is³、a⁵、a⁷And a⁸Independently is an aliphatic, polar hydrophilic or hydrophobic amino acid residue; a is⁴Is a polar hydrophilic amino acid residue; a is⁶Is an aliphatic or hydrophobic amino acid residue; a is⁹Absent, or aliphatic or polar hydrophilic amino acid residues; a is¹⁰Is an aliphatic, aromatic or hydrophobic amino acid residue;

b) a CDR2 region comprising the amino acid sequence of the formula:

b¹b²b³b⁴b⁵b⁶b⁷

wherein:

b¹is an aliphatic, polar hydrophobic or hydrophobic amino acid residue; b²Is an aliphatic or hydrophobic amino acid residue; b³And b⁴Independently is a polar hydrophilic, aliphatic or hydrophobic amino acid residue; b⁵Is a polar hydrophilic or aliphatic hydrophobic amino acid residue; b⁶Is a polar hydrophilic or aliphatic hydrophobic amino acid residue; b⁷Is a polar hydrophilic amino acid residue;

c) a CDR3 region comprising the amino acid sequence of the formula:

c¹c²c³c⁴c⁵c⁶c⁷c⁸c⁹c¹⁰c¹¹c¹²c¹³c¹⁴c¹⁵c1⁶c¹⁷

wherein:

c¹and c²Independently is a polar hydrophilic amino acid residue; c. C³Is a polar hydrophilic, aliphatic or hydrophobic amino acid residue; c. C⁴、c⁵And c⁶Independently is an aliphatic, polar hydrophilic or hydrophobic amino acid residue; c. C⁷Either absent or polar hydrophilic or aliphatic hydrophobic amino acid residues; c. C⁸Is a polar hydrophilic or hydrophobic amino acid residue; c. C⁹Is a polar hydrophilic amino acid residue, and wherein the antibody or fragment is present at about 1x10^-9Or lower K_DDisassociation from human NGF polypeptide at about 1X10 in standard in vitro assays^-8IC of M or less₅₀Neutralize human NGF biological activity.

In one aspect, a¹、a³、a⁴、a⁷And a⁸Independently is a polar hydrophilic amino acid residue; a is²、a⁶、a¹¹And a¹²Independently is an aliphatic hydrophobic amino acid residue; a is⁵Is a polar hydrophilic or aliphatic amino acidA residue; a is⁹Absent, or aliphatic or polar hydrophilic amino acid residues; a is¹⁰Is an aliphatic or aromatic amino acid residue; b¹Is an aliphatic, polar hydrophobic or hydrophobic amino acid residue; b²Is an aliphatic hydrophobic amino acid residue; b³、b⁴And b⁷Independently is a polar hydrophilic amino acid residue; b⁵And b⁶Independently is a polar hydrophilic or aliphatic hydrophobic amino acid residue; c. C¹And c²Independently is a polar hydrophilic amino acid residue; c. C³Is a polar hydrophilic, aliphatic or hydrophobic amino acid residue; c. C⁴、c⁵And c⁶Independently is an aliphatic, polar hydrophilic or hydrophobic amino acid residue; c. C⁷Absent or aliphatic hydrophobic amino acid residues; c. C⁸Is a hydrophobic amino acid residue; c. C⁹Are polar hydrophilic amino acid residues.

In a particular aspect, a¹、a³、a⁴And a⁷Arg, Ser, Gln and Ser, respectively; a is²Is Ala; a is⁵Is Gly or Ser; a is⁸Is Ser or Ile; a is⁹Is absent, Ser or Gly; a is¹⁰Is Ala, Tyr, Trp or Phe; b¹Asp, Gly, Ala or Val; b²And b³Ala and Ser, respectively; b⁴Is Ser or Asn; b⁵Is Leu or Arg; b⁶Is Glu, Ala or Gln; b⁷Is Ser or Thr; c. C¹And c²Is Gln; c. C³Is Phe, Tyr, Arg, or Ala; c. C⁴Is Asn, Gly or Ser; c. C⁵Is Ser or Asn; c. C⁶Is Tyr, Ser, Trp or Phe; c. C⁷Is absent, Pro or His; c. C⁸Is Leu, Trp, Tyr or Arg; c. C⁹Is Thr.

In another specific aspect, a¹、a²、a³、a⁴And a⁷Arg, Ala, Ser, gin and Ser, respectively; a is⁵Is Gly or Ser; a is⁸Is Ser or Ile; a is⁹Is absent, Ser or Gly; a is¹⁰Is Ala or Tyr; b¹Is Asp or Gly; b²And b³Are Ala andSer；b⁴is Ser or Asn; b⁵Is Leu or Arg; b⁶Is Glu, Ala or Gln; b⁷Is Ser or Thr; c. C¹And c²Is Gln; c. C³Is Phe, Tyr, Arg, or Ala; c. C⁴Is Asn, Gly or Ser; c. C⁵Is Ser or Asn; c. C⁶Is Tyr, Ser, Trp or Phe; c. C⁷Is absent, Pro or His; c. C⁸Is Leu, Trp, Tyr or Arg; c. C⁹Is Thr.

In other particular aspects:

a) light chain CDR1 has the amino acid sequence of SEQ ID NO: 24, and a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence represented by seq id no;

b) light chain CDR1 has the amino acid sequence of SEQ ID NO: 95, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 96, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 97, or a pharmaceutically acceptable salt thereof;

c) light chain CDR1 has the amino acid sequence of SEQ ID NO: 101, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 102, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 103, or a pharmaceutically acceptable salt thereof;

d) light chain CDR1 has the amino acid sequence of SEQ ID NO: 107, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 108, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 109, or a pharmaceutically acceptable salt thereof;

e) light chain CDR1 has the amino acid sequence of SEQ ID NO: 113, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 114, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 115, or a pharmaceutically acceptable salt thereof;

f) light chain CDR1 has the amino acid sequence of SEQ ID NO: 119, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 120, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 121, or a pharmaceutically acceptable salt thereof;

g) light chain CDR1 has the amino acid sequence of SEQ ID NO: 122, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 123, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence represented by seq id no;

h) light chain CDR1 has the amino acid sequence of SEQ ID NO: 125, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 126, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 127, or a pharmaceutically acceptable salt thereof;

i) light chain CDR1 has the amino acid sequence of SEQ ID NO: 128, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 129, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence represented by seq id no;

j) light chain CDR1 has the amino acid sequence of SEQ ID NO: 132, and a light chain CDR2 having the amino acid sequence of SEQ ID NO: 133, and a light chain CDR3 having the amino acid sequence of SEQ ID NO: 134, or a pharmaceutically acceptable salt thereof.

Also part of the invention are polynucleotide sequences encoding novel anti-human NGF human antibodies, vectors comprising polynucleotide sequences encoding anti-human NGF human antibodies, host cells transformed with vectors incorporating polynucleotides encoding anti-human NGF human antibodies, formulations comprising anti-human NGF human antibodies and methods of making and using the same.

The invention also provides a method of detecting NGF levels in a biological sample, the method comprising the step of contacting the sample with an antibody, or antigen-binding fragment thereof, of the invention. The anti-NGF antibodies of the present invention can be applied to any known analytical assay methods, such as competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays, and enzyme-linked immunosorbent assays (ELISA) (see Sola, 1987,Monoclonal Antibodies：A Manual of Techniquespage 147-. Antibodies of the invention bind NGF with an affinity suitable for the assay employed.

Furthermore, the present invention provides a method of treating a disease associated with increased NGF production or increased sensitivity to NGF, comprising the step of administering to an individual in need thereof a pharmaceutically effective amount of a pharmaceutical composition comprising at least one antibody or antigen-binding fragment or immunologically functional immunoglobulin fragment thereof of the present invention.

Specific preferred embodiments of the present invention will become apparent from the following more detailed description of certain preferred embodiments and the claims.

Brief Description of Drawings

Figure 1 depicts a graph showing the neutralization of NGF activity by 4D4 monoclonal antibody purified from hybridoma conditioned media in a DRG neuron-based neutralization bioassay.

Figure 2 depicts a graph showing VR1 expression stimulated by human NGF activity, and the neutralizing effect on NGF activity of anti-NGF monoclonal antibody (4D4) purified from hybridoma conditioned media in a DRG neuron-based neutralization bioassay.

Figure 3 depicts a graph showing the neutralization of NGF activity by transiently expressing recombinant anti-NGF 4D4 monoclonal antibodies expressed as IgG1 or IgG2 in roller bottle (R) or spinner bottle (S) cultured cells in a DRG neuron-based neutralization bioassay.

Figure 4 depicts a sequence alignment of neurotrophins. The numbering and secondary structural elements above the sequence refer to mature human NGF. Conserved residues are indicated by asterisks and regions with lower sequence homology are indicated by shading. Human NGF is SEQ id no: 135 of the total weight of the raw materials; mouse NGF is seq id NO: 136; BDNF is SEQ ID NO: 137; NT3 is SEQ ID NO: 138.

FIG. 5 shows the anti-NGF CDR1 heavy chain alignment and percent identity for the 14D10(SEQ ID NO: 98), 6H9(SEQ ID NO: 104), 7H2(SEQ ID NO: 110), 4G6(SEQ ID NO: 116), 14D11(SEQ ID NO: 92) and 4D4(SEQ ID NO: 22) antibodies.

FIG. 6 shows the anti-NGF CDR2 heavy chain alignment and percent identity for the 14D10(SEQ ID NO: 99), 6H9(SEQ ID NO: 105), 7H2(SEQ ID NO: 111), 4G6(SEQ ID NO: 117), 14D11(SEQ ID NO: 93) and 4D4(SEQ ID NO: 18) antibodies.

FIG. 7 shows the anti-NGF CDR3 heavy chain alignment and percent identity for the 14D10(SEQ ID NO: 100), 6H9(SEQ ID NO: 106), 7H2(SEQ ID NO: 112), 4G6(SEQ ID NO: 118), 14D11(SEQ ID NO: 94) and 4D4(SEQ ID NO: 14) antibodies.

FIG. 8 shows the anti-NGFCDR 1 light chain alignment and percent identity of the antibodies 14D10(SEQ ID NO: 95), 6H9(SEQ ID NO: 107), 7H2(SEQ ID NO: 113), 4G6a (SEQ ID NO: 119), 4G6b (SEQ ID NO: 122), 4G6c (SEQ ID NO: 125), 4G6D (SEQ ID NO: 128), 4G6e (SEQ ID NO: 132), 14D11(SEQ ID NO: 95) and 4D4(SEQ ID NO: 24) (20031028340 in each figure means 4G6 a; 20031028351 in each figure means 4G6 b; 20031071526 in each figure means 4G6 c; 20031028344 in each figure means 4G 6D; 20031000528 in each figure means 4G6 e).

FIG. 9 shows the anti-NGFCDR 2 light chain alignment and percent identity of the antibodies 14D10(SEQ ID NO: 96), 6H9(SEQ ID NO: 108), 7H2(SEQ ID NO: 114), 4G6a (SEQ ID NO: 120), 4G6b (SEQ ID NO: 123), 4G6c (SEQ ID NO: 126), 4G6D (SEQ ID NO: 129), 4G6e (SEQ ID NO: 133), 14D11(SEQ ID NO: 96) and 4D4(SEQ ID NO: 20) (20031028340 in each figure means 4G6 a; 20031028351 in each figure means 4G6 b; 20031071526 in each figure means 4G6 c; 20031028344 in each figure means 4G 6D; 20031000528 in each figure means 4G6 e).

FIG. 10 shows the anti-NGFCDR 3 light chain alignment and percent identity of the antibodies 14D10(SEQ ID NO: 97), 6H9(SEQ ID NO: 109), 7H2(SEQ ID NO: 115), 4G6a (SEQ ID NO: 121), 4G6b (SEQ ID NO: 124), 4G6c (SEQ ID NO: 127), 4G6D (SEQ ID NO: 130), 4G6e (SEQ ID NO: 134), 14D11(SEQ ID NO: 97) and 4D4(SEQ ID NO: 16) (20031028340 in each figure means 4G6 a; 20031028351 in each figure means 4G6 b; 20031071526 in each figure means 4G6 c; 20031028344 in each figure means 4G 6D; 20031000528 in each figure means 4G6 e).

FIG. 11 shows the anti-NGF light chain alignment and percentage identity of the antibodies 14D10(SEQ ID NO: 82), 6H9(SEQ ID NO: 84), 7H2(SEQ ID NO: 86), 4G6a (SEQ ID NO: 88), 4G6b (SEQ ID NO: 89), 4G6c (SEQ ID NO: 90), 4G6D (SEQ ID NO: 91), 4G6e (SEQ ID NO: 131), 14D11(SEQ ID NO: 80) and 4D4(SEQ ID NO: 12) (20031028340 in each figure refers to 4G6 a; 20031028351 in each figure refers to 4G6 b; 20031071526 in each figure refers to 4G6 c; 20031028344 in each figure refers to 4G 6D; 20031000528 in each figure refers to 4G 5396 6 e).

FIG. 12 shows the anti-NGF heavy chain alignment and percent identity for the 4D4(SEQ ID NO: 10), 4G6(SEQ ID NO: 87), 14D10(SEQ ID NO: 81), 14D11(SEQ ID NO: 79), 7H2(SEQ ID NO: 85), and 6H9(SEQ ID NO: 83) antibodies.

Detailed description of certain preferred embodiments

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are incorporated herein by reference for any purpose.

Definition of

For recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) conventional techniques may be used. Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or in a manner commonly used in the art or as described herein. The techniques and methods described above can generally be performed according to methods well known in the art, or as described in various general and more specific references that are cited and discussed in the present specification. See, e.g., Sambrook et al, 2001, MOLECULAR CLONING: a LAB ORATORY MANUAL, 3 rd edition, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature and laboratory procedures and techniques described herein, which relate to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry, are well known and commonly used in the art. Likewise, conventional techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

The following terms used in accordance with the present disclosure, unless otherwise indicated, shall be understood to have the following meanings: the phrases "biological properties", "biological characteristics", and the term "activity" with respect to the antibodies of the invention are used interchangeably herein and include, but are not limited to, epitope affinity and specificity (e.g., binding of human antibodies to human NGF), ability to antagonize the activity of the target polypeptide (e.g., NGF activity), in vivo stability of the antibody, and immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies recognized in the art include, for example, cross-reactivity (i.e., generally cross-reactivity with non-human homologs of the target polypeptide or with other proteins or tissues), and the ability to maintain high levels of expression of the protein in mammalian cells. The above properties or characteristics can be observed or measured using art recognized techniques, including but not limited to ELISA, competitive ELISA, surface plasmon resonance analysis, in vitro and in vivo neutralization assays (e.g., example 2), and immunohistochemical analysis using tissue sections from different sources, including humans, primates, or any other source as may be desired. The specific activity and biological properties of human antibodies against human NGF are described in more detail in the examples below.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, due to its origin, is (1) unrelated to all or a portion of the polynucleotide in which it is naturally found, (2) linked to a polynucleotide not naturally associated therewith, or (3) not naturally occurring as part of a larger sequence.

The term "isolated protein" as referred to herein means that the subject protein is (1) free of at least some other proteins with which it is ordinarily found associated, (2) substantially free of other proteins of the same origin (e.g., the same species), (3) expressed by cells of a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other materials with which it is naturally associated, (5) not associated with the portion of the protein to which the "isolated protein" is naturally associated (via covalent or non-covalent interactions), (6) operatively associated with a polypeptide with which it is not naturally associated (via covalent or non-covalent interactions), or (7) not found in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated protein is substantially separated from proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or other use).

An "isolated" antibody is one that has been identified and separated from and/or recovered from a component of its natural environment. Contaminating components in the natural environment of an antibody are substances that can interfere with its diagnostic or therapeutic use and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody will be purified (1) to more than 95% by weight, most preferably more than 99% by weight of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15N-terminal or internal amino acid residue sequences (by using a spinning cup sequencer), and (3) to homogeneity (by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue staining or preferably silver staining). Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment is not present.

The term "polypeptide" or "protein" refers to a molecule having the sequence of a native protein, i.e., a protein produced by natural cells and particularly non-recombinant cells, or genetically engineered or recombinant cells, including molecules having the amino acid sequence of a native protein, or molecules that have deletions from, insertions into, and/or substitutions of one or more amino acids of a native sequence. The terms "polypeptide" and "protein" expressly encompass anti-NGF antibodies or sequences lacking, inserting and/or replacing one or more amino acids of an anti-NGF antibody.

The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion, and/or an internal deletion. In certain embodiments, fragments are at least 5 to about 500 amino acids in length. It is understood that in certain embodiments, fragments are at least 5,6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains, including binding domains. In the case of anti-NGF antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of heavy or light chains, a portion of an antibody chain or just its variable region comprising two CDRs, and the like.

The term "specific binder" refers to a natural or unnatural molecule that specifically binds to a target. Examples of specific binders include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids. In certain embodiments, the specific binding agent is an antibody.

The term "specific binding agent for NGF" refers to a specific binding agent that specifically binds to any part of NGF. In certain embodiments, the specific binder for NGF is an antibody that specifically binds NGF.

The term "immunologically functional immunoglobulin fragment" as used herein refers to a polypeptide fragment that contains at least the CDRs of an immunoglobulin heavy and light chain. The immunologically functional immunoglobulin fragments of the invention are capable of binding an antigen. In a preferred embodiment, the antigen is a ligand that specifically binds to the receptor. In these embodiments, the binding of the immunologically functional immunoglobulin fragments of the invention prevents the binding of the ligand to its receptor, blocking the biological response that results from the binding of the ligand to the receptor. Preferably, the immunologically functional immunoglobulin fragments of the invention specifically bind NGF. Most preferably, the fragment specifically binds human NGF.

As used herein, "native" as applied to an object means that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is native.

The term "operably linked" means that the components to which the term refers are in a relationship that allows them to perform their inherent function under appropriate conditions. For example, a control sequence "operably linked" to a protein coding sequence is linked to the coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence.

The term "control sequences" as used herein refers to polynucleotide sequences capable of effecting the expression, processing or intracellular localization of the coding sequences to which they are ligated. The nature of such control sequences may depend on the host organism. In particular embodiments, the control sequences of prokaryotes may include a promoter, a ribosome binding site, and a transcription termination sequence. In other embodiments, eukaryotic control sequences may include a promoter comprising one or more recognition sites for transcription factors, a transcription enhancing sequence, a transcription termination sequence, and a polyadenylation sequence. In certain embodiments, a "control sequence" may include a leader sequence and/or a fusion partner sequence.

The term "polynucleotide" as referred to herein means a single-or double-stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide may be ribonucleic or deoxyribonucleic acids or modified forms of any type of nucleotide. Such modifications include, for example, base modifications such as bromouridine (bronuridine), ribose modifications such as arabinoside and 2 ', 3' -dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanillylate, and phosphoramidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

The term "oligonucleotide" as referred to herein includes natural and modified nucleotides linked together by natural and/or non-natural oligonucleotide linkages. Oligonucleotides are a subset of polynucleotides, comprising members that are typically single-stranded and 200 nucleotides or less in length. In certain embodiments, the oligonucleotide is 10-60 nucleotides in length. In certain embodiments, the oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 nucleotides in length. Oligonucleotides may be single-stranded or double-stranded, e.g., for use in the construction of genetic mutants. The oligonucleotides of the invention may be sense or antisense oligonucleotides to a protein coding sequence.

The term "natural nucleotide" includes deoxyribonucleic acid and ribonucleic acid. The term "modified nucleotide" includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkage" includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilide, phosphoroamidate, and the like. See, e.g., LaP1 ache et al, 1986, nucl.14: 9081; stec et al 1984, J.am.chem.Soc.,106: 6077; stein et al, 1988, nucleic acids res,16: 3209; zon et al, 1991, Anti-Cancer Drug Design,6: 539; zon et al, 1991, OLIGONUCLEOTIDES AND ANALOGUES: PRACTICALAPPROACH, pages 87-108 (f. eckstein editors), Oxford University Press, Oxford England; stec et al, U.S. patent No. 5,151,510; uhlmann and Peyman, 1990, Chemical Reviews,90: 543, the disclosures of which are incorporated herein by reference for all purposes. The oligonucleotide may include a detectable label to enable detection of the oligonucleotide or hybridization thereof.

The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular strand-taking DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as

A "recombinant expression vector" (or simply "expression vector"). Generally, expression vectors useful in recombinant DNA techniques are often presented in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, the invention encompasses such other types of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.

The phrase "recombinant host cell" (or simply "host cell") includes cells into which a recombinant expression vector has been introduced. Those skilled in the art will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either variation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. There are a wide variety of host expression systems that can be used to express the antibodies of the invention, including bacterial, yeast, baculovirus and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC 19. For recombinant expression of the antibody, the host cells are transfected with one or more recombinant expression vectors carrying DNA segments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cells and preferably secreted into the medium in which the host cells are cultured, from which medium the antibody can be recovered. The heavy and light chain genes are obtained by standard recombinant DNA methods, incorporated into recombinant expression vectors, and the vectors are introduced into host cells, for example as described in Sambrook et al, 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold spring Harbor Laboratories, Ausubel, F.M. et al (eds.) Current Protocols in MOLECULAR biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 to Boss et al.

The term "host cell" is used to refer to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence, and is then capable of expressing a selected gene of interest. The term includes progeny of the parent cell, whether or not the progeny are identical in morphology or genetic structure to the original parent, so long as the selected gene is present.

The term "transduction" is used to refer to the transfer of a gene from one bacterium to another, typically by phage transfer. "transduction" also refers to the acquisition and transfer of eukaryotic cell sequences by retroviruses.

The term "transfection" is used to refer to the uptake of foreign or exogenous DNA by a cell, which is "transfected" when the exogenous DNA has been introduced into the cell membrane. A variety of transfection techniques are known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52: 456; sambrook et al, 2001, Molecula clone, ALABORATORY MANUAL, Cold Spring Harbor Laboratories; davis et al, 1986, BASIC METHODS IN MOLECULARBIOLOGY, Elsevier; and Chu et al, 1981, Gene13: 197. this technique can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

The term "transformation" is used herein to refer to a change in the genetic characteristics of a cell, which is transformed when the cell has been modified to contain new DNA. For example, in the case of a cell that is genetically modified from its native state, the cell is transformed. Following transfection or transduction, the transforming DNA may recombine with the DNA of the cell by physically integrating into the chromosome of the cell, or may be maintained transiently as an episomal element that does not replicate, or may replicate independently as a plasmid. When the transforming DNA replicates as the cell divides, the cell is considered to have been stably transformed.

The term "naturally occurring" or "native" when used with respect to biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials that can be found in nature and are not manipulated by man. Likewise, "non-naturally occurring" or "non-natural" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, which can additionally be used in an animal to produce an antibody that can bind an epitope of the antigen. An antigen has one or more epitopes.

The term "identity" as is well known in the art refers to the relationship between two or more polypeptide molecules or two or more nucleic acid molecules as determined by comparing the sequences of the sequences. In the art, "identity" also refers to the degree of sequence relatedness between nucleic acid molecules or between polypeptides, as the case may be, as determined by the pairing between two or more nucleotide sequence strands or between two or more amino acid sequence strands. An "identity" determination is the percentage of identical pairings of the (common) smaller of two or more sequences, and gap alignments (if any) are processed through special mathematical models or computer programs (i.e., "algorithms").

The term "similarity" is used in the art to refer to related concepts, but unlike "identity," similarity "refers to a measure of relatedness, which includes identical pairs and pairs of conservative substitutions. If two polypeptide sequences have the same amino acid, e.g., 10/20, and the remainder are non-conservative substitutions, the percent identity and percent similarity are both 50%. In the same example, if there are 5 more conservative substitutions, the percent identity remains 50%, but the percent similarity will be 75% (15/20). Thus, in the presence of conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between the two polypeptides.

The identity and similarity of related nucleic acids and polypeptides can be readily calculated by well-known methods. Such methods include, but are not limited to, composition mol lecularbiogy, (Lesk, editors a.m.), 1988, oxford university Press, new york; BIOCOMPUTING: INFORMATICS AND GENOME projection, (Smith, d.w. editions), 1993, Academic Press, New York; compouteralysis OF SEQUENCE DATA, Part1, (Griffin, a.m. and Griffin, h.g. editions), 1994, human Press, New Jersey; von Heinje, g., SEQUENCEANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; sequenceanalisis PRIMER, (Gribskov, m. and Devereux, j. ed), 1991, m.stockton Press, new york; the molecular weight of Carillo et al, 1988,SIAM J.AppliedMath.，48: 1073; and Durbin et al, 1998, BIOLOGICALSEQUENCEANALYSIS, Cambridge University Press.

Preferred identity determination methods are designed to produce the greatest degree of pairing between test sequences. The identity determination method is described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucl. acid. Res.,12: 387; genetics computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J.mol.biol.,215: 403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information, NCBI, and other sources (BLAST handbook, Altschul et al NCB/NLM/NIH Bethesda, MD 20894; Altschul et al 1990, supra). The well-known Smith Waterman algorithm can also be used to determine consistency.

Certain alignment schemes for aligning two amino acid sequences may result in the pairing of only a short region of the two sequences, and this smaller aligned region may have very high sequence identity, although there is no significant relationship between the two full-length sequences. Thus, in certain embodiments, the selected alignment method (GAP program) will result in an alignment of at least 50 contiguous amino acids across the target polypeptide.

For example, the GAP Computer algorithm (Genetics Computer Group, University of Wisconsin, Madison, Wis.) is used to align two polypeptides to be determined for percent sequence identity for optimal matching of their respective amino acids. ("pairing Range", determined by the algorithm). In certain embodiments, a gap opening penalty (to be calculated as 3 times the average diagonal; where the "average diagonal" is the average of the diagonals of the comparison matrix used; the "diagonal" is the score or value assigned to each complete amino acid pair by a particular comparison matrix) and a gap propagation penalty (which is typically one tenth the gap opening penalty), as well as the comparison momentsAn array such as PAM250 or BLOSUM 62 is used with the algorithm. In certain embodiments, standard comparison matrices are also used in the algorithm (PAM 250 comparison matrix see Dayhoff et al, 1978, Atlas of protein Sequence and Structure,5: 345-352; BLOSUM 62 comparison matrix is described in Henikoff et al, 1992, Proc. Natl. Acad. Sci USA,89：10915-10919)。

in certain embodiments, the parameters for polypeptide sequence comparison include the following:

the algorithm is as follows: needleman et al, 1970, J. mol. biol.,48：443-453；

comparing the matrixes: BLOSUM 62 from Henikoff et al, 1992 (supra);

gap penalties: 12

Gap length penalty: 4

Similarity threshold: 0

The GAP program may use the above parameters. In certain embodiments, the above parameters are default parameters for polypeptide comparisons using the GAP algorithm (no penalty for end GAPs).

The term "homology" refers to the degree of similarity between protein or nucleic acid sequences. Homology information is used to understand the genetic relatedness of certain protein or nucleic acid species. Homology can be determined by aligning and comparing sequences. Typically, to determine amino acid homology, protein sequences are compared to a database of known protein sequences. Homologous sequences share a common functional identity somewhere in their sequence. A higher degree of similarity or identity generally indicates homology, although a lower degree of similarity or identity does not necessarily indicate a lack of homology.

There are several methods available to compare amino acids of one sequence with those of another sequence to determine homology. Generally, these methods fall into two categories: (1) comparing physical characteristics such as polarity, charge, and van der waals volume to generate a similarity matrix; (2) based on observations of many protein sequences from known homologous proteins, amino acids in the sequences were compared for possible substitution by any other amino acid to generate an acceptable point mutation matrix (PAM).

Percent identity can also be calculated using the program needle (EMBOSS package) or stretcher (EMBOSS package) or program align X as a module of the Vector NTI suite 9.0.0 software package, using default parameters (e.g., gap penalty of 5, gap open penalty of 15, gap extension penalty of 6.6).

The twenty common amino acids and their abbreviations used herein follow conventional usage. See IMMUNOLOGY-ASYNTHESIS, 2 nd edition, (e.s.golub and d.r.gren, editions), Sinauer Associates: sunderland, MA, 1991, which is incorporated herein by reference for any purpose. Stereoisomers of twenty common amino acids (e.g., D-amino acids); unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid and other unusual amino acids may also be suitable components of the polypeptides of the invention. Examples of unusual amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, -N, N, N-trimethyllysine, -N-acetyl lysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, alpha-N-methyl arginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, according to standard usage and convention.

Natural residues can be classified according to common side chain properties as follows:

1) hydrophobic residue: norleucine (Nor), Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;

2) polar hydrophilic residues: arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr;

3) aliphatic residue: ala, Gly, Ile, Leu, Val, Pro;

4) aliphatic hydrophobic residues: ala, Ile, Leu, Val, Pro;

5) neutral hydrophilic residues: cys, Ser, Thr, Asn, Gln;

6) acidic residue: asp, Glu;

7) basic residue: his, Lys, Arg;

8) residues that affect the orientation of the chain: gly, Pro;

9) aromatic residue: his, Trp, Tyr, Phe;

10) aromatic hydrophobic residues: phe, Trp, Tyr.

Conservative amino acid substitutions may involve the interchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may include unnatural amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other inverted or inverted forms of amino acid moieties.

Non-conservative substitutions may include the interchange of a member of one of these classes with a member of another class. Such replacement residues may be introduced into a region of a human antibody that is homologous to a non-human antibody, or into a non-homologous region of the molecule.

According to certain embodiments, the hydropathic index of an amino acid may be considered in making such a change. Each amino acid is given a hydrophilicity index according to its hydrophobicity and charge characteristics. They are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferring active biological function on a protein is recognized in the art (see examples)E.g., Kyte et al, 1982, J.157: 105-131). It is known that other amino acids having similar hydrophilicity indices or fractions can be substituted with certain amino acids and still retain similar biological activity. Where such changes are made according to hydropathic index, in certain embodiments, amino acid substitutions are included whose hydropathic index is within ± 2. In certain embodiments, amino acid substitutions having a hydropathic index within ± 1 are included, and in certain embodiments, amino acid substitutions having a hydropathic index within ± 0.5 are included.

It is also recognized in the art that substitution of like amino acids can be made effectively based on hydrophilicity, as disclosed herein, particularly where the created biofunctional protein or peptide is intended for use in immunological embodiments. In certain embodiments, the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

These amino acid residues have been assigned the following hydrophilicity values: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). Where changes are made according to similar hydrophilicity values, in certain embodiments amino acid substitutions having a hydrophilicity index within ± 2 are included, in certain embodiments amino acid substitutions having a hydrophilicity index within ± 1 are included, and in certain embodiments amino acid substitutions having a hydrophilicity index within ± 0.5 are included. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions".

Exemplary amino acid substitutions are shown in table 1.

TABLE 1

Amino acid substitution

Original amino acid	Exemplary permutations	Preference is given to substitution
			Ala	Val，Leu，Ile	Val
Arg	Lys，Gln，Asn	Lys
			Ash	Gln	Gln
Asp	Glu	Glu
			Cys	Ser，Ala	Ser
Gln	Asn	Asn
			Glu	Asp	Asp
Gly	Pro，Ala	Ala
			His	Asn，Gln，Lys，Arg	Arg
Ile	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu	Norleucine, Ile, Val, Met, Ala, Phe	Ile

Lys	Arg, 1, 4-diaminobutyric acid, Gln, Asn	Arg
			Met	Leu，Phe，Ile	Leu
Phe	Leu，Val，Ile，Ala，Tyr	Leu
			Pro	Ala	Gly
Ser	Thr，Ala，Cys	Thr
			Thr	Ser	Ser
Trp	Tyr，Phe	Tyr
			Tyr	Trp，Phe，Thr，Ser	Phe
Val	Ile, Met, Leu, Phe, Ala, norleucine	Leu

The skilled person will be able to determine suitable variants of the polypeptides represented herein using well known techniques. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity. In other embodiments, the skilled artisan is able to identify residues and molecular portions that are conserved among similar polypeptides. In other embodiments, even regions of interest for biological activity or for structure may be conservatively substituted for amino acids without destroying biological activity or adversely affecting the structure of the polypeptide.

In addition, one skilled in the art can review structure-function studies relating to identifying residues in similar polypeptides that are important for activity or structure. From this comparative study, the skilled person is able to predict the importance of amino acid residues in a protein, which correspond to amino acid residues of similar proteins that are important for activity or structure. Those skilled in the art can select chemically similar amino acid substitutions for such amino acid residues of predicted importance.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence by reference to the three-dimensional structure in similar polypeptides. From this information, those skilled in the art can predict the arrangement of amino acid residues of an antibody with reference to its three-dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be located on the surface of a protein, as such residues may be involved in interactions with other molecules. In addition, one skilled in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. Variants can then be screened using activity assays well known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, variants with such changes can be avoided if a change in a particular amino acid residue is found to result in a disruption, undesirable reduction, or inappropriate activity. In other words, based on the information gleaned from such routine experiments, the skilled person can easily determine amino acids for which further substitutions should be avoided, either alone or together with other mutations.

There are many scientific publications that have been devoted to studying predictions of secondary structure. See Moult, 1996, curr. op. in biotech.7: 422 and 427; chou et al, 1974, Biochemistry13: 222-245; chou et al, 1974, Biochemistry113: 211-222; chou et al, 1978, adv.enzymol.Relat.areas mol.biol.47: 45-148; chou et al, 1979, Ann. Rev. biochem.47: 251-276; and Chou et al, 1979, Bio phys.J.26: 367-384. In addition, computer programs are also currently available to help predict secondary structures. One method of predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity typically have similar topologies. Recent developments in protein structure databases (PDBs) have led to improved predictability of secondary structure, including the number of potential folds within a polypeptide or protein structure. See Holm et al, 1999, nucl.27: 244-247. It has been proposed (Brenner et al, 1997, curr. Op. struct. biol.7: 369-376) that in a particular polypeptide or protein, the number of folds is limited and that once the critical number of structures is resolved, the structure prediction will become significantly more accurate.

Additional secondary structure prediction methods include the "string algorithm" (Jones, 1997, curr. opin. struct. biol).7: 377-87; sippl et al, 1996, Structure4: 15-19), "Profile analysis" (Bowie et al, 1991, Science)253: 164-170; gribskov et al, 1990, meth.183: 146- > 159; gribskov et al, 1987, proc.nat.acad.sci.84: 4355-4358) and "evolutionary linkage" (see Holm, 1999, supra; and Brenner, 1997, supra).

In certain embodiments, antibody variants include glycosylation variants in which the number and/or type of glycosylation sites has been altered as compared to the amino acid sequence of the parent polypeptide. In certain embodiments, protein variants comprise a greater or lesser number of N-linked glycosylation sites than the native protein. The N-linked glycosylation sites are characterized by the following sequences: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue labeled X can be any amino acid residue other than proline. The substitution of amino acid residues to create such a sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, elimination of the sequence substitution will remove the existing N-linked carbohydrate chain. Rearrangement of the N-linked carbohydrate chains is also provided, wherein one or more N-linked glycosylation sites (typically native N-linked glycosylation sites) are removed, and one or more new N-linked sites are created. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine variants may be useful when the antibody must be refolded into a biologically active conformation (such as after isolation of insoluble inclusion bodies). Cysteine variants typically have fewer cysteine residues than the native protein, and are usually an even number to minimize interactions resulting from unpaired cysteines.

In additional embodiments, antibody variants may include antibodies comprising modified Fc fragments or modified heavy chain constant regions. The Fc fragment (which represents a crystallizable fragment) or the heavy chain constant region may be modified by mutation to confer altered binding characteristics to the antibody. See, e.g., Burton and Woof, 1992, Advances in Immunology51: 1 to 84; ravatch and Bolland, 2001, annu.19: 275 to 90; chem is Shields et al, 2001, Journal of biol276: 6591 and 6604; telleman and Junghans, 2000, Immunology100: 245-251; medesan et al, 1998, Eur.J. Immunol.28: 2092-2100; all of which are incorporated herein by reference). Such mutations can include substitutions, insertions, deletions or any combination thereof, typically by site-directed mutagenesis using one or more mutagenic oligonucleotides according to the METHODS described herein, and according to METHODS well known IN the art (see, e.g., Sambrook et al, MOLECULAR CLONING: A LABORATORYMANUAL, 3 rd edition, 2001, Cold Spring Harbor, N.Y., and Berger and Kimmel, METHODS IN ENZYMOLOGY, Vol. 152, Guide to MOLECULAR CLONING Techniques, 1987, Academic Press, Inc., San Diego, CA, which are incorporated herein by reference).

According to certain embodiments, the amino acid substitutions are as follows: (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering the binding affinity for formation of a protein complex, (4) altering the binding affinity and/or (5) conferring or modifying other physiochemical or functional properties to such a polypeptide. According to certain embodimentsSingle or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made in the native sequence (in some embodiments, in portions of the polypeptide outside the domains that form intermolecular contacts). In preferred embodiments, conservative amino acid substitutions generally do not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not tend to disrupt the helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized secondary and tertiary STRUCTURES of polypeptides are described in protins, stuctcurs and anolecular PRINCIPLES, (edited by creeton), 1984, w.h.freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C.Branden and J.Tooze, eds.), 1991, Garland Publishing, New York, N.Y.; and Thornton et al, 1991, Nature354: 105, each of which is incorporated herein by reference.

Peptide analogs are often used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptidomimetics". See faucher, 1986, adv.15：29；Veber&Freidinger, 1985, TINS, p.392; and Evans et al, 1987, J.Med.chem.30: 1229, which are incorporated herein by reference for any purpose. Such compounds are typically developed by means of computer molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or prophylactic effects. Generally, peptidomimetics are similar in structure to model polypeptides (i.e., polypeptides having certain biochemical or pharmaceutical activities), such as human antibodies, but have one or more peptide bonds optionally replaced by a bond selected from the group consisting of: -CH₂-NH-、-CH₂-S-、-CH₂-CH₂-, -CH-CH- (cis and trans) -, -COCH₂-、-CH(OH)CH₂-and-CH₂SO-. Systematic substitution of one or more amino acids of the consensus sequence with a D-amino acid of the same type (e.g., D-lysine for L-lysine) can be used in certain embodiments to produce more stable peptides. In addition, comprising a consensus sequenceOr substantially identical consensus variants, may be generated using methods well known in the art (Rizo)&Gierasch，1992，Ann.Rev.Biochem.61: 387, incorporated herein by reference for any purpose); for example, by adding an internal cysteine residue capable of cyclizing the peptide to form an intramolecular disulfide bridge.

"antibody" or "antibody peptide" refers to an intact antibody or a binding fragment that competes for specific binding with an intact antibody. In certain embodiments, the binding fragments are produced by recombinant DNA techniques. In additional embodiments, the binding fragments are generated by enzymatic or chemical cleavage of an intact antibody. Binding fragments include, but are not limited to, F (ab), F (ab')₂Fv, and single chain antibodies.

The term "heavy chain" includes any immunoglobulin polypeptide having sufficient variable region sequence to confer NGF specificity. The term "light chain" includes any immunoglobulin polypeptide having sufficient variable region sequence to confer NGF specificity. The full-length heavy chain comprises a variable region domain V_HAnd three constant region domains C_H1、C_H2 and C_H3。V_HThe Domain is located at the amino terminus of the polypeptide, C_HThe 3 domain is located at the carboxy terminus. The term "heavy chain" as used herein encompasses full-length heavy chains and fragments thereof. The full-length light chain comprises a variable region domain V_LAnd a constant region domain C_L. Like the heavy chain, the variable region domain of the light chain is located at the amino terminus of the polypeptide. The term "light chain" as used herein encompasses full-length light chains and fragments thereof. F (ab) fragment consisting of C of one light chain and one heavy chain_H1 and variable regions. The heavy chain of the f (ab) molecule is unable to form a disulfide bond with another heavy chain molecule. The F (ab') fragment contains a light chain and a heavy chain, the heavy chain containing C_H1 and C_H2 domain such that an interchain disulfide bond is formed between the two heavy chains, forming F (ab')₂A molecule. The Fv region comprises variable regions derived from the heavy and light chains, but lacks the constant regions. Single chain antibodies are antibodies in which the heavy and light chain variable regions have been flexibly linkedThe groups are linked to form an Fv molecule of a single polypeptide chain that forms the antibody binding region. Single chain antibodies are described in more detail in International patent application publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies are understood to comprise binding sites having the same antigenic specificity.

In assessing antibody binding and specificity according to the invention, an antibody substantially inhibits adsorption of a ligand to a receptor when an excess of antibody reduces the amount of ligand bound to the receptor by at least about 20%, 40%, 60%, 80%, 85% or more (particularly using an in vitro competitive binding assay).

"neutralizing antibody" refers to an antibody molecule that is capable of blocking or substantially reducing the effector function of a target antigen to which it binds. Thus, a "neutralizing" anti-NGF antibody can block or substantially reduce NGF effector functions, such as receptor binding and/or initiation of a cellular response. By "substantially reduce" is meant reducing effector function of a target antigen (e.g., human NGF) by at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, yet more preferably at least about 85%, and most preferably at least about 90%.

The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of the molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of an antigen that can be bound by an antibody. In certain embodiments, an antibody can be said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In a preferred embodiment, when the equilibrium dissociation constant is ≦ 10^-8M, more preferably when the equilibrium dissociation constant is 10 or less^-9M, most preferably when balancedDissociation constant less than or equal to 10^-10M, the antibody can be said to specifically bind to the antigen.

An antibody binds "substantially the same epitope" as a reference antibody when both antibodies recognize the same epitope or spatially overlapping epitopes. The most common and rapid method to determine whether two antibodies bind to the same epitope or spatially overlapping epitopes is a competitive assay, which uses either a labeled antigen or a labeled antibody, that can be assembled in all numbers of different formats. Typically, the antigen is immobilized on a matrix and the ability of the unlabeled antibody to block the binding of the labeled antibody is determined using a radiolabel or an enzymatic label.

The term "substrate" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from a biological material.

The term "label" or "labeled" as used herein refers to the incorporation of a detectable label, for example by incorporating a radiolabeled amino acid or attaching a biotin moiety to a polypeptide which is detectable by a labeled avidin (e.g., streptavidin, preferably comprising a detectable label such as a fluorescent label, chemiluminescent label, or enzymatic activity detectable by optical or colorimetric methods). In certain embodiments, the marker is also therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be conveniently used in the methods disclosed herein. Examples of polypeptide tags include, but are not limited to, the following: radioisotopes or radionuclides (e.g. of the type³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、^99mTc、¹¹¹In、¹²⁵I、¹³¹I) Fluorescent labels (e.g. fluorescein isothiocyanate or FITC, rhodamine or lanthanide phosphates)), enzymatic labels (e.g. horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, hapten labels such as a biotin group and predetermined polypeptide epitopes recognized by a second reporter molecule (e.g. leucine zipper pair sequence, binding site for a second antibody, metal binding domain or epitope tag)In some embodiments, the label is passed through spacer arms of various lengths (e.g., (CH)₂)_nWhere n < about 20) to reduce potential steric hindrance.

The term "biological sample" as used herein includes, but is not limited to, any number of substances from a living organism or a past living organism. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Including, but not limited to, blood, serum, urine, cells, organs, tissues, bone marrow, lymph nodes, and skin.

The term "drug" as used herein refers to a chemical compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a patient. The expression "therapeutically effective amount" in relation to a pharmaceutical composition comprising one or more antibodies of the invention is to be understood as meaning, according to the invention, an amount of said pharmaceutical composition which is capable of eliminating, in a patient in need thereof, a decrease in the sensitivity threshold to external stimuli, bringing this sensitivity threshold back to a level comparable to the threshold observed in healthy subjects.

A "disorder" is any disease that can benefit from treatment according to the present invention. "disorder" and "disease" are used interchangeably herein and include chronic and acute NGF-mediated disorders or NGF-mediated diseases, including pathological conditions that predispose a mammal to the disorder in question.

The terms "NGF-mediated disease" and "NGF-mediated disorder" encompass any medical disease or disorder associated with elevated levels of NGF or increased sensitivity to NGF, including, but not limited to, acute pain, dental pain, pain resulting from trauma, surgical pain, pain resulting from amputation or abscess, causalgia, demyelinating disease, trigeminal neuralgia, cancer, chronic alcoholism, stroke, thalamic syndrome, diabetes, acquired immunodeficiency syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster headache, mixed vascular and non-vascular syndromes (mixed-vascular or non-vascular syndromes), tension headache, general inflammation, arthritis, rheumatism, lupus, osteoarthritis, inflammatory bowel disease, irritable bowel syndrome, inflammatory eye disease, inflammatory or unstable bladder dysfunction, psoriasis, skin discomfort due to inflammatory components, skin irritation, demyelinating diseases, and the like, Sunburn, myocarditis, dermatitis, myositis, neuritis, collagen vascular disease, chronic inflammatory disease, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, diabetic neuropathic pain, causalgia, sympathetically maintained pain, afferent nerve blocking syndrome, asthma, epithelial tissue injury or dysfunction, herpes simplex, visceral dysfunction in the respiratory, urogenital, gastrointestinal or vascular regions, trauma, burns, allergic skin reactions, pruritus, vitiligo, general gastrointestinal dysfunction, colitis, gastric ulcer, duodenal ulcer, vasomotor or allergic rhinitis or bronchopathy, dysmenorrhea, dyspepsia, gastroesophageal reflux, pancreatitis and visceral pain.

The terms "effective amount" and "therapeutically effective amount" as used herein, when used in reference to a vehicle or pharmaceutical composition comprising one or more human antibodies against human NGF, refer to an amount or dose sufficient to produce a desired effect (i.e., a desired effect such as a reduction in inflammation and/or pain for treatment with a vehicle or human antibody against human NGF of the present invention) or to facilitate an observable decrease in the level of one or more biological activities of NGF. More specifically, a therapeutically effective amount is an amount of human antibody against human NGF that is sufficient to inhibit, for a period of time, one or more clinically defined pathological processes associated with the disease in question (e.g., inflammation or pain) in a subject for in vivo treatment. In the present invention, an "effective amount" of an anti-human NGF antibody can prevent, stop, control or reduce the perception of pain associated with any painful medical condition. In the methods of the invention, the term "control" and grammatical variations thereof is used to refer to the prevention, partial or complete inhibition, reduction, delay or slowing of an unwanted event, such as pain. The effective amount may vary depending on the particular vehicle or human anti-human NGF antibody selected, and on various factors and conditions associated with the patient to be treated, as well as the severity of the condition. For example, if the vehicle or human antibody against human NGF is administered in vivo, factors to be considered are the age, weight and health of the patient, and dose-response curves and toxicity data obtained in preclinical animal testing, among others. If the agent is to be contacted with cells in vitro, multiple preclinical in vitro studies are also designed to assess parameters such as uptake, half-life, dose, toxicity, etc. Determination of an effective or therapeutically effective amount of a given agent is well within the ability of those skilled in the art.

The terms "nerve growth factor" and "NGF" as used herein are defined as the native sequence NGF of all mammalian species, including SEQ ID NO: 30, and recombinant human 1-120.

As used herein, "substantially pure" or "substantially purified" means a compound or species that is present as the predominant species (i.e., it is much more abundant on a molar basis than any other individual species in the composition). In certain embodiments, a substantially purified fraction is a composition in which certain species comprise at least about 50% (on a molar basis) of all macromolecular species present. In certain embodiments, the substantially pure composition comprises more than about 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition. In certain embodiments, the species is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

The term "patient" includes both human and animal subjects.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include subjects already suffering from the disorder, as well as subjects susceptible to or in need of prevention of the disorder.

Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

According to certain embodiments of the invention, NGF-directed antibodies may be used to treat neuropathic and inflammatory pain and NGF-mediated diseases, including but not limited to the pain and diseases described above.

One aspect of the present invention provides fully human monoclonal antibodies raised against human NGF and having biological and immunological specificity for binding to human NGF. In another aspect, the invention provides nucleic acids comprising nucleotide sequences encoding the amino acid sequences of heavy and light chain immunoglobulin molecules, particularly sequences corresponding to the variable regions thereof. Particular embodiments of this aspect of the invention are sequences corresponding to the Complementarity Determining Regions (CDRs), specifically the CDRs 1 through 3 of the heavy and light chains provided herein. In yet another aspect of the invention, hybridoma cells and cell lines are provided which express immunoglobulin molecules and antibodies, preferably monoclonal antibodies of the invention. The invention also provides biologically and immunologically purified preparations of antibodies, preferably monoclonal antibodies raised against human NGF and having biological and immunological specificity for binding human NGF.

The ability to clone and reconstitute megabase-sized human loci in Yeast Artificial Chromosomes (YACs) and introduce the loci into the mouse germline provides an advantageous method for elucidating the functional components of very large or roughly mapped loci and for generating useful models of human disease. In addition, the use of this technique of replacing mouse loci with human counterparts provides unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease initiation and development.

An important practical application of this strategy is the "humanization" of the mouse humoral immune system. Mice into which the endogenous Ig genes of the human immunoglobulin (Ig) locus have been inactivated provide an opportunity to study the underlying mechanisms of programmed expression and assembly of antibodies and the role of antibodies in B cell development. In addition, this strategy provides a source for the production of fully human monoclonal antibodies (MAbs).

The term "human antibody" includes antibodies having variable and constant regions that substantially correspond to human germline immunoglobulin sequences. In certain embodiments, human antibodies are produced in non-human mammals, including but not limited to rodents such as mice and rats, and lagomorphs such as rabbits. In certain embodiments, the human antibody is produced in a hybridoma cell. In certain embodiments, the human antibody is produced recombinantly.

The term "recombinant" in relation to an antibody includes an antibody produced, expressed, created or isolated by recombinant means. Illustrative examples include antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from libraries of recombinant combinatorial human antibodies, antibodies isolated from animals (e.g., mice) transfected with human immunoglobulin genes (see, e.g., Taylor, L.D., et al, Nucl. acids Res.20: 6287 6295, (1992)), or antibodies prepared, expressed, created, or isolated by any means involving splicing of human immunoglobulin gene sequences to other DNA sequences.

Human antibodies have at least three advantages when used in human therapy compared to non-human antibodies and chimeric antibodies:

1) since the effector portion of an antibody is a human effector, it may better interact with other parts of the human immune system (e.g., more efficiently destroy target cells through complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC));

2) the human immune system does not recognize human antibodies as foreign antibodies, and therefore the antibody response to such injected antibodies is less than that of fully foreign non-human antibodies or partially foreign chimeric antibodies;

3) injected non-human antibodies are reported to have a much shorter half-life in the human circulation than human antibodies. The half-life of an injected human antibody is substantially the same as that of a natural human antibody, so that a smaller dose and a smaller number of times can be administered.

Thus, it is expected that fully human antibodies will minimize the immunogenic and allergic reactions inherent in mouse mabs or mouse-derivatized mabs, thereby increasing the efficacy and safety of the administered antibodies. Thus, fully human antibodies of the invention are useful in the treatment of chronic and recurrent pain that requires repeated administration of the antibody. Thus, a particular advantage of the anti-NGF antibodies of the invention is that the antibodies are fully human and can be administered to a patient in a non-acute (non-acute) manner while minimizing adverse reactions that typically accompany human anti-mouse antibodies or other previously described non-fully human antibodies from non-human species.

One skilled in the art can engineer mouse strains deficient in mouse antibody production with large fragments of human Ig loci so that the mice produce no mouse antibodies but human antibodies. Large human Ig fragments can maintain large variable gene diversity and correct regulation of antibody production and expression. The repertoire of human antibody immune cells in these mouse strains produces antibodies with high affinity for any antigen of interest (including human antigens) by exploiting the mouse cellular mechanisms of antibody diversification and selection and the lack of immune tolerance to human proteins. Antigen-specific human MAbs with the desired specificity can be produced and selected using hybridoma technology.

Transgenic animals (e.g., mice) that are capable of producing a complete immune cell repertoire of human antibodies without producing endogenous immunoglobulins upon immunization can be used. Transfer of a human germline immunoglobulin gene array into such a germline mutant mouse results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90: 2551-, monoclone Antibodies and cancer therap, Alan r. loss, page 77 (1985) and Boerner et al, j.immunol., 147 (1): 86-95(1991)).

Recombinant human antibodies can also be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals transgenic for human Ig sequences), and thus the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, while derived from sequences related to human germline VH and VL sequences, may not naturally exist in the human antibody germline cell repertoire in vivo.

In certain embodiments, the skilled artisan can use constant regions from species other than human, together with human variable regions, in such mice to generate chimeric antibodies.

Structure of natural antibody

The structural units of a natural antibody typically comprise tetramers. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full length "light" (typically about 25kDa in molecular weight) and one full length "heavy" (typically about 50-75kDa in molecular weight). The amino-terminal portion of each of the light and heavy chains typically includes a variable region of about 100-110 or more amino acids typically responsible for antigen recognition. The carboxy terminus of each chain typically defines a constant region responsible for effector function. Human light chains are generally classified into kappa and lambda light chains. Heavy chains are generally classified as μ, γ, α or heavy chains and determine the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG 4. Subclasses of IgM include, but are not limited to, IgM1 and IgM 2. IgA can be similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA 2. Typically within full-length light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, e.g., fundamantalmunology, chapter 7, 2 nd edition, (Paul, w. editions), 1989, Raven Press, n.y. (which is incorporated herein by reference in its entirety for any purpose). The variable region of each light/heavy chain pair typically forms an antigen binding site.

The variable regions typically exhibit the same general structure, i.e., a relatively conserved Framework Region (FR) is linked to three hypervariable regions (also known as complementarity determining regions or CDRs). The CDRs of both chains of each pair are typically aligned by framework regions, which enable binding to a particular epitope. The variable regions of light and heavy chains typically comprise, from N-terminus to C-terminus, the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The amino acid arrangement of each domain is typically according to Kabat Sequences of Proteins of immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.) or Chothia&Lesk, 1987, j.mol.biol.196: 901-; chothia et al, 1989, Nature342: 878-883. Bispecific or bifunctional antibodies

Bispecific or bifunctional antibodies are typically artificial hybrid antibodies with two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including but not limited to fusion of hybridomas or ligation of F (ab') fragments. See, e.g., Songsivilai&Lachmann，1990，Clin.Exp.Immunol.79: 315- > 321; kostelny et al, 1992, J.Immunol148：1547-1553。

Preparation of antibodies

The present invention provides antibodies that bind human NGF. These antibodies can be produced by immunization with full-length NGF or a fragment thereof. The antibody of the invention may be a polyclonal antibody or a monoclonal antibody, and/or may be a recombinant antibody. In a preferred embodiment, the antibody of the invention is a human antibody, for example, prepared by immunizing a transgenic animal capable of producing human antibodies (see, for example, International patent application publication WO 93/12227).

The Complementarity Determining Regions (CDRs) of the light chain variable region and the heavy chain variable region of the anti-NGF antibody of the present invention may be grafted to the Framework Regions (FRs) of the same species or another species. In certain embodiments, the CDRs of the light and heavy chain variable regions of the anti-NGF antibody can be grafted to consensus human FRs. To create consensus human FRs, FRs in several human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. The FRs of the heavy or light chain of an anti-NGF antibody may be replaced with FRs from a different heavy or light chain. The rare amino acids in the FRs of the heavy and light chains of an anti-NGF antibody are typically not substituted, while the remaining FR amino acids may be substituted. Rare amino acids are special amino acids, which usually cannot find their position in the FR. The grafted variable regions of the anti-NGF antibodies of the invention may be used with constant regions that are different from the constant regions of the anti-NGF antibodies. Alternatively, the grafted variable region is part of a single chain Fv antibody and CDR grafting is described, for example, in U.S. Pat. nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference for any purpose.

The antibodies of the invention are preferably prepared using transgenic mice having antibody-producing cells into which a substantial portion of the human antibody-producing locus has been inserted and which are further engineered to be deficient in the production of endogenous murine antibodies. Such mice are capable of producing human immunoglobulin molecules and antibodies, with no or substantially reduced amounts of murine immunoglobulin molecules and antibodies. Techniques to achieve this result are disclosed in the patents, patent applications, and references disclosed in this specification. In a preferred embodiment, the skilled person may employ a method as disclosed in international patent application publication WO 98/24893, which is incorporated herein by reference for any purpose. See also Mendez et al, 1997, Nature Genetics15: 146-' 156, which is incorporated herein by reference for any purpose.

The monoclonal antibodies (mAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein (1975, Nature)256: 495). Although the somatic cell hybridization procedure is preferred, in principle other techniques for producing monoclonal antibodies, such as B-lymphocyte viruses or cancer, can also be usedAnd (4) gene transformation.

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

In a preferred embodiment, human monoclonal antibodies directed to NGF may be produced using transgenic mice carrying portions of the human immune system rather than the mouse immune system. These transgenic mice, referred to herein as "HuMab" mice, contain a human immunoglobulin gene minilocus encoding unrearranged human (mu and gamma) heavy and kappa light chain immunoglobulin sequences, and targeted mutations that inactivate endogenous u and kappa chain loci (Lonberg et al, 1994, Nature)368: 856-859). Thus, mice express reduced mouse IgM or kappa and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce a high affinity human IgG kappa monoclonal antibody (Lonberg et al, supra; Lonberg and Huszar, 1995, Intern.Rev.Immunol.13: 65-93; harding and Lonberg, 1995, ann.n.y.acad.sci.764: 536-546). HuMab mice were prepared in Taylor et al, 1992, Nucleic Acids Res.20: 6287-6295; chen et al, 1993, International Immunology5: 647-656; tuaillon et al, 1994, J.152: 2912-2920; lonberg et al, 1994, Nature368：856-859；Lonberg，1994，Handbook ofExp.Pharmacology113: 49-101; taylor et al, 1994, International Immunology6：579-591；Lonberg&Huszar，1995，Intern.Rev.Immunol.13：65-93；Harding&Lonberg，1995，Ann.N.Y. Acad.Sci764: 536-546; fishwild et al, 1996, Nature Biotechnology14: 845-851 are described in detail, and the contents of all of these documents are incorporated by reference herein in their entirety. See also U.S. Pat. nos. 5,545,806 to Lonberg and Kay; U.S. Pat. No. 5,569,825; 5,625,126 No; 5,633,425 No; 5,789,650 No; 5,877,397 No; 5,661,016 No; nos. 5,814,318; 5,874,299 th and 5,7 th70,429, and U.S. Pat. No. 5,545,807 to Surani et al; international patent application publication WO93/1227, 6/24/1993; WO 92/22646 published on 23.12.1992; and WO92/03918, published 3/19/1992, the disclosures of all of which are incorporated herein by reference in their entirety. Alternatively, the Hco7, Hco12, and KM transgenic mice described in the examples below can be used to generate human anti-NGF antibodies.

The present invention provides human monoclonal antibodies specific for and capable of neutralizing biologically active human NGF polypeptides. The invention also provides antibody heavy and light chain amino acid sequences that are highly specific for, and capable of neutralizing, NGF polypeptides when bound thereto. This high specificity makes human antibodies against human NGF and human monoclonal antibodies with similar specificity effective immunotherapies for NGF-related diseases.

In one aspect, the invention provides isolated human antibodies that bind the same or substantially the same epitope as the 4D4 antibodies provided herein.

In one aspect, the invention provides a polypeptide comprising at least SEQ ID NO: 10. 12, 14, 16, 18, 20, 22, 24, and 79-130, which antibody binds an epitope of an NGF polypeptide with high affinity and has the ability to antagonize an NGF polypeptide activity. Preferably, the epitope to which these antibodies bind is the same or substantially the same as the 4D4 antibody provided herein.

In a preferred embodiment, the human antibody is isolated as 1x10^-9Dissociation constant (K) of M or less_D) NGF-binding polypeptides and neutralizing assays in vitro at 1X10^-7IC of M or less₅₀Inhibits NGF-induced survival. In a more preferred embodiment, the human antibody is isolated as 1x10^-10Dissociation constant (K) of M or less_D) NGF polypeptide binding and neutralization assay at 1X10 in vitro^-8IC of M or less₅₀Inhibits NGF-induced survival. In an even more preferred embodiment, the anti-NGF human antibody is isolated as a 1x10^-11Dissociation constant (K) of M or less_D) Binds human NGF polypeptide, and in vivo1x10 in the external neutralization test^-9IC of M or less₅₀Inhibits NGF-induced survival. Examples of human antibodies against human NGF that meet the above binding and neutralization criteria are provided herein.

The most preferred anti-human NGF human antibodies of the invention are referred to herein as 4D4, having the amino acid sequence of seq id NO: 12 and SEQ ID NO: 10, respectively, a VL polypeptide sequence and a VH polypeptide sequence. The polynucleotide sequences encoding VL and VH of 4D4 are shown in seq id NOs: 1 and SEQ ID NO: shown at 9. The properties of the human antibodies of the invention against human NGF are explicitly disclosed in the examples. The high affinity for NGF polypeptides and the high ability to antagonize NGF polypeptide activity shown herein are particularly noteworthy.

Dissociation constant (K) of human antibodies against human NGF as generally described in example 9_D) The measurement can be performed by surface plasmon resonance. In general, surface plasmon resonance analysis measures the real-time binding interaction between a ligand (recombinant NGF polypeptide immobilized on a Biosensor matrix) and an analyte (antibody in solution) by Surface Plasmon Resonance (SPR) using the BIAcore system (Pharmacia Biosensor, Piscataway, NJ). Surface plasmon analysis is also performed by immobilizing the analyte (antibodies on the biosensor matrix) and presenting the ligand (recombinant V in solution). Dissociation constant (K) of human antibody against human NGF_D) This can also be determined by the KinExA method. In certain embodiments of the invention, the antibody is present at about 10^-8M-10^-12K of M_DBinds NGF. The term "K" as used herein_D"means the dissociation constant for a particular antibody-antigen interaction. For the purposes of the present invention, K_DThe assay was performed as described in example 9.

In preferred embodiments, the antibodies of the invention are of the IgG1, IgG2, IgG3 or IgG4 isotype. Preferably the antibody is of the IgG3 isotype. More preferably the antibody is of the IgG1 isotype. Most preferred are antibodies of the IgG2 isotype. In other embodiments, the antibodies of the invention are of the IgM, IgA, IgE or IgD isotype. In a preferred embodiment of the invention, the antibody comprises a human kappa light chain and a human IgG1, IgG2, IgG3 or IgG4 heavy chain. Expression of antibodies of the invention comprising an IgG1 or IgG2 heavy chain constant region is described in the examples below. In particular embodiments, the variable region of the antibody is linked to a constant region other than that of the IgG1, IgG2, IgG3, or IgG4 isotype. In certain embodiments, the antibodies of the invention have been cloned for expression in mammalian cells.

In certain embodiments, conservative modifications to the heavy and light chains of an anti-NGF antibody (and corresponding modifications to the encoding nucleotides) will result in an anti-NGF antibody having similar functional and chemical characteristics as the anti-NGF antibodies disclosed herein. In contrast, substantial modification of the functional and/or chemical characteristics of an anti-NGF antibody can be achieved by selecting substitutions in the amino acid sequences of the heavy and light chains that differ greatly in their roles in maintaining (a) the molecular backbone structure, e.g., the folded or helical conformation, at the site of substitution, (b) the molecular charge or hydrophobicity at the target site, or (c) the size of the side chain.

For example, a "conservative amino acid substitution" may involve the replacement of a natural amino acid residue with a non-natural amino acid residue, resulting in little or no effect on the polarity or charge of the amino acid residue at the replacement position. In addition, any natural residue in a polypeptide can also be replaced with alanine, as described above for "alanine scanning mutagenesis".

Desired amino acid substitutions, whether conservative or non-conservative, may be determined by one of skill in the art at the time such a substitution is desired. In certain embodiments, amino acid substitutions can be used to identify important residues of an anti-NGF antibody, or to increase or decrease the affinity of an anti-NGF antibody described herein.

It is well known that minor changes in the amino acid sequence, such as deletion, insertion or substitution of one, a few or even a few amino acids, can result in the appearance of allelic forms of the original protein that have essentially the same properties. Thus, in addition to the antibodies specifically described herein, other "substantially homologous" antibodies can be readily designed and prepared using a variety of recombinant DNA techniques well known to those skilled in the art. In general, modification of genes can be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis. Thus, the present invention contemplates "mutated" or "mutated" anti-NGF human antibodies having substantially similar characteristics as the anti-NGF human antibodies disclosed herein (see, e.g., WO00/56772, the entire contents of which are incorporated herein by reference). Thus, the term "variant" or "mutant" relating to an anti-NGF human antibody means any binding molecule (molecule X) (i) wherein the hypervariable regions of the heavy chain CDR1, CDR2 and CDR3 or of the light chain CDR1, CDR2 and CDR3 as a whole respectively have the amino acid sequence of SEQ ID NO: 14. 18 and 22 or SEQ ID NO: 16. 20 and 24, and (ii) wherein said variant or mutant is capable of inhibiting the activity of human NGF to the same extent as a reference anti-NGF human antibody having framework regions identical to those of molecule X.

Generally, the light and/or heavy chain CDRs of an anti-NGF human antibody variant as a whole will be identical to SEQ ID NO: 14. 18 and 22 and/or SEQ ID NOS: 16. 20 and 24 has at least about 80% amino acid sequence identity, preferably at least about 85% sequence identity, even more preferably at least about 90% sequence identity, yet more preferably at least about 91% sequence identity, yet more preferably at least about 92% sequence identity, yet more preferably at least about 93% sequence identity, yet more preferably at least about 94% sequence identity, yet more preferably at least about 95% sequence identity, yet more preferably at least about 96% sequence identity, yet more preferably at least about 97% sequence identity, yet more preferably at least about 98% sequence identity, and yet more preferably at least about 99% amino acid sequence identity.

More preferably, the light chain variable region of the anti-NGF human antibody variant as a whole will be identical to seq id NO: 12. 80, 82, 84, 86, 88, 89, 90, or 91, has at least about 80% amino acid sequence identity, more preferably at least about 81% sequence identity, even more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, even more preferably at least about 94% sequence identity, still more preferably at least about 95% sequence identity, still more preferably at least about 96% sequence identity, still more preferably at least about 97% sequence identity, still more preferably at least about 98% sequence identity, still more preferably at least about 99% amino acid sequence identity, and/or the heavy chain variable region as a whole will have a sequence identity to SEQ ID NO: 10. 81, 83, 85, or 87, having at least about 70% amino acid sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% sequence identity, even more preferably at least about 81% sequence identity, even more preferably at least about 82% sequence identity, even more preferably at least about 83% sequence identity, even more preferably at least about 84% sequence identity, even more preferably at least about 85% sequence identity, even more preferably at least about 86% sequence identity, even more preferably at least about 87% sequence identity, even more preferably at least about 88% sequence identity, even more preferably at least about 89% sequence identity, even more preferably at least about 90% sequence identity, even more preferably at least about 91% sequence identity, even more preferably at least about 92% sequence identity, even more preferably at least about 93% sequence identity, still more preferably at least about 94% sequence identity, still more preferably at least about 95% sequence identity, still more preferably at least about 96% sequence identity, still more preferably at least about 97% sequence identity, still more preferably at least about 98% sequence identity, and still more preferably at least about 99% amino acid sequence identity.

By "variant" with respect to a polynucleotide is meant a nucleic acid molecule having at least about 75% nucleic acid sequence identity to a polynucleotide sequence of the present invention. Generally, a variant of a polynucleotide will have at least about 75% nucleic acid sequence identity, more preferably at least about 80% nucleic acid sequence identity, still more preferably at least about 81% nucleic acid sequence identity, still more preferably at least about 82% nucleic acid sequence identity, still more preferably at least about 83% nucleic acid sequence identity, still more preferably at least about 84% nucleic acid sequence identity, still more preferably at least about 85% nucleic acid sequence identity, still more preferably at least about 86% nucleic acid sequence identity, still more preferably at least about 87% nucleic acid sequence identity, still more preferably at least about 88% nucleic acid sequence identity, still more preferably at least about 89% nucleic acid sequence identity, still more preferably at least about 90% nucleic acid sequence identity, still more preferably at least about 91% nucleic acid sequence identity, still more preferably at least about 92% nucleic acid sequence identity to the novel nucleic acid sequences disclosed herein, still more preferably at least about 93% nucleic acid sequence identity, still more preferably at least about 94% nucleic acid sequence identity, still more preferably at least about 95% nucleic acid sequence identity, still more preferably at least about 96% nucleic acid sequence identity, still more preferably at least about 97% nucleic acid sequence identity, still more preferably at least about 98% nucleic acid sequence identity, still more preferably at least about 99% nucleic acid sequence identity.

In particular embodiments, the invention provides an antibody that has a portion of identity to an antibody of the invention, or an antibody that comprises a heavy chain variable region, light chain variable region, CDR1, CDR2, or CDR3 region that has a portion of identity to a heavy chain variable region, light chain variable region, CDR1, CDR2, or CDR3 region of the invention as set forth in example 10 herein and in fig. 5-10.

In certain embodiments, the present invention provides an isolated human antibody that specifically binds nerve growth factor comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising the amino acid sequence: which is similar to SEQ ID NO: 10 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 75% identity; and SEQ ID NO: 81 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof having at least 70%, 80%, 85% or 95% homology; and SEQ ID NO: 83 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70%, 80%, 85%, or 95% identical; and SEQ ID NO: 85 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 80%, or 85% identity; and SEQ ID NO: 87 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, wherein the amino acid sequence represented by 87 is at least 70%, 75% or 80% identical; and SEQ ID NO: 79 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof has at least 56% identity.

In certain embodiments, the present invention provides an isolated human antibody that specifically binds nerve growth factor comprising a heavy chain and a light chain, wherein the light chain comprises a light chain variable region comprising the amino acid sequence: which is similar to SEQ ID NO: 12 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 70%, 75%, 80% or 90% identical; and SEQ ID NO: 80 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 85%, or 90% identity; and SEQ ID NO: 88 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 74%, 90%, or 94% identity; and SEQ ID NO: 89, or an antigen-binding fragment thereof, or an immunologically functional immunoglobulin fragment thereof, is at least 70%, 80%, 85%, or 87% identical; and SEQ ID NO: 90 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70%, 85%, 90%, or 94% identical; and SEQ ID NO: 91 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 85%, 90%, 95%, or 99% identity; and SEQ ID NO: 82 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 80%, 90%, 95%, or 96% identity; and SEQ ID NO: 84 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 70%, 85%, 90%, 95%, 98%, or 99% identical; or to SEQ ID NO: 86 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 70%, 85%, 90%, 95%, 98% or 99% identical.

In certain other embodiments, the invention provides an isolated human antibody that specifically binds nerve growth factor comprising a human heavy chain CDR1, wherein the heavy chain CDR1 is an amino acid sequence that hybridizes to SEQ ID NO: 98. SEQ ID NO: 105. SEQ ID NO: 110 or SEQ ID NO: 22 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof has at least 40% or 60% identity.

In other embodiments, the invention provides an isolated human antibody that specifically binds nerve growth factor comprising a human heavy chain CDR2, wherein the heavy chain CDR2 is the amino acid sequence: which is similar to SEQ ID NO: 99 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70%, 82%, or 94% identity; and SEQ ID NO: 106 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70% or 76% identity; and SEQ ID NO: 18 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 59% identical; and SEQ ID NO: 117 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70% identical; and SEQ ID NO: 111 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof has at least 70%, 75% or 80% identity.

In still other embodiments, the invention provides an isolated human antibody that specifically binds nerve growth factor comprising a human light chain CDR1, wherein the CDR1 is the amino acid sequence of: which is similar to SEQ ID NO: 101 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70% or 80% identity; and SEQ ID NO: 95 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70%, 75%, 80%, or 90% identity; and SEQ ID NO: 119 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 75%, 80% or 90% identity; and SEQ ID NO: 122 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 75%, 80%, or 90% identical; and SEQ ID NO: 125 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 80% identical; and SEQ ID NO: 24 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof that is at least 75%, 80%, or 90% identical; and SEQ ID NO: 107 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 80% identity; or to SEQ ID NO: 113 or an antigen-binding fragment or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 80% identity.

In additional embodiments, the invention provides an isolated human antibody that specifically binds nerve growth factor comprising a human light chain CDR2, wherein the CDR2 is the amino acid sequence of: which is similar to SEQ ID NO: 102 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 96 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 70% identical; and SEQ ID NO: 120 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70% identical; and SEQ ID NO: 123 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70% identical; and SEQ ID NO: 126 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 129 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 20 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 70% identical; and SEQ ID NO: 108 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 133 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% identity; or with SEQ ID NO: 114 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, is at least 70% or 85% identical.

In other embodiments, the invention provides an isolated human antibody that specifically binds nerve growth factor comprising a human light chain CDR3, wherein the CDR3 is the amino acid sequence of: which is similar to SEQ ID NO: 103 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, with at least 70% or 85% identity; and SEQ ID NO: 97 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 121 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof has at least 70% or 78% identity; and SEQ ID NO: 127 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, wherein the amino acid sequence is at least 70% or 78% identical; and SEQ ID NO: 130 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 78% identity; and SEQ ID NO: 16 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 78% identity; and SEQ ID NO: 109 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof, having at least 70% or 85% identity; and SEQ ID NO: 134 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 78% identical; or to SEQ ID NO: 115 or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof is at least 85% identical.

The sequences of the heavy chain variable region and the light chain variable region of the 4D4 antibody are set forth in SEQ ID NOs: 10 and 12. However, many potential CDR-contacting residues are susceptible to substitution by other amino acids and still allow the antibody to retain comparable affinity for the antigen. Likewise, many framework residues not in contact with the CDRs in the heavy and light chains can accommodate replacement of the amino acid at the corresponding position with a human consensus amino acid from other human antibodies or an amino acid from other mouse antibodies without significant loss of affinity or non-immunogenicity of the human antibody. Various additional amino acids can be selected to produce various forms of the anti-NGF antibodies and fragments thereof disclosed herein, with varying combinations of affinity, specificity, non-immunogenicity, ease of preparation, and other desirable properties.

In another embodiment, the antibodies of the invention may be expressed in a cell line other than a hybridoma cell line. In these embodiments, sequences encoding particular antibodies may be used to transform a suitable mammalian host cell. According to these embodiments, transformation can be accomplished by any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide in a virus (or into a viral vector) and transducing the host cell with the virus (or vector), or by transfection methods well known in the art, such as those exemplified in U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (all of which are incorporated herein by reference for any purpose). In general, the transformation method used may depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, and direct microinjection of the DNA into the nucleus.

Nucleic acid molecules encoding the amino acid sequence of the heavy chain constant region, heavy chain variable region, light chain constant region, or light chain variable region of an NGF antibody of the invention are inserted into an appropriate expression vector using standard ligation techniquesIn (1). In a preferred embodiment, the anti-NGF antibody heavy or light chain constant region is appended to the C-terminus of the appropriate variable region and ligated into an expression vector. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host machinery such that amplification of the gene and/or expression of the gene can occur). For a review of expression vectors see meth.185(edited by Goeddel), 1990, Academic Press.

In general, an expression vector for use in any host cell will contain sequences for maintaining the plasmid and cloning and expression of the exogenous nucleotide sequence. Such sequences, collectively referred to as "flanking sequences" in certain embodiments, typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a polypeptide secretion leader sequence, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a "marker" coding sequence, i.e., an oligonucleotide molecule located at the 5 'or 3' end of the anti-NGF antibody polypeptide coding sequence; the oligonucleotide sequence encodes a polyhistidine (e.g., hexa-histidine) or another "tag" such as FLAG, HA (hemagglutinin influenza virus) or myc (for which there are commercially available antibodies). Such a tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification and detection of NGF antibodies in the host cell. Affinity purification can be achieved by column chromatography, for example, using antibodies against the label as an affinity matrix. Optionally, the label may then be removed from the purified anti-NGF antibody polypeptide by various means, such as using certain peptidases for cleavage.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in and capable of being activated by the host cell machinery.

The flanking sequences for use in the vectors of the present invention may be obtained by any of several methods well known in the art. Typically, the flanking sequences used herein have been previously identified by mapping and/or restriction endonuclease digestion and may therefore be isolated from a suitable tissue source using an appropriate restriction endonuclease. In some cases, the complete nucleotide sequence of the flanking sequences may be known. In this regard, the flanking sequences may be synthesized using the nucleic acid synthesis or cloning methods described herein.

Whether all or only a portion of the flanking sequences are known, they may be obtained by using the Polymerase Chain Reaction (PCR) and/or by screening a genomic library with suitable probes, such as oligonucleotides and/or fragments of the flanking sequences from the same or another species. In the case where the flanking sequences are not known, the DNA segment containing the flanking sequences may be isolated from a larger piece of DNA which may contain, for example, the coding sequence or even another gene or genes. The separation can be achieved as follows: digesting with restriction endonuclease to produce suitable DNA fragment, purifying with agarose gel,Separation is carried out by column chromatography (Chatsworth, CA) or other methods known to the skilled person. The selection of suitable enzymes to achieve this is readily apparent to those of ordinary skill in the art.

The origin of replication is usually part of a commercially available prokaryotic expression vector, which facilitates the amplification of the vector in a host cell. If the vector selected does not contain an origin of replication site, a site can be chemically synthesized from a known sequence and ligated into the vector. For example, the origin of replication of the plasmid pBR322(New England Biolabs, Beverly, Mass.) is suitable for most gram-negative bacteria, and various viral origins (e.g., the origin of SV40, polyoma, adenovirus, Vesicular Stomatitis Virus (VSV) or papillomavirus HPV or BPV) can be used to clone vectors in mammalian cells. In general, the origin of replication component is not essential for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains a viral early promoter).

Transcription termination sequences are typically located 3' to the end of the polypeptide coding region to terminate transcription. In general, the transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. Although this sequence can be readily cloned from a library, or even commercially available as part of a vector, it can also be readily synthesized using the nucleic acid synthesis methods described herein.

Selectable marker genes encode proteins that are essential for the survival and production of host cells grown in selective media. Typical selectable marker genes encode proteins that function as follows: (a) conferring resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin) to prokaryotic host cells; (b) complement the auxotrophy of the cell; or (c) supply critical nutrients not available from synthetic or defined media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The neomycin resistance gene may also be advantageously used in the selection of prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene to be expressed. Amplification is a process in which genes required for the production of proteins critical to cell growth or survival are repeated in tandem in the chromosomes of successive generations of recombinant cells. Examples of suitable mammalian selectable markers include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. Mammalian cell transformants are placed under selection pressure, wherein only transformants can survive exclusively on the selection gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selection factor in the medium is successively increased, resulting in the simultaneous amplification of the selective gene and the DNA encoding another gene product, such as an antibody that binds to an NGF polypeptide. As a result, the amount of a polypeptide synthesized from the amplified DNA, such as an anti-NGF antibody, increases.

Ribosome binding sites are usually required for translation initiation of mRNA and are characterized by Shine-Dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). The site element is typically located 3 'to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, various pre-sequences can be manipulated to enhance glycosylation or increase yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or a pre-sequence added, which can also affect glycosylation. The final protein product may have one or more additional amino acids at the-1 position (relative to the first amino acid of the mature protein) that are produced upon expression and that have not been completely removed. For example, the final protein product may have one or more amino acid residues found at the peptidase cleavage site attached to the amino terminus. Alternatively, the use of certain enzymatic cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cleaves in such a region within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and is operably linked to a molecule encoding an anti-NGF antibody. Promoters are non-transcribed sequences (typically within about 100 to 1000 bp) located upstream (i.e., 5') to the start codon of a structural gene that control transcription of the structural gene. Promoters can generally fall into one of two categories: inducible promoters and constitutive promoters. Inducible promoters trigger an increase in the level of transcription of DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature. Constitutive promoters, on the other hand, transcribe all their operably linked genes uniformly, i.e., with little or no control over gene expression. Many promoters are known which are recognized by a variety of potential host cells. Suitable promoters are operably linked to DNA encoding an anti-NGF antibody (comprising either a heavy chain or a light chain) of the invention as follows: the promoter is removed from the source DNA by digestion with restriction enzymes and the desired promoter sequence is inserted into the vector.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers may be advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and most preferably monkey virus 40(SV 40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Additional promoters that may be of interest include, but are not limited to: SV40 early promoter (Bernoist and Chambon, 1981, Nature)290: 304-10); CMV promoter (Thomsen et al, 1984, Proc. Natl. Acad. USA)81: 659-; promoters contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell)22: 787-97); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Nad. Acad. Sci. U.S.A).78: 1444-45); promoters and regulatory sequences in the metallothionein Gene (Brinster et al, 1982, Nature)29639-42), and prokaryotic promoters such as β -lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl.Acad. Sci.U.S.A.,75: 3727-31); or the tac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. u. s. A.,80: 21-25). Also of importance are the following animal transcriptional control regions that exhibit tissue specificity and have been applied to transgenic animals: elastase I gene control region active in pancreatic acinar cells (Swift et al, 1984, Cell)38: 639-46; ornitz et al, 1986, Cold spring harbor Symp. Quant. biol.50：399-409(1986)；MacDonald，1987，Hepatology7425-515) insulin gene control region active in pancreatic β cells (Hanahan, 1985, Nature)315: 115-22); immunoglobulin gene control region active in lymphoid cells (Grosschedl et al, 1984, Cell)38: 647-58; adames et al, 1985, Nature318: 533-38; alexander et al, 1987, mol.cell.biol.,7: 1436-44); mouse mammary cancer virus control region active in testicular, mammary, lymphoid and mast cells (Leder et al, 1986, Cell)45: 485-95); the albumin gene control region is active in the liver (Pinkert et al, 1987, Genes and Devel.1: 268-76); the alpha-fetoprotein gene control region that is active in the liver (Krumlauf et al, 1985, mol. cell. biol.,5: 1639-48; hammer et al, 1987, Science23553-58), α 1-antitrypsin gene control region active in the liver (Kelsey et al, 1987, Genes and Devel.1161-71), β globin gene control region active in myeloid cells (Mogram et al, 1985, Nature)315: 338-40; kollias et al, 1986, Cell46: 89-94); myelin basic protein gene control region active in oligodendrocytes in the brain (Readhead et al, 1987, Cell)48: 703-12); myosin light chain-2 Gene control region active in skeletal muscle (Sani, 1985, Nature)314: 283-86); and the gonadotropin-releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science)234：1372-78)。

Enhancer sequences may be inserted into the vector to enhance transcription of the DNA encoding the anti-NGF antibody (including light or heavy chains) of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually 10-300bp in length, that act on a promoter to increase transcription. Enhancers are relatively orientation and position independent, and have been found in the 5 'and 3' positions of transcription units. Several enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and the adenovirus enhancer, which are well known in the art, are exemplary enhancer elements used to activate eukaryotic promoters. While the enhancer may be located 5 ' and 3 ' to the coding sequence in the vector, it is usually located 5 ' to the promoter.

The expression vectors of the present invention can be constructed from starting vectors such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. In the case where one or more of the flanking sequences described herein are no longer present in the vector, the flanking sequences may be obtained separately and ligated into the vector. Methods for obtaining each of the flanking sequences are well known to those skilled in the art.

After the vector has been constructed and the nucleic acid molecules encoding the light, heavy or both light and heavy chains that make up the anti-NGF antibody have been inserted into the correct location in the vector, the completed vector can be inserted into a suitable host cell for amplification and/or polypeptide expression. The expression vector for the anti-NGF antibody can be transformed into the selected host cell by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection or other known techniques. The method of choice is often determined in part by the type of host cell to be used. These and other suitable methods are well known to the skilled artisan and are set forth, for example, in Sambrook et al, supra.

When cultured under appropriate conditions, the host cells synthesize anti-NGF antibodies, which can then be collected from the culture medium (if the host cells secrete the antibodies into the culture medium) or directly from the host cells producing the antibodies (if the antibodies are not secreted). The choice of an appropriate host cell will often depend on various factors such as the desired expression level, the polypeptide modifications required or necessary for activity (e.g., glycosylation and phosphorylation), and the ease of folding into a biologically active molecule.

Mammalian cell lines useful as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Center (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a variety of other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines have high expression levels and are capable of constitutively producing antibodies having NGF binding properties. In another embodiment, a cell line of B cell lineage that does not produce autoantibodies, but has the ability to produce and secrete heterologous antibodies, may be selected.

The antibodies of the invention are useful for detecting NGF in a biological sample and for identifying cells or tissues capable of producing NGF protein. Antibodies of the invention that specifically bind NGF may be used in the treatment of NGF-mediated diseases. The antibodies can be used in binding assays to detect NGF and inhibit the formation of a complex of NGF with NGF receptors. Such antibodies that bind NGF and block interaction with other binding compounds may have therapeutic utility in modulating NGF-mediated diseases. In preferred embodiments, anti-NGF antibodies can block NGF binding to its receptor, which may result in disruption of the NGF-induced signaling cascade.

The invention also relates to the use of one or more antibodies of the invention in the manufacture of a medicament for treating a pain disorder or disease (e.g., any of the disorders or diseases disclosed herein) in a patient caused by increased expression of NGF or increased sensitivity to NGF.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies of the invention and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. Preferably, acceptable formulation materials are non-toxic to recipients at the dosages and concentrations employed. In a preferred embodiment, a pharmaceutical composition comprising a therapeutically effective amount of an anti-NGF antibody is provided.

In certain embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed.

In certain embodiments, the pharmaceutical composition may contain formulation materials to adjust, maintain or maintain, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration rate of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, sodium bisulfite); buffering agents (such as borate, bicarbonate, Tris-HCl, citrate, phosphate or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; a disaccharide; and other carbohydrates (e.g., glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; an emulsifier; hydrophilic polymers (such as polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., poloxamers, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); a transmission medium; a diluent; excipients and/or pharmaceutical adjuvants. See REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition (edited by A.R. Gennaro), 1990, Mack Publishing Company.

In certain embodiments, optimal pharmaceutical compositions are often determined by one of skill in the art based on, for example, the intended route of administration, delivery form, and desired dosage. See, e.g., REMINGTON' S pharmaceutical sciences, supra. In certain embodiments, such compositions may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibodies of the invention.

In certain embodiments, the primary carrier or vehicle in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable carrier or vehicle may be water for injection, a physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly used in parenterally administered compositions. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. In a preferred embodiment, the pharmaceutical composition of the present invention comprises a Tris buffer at a pH of about 7.0-8.5, or an acetate buffer at a pH of about 4.0-5.5, and may further comprise sorbitol, sucrose, Tween-20 and/or suitable substitutes thereof. In certain embodiments of the invention, anti-NGF antibody compositions may be prepared for storage as follows: the selected composition of the desired purity is mixed with an optional formulation (REMINGTON's dental SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution. Furthermore, in certain embodiments, the anti-NGF antibody product can be formulated as a lyophilizate with a suitable excipient, such as sucrose.

The pharmaceutical compositions of the present invention may be selected for parenteral delivery. In addition, the compositions may also be selected for inhalation, or for delivery through the digestive tract, such as oral delivery. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation ingredients are preferably present at concentrations acceptable to the site of administration. In certain embodiments, the composition is maintained at physiological pH or a slightly lower pH, typically in the pH range of about 5 to about 8, with a buffering agent.

When parenteral administration is contemplated, the therapeutic compositions for use in the present invention may be provided in the form of pyrogen-free, parenterally acceptable aqueous solutions comprising the desired anti-NGF antibody in combination with a pharmaceutically acceptable carrier. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the anti-NGF antibody is formulated as a sterile isotonic solution and suitably stored. In certain embodiments, the formulation may involve formulating the desired molecule with a medium that provides controlled or sustained release for the product (which can be delivered by injection of a long acting formulation), such as injectable microspheres, bioerodible particles, polymers (such as polylactic or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid may also be used which has the effect of increasing duration in the systemic circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antibody molecules.

The pharmaceutical compositions of the present invention may be formulated for inhalation. In these embodiments, the anti-NGF antibody can be conveniently formulated as an inhalable dry powder. In a preferred embodiment, an anti-NGF antibody inhalation solution may also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be atomized. Pulmonary administration and methods of formulation are further described in International patent application PCT/US94/001875, which is incorporated herein by reference, which describes pulmonary delivery of chemically modified proteins.

It is also contemplated that the formulation may be administered orally. anti-NGF antibodies administered in this manner may or may not be formulated with carriers commonly used to formulate solid dosage forms, such as tablets and capsules. In certain embodiments, the capsule can be designed to release the active portion of the formulation in the gastrointestinal tract at a point where bioavailability is maximized and degradation is minimized prior to systemic circulation. Additional mediators can be included to facilitate the uptake of anti-NGF antibodies. Diluents, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binding agents may also be used.

Preferably, pharmaceutical compositions of the invention are provided comprising an effective amount of one or more anti-NGF antibodies in admixture with non-toxic excipients suitable for the manufacture of tablets. The solution may be formulated into unit dosage forms by dissolving the tablets in sterile water or another suitable vehicle. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or a binder, such as starch, gelatin or acacia; or a lubricant such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving sustained or controlled release delivery of anti-NGF antibodies. Techniques for formulating a variety of other sustained or controlled release delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and long acting injections, are also well known to those skilled in the art. See, for example, international patent application PCT/US93/00829 (which is incorporated herein by reference) which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained release formulations may include a semipermeable polymeric matrix in the form of a shaped article, for example a film or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European patent application publication EP 058481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and ethyl-gamma-L-glutamate (Sidman et al, 1983, Biopolymers)22: 547-556), poly (2-hydroxyethyl methacrylate) (Langer et al, 1981, J.biomed.Mater.Res.15: 167-.12: 98-105), ethylene vinyl acetate (Langer et al, supra) or poly-D (-) -3-hydroxybutyric acid (European patent application publication EP133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, 1985, Proc. Natl. Acad. Sci. UNA82: 3688-; european patent application publication EP036,676; EP 088,046 and EP143,949, which are incorporated herein by reference.

Pharmaceutical compositions for in vivo administration are generally provided as sterile formulations. Sterilization may be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization can be performed in this manner either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Parenteral compositions are typically filled into containers having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition is formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored in a ready-to-use form or in a form that can be reconstituted prior to administration (e.g., lyophilized form).

The invention also provides kits for producing single dosage units. Each kit of the invention may contain a first container containing a dried protein and a second container containing an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

The effective amount of the anti-NGF antibody-containing pharmaceutical composition to be therapeutically applied depends on, for example, the therapeutic content and the objective. One skilled in the art will recognize that the appropriate dosage level for treatment will vary, in part, depending on the molecule delivered, the indication for which the anti-NGF antibody is being used, the route of administration, and the size (body weight, body surface area or organ size) and/or state (age and general health) of the patient. In certain embodiments, the clinician may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Depending on the factors described above, typical dosages may range from about 0.1 μ g/kg up to about 30mg/kg or more. In a preferred embodiment, the dose may range from 0.1 μ g/kg to about 30 mg/kg; more preferably in the range of 1 μ g/kg to about 30 mg/kg; or even more preferably in the range of 5 μ g/kg to about 30 mg/kg.

The frequency of dosing often depends on the pharmacokinetic parameters of the particular anti-NGF antibody in the formulation used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or in two or more doses (which may or may not contain the same amount of the desired molecule) over time, or by continuous infusion through an implanted device or cannula. Further modifications of appropriate dosages can be routinely made by those of ordinary skill in the art, within the scope of their routine performance tasks. Appropriate dosages can be determined by using appropriate dose-response data. In certain embodiments, the antibodies of the invention can be administered to a patient for an extended period of time. Chronic administration of the antibodies of the invention minimizes adverse immune or allergic reactions that are typically associated with antibodies raised against human antigens in non-human animals, such as non-fully human antibodies produced in non-human species.

The route of administration of the pharmaceutical composition is in accordance with known methods, e.g., oral administration; administration by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal (intraportal) or intralesional (intradivision) routes; administration is by a slow release system or by an implanted device. In certain embodiments, the composition may be administered by bolus injection or continuous infusion, or by an implanted device.

The compositions may also be administered topically by implanting a film, sponge, or other suitable material that has absorbed or encapsulated the desired molecule. In certain embodiments using an implant device, the implant device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be by diffusion, timed release bolus, or continuous administration.

It is also desirable to use the anti-NGF antibody pharmaceutical compositions of the present invention in vitro. In this case, cells, tissues or organs removed from the patient are exposed to the anti-NGF antibody pharmaceutical composition, and then the cells, tissues and/or organs are transplanted back into the patient.

Specifically, anti-NGF antibodies can be delivered by implanting cells genetically engineered with the methods described herein to express and secrete the polypeptide. In certain embodiments, such cells may be animal cells or human cells, and may be autologous cells, heterologous cells, or xenogeneic cells. In certain embodiments, the cell may be an immortalized cell. In other embodiments, to reduce the likelihood of an immune response, cells may be encapsulated to avoid infiltration of surrounding tissue. In other embodiments, the encapsulating material is typically a biocompatible, semi-permeable polymeric wrap or film that allows the release of the protein product, but prevents the cells from being damaged by the patient's immune system or by other deleterious factors from the surrounding tissue.

Examples

The following examples, including experiments conducted and results obtained, are provided for illustrative purposes only and are not to be construed as limiting the invention.

Example 1

Production of human NGF protein from E.coli cells

Cloning of rHu-NGF (1-120)

Using a nucleic acid having SEQ ID NO: 27 and SEQ ID NO: 28 and standard PCR techniques to amplify from cDNA a nucleotide sequence encoding human NGF. The 5' primer generated an NdeI restriction site and a methionine start codon immediately preceding codon 1 (serine) of the mature sequence. The 3' primer creates a BamHI restriction site immediately after the stop codon. The resulting PCR product was gel-purified, digested with the restriction endonucleases NdeI and BamHI and then ligated into the vector pCFM1656, which was also digested with NdeI and BamHI. The ligated DNA was transformed into E.coli 657 strain competent host cells. Clones were screened for their ability to produce recombinant protein products and to possess plasmids carrying the correct nucleotide sequence (i.e., SEQ ID NO: 29). The amino acid sequence of the recombinant human NGF 1-120 is shown as SEQ ID NO: shown at 30.

Expression vector pCFM1656(ATCC #69576) was derived from the expression vector system described in U.S. Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the pCFM836 plasmid (patent No. 4,710,473) as described below: (a) disruption by end filling with T4 polymerase followed by blunt end ligationTwo endogenous NdeI restriction sites; (b) replacement of synthetic P-containing region between unique AatII and ClaI restriction sites with a similar fragment containing the PL promoter from pCFM636 (patent No. 4,710,473)_LThe DNA sequence of the promoter; then (c) converting the amino acid sequence of SEQ ID NO: 31 and SEQ ID NO: 32, replacing the small DNA sequence between the unique ClaI and KpnI restriction sites.

The E.coli K12 host strain (Amgen 657 strain) is a derivative of E.coli W1485 (strain K12) obtained from the E.coli genetic Stock Center (E.coli University, Yale University, New Haven, CT (CGSC6159 strain).

Expression of rHu-NGF (1-120)

Coli cells containing NGF expression constructs (as described above) were fermented in a fed-batch mode in rich medium. Cells were grown at 30 ℃ to an OD (at 600 nm) of 49 and then induced by temperature shift to 42 ℃. Cells were harvested by centrifugation 4 hours after induction. The final OD was 75. The expression yield was determined to be about 0.15 g/L.

refolding and purification of rHu-NGF (1-120)

The cell paste was lysed in a Microfluidizer (Microfluidizer), centrifuged at 10,000X g for 30 minutes, the pellet washed with 1% deoxycholic acid, centrifuged as above, and the resulting pellet washed with cold water and centrifuged again. The resulting pellet (WIB-washed inclusion bodies) was resuspended in the denaturant 8M guanidine hydrochloride, 50mM Tris pH 8.5 (containing 10mM DTT), solubilized at room temperature for 1 hour, centrifuged at 10,000X g for 30 minutes, the supernatant carefully decanted, and then diluted 25-fold in an aqueous buffer containing redox pairs at 4 ℃ for 5 days. The resulting refolded material was then titrated to pH 3.0 and filtered through a 0.45uM filter. The refolded material was purified with a standard NaCl gradient using an Sp-Sepharose fast flow column. The pool from the cation exchange column was then concentrated and aliquots were frozen at-80 ℃. The purity of the protein was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie blue staining. The purified protein obtained by this method is more than 90% of the main band.

Example 2

Production of human monoclonal antibodies to Nerve Growth Factor (NGF)

Transgenic HuMab and KM mice

Transgenic mouse HCo7, HCo12, HCo7+ HCo12, and KM strains, each expressing human antibody genes, were used to prepare fully human monoclonal antibodies against NGF. In each of these mouse strains, the endogenous mouse kappa light chain gene has been described by Chen et al (1993, EMBO J.12: 811-820), and the endogenous mouse heavy chain gene has been homozygously disrupted as described in example 1 of international patent application publication WO01/09187 (incorporated herein by reference). Each of these mouse strains carries, for example, Fishwild et al (1996, Nature Biotechnology)14: 845-. The HCo7 strain carries the HCo7 human heavy chain transgene as described in U.S. Pat. nos. 5,545,806, 5,625,825 and 5,545,807 (incorporated herein by reference). The HCo12 strain carries the HCo12 human heavy chain transgene as described in example 2 of international patent application publication WO01/09187 (incorporated herein by reference). The HCo7+ HCo12 line carries the HCo7 and HCo12 heavy chain transgenes, which are semi-homozygous for each transgene. KM mice contain the SC20 heavy chain transgene as described by Tomizuka et al (1997, Nature Genet.16, 133-143 and 2000, Proc. Natl. Acad. Sci, 97, 722-727). The transgene is not integrated into the mouse chromosome, but instead proliferates as an independent chromosome segment. This fragment includes approximately 15MB of human chromosome 14. It encompasses the entire human heavy chain locus, including all VH, D and JH gene segments and all heavy chain constant region isoforms. All of these strains are referred to herein as HuMab mice.

HuMab immunization:

purified recombinant N from E.coli cells for production of fully human monoclonal antibodies against NGFGF was used as antigen (example 1) to immunize HuMab mice. A general immunization scheme for HuMab mice is described in Lonberg et al (1994, Nature)368: 856-859; fisherworld et al, supra, and international patent application publication WO 98/24884, the teachings of each of which are incorporated herein by reference). Mice were 6-16 weeks old at the first infusion of antigen. HuMab mice were immunized Intraperitoneally (IP) or Subcutaneously (SC) with purified recombinant NGF antigen preparations (25-100. mu.g).

Immunization of HuMab transgenic mice was achieved as follows: two injections were performed with antigen in complete Freund's adjuvant followed by IP immunization with antigen in complete Freund's adjuvant for 2-4 weeks (up to 9 immunizations). Several mice were immunized with each antigen. A total of 118 mice of HCo7, HCo12, HCo7+ HCo12 and KM strains were immunized with NGF antigen. Immune responses were monitored by retroorbital bleeds.

To select HuMab mice that produce antibodies that bind human HGF, sera from immunized mice were tested by ELISA as described by fisherwild et al (supra). Briefly, microtiter plates (50. mu.L/well) were coated with purified recombinant NGF from E.coli (example 1) in 1-2. mu.g/mL PBS, incubated overnight at 4 ℃ and then blocked with 200. mu.L/well of 5% chicken serum in PBS/Tween (0.05%). Plasma dilutions of NGF immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The microtiter plates were washed with PBS/Tween and then incubated with goat anti-human IgG Fc-specific polyclonal reagents conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing of the microtiter plates, developed with ABTS substrate (Sigma Chemical Co., St. Louis, MO, cat. No. A-1888, 0.22mg/mL) and analyzed spectrophotometrically by measuring Optical Density (OD) at 415-495 nm. Mice with sufficient titers of anti-NGF human immunoglobulin were used to produce monoclonal antibodies as described below. Generation of hybridomas producing anti-NGF human monoclonal antibodies

Mice for monoclonal antibody production were prepared by intravenous boosting with antigen 2 days before mice sacrifice, after which their spleens were removed. Mouse splenocytes were isolated from HuMab mice and fused with PEG to mouse myeloma cell lines using standard protocols. Typically, for each antigen, 10-20 fusions are performed.

Briefly, a single cell suspension of splenic lymphocytes from immunized mice was fused to a quarter number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, accession number CRL 1580) using 50% PEG (Sigma). At about 1x10⁵Cells were plated in flat-bottomed microtiter plates, followed by incubation for about 2 weeks in selective medium containing 10% fetal bovine serum, 10% P388D1- (ATCC, accession number CRL TIB-63) conditioned medium, 3-5% origen (IGEN) in DMEM (Mediatech, catalog number CRL 10013, with high levels of glucose, L-glutamine and sodium pyruvate) plus 5mM HEPES, 0.055mM 2-mercaptoethanol, 50mg/mL gentamicin and 1 XHAT (Sigma, catalog number CRL P-7185). After 1-2 weeks, cells were cultured in medium in which HAT was replaced with HT.

The resulting hybridomas are screened to find hybridomas producing antigen-specific antibodies. Individual wells were screened by ELISA (as described above) to find human anti-NGF monoclonal IgG antibodies. Once a large number of hybridoma growths have occurred, the media is typically monitored after 10-14 days. Hybridomas secreting antibody were replated, screened again, and if still positive for human IgG, anti-NGF monoclonal antibody was subcloned at least twice by limiting dilution. The stable subclones were then cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

Selection of human monoclonal antibodies that bind NGF

Hybridomas that showed positive reactivity to NGF immunogen were screened using the ELISA assay described above. Hybridomas secreting monoclonal antibodies that bind NGF with high affinity were subcloned and further characterized. One clone that retained parental cell reactivity (as measured by ELISA) was selected from each hybridoma and used to prepare a 5-10 vial cell bank and stored in liquid nitrogen.

Isotype-specific ELISAs were performed to determine the isotypes of monoclonal antibodies produced as disclosed herein. In these experiments, 1 u g/mL mouse anti human kappa light chain PBS solution, 50u L/hole coated microtiter plate, and at 4 degrees C were incubated overnight. Microtiter plates were blocked with 5% chicken serum and reacted with supernatants from each monoclonal antibody tested and purified isotype controls. The microtiter plates were incubated at ambient temperature for 1-2 hours. Each well was then reacted with various human IgG-specific horseradish peroxidase-conjugated goat anti-human polyclonal antisera, and microtiter plates were developed and analyzed as described above.

Monoclonal antibodies purified from hybridoma supernatants that showed significant binding to NGF by ELISA assay were further tested for biological activity using various bioassays described below.

Example 3

Selection and cloning of anti-NGF antibodies with potent NGF-neutralising Activity

The effectiveness of antibodies, originally identified in example 2 as inhibitors of NGF activity (i.e., NGF "neutralization"), was evaluated by assaying each modified peptide for its ability to block NGF induced vanilloid receptor-1 (VR1) expression.

Dorsal root ganglion neuron culture

Dorsal Root Ganglia (DRGs) were dissected one by one from all spinal cord segments of 19-day-old (E19) embryonic rats surgically removed from the uterus of periodically pregnant, periodically anesthetized Sprague-Dawley rats (Charles River, Wilmington, MA) under sterile conditions. DRG was collected in L-15 medium (GibcoBRL, Grand Island, NY) containing 5% heat-inactivated horse serum (GibcoBRL) pre-chilled with ice to remove any loose connective tissue and blood vessels. DRG in the absence of Ca²⁺And Mg²⁺In Dulbecco's Phosphate Buffered Saline (DPBS), pH 7.4 (GibcoBRL). DRG was then dissociated into single cell suspensions using a papain dissociation system (Worthington Biochemical corp., Freehold, NJ). Briefly, DRG was dissolved in a digestive solution (containing 20U/ml papaya)Earle Balanced Salt Solution (EBSS)) of protease, and incubated at 37 ℃ for 50 minutes. Cells were dissociated by grinding with fire-cut pasteur pipettes in dissociation medium consisting of MEM/Ham's F12 (1: 1), 1mg/ml ovomucoid inhibitor and 1mg/ml ovalbumin, and 0.005% DNase I (DNase).

Dissociated cells were pelleted for 5 minutes at 200x g and then resuspended in EBSS containing 1mg/ml ovomucoid inhibitor, 1mg/ml ovalbumin and 0.005% DNase. The cell suspension was centrifuged at 200x g through a gradient solution containing 10mg/ml ovomucoid inhibitor, 10mg/ml ovalbumin for 6 minutes to remove cell debris, and then filtered through an 88- μm nylon screen (Fisher Scientific, Pittsburgh, Pa.) to remove any clots. Cell number was determined by hemocytometer at 10X 10 in complete medium³Cells/well cells were seeded in 96-well plates coated with polyornithine 100 μ g/mL (Sigma, st. louis, MO) and mouse laminin 1 μ g/mL (gibcobrl). The complete medium consisted of Minimal Essential Medium (MEM) and Ham's F12 (1: 1), penicillin (100U/ml), streptomycin (100. mu.g/ml) and 10% heat-inactivated horse serum (GibcoBRL). The culture was maintained at 37 ℃ with 5% CO₂And 100% humidity. To control the growth of non-neuronal cells, 5-fluoro-2' -deoxyuridine (75, uM) and uridine (180, cEM) were included in the medium.

Treatment with NGF and anti-NGF

Two hours after plating, cells were treated with either recombinant human β -NGF (Amgen) or recombinant rat β -NGF (R & D Systems, Minneapolis, MN) at a concentration of 10ng/ml (0.38 nM). Positive controls containing serial dilutions of anti-NGF antibodies (R & DSystems) were applied to each plate. Ten concentrations of test antibody were added at 3.16-fold serial dilutions. All samples were diluted in complete medium before addition to the culture. The incubation time was 40 hours, after which the expression of VR1 was measured.

Determination of VR1 expression in DRG neurons

Cultures were fixed with Hanks balanced salt solution containing 4% paraformaldehyde for 15 min, blocked with Superblock (Pierce, Rockford, IL) and permeabilized with 0.25% NonidetP-40(Sigma) in Tris-hcl (Sigma) -buffered saline (TBS) for 1 h at room temperature. Cultures were rinsed once with TBS containing 0.1% tween 20(Sigma), followed by incubation with rabbit anti-VR 1IgG for one and a half hours at room temperature, and then incubated with Eu-labeled anti-rabbit secondary antibody (Wallac Oy, Turku, finland) for 1 hour at room temperature. After each antibody incubation, washing was performed with TBS (3 × 5 min, gentle shaking). A strengthening solution (150. mu.l/well, Wallac Oy) was added to the cultures. The fluorescence signal was then measured with a time-resolved fluorometer (Wallac Oy). VR1 expression was determined in samples treated with the modified peptide by comparison to a standard curve (0-1000ng/ml) for NGF titration. The percent inhibition of VR1 expression in DRG neurons by NGF (compared to the maximum possible inhibition) was determined by comparison to controls not treated with NGF. The results are given in tables 2 and 5.

Cell lines are designated #110- # 129. Antibodies from cell lines #119, #124 and #125 showed very strong NGF neutralization activity (figure 1). The #124 cell line is the parental cell line, also known as 4D4. The #119 and #125 cell lines were subclones of the 4D4 parent. An additional sample in the original vial containing hybridoma #124(4D4) was grown and labeled #167(4D 4).

Antibodies produced by hybridoma #167(4D4) were subjected to the same DRG neuron-based NGF neutralization assay as the previous samples. Antibody #167(4D4) showed strong anti-NGF activity, IC₅₀This was 0.50nM (FIG. 2), which is consistent with the activity of samples #119, #124 and # 125.

The activity of these four samples is shown in table 2.

TABLE 2

N-terminal sequencing and Mass Spectrometry

Purified anti-NGF hybridoma antibody samples were prepared for protein sequencing and LC/MS analysis. Antibodies were purified from conditioned media by concentrating the media to a volume of less than 15ml with Amiconcentriprep-30. One batch of rproa (pharmacia) resin 4x was washed with PBS and made into a 50% slurry with PBS after the last wash. An appropriate amount of rProA resin (approximately 5ug antibody/ul resin, but not less than 50ul resin used) was added to the antibody sample and incubated overnight at 4 ℃. The Ab-resin mixture was centrifuged and the unbound fraction was collected. 0.5ml PBS was added and the sample was transferred to a 0.45um Spin-X (CoStar) tube and then centrifuged at 10000rpm for 3 minutes. The resin was then washed at least 3 times with 0.5ml PBS, then 1.5x resin volume of 0.1M glycine (pH 2.7) was added, incubated at room temperature for 10 minutes, then centrifuged again at 10000rpm for 3 minutes and the supernatant collected. This elution step was repeated two more times and the combined supernatants were then neutralized with one twenty-fifth volume of 1.0M tris (pH 9.2).

After the last filtration step with a new Spin-x tube (0.2um), the antibodies were quantified using either a standard Bradford assay with human IgG as standard or absorbance at 280 (for larger samples). 2ug of each sample was also run with 2ug of human IgG1, k (Sigma). For mass spectrometry, 4 μ g of the sample was deglycosylated, reduced and loaded onto HPLC (HP1090) connected online to a finegan LCQ mass spectrometer. The light chain was separated from the heavy chain by reverse phase HPLC. Light and heavy chains were also collected for N-terminal protein sequencing analysis.

The N-terminal sequences of the light and heavy chains of the anti-NGF #167(4D4) antibody sample were identical to the two N-terminal sequences of the anti-NGF #119(4D4) antibody sample. In addition, the measured quality of the antibodies indicated that the isolated antibodies from the #167 and #119 hybridomas were identical. The deconvolution measured mass (23096) of the light chain of anti-NGF #167 coincided with the measured mass (23096) of the light chain of anti-NGF Ab # 119.

Cloning of anti-NGF antibody heavy and hydroxyl chains

Hybridoma 4D4.D7 expressing the strongest NGF-binding monoclonal antibody was used as the starting material, andreagents (Invitrogen) Total RNA was isolated. First strand cDNA was synthesized using random primers with overhang adapters (5 '-GGC CGG ATA GGC CTC CAN NNN NNT-3') (SEQ ID NO: 33) using GeneRacer^TMThe 5 'RACE (Rapid amplification of cDNA Ends) preparation assay was performed with the kit (Invitrogen) according to the manufacturer's instructions. To prepare the complete light chain encoding cDNA, the forward primer was used with GeneRacer^TMNested primers, reverse primer 5'-GGG GTC AGG CTG GAA CTG AGG-3' (SEQ ID NO: 34). To prepare cDNA encoding the variable region of the heavy chain, GeneRacer was used as the forward primer^TMNested primers, reverse primer 5'-TGA GGA CGC TGA CCA CAC G-3' (SEQ ID NO 35). RACE products were cloned into pCR4-TOPO (Invitrogen) and sequenced. Primers for full-length antibody chain PCR amplification were designed using the consensus sequences.

To prepare a cDNA encoding an anti-NGF 4D4. D71. kappa. light chain, the 5 ' PCR primers encoded the amino terminus of the signal sequence, an XbaI restriction site, and an optimized Kozak sequence (5'-CAGCAG AAG CTT CTA GAC CAC CAT GGACAT GAG GGT GCC CGCTCA GCT CCT GGG-3'; SEQ ID NO: 36). The 3 ' primer encodes a carboxy terminus and a stop codon, as well as a SalI restriction site (5'-CTT GTC GAC TCA ACA CTCTCC CCT GTT GAA GCT C-3'; SEQ ID NO: 37). The resulting PCR product fragment was purified, digested with XbaI and SalI, then gel separated and ligated to the mammalian expression vector pDSR α 20 (see international application publication WO 90/14363, which is incorporated herein by reference for any purpose). pDSR α 20 was produced by changing nucleotide 2563 in pDSR α 19 from "guanosine" to "adenosine" using site-directed mutagenesis. ).

To prepare the cDNA encoding the anti-NGF 4D4.D7 heavy chain, the 5 ' PCR primers encoded the amino terminus of the signal sequence, an XbaI restriction site, and an optimized Kozak sequence (5'-CAGCAG AAG CTT CTA GAC CAC CAT GGA GTTGGG GCT GTG CTGGGT TTT CCT TGT T-3'; SEQ ID NO: 38). The 3 ' primer encodes a carboxy terminus and a stop codon, as well as a SalI restriction site (5'-GCA TGT CGA CTC ATT TACCCG GAG ACA GGG AGA G-3'; SEQ ID NO: 39). The resulting product was purified, digested with XbaI and SalI, gel separated, and ligated to pDSR α 20 vector.

Calculated mass of the DNA sequence of the light chain of the anti-NGF Ab 4D4 clone (23099) was determined by translating the nucleotide sequence into predicted amino acids and adding the molecular weights of the amino acids together, consistent with the measured mass measured by mass spectrometry. Within instrumental variation, the measured mass for de-folding of the heavy chain of anti-NGF Ab #167 (49479) coincided with the measured mass of the heavy chain of anti-NGFAb #119 (49484) and also with the theoretical mass of the DNA sequence of the heavy chain of anti-NGF Ab 4D4 clone (49484) (table 3).

The N-terminal protein sequence and LC/MS data confirm that hybridoma #19 expresses the same antibody as hybridoma # 167. In addition, sequence-based antibody mass calculations further corroborate this observation.

TABLE 3 summary of Mass Spectrometry results

Example 4

Expression of anti-NGF antibodies in Chinese Hamster Ovary (CHO) cells

Stable expression of the 4D4 anti-NGF mAb was achieved by co-transfection of the 4D4 heavy chain/pDSRa 19IgG2 or 4D 4-heavy chain/pDSRa 19IgG1 and NGF-kappa/pDSRa 19 plasmids into dihydrofolate reductase-deficient (DHFR-) serum-free adapted Chinese Hamster Ovary (CHO) cells using the calcium phosphate method. Transfected cells were selected in medium containing dialyzed serum but not containing hypoxanthine-thymidine to ensure growth of cells expressing HDFR enzyme. Transfected clones are screened using assays such as ELISA to detect expression of 4D4 anti-NGF mAb in conditioned media. HDFR amplification was performed by exposing the highest expressing clones to increasing concentrations of Methotrexate (MTX). MTX amplified clones are screened using assays such as ELISA to detect the more highly expressed 4D4 anti-NGF mAb in conditioned media. The clones with the highest expression were subcloned to obtain a uniform population, and a cell bank was created.

The recombinant anti-NGF antibodies of the invention may be produced in HDFR deficient Chinese hamster ovary cells using the same protocol as described above for the production of anti-NGF monoclonal antibodies. The DNA sequences encoding the complete heavy or light chain of each anti-NGF antibody of the invention were cloned into expression vectors. CHO deficient cells were co-transfected with an expression vector capable of expressing the full heavy chain of an appropriate anti-NGF antibody and an expression vector expressing the full light chain of an appropriate anti-NGF antibody. For example, to generate an anti-NGF antibody, a polypeptide capable of expressing an antibody comprising SEQ ID NO: 40 and a vector capable of expressing a full heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 44, and a vector that is a complete light chain of the amino acid sequence set forth in seq id no. Table 4 summarizes the complete heavy and complete light chains of the 4D4 antibody with various IgG heavy chain constant regions.

TABLE 4

Example 5

Identification of Activity of anti-NGF 4D4 antibodies

Transiently expressed anti-NGF 4D4 antibodies produced in cells grown in spinner culture (S) or roller bottle culture (R) conditions were tested to confirm their ability to neutralize NGF in DRG neuron-based NGF neutralization bioassays performed as described above (example 3).

NGF antibodies were transiently expressed in serum-free suspension adapted 293T cells. Transfection was performed with 500mL or 1L of culture. Briefly, cell inoculum (5.0X 10) was centrifuged at 2,500RPM at 4 deg.C⁵Individual cells/mL X culture volume) for 10 minutes, and the conditioned media was removed. The cells were resuspended in serum-free DMEM and centrifuged again at 2,500RPM for 10 minutes at 4 ℃. After the washing solution is aspirated, in1L or 3L spinner flasks, the cells were resuspended in growth medium [ DMEM/F12 (3: 1) +1X insulin-transferrin-selenium supplement +1X Pen Strep Glut +2mM L-glutamine +20mM HEPES + 0.01% Pluronic F68]. The spinner flask culture was maintained at 125RPM on a magnetic stir plate placed at 37 ℃ and 5% CO₂In a humidified incubator. Plasmid DNA was complexed with transfection reagents in 50mL Erlenmeyer flasks. The DNA-transfection reagent complexes were prepared in serum-free DMEM at a volume of 5% of the final culture volume. First 1. mu.g plasmid DNA/mL culture was added to serum-free DMEM, followed by 1. mu. l X-TremeGeneRO-1539/mL culture. The complexes were incubated at room temperature for approximately 30 minutes and then added to the cells in the spinner flask. After 7 days of transfection/expression, conditioned media was harvested by centrifugation at 4,000RPM for 60 minutes at 4 ℃.

For roller bottle transient transfection, we used 293T adherent cells grown and maintained in DMEM supplemented with 5% FBS +1X non-essential amino acids +1X PenStrep Glut +1X sodium pyruvate. Will be about 4-5X 10⁷293T cells were seeded at 850cm²The bottles were rolled overnight. The next day, previously seeded cells were then transfected with FuGene6 transfection reagent. The DNA-transfection reagent mixture was prepared with approximately 6.75mL serum-free DMEM. 675. mu.l of FuGene6 transfection reagent were added first, followed by 112.5. mu.g of plasmid DNA. The resulting complex was incubated at room temperature for 30 minutes. The entire mixture was then added to the roller bottle. 5% CO for roller bottle₂The gas mixture was aerated, capped tightly, and placed in a 37 ℃ incubator on a roller rack rotating at 0.35 RPM. 24 hours after transfection, the medium was replaced with 100mL DMEM +1X insulin-transferrin-selenium supplement +1X Pen Strep Glut +1X non-essential amino acids +1X sodium pyruvate. Typically, two 100ml 48 hour harvests per roller bottle were obtained. The harvested serum-free conditioned medium was pooled and centrifuged at 4,000RPM for 30 minutes at 4 ℃.

4D4.IgG1 and 4D4.IgG2 show strong activity on human NGF, IC₅₀Values were about 0.14nM to about 0.2nM (FIG. 2). The results of the activity tests are summarized in table 5. These antibodies were directed against rat NGFShowed little activity (figure 3). These results are similar to the antibody activity directly detected from the above hybridomas.

TABLE 5

Ab	IC50@hNGF(nM)	IC50@rNGF(nM)
			4D4.IgG1.R	0.1488	＞34nM
4D4.IgG1.S	0.1587	＞45nM
			4D4.IgG2.R	0.2047	＞59nM
4D4.IgG2.S	0.2063	＞37nM

hNGF ═ human NGF, rNGF ═ rat NGF, R ═ roller bottle culture, S ═ spinner culture

Example 6

Production of anti-NGF antibodies

anti-NGF antibodies were produced by expression in a clonal line of CHO cells. For each production run, cells in a single vial were thawed into serum-free cell culture medium. Cells were grown in T-flasks, then spinner flasks, and then stainless steel reactors, gradually scaled up to 2000L bioreactors. Production was carried out in a 2000L bioreactor in a fed batch mode, with nutrient feed containing concentrated media components added to the reactor to maintain cell growth and culture viability. Production continues for approximately two weeks during which time anti-NGF antibodies are constitutively produced by the cells and secreted into the cell culture medium.

The production reactor is controlled at a predetermined pH, temperature and dissolved oxygen level: the pH is controlled by adding carbon dioxide gas and sodium carbonate; dissolved oxygen is controlled by introducing air, nitrogen and oxygen.

At the end of production, the cell culture broth is fed into a disk centrifuge and the culture supernatant is separated from the cells. The concentrate was further clarified by passing through a depth filter followed by a 0.2 μm filter. The clarified conditioned medium was then concentrated by tangential flow ultrafiltration. The conditioned medium was concentrated 15 to 30 times. The resulting concentrated conditioned medium is then processed for purification or frozen for later purification.

Example 7

Cross-reactivity with other neurotrophins

The cross-reactivity of the 4D4 antibody to human NT3 or human BDNF was tested in different biological assays, including a DRG neuron survival assay for human NT3, and an assay for DA uptake in cultured DA neurons for human BDNF.

Treatment of DRG cultures with NT3, anti-NT 3 and anti-NGF

Two hours after plating, DRG cells were treated with recombinant hNT-3100ng/ml (3.8nM) (isolation procedure described in example 3 above). Serial dilutions of anti-hNT 3 antibody (R & D) were used as positive control samples. Unknown samples (anti-NGF Ab samples) were added at various concentrations at 3.16-fold serial dilutions of 10 points. All samples were diluted in complete medium before being added to the culture.

Determination of MAP2 expression in DRG neurons

Cultures were fixed with Hanks balanced salt solution containing 4% paraformaldehyde for 15 min, blocked with superblock (Pierce) for 1 h, and permeabilized with 0.25% Nonidet P-40(Sigma) in Tris-HCl (Sigma) -buffered saline (TBS) for 1 h at Room Temperature (RT). Cultures were rinsed once with TBS containing 0.1% tween 20(Sigma), then incubated with mouse anti-MAP 2IgG (Chemicon, Temecula, CA) for 1.5 hours at room temperature, and then with Eu-labeled anti-mouse secondary antibody (Wallac Oy, Turku, finland) for 1 hour at room temperature. After each antibody incubation, washes were performed with TBS (3x5 min, gentle shaking). An enhancing solution (150. mu.l/well, Wallac Oy) was added to the culture, and then the fluorescence signal was measured with a time-resolved fluorometer (Wallac Oy).

Embryonic midbrain culture

Sprague-Dawley rats (Jackson Labs) were used as 19 day old (E19) embryos. Ventral midbrain tissue enriched for dopaminergic neurons was excised and transferred to cold Dulbecco Phosphate Buffered Saline (DPBS), pH 7.4, Ca free⁺⁺And Mg⁺⁺(Gibco). Tissue fragments were dissociated into single cell suspensions using a papain dissociation system (Worthington Biochemical corp., Freehold, NJ). Briefly, the tissue fragments were incubated in digestion solution (containing 20 units/ml papain in Earle Balanced Salt Solution (EBSS)) for 50 minutes at 37 ℃. Cells were dissociated by grinding with fire-cut pasteur pipettes in dissociation medium consisting of MEM/Ham's F12 (1: 1), 1mg/ml ovomucoid inhibitor and 1mg/ml ovalbumin, and 0.005% DNase I (DNase). Dissociated cells were pelleted for 5 minutes at 200x g and then resuspended in EBSS containing 1mg/ml ovomucoid inhibitor, 1mg/ml ovalbumin and 0.005% DNase. To be provided with200Xg the cell suspension was centrifuged through a gradient solution containing 10mg/ml ovomucoid inhibitor, 10mg/ml ovalbumin for 6 minutes to remove cell debris; then filtered through a 25 μ g nitex nylon sieve (Tetko, Inc.) to remove clots. At a rate of 100,000/cm²Plating the dissociated cells in a tissue culture plate. Tissue culture plates were pre-coated with polyornithine 100. mu.g/ml (Sigma) and mouse laminin 1. mu.g/ml (GibcoBRL) as described previously (LouisJC et al, J. Pharmacol. exp. Ther. 1992; 262: 1274-one 1283.). The medium consisted of Minimal Essential Medium (MEM)/Ham's F12 (1: 1), 12% horse serum (GibcoBRL), 100. mu.g/ml transferrin and 2.5. mu.g/ml insulin (Sigma). Culture at 37 deg.C with 5% CO₂And held at 100% humidity for 6 days.

Treatment of midbrain cultures with BDNF and anti-BDNF or anti-NGF

Two hours after plating, BDNF was added to the cells at a concentration of 10ng/ml, followed by a series of concentrations of anti-NGF Ab samples. An anti-BDNF antibody (produced by Amgen) was used as a positive control sample.

DA uptake in neurons of the middle brain

Dopamine uptake assays were performed as described previously (Friedman, L. and Mytilineou, C., Neuroscience Letters 1987; 79: 65-72). On day 6, the culture was washed once with pre-warmed Krebs-Ringer phosphate buffer (pH 7.4) containing 5.6mM glucose, 1.3mM EDTA, and 0.5mM pargyline (monoamine oxidase inhibitor). The culture was incubated at 37 ℃ in a medium containing 50nM [ alpha ], [ beta ], [³H]Da (nen) was incubated for 60 minutes in uptake buffer. Uptake was stopped by removing the uptake buffer and the cultures were washed three times with Krebs-Ringer phosphate buffer. Lysing the cells to release the [ 2 ] by adding a liquid scintillation mixture, OptiPhaseSuperMix (Wallac), directly to the culture³H]And D, DA. The radioactivity of the cell lysates was then calculated using a Microbeta-Plus liquid scintillation counter (Wallac, Inc.). By adding 0.5mM GBR12909 (specific inhibitor of high affinity DA uptake site, Heikkila RE and Mazino L, European Journal of Pharmacology 1984; 103: 241-8) to the uptakeIn buffer, low affinity DA uptake was estimated and then subtracted from the total uptake to give a high affinity DA uptake value.

TABLE 6

Example 8

Identification of epitopes for anti-NGF antibody binding

Epitope mapping by limited proteolysis

5 micrograms (. mu.g) of NGF were incubated with 4D4 (11. mu.g) in 0.1M Tris buffer (pH 7.5) for 30 minutes at 4 ℃. The resulting complex was then digested with 1. mu.g protease (subtilisin) at 37 ℃. The HPLC peptide plots were compared to each other to find the peptide protected by the 4D4 antibody. Limited proteolysis of NGF has shown that there are several major peptides initially released from NGF. Of particular interest, peptides S18.3, S18.5 and S34.4 were produced and protected from proteolysis by antibodies. Other peaks were not significantly formed or protected. The protected peptides in both experiments (1 hour and 2 hour digestion) are shown in table 7.

TABLE 7

Percent protection was calculated from the peak height of the peptide. S18.5 contains two peptides, but absorbance measurements at 280nm found that only one peptide (SSSHPIFHR; SEQ ID NO: 46) was protected by the 4D4 antibody, since the peak of the other peptide (HWNSY; SEQ ID NO: 47) remained unchanged after addition of the 4D4 antibody. Peptide S18.3 is the C-terminal portion of S34.4, both from the same loop region. The N-terminal and central loop regions are also possible epitopes.

Microcon isolation of digested peptides

Subtilisin-digested material (3. mu.g each) was incubated with active 4D4 antibody and inactivated monoclonal antibody (#162) (8. mu.g) in 0.1M Tris buffer (pH 7.5) for 30 min at 4 ℃. Bound/unbound peptides were separated by Microcon 10(Millipore Corp., Bedford, Mass) and the two fractions (bound and unbound) were analyzed by HPLC to find antibody-bound peptides. After treatment with 4D4 antibody and #162 and separation with Microcon, the unbound fractions were compared by HPLC, identifying two depleted peaks that were recovered, which represent the peptides to which the antibodies bound. The 4D4 binding peptide was:

s1(4.4) - - - -SRKAVRR (113-119) (SEQ ID NO: 49), C-terminal;

s2(28.3) - - - -EVMVL (35-39) (SEQ ID NO: 50), loop region.

NGF samples were additionally digested with Lys-C (K) for 24 hours. Cysteine residues are reduced and carboxymethylated without the addition of denaturants. The samples were incubated with monoclonal antibody 4D4 and AMG162, followed by separation with Microcon 100. Bound and unbound fractions were analyzed by reverse phase HPLC. Only two peptides shown below were identified as antibody-binding K peptides. The calculated masses of the two peptides determined by sequence analysis were consistent with their mass spectral observations. These two peptides are located at the N-terminal and C-terminal regions as shown below.

K1(37.6)----SSSHPIFHRGEFSVCDSVSVWVGDK(SEQ ID NO：51)

Calculating the mass to be 2821; observed mass 2828.2; n-terminal

K2(39.5)----QAAWRFIRIDTACVCVLSRK(SEQ ID NO：52)

Calculating the mass as 2452; observed mass 2459.5; c-terminal

The foregoing epitope mapping experiments showed that at least three regions were possible epitopes for the 4D4 antibody, including the N-terminal region (1-9), the inner region (46-57) and the C-terminal region (96-98). Furthermore, AspN digestion revealed that the peptide fragment consisting of- - -SSHPIFHRGEFSVC- - - (SEQ ID NO: 53) was protected by the 4D4 antibody, whereas trypsin digestion revealed that the peptide fragment consisting of- - -SSHPIFHR- - - (SEQ ID NO: 54) was not protected by the 4D4 antibody. Thus, at the N-terminus, the sequence GEFSVC (SEQ ID NO: 55) is most important for binding to the 4D4 antibody.

To more clearly define the epitope for the anti-NGF antibody 4d4.igg1, a total of 23 peptides were generated synthetically using standard techniques based on the entire human mature NGF (hngf) sequence (table 8). These peptides are 15 amino acids long, overlap by up to 10 amino acids, and terminate in a cysteine at the C-terminus for conjugation to a substrate. The human anti-hNGF Ab 4d4.igg1 was used for mapping experiments.

TABLE 8

Peptide #	Sequence of	SEQ ID NO
			33582-27-01	SSSHPIFHRGEFSVC(1-15)	56
33582-27-02	IFHRGEF SVADSVSVC(6-20)	57
			33582-27-03	EFSVADSVSVWVGDKC(11-25)	58
33582-27-04	DSVSVWVGDKTTATDC(16-30)	59
			33582-27-05	WVGDKTTATDIKGKEC(21-35)	60
33582-27-06	TTATDIKGKEVMVLGC(26-40)	61
			33582-27-07	IKGKEVMVLGEVNIN (31-45)	62
33582-27-08	VMVLGEVNINNSVFKC(36-50)	63
			33582-27-09	EVNINNSVFKQYFFEC(41-55)	64
33582-27-10	NSVFKQYFFETKARDC(46-60)	65
			33582-27-11	QYFFETKARDPNPVDC(51-65)	66
33582-27-12	TKARDPNPVDSGARDC(56-70)	67
			33582-27-13	PNPVDSGARDIDSKHC(61-75)	68
33582-27-14	SGARDIDSKHWNSYC(66-80)	69
			33582-27-15	IDSKHWNSYATTTHTC(71-85)	70
33582-27-16	WNSYATTTHTFVKALC(76-90)	71
			33582-27-17	TTTHTFVKALTMDGKC(81-95)	72
33582-27-18	FVKALTMDGKQAAWRC(86-100)	73
			33582-27-19	TMDGKQAAWRFIRIDC(91-105)	74
33582-27-20	QAAWRFIRIDTAAVC(96-110)	75
			33582-27-21	FIRIDTAAVAVLSRKC(101-115)	76
33582-27-22	TAAVAVLSRKAVRRAC(106-120)	77
			33582-27-23	CAAVAVLSRKAVRRA(107-120)	78

Human NGF peptide fragments were diluted in PBS with 5% DMSO, 1mM EDTA, pH 6.23. The final peptide concentration was normalized to the same molar concentration of 55. mu.M (about 100. mu.g/ml). The peptides were incubated at 100. mu.l/well in a reaction-Bind maleimide activated 96-well microtiter plate (Pierce Cat. No. 15150) at room temperature for 2 hours, followed by overnight incubation with stirring at 4 ℃. Human NGF (100. mu.g/ml) was used as a positive control sample. The microtiter plates were washed with wash buffer (KPL), blocked with 0.2% skim milk powder (in PBS-EDTA buffer, pH 6.23) for 2 hours at room temperature, followed by another 1 hour blocking with 5% BSA. Microtiter plates were then incubated with various concentrations (0, 3, 10, 30. mu.g/ml) of human anti-NGF antibody, followed by goat anti-hFcAb-HRP (KPL) for 2 hours. The signal was visualized with TMB substrate and after addition of stop solution (KPL) read at 450 nm.

Among the 23 human NGF peptides, at least 4 major peaks were observed, showing 4D4 binding. These peaks correspond to the following peptides: peptide #1(SEQ ID NO: 56), SSSHPIFHRGEFSVC (1-15); peptide #10(SEQ ID NO: 65), NSVFKQYFFETKARD (46-60); peptide #16-17(SEQ ID NO: 71-SEQ ID NO: 72), WNSYATTTHTFVKAL- - (76-95); and peptides #18-21(SEQ ID NO: 73-SEQ ID NO: 76), TTTHT- - -LSRKC (100-.

Such as in Weismann et al (1999, Nature)401: 184-8), the four binding peaks of 4D4 were located at the N-terminus, C-terminus, internal domain, and loop L2 and loop L4 in NGF.

These results are summarized in table 9.

TABLE 9

Wiesmann et al disclose the crystal structure of trkA receptor-binding hNGF, showing that the N-terminus (residues 2-9) is important for receptor binding (Wiesmann et al, 1999, Nature)401: 184-8). Residues of this segment in NGF are also important for their greater specificity for trkA than for the trkB or trkC receptors. Antibody 4D4 was more selective for human NGF than for mouse/rat NGF and BDNF and NT-3, probably due to the N-terminal differences between human NGF and other neurotrophins.

Antibody 4D4 binds to peptide #10(SEQ ID NO: 65) (NSVFK- -, 46-60) and peptide #17(SEQ ID NO: 72) (TTTHTFVKALTMDGKC, 81-95), which correspond to loop L2 and loop L4, respectively, which represent two of seven distinct regions of neurotrophins in which the sequence diversity is higher than average. Exchange experiments of these seven regions between NGF and BDNF showed that L2 and L4 are important for the biological activity of NGF. In addition, replacement of five NT3 residues in loop L2 and loop L4 with residues in NGF introduced NGF-like activity while maintaining NT3 activity. Thus, L2 and L4 may be regions of the antibody 4D4 that selectively bind NGF rather than BDNF or NT-3.

Antibody 4D4 also bound to peptide #16(SEQ ID NO: 71) (WNSYATTTHTFVKAL, 76-90), consistent with the internal domains of the NGF crystal structure. This region is 100% homologous between human NGF and mouse NGF, but is distinct from other neurotrophins. 4D4 was much less active on rat/mouse NGF than it was on human NGF. Thus, binding to this part of NGF is likely not critical for species specificity, but is important for selectivity among neurotrophins.

Antibody 4D4 also bound the C-terminal region of NGF (peptide #19-21(SEQ ID NO: 74-SEQ ID NO: 76) TMDGK- - -LSRKC, 91-115), one of the regions in human NGF that distinguishes NGF from other neurotrophins (BDNF and NT 3). Binding to this region helps explain why 4D4 is not active on other neurotrophins. Furthermore, there was a single amino acid difference between human NGF and mouse NGF at the C-terminus, suggesting that this single amino acid may be one of the reasons for the stronger selectivity of 4D4 for human NGF over rat/mouse NGF, similar to the species difference observed at the N-terminus.

Finally, 4D4 also interacted with the internal domain of human NGF represented by peptide #10(SEQ ID NO: 65) (- -KARDC, 50-60), a region of importance for NGF to preferentially bind trkA rather than trkB or trkC, further explaining the selective neutralizing activity of 4D4 on human NGF.

Example 9

Determination of the affinity of monoclonal antibodies by KinExA

Binding of Ab 4D4(38859-80) to huNGF (29714-91) was determined by KinExA. Briefly, reaction-Gel 6x (Pierce) was pre-coated with huNGF and blocked with BSA. Samples of Ab 4D4 at 10pM and 30pM were incubated with various concentrations of huNGF (amgen) for 8 hours at room temperature, then flowed through huNGF-coated beads. The amount of bead bound antibody was quantified with a goat anti-human IgG antibody (Jackson Immune Research) labeled with fluorescence (Cy 5). The binding signal is proportional to the concentration of free antibody at equilibrium. Using a hyperbolic single-point homologation model (Dual-curve one-site homology binding model) (KinEx)^TMSoftware) to perform nonlinear regression on the competition curves to obtain the dissociation equilibrium constant (K)_D). For Ab 4D4 binding to huNGF, K_DAbout 4 pM.

Example 10

Identification of additional anti-NGF antibodies

Additional anti-NGF antibodies (designated 14D10, 6G9, 7H2, 14F11 and 4G6) produced and identified as described above in examples 2 and 3 were selected for further study. Briefly, conditioned media were tested for binding activity. The antibodies in the medium were purified and sequenced. The predicted mass of the antibody in the conditioned media is compared to its mass spectrometry data. Cloning the antibody. Two of these clones were expressed in CHO cells and tested for activity as described above.

The results are shown in Table 10.

Watch 10

The sequences of the light chain variable region and the heavy chain variable region of these antibodies were then compared to the 4D4 antibody sequences, while comparing to each other (fig. 5 and 6). The percent homology of the heavy chain variable regions identified from these comparisons is shown in table 11. The percent homology of the light chain variable regions is shown in table 12. In addition, the percent homology of the CDR regions of the various antibodies is shown in FIGS. 5-10.

TABLE 11

4D4VH

14D10VH

6H9VH

7H2VH

14D11VH

4G6VH

4D4VH

100％

70.9％

70.1％

75.6％

47.2％

73.4％

14D10VH

100％

95.3％

85％

54.3％

81.1％

6H9VH

100％

86.6％

54.3％

81.1％

7H2VH

100％

51.2％

79.8％

14D11VH

100％

56.8％

4G6VH

100％

TABLE 12

It should be understood that the foregoing disclosure emphasizes specific embodiments of the invention and all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims (18)

1. An isolated nucleic acid molecule consisting of the sequence:

(a) SEQ ID NO: 9; or

(b)SEQ ID NO：11。

2. An isolated nucleic acid molecule encoding a human antibody that specifically binds human Nerve Growth Factor (NGF), comprising:

(a) SEQ ID NO: 21. SEQ ID NO: 17 and SEQ ID NO: 13; and

(b) SEQ ID NO: 23. SEQ ID NO: 19 and SEQ ID NO: 15.

3. a host cell comprising a nucleic acid encoding a human antibody that specifically binds NGF, wherein the nucleic acid consists of SEQ ID NO: 9 and SEQ ID NO: 11, or a nucleic acid molecule according to claim 2.

4. A cell line expressing a human antibody that specifically binds NGF, wherein the antibody is encoded by a nucleic acid consisting of the amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 11, or a nucleic acid molecule according to claim 2.

5. An isolated nucleic acid molecule encoding a human antibody that specifically binds human Nerve Growth Factor (NGF), consisting of a nucleotide sequence encoding:

(a) SEQ ID NO: 10 and SEQ ID NO: 12; or

(b) SEQ ID NO: 40 and the heavy chain of SEQ ID NO: 44, a light chain; or

(c) SEQ ID NO: 41 and the heavy chain of SEQ ID NO: 44, a light chain; or

(d) SEQ ID NO: 42 and the heavy chain of SEQ ID NO: 44, a light chain; or

(e) SEQ ID NO: 43 and the heavy chain of SEQ ID NO: 44, or a light chain as shown.

6. An isolated nucleic acid molecule encoding a human antibody that specifically binds human Nerve Growth Factor (NGF), comprising a nucleotide sequence encoding:

(a) SEQ ID NO: 22, the heavy chain CDR1 region shown in SEQ ID NO: 18 and the heavy chain CDR2 region shown in SEQ ID NO: 14, the heavy chain CDR3 region; and

(b) SEQ ID NO: 24, the light chain CDR1 region shown in SEQ ID NO: 20 and the light chain CDR2 region shown in SEQ ID NO: 16, the light chain CDR3 region.

7. A host cell comprising nucleic acid encoding a human antibody that specifically binds NGF, wherein the nucleic acid is the nucleic acid molecule of claim 5 or 6.

8. A cell line expressing a human antibody that specifically binds NGF, wherein the antibody is encoded by the nucleic acid molecule of claim 5 or 6.

9. An isolated nucleic acid molecule encoding a Nerve Growth Factor (NGF) -specific antibody or antigen-binding fragment thereof, wherein the specific antibody or antigen-binding fragment thereof comprises

(a) SEQ ID NO: 10 and SEQ ID NO: 12; or

(b) SEQ ID NO: 40 and the heavy chain of SEQ ID NO: 44, a light chain; or

(c) SEQ ID NO: 41 and the heavy chain of SEQ ID NO: 44, a light chain; or

(d) SEQ ID NO: 42 and the heavy chain of SEQ ID NO: 44, a light chain; or

(e) SEQ ID NO: 43 and SEQ ID NO: 44, or a light chain as shown.

10. An isolated nucleic acid molecule encoding a Nerve Growth Factor (NGF) -specific antibody, wherein the specific antibody comprises SEQ ID NO: 22, the heavy chain CDR1 region shown in SEQ ID NO: 18, the heavy chain CDR2 region shown in SEQ ID NO: 14, the heavy chain CDR3 region shown in SEQ ID NO: 24, the light chain CDR1 region shown in SEQ ID NO: 20 and the light chain CDR2 region shown in SEQ ID NO: 16, the light chain CDR3 region.

11. A host cell comprising nucleic acid encoding a human antibody that specifically binds NGF, wherein the nucleic acid is the nucleic acid molecule of claim 9 or 10.

12. A cell line expressing a human antibody that specifically binds NGF, wherein the antibody is encoded by the nucleic acid molecule of claim 9 or 10.

13. An antibody or antigen-binding fragment thereof, comprising:

(a) consisting of SEQ ID NO: 41 and the heavy chain represented by SEQ ID NO: 44, a light chain; or

(b) Consisting of SEQ ID NO: 42 and the heavy chain set forth by SEQ ID NO: 44, a light chain; or

(c) Consisting of SEQ ID NO: 43 and the heavy chain set forth by SEQ ID NO: 44, a light chain;

wherein the antibody or antigen binding fragment thereof binds Nerve Growth Factor (NGF) and is selected from the group consisting of: f (ab), F (ab')₂Fv, and single chain antibodies.

14. An isolated monoclonal antibody that specifically binds to an epitope on human Nerve Growth Factor (NGF), wherein the isolated monoclonal antibody comprises:

(a) consisting of SEQ ID NO: 9, and the heavy chain variable region encoded by the nucleic acid molecule set forth in SEQ ID NO: 11, or a light chain variable region encoded by a nucleic acid molecule set forth in seq id no; or

(b) Consisting of SEQ ID NO: 21. SEQ ID NO: 17 and SEQ ID NO: 13, and the heavy chain CDR1, CDR2, CDR3 regions encoded by the isolated nucleic acid molecule set forth in SEQ ID NO: 23. SEQ ID NO: 19 and SEQ ID NO: 15, and a light chain CDR1, CDR2, CDR3 region encoded by the isolated nucleic acid molecule set forth in seq id no.

15. The monoclonal antibody of claim 14, wherein the isolated monoclonal antibody binds a polypeptide selected from the group consisting of SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 65. SEQ ID NO: 71 and SEQ ID NO: 75 of at least one NGF peptide.

16. The isolated monoclonal antibody of claim 14, wherein the epitope comprises a heavy chain variable region comprising SEQ ID NO: 55. SEQ ID NO: 56. SEQ ID NO: 65. SEQ ID NO: 71 or SEQ ID NO: 75 amino acid sequence of a plurality of NGF peptides.

17. The isolated monoclonal antibody of claim 14, wherein the isolated monoclonal antibody is a fully human antibody.

18. The isolated monoclonal antibody of claim 14, wherein the isolated monoclonal antibody inhibits NGF signaling.