KR100894191B1 - Human anti-ngf neutralizing antibodies as selective ngf pathway inhibitors and a pharmaceutical composition comprising these antibodies - Google Patents

Abstract

The present invention relates to antibodies that interact with or bind to nerve growth factor (NGF) to neutralize the function of NGF. The present invention also relates to a pharmaceutical composition comprising said antibody and a method for neutralizing the function of NGF, in particular a method for treating NGF-related diseases (eg chronic pain) by administering a pharmaceutically effective amount of an anti-NGF antibody. will be. The invention also relates to a method of detecting the amount of NGF in a sample using an anti-NGF antibody.

NGF-related diseases including nerve growth factor (NGF), anti-human NGF human antibodies, chronic pain

Description

HUMAN ANTI-NGF NEUTRALIZING ANTIBODIES AS SELECTIVE NGF PATHWAY INHIBITORS AND A PHARMACEUTICAL COMPOSITION COMPRISING THESE ANTIBODIES}

This application is related to and claims priority in US Provisional Serial No. 60 / 487,431, filed Jul. 15, 2003, which is incorporated herein by reference.

The present invention relates to human monoclonal antibodies that bind to nerve growth factor (NGF). The invention also relates to pharmaceutical compositions for treating pain and pain-related disorders.

More than two million people in the United States suffer from chronic pain every day [Jessell and Kelly, 1991, "Pain and Analgesia" in PRINCIPLES OF NEURAL SCIENCE, 3 ^rd Ed., (Kandel, Schwartz, and Jessell, ed.), Elsevier , New York]. Unfortunately, the treatment of pain at present is only partially effective and can lead to debilitating or dangerous side effects. For example, non-steroidal anti-inflammatory drugs ("NSAIDs"), such as aspirin, ibuprofen, and indomethacin, effectively alleviate inflammatory pain, but these drugs are also toxic to the kidneys and, at high doses, gastrointestinal hypersensitivity, It tends to cause ulceration, bleeding and confusion. Patients treated with opioids also frequently experience confusion and prolonged use of opioids is associated with tolerance and dependence. Local anesthetics such as lidocaine and mexilitine cause simultaneous pain inhibition and loss of normal sensation.

Pain is a perceptual response based on signals that the nervous system accepts and transmits from the environment (see Millan, 1999, Prog. Neurobiol. 57: 1-164). Harmful stimuli such as heat and contact give rise to specialized skin sensory receptors for signaling to the central nervous system ("CNS"). This process is called nociception, and the peripheral sensory neurons that mediate it are trauma receptors. Depending on the strength of the signal from the trauma receptor and the extraction and generation of that signal by the CNS, a person may or may not feel painful stimuli. When the perception of pain is appropriately determined for the intensity of the stimulus, pain provides its intended preventive action. However, certain types of muscle damage cause a phenomenon known as hyperalgesia or pronociception, which perceives relatively harmless stimuli as strong pain, because the threshold of human pain is lowered. Both inflammation and nerve damage can induce hyperalgesia. People with inflammatory symptoms such as sunburn, osteoarthritis, colitis, heartitis, dermatitis, myositis, neuritis, collagen vascular diseases (including rheumatoid arthritis and lupus) often experience increased sensory pain. Similarly, trauma, surgery, amputation, abscess, burning pain, collagen vascular disease, dehydration disease, trigeminal neuralgia, cancer, chronic alcoholism, seizures, hypothalamic syndrome, diabetes, herpes infection, acquired immunodeficiency syndrome ("AIDS") Toxins and chemotherapy cause nerve damage that causes excessive pain.

As the mechanism by which trauma receptors introduce external signals under normal and hyperalgesia conditions is better understood, processes involving hyperalgesia can be aimed at reducing the level of pain by inhibiting the lowering of pain thresholds. have.

Neurotrophic factors have been shown to play a prominent role in the transmission of physiological and pathological pain. NGF is found to be particularly important (see McMahon, 1996, Phil. Trans.R. Soc. Lond. 351: 431-40; and Apfel, 2000, The Clinical Journal of Pain 16: S7-S11). . Local and systemic administration of NGF has been shown to induce hyperalgesia and allodynia (see Lewin et al., 1994, Eur. J. Neurosci. 6: 1903-1912). Intravenous injection of NGF into the human body causes systemic myalgia, whereas topical administration not only causes hyperalgesia and pain at the injection site, but also has a systemic effect (see Apfel et al. 1998, Neurology 51: 695-702). . There is also evidence that symptoms with pain as the main feature are associated with endogenous NGF. For example, NGF is upregulated in dorsal root ganglion (DRG) Schwann cells for at least two months after it has been reported that peripheral nerve damage and increased levels are associated with animal models suffering from various arthritis (eg, Aloe et al. 1993, Growth Factors 9: 149-155). In humans, NGF levels are increased in synovial fluid obtained from patients with rheumatoid or other types of arthritis (see, eg, Aloe et al. 1992, Arthritis and Rheumatism 35: 351-355). In addition, antagonism of NGF function prevents hyperalgesia and allodynia in neuropathic and chronic inflammatory pain models. For example, systemic injection of NGF-binding neutralizing antibodies in neuropathic pain (eg, neural stem or spinal nerve ligation) animal models prevents allodynia and hyperalgesia (Ramer et al. 1999, Eur. J.). Neurosci. 11: 837-846; and Ro et al., 1999, Pain 79: 265-274). Examples of anti-NGF antibodies known in the art include, for example, PCT Publication Nos. WO 01/78698, WO 01/64247, WO 02/096458 and WO 2004/032870; US Patent Nos. 5,844,092, 5,877,016 and 6,153,189; 2000, Hybridoma 19: 215-227; 1993, Cell. Mol. Biol. The antibody described in 13: 559-568; And GenBank Accession Nos. U39608, U39609, L17078 or L17077.

Clearly, it is necessary to target new, safe and effective treatments for pain, especially small molecule mediators or exacerbators 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 present invention provides monoclonal antibodies that bind to nerve growth factor (NGF). Preferably, the monoclonal antibody is a human monoclonal antibody, which antibody is useful for neutralizing the biological activity of NGF and improving the effects of NGF-mediated pain response. The invention also provides cells which produce the monoclonal antibodies of the invention and most preferably secrete said antibodies into cell culture medium. The antibodies of the present invention are not only used to treat and manage pain, but also to treat neuropathic and inflammatory pain-related responses.

The invention also relates to sequences of antibody Fc regions, and 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 A fusion protein comprising one or more of the sequences set forth in: 22 and SEQ ID NOs: 79-130. Such molecules can be prepared using the methods as described, for example, in International Patent Application Publication No. WO 00/24782, which is incorporated herein by reference. Such molecules can be expressed, for example, in mammalian cells (eg, Chinese hamster ovary cells) or bacterial cells (eg, E. coli cells).

In certain aspects, the present invention provides antibodies comprising heavy and light chains, preferably monoclonal antibodies, most preferably human antibodies and human monoclonal antibodies, wherein the heavy chains are SEQ ID NO: 2, SEQ ID NO: 4 or an amino acid sequence set forth in SEQ ID NO: 6, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment, wherein the heavy chain variable region is an amino acid sequence set forth in SEQ ID NO: 10, or an antigen thereof -Immunoglobulin fragments that are binding fragments or are immunologically functional. Preferably, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 4.

In certain aspects, the present invention provides antibodies, preferably human antibodies, more preferably monoclonal antibodies, most preferably human monoclonal antibodies, comprising heavy and light chains, which heavy chains are incorporated herein by reference. Heavy chain constant regions selected from among IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE heavy chain constant regions or all allelic variants thereof (Kabat et al. 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human) Service, NIH Publication No. 91-3242), wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 10, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. Include. Preferably, the antibody of the present invention comprises the amino acid sequence of the IgG2 heavy chain constant region set forth in SEQ ID NO: 4, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the present invention provides antibodies, preferably human antibodies, more preferably monoclonal antibodies, most preferably human monoclonal antibodies, comprising heavy and light chains, wherein the light chains are described in SEQ ID NO: 8 An immunoglobulin fragment, or an immunologically functional immunoglobulin fragment thereof, wherein the light chain variable region is an amino acid sequence set forth in SEQ ID NO: 12, or an antigen-binding fragment thereof, or immunologically Functional immunoglobulin fragments.

In certain aspects, the antibodies of the invention comprise heavy and light chains, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 10, or an antigen-binding fragment thereof, or an immunologically functional immunoglobulin fragment. . In another aspect, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, or an antigen-binding fragment thereof, or an immunologically functional immunoglobulin fragment. In an additional aspect, the heavy chain comprises the amino acid sequence set forth in any one of SEQ ID NO: 14, SEQ ID NO: 18 and SEQ ID NO: 20, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. . In a still further aspect, the light chain comprises an amino acid sequence set forth in any one of SEQ ID NO: 16, SEQ ID NO: 20 and SEQ ID NO: 24, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. Include.

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

The invention also provides an isolated human antibody that specifically binds to NGF, comprising (a), (b), (c) or (d): (a) as described in SEQ ID NO: 79 A heavy chain having a heavy chain variable region comprising an amino acid sequence, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 80, or an antigen-binding fragment thereof, or immunological A light chain having a light chain variable region comprising a functional immunoglobulin fragment; (b) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 81, or an antigen-binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 82, Or a light chain having a light chain variable region comprising an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment; (c) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 83, or an antigen-binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 84, Or a light chain having a light chain variable region that is an antigen-binding fragment thereof or comprises an immunologically functional immunoglobulin fragment; Or (d) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 86, or an antigen-binding fragment thereof or comprising an immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 87 Or a light chain having a light chain variable region comprising an immunoglobulin fragment or an antigen-binding fragment thereof.

In certain aspects, the invention also provides antibodies that specifically bind to NGF, comprising heavy and light chains, wherein the heavy chains are at least 75%, preferably 80%, more than the amino acid sequence set forth in SEQ ID NO: 10 Preferably, 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% have the same amino acid sequence A heavy chain variable region comprising: the light chain comprising at least 80%, preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94 with the amino acid sequence set forth in SEQ ID NO: 12 %, 95%, 96%, 97%, 98%, most preferably about 99% comprise light chain variable regions comprising a sequence having the same amino acid sequence.

The present invention also provides an antibody that specifically binds to NGF, wherein the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 14, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment, The light chain comprises an aminoglobin sequence set forth in SEQ ID NO: 16, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the invention provides antibodies that specifically bind to NGF, including heavy and light chains, wherein the heavy chains are in any one of SEQ ID NO: 14, SEQ ID NO: 18 and SEQ ID NO: 22. At least 75%, preferably 80%, more preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the amino acid sequence described %, Most preferably about 99% comprises a heavy chain variable region comprising a sequence having the same amino acid sequence, wherein the light chain is at least 80%, preferably at least 85%, more preferably an amino acid sequence set forth in SEQ ID NO: 16 At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, most preferably about 99% comprises a light chain variable region comprising a sequence having the same amino acid sequence do.

The invention also provides single chain antibodies, single chain Fv antibodies, F (ab) antibodies, F (ab) 'antibodies and (Fab') ₂ antibodies.

In certain aspects, the present invention provides a light chain comprising an aminoglobin sequence set forth in SEQ ID NO: 16, or an antigen-binding fragment thereof, or an immunologically functional immunoglobulin fragment.

The present invention also provides a heavy chain comprising an amino acid sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 18 and SEQ ID NO: 22, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. To provide.

The invention also relates to an isolated human antibody that specifically binds to NGF, comprising the following (a) and (b): (a) human heavy chain outline region, human heavy chain CDR1 region, human heavy chain CDR2 region And human heavy chain CDR3 region; And (b) human light chain outline region, human light chain CDR1 region, human light chain CDR2 region and human light chain CDR3 region. In certain aspects, the human heavy chain CDR1 region is the heavy chain CDR1 region of the monoclonal antibody (mAb) named 4D4 described in SEQ ID NO: 22, and the human light chain CDR1 region is the light chain CDR1 of mAb 4D4 described in SEQ ID NO: 24. Area. In another aspect, the human heavy chain CDR2 region is the heavy chain CDR2 region of mAb 4D4 described in SEQ ID NO: 18 and the human light chain CDR2 region is the light chain CDR2 region of mAb 4D4 described in SEQ ID NO: 20. In another aspect, the human heavy chain CDR3 region is the heavy chain CDR3 region of mAb 4D4 described in SEQ ID NO: 14 and the human light chain CDR3 region is the light chain CDR3 region of mAb 4D4 described in SEQ ID NO: 16.

The invention also provides isolated human antibodies that specifically bind to nerve growth factors, including heavy and light chains, wherein the heavy chains are SEQ ID NO: 10, SEQ ID NO: 79, SEQ ID NO: 81, SEQ A heavy chain variable region comprising an amino acid sequence set forth in ID NO: 83, SEQ ID NO: 85 or SEQ ID NO: 87, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

The invention also provides isolated human antibodies that specifically bind to NGF, including heavy and light chains, wherein the light chains are 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 thereof Or a light chain variable region comprising an immunologically functional immunoglobulin fragment.

Antibodies of the invention are characterized by the ability to antagonize at least one ex vivo and / or in vivo activity associated with an NGF polypeptide. Preferably, the present invention provides an isolated anti-human NGF human antibody that binds with high affinity to an NGF polypeptide, wherein the antibody binds to a human NGF polypeptide and is measured at about 50 × 10 ⁻¹² M as measured using KinExA. Or less than a dissociation constant (K _D ) or isolated from human NGF polypeptides or inhibits NGF induced survival in an in vitro neutralization assay with an IC ₅₀ of about 1 × 10 ⁻⁸ M or less.

In a preferred embodiment, the invention provides an isolated anti-human NGF human antibody having the following characteristics (a), (b) and (c): (a) about 1 × 10 ⁻⁹ M or less IC ₅₀ inhibits NGF induced survival in in vitro neutralization assay; (b) has a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (c) has a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

The present invention also provides isolated human antibodies, or antigen-binding fragments thereof, or immunologically functional immunoglobulin fragments that specifically bind to NGF with high affinity, wherein (a), (b) and ( c) said antibody or fragment comprises a heavy chain variable region containing about 1 × 10 ^-9 or less are separated from the human NGF polypeptide with a K _D of about 1 × 10 ^-8 M in vivo or IC ₅₀ of less Neutralize human NGF bioactivity in an external standard assay: (a) CDR1 region a ¹ a ² a ³ a ⁴ a ⁵ comprising an amino acid sequence of the formula a ¹ is a polar hydrophilic amino acid residue; a ² is an aromatic amino acid residue; a ³ is an aliphatic, polar hydrophobic, aromatic amino acid residue; a ⁴ is a neutral hydrophobic or aliphatic amino acid residue; a ⁵ is an aliphatic or polar hydrophilic amino acid residue; (b) a CDR2 region b ¹ b ² b ³ b ⁴ b ⁵ b ⁶ b ⁷ b ⁸ b ⁹ b ¹⁰ b ¹¹ b ¹² b ¹³ b ¹⁴ b ¹⁵ b ¹⁶ b ¹⁷ comprising the amino acid sequence of the formula: b ¹ is an aliphatic, polar hydrophobic or aromatic amino acid residue; b ² is an aliphatic hydrophobic amino acid residue; b ³ is a polar hydrophilic or aromatic amino acid residue; b ⁴ is a polar hydrophilic, hydrophobic or aromatic amino acid residue; b ⁵ -b ⁹ are independently polar hydrophilic or aliphatic amino acid residues; b ¹⁰ Is a polar hydrophilic, aromatic or aliphatic amino acid residue; b ¹¹ is an aromatic or hydrophobic amino acid residue; b ¹² is an aliphatic hydrophobic or polar hydrophilic amino acid residue; b ¹³ is an aliphatic, hydrophobic or polar hydrophilic amino acid residue; b ¹⁴ and b ¹⁶ are independently polar hydrophilic amino acid residues; b ¹⁵ is an aliphatic or aromatic hydrophobic amino acid residue; b ¹⁷ is an aliphatic acidic amino acid residue; (c) CDR3 region c ¹ c ² c ³ c ⁴ c ⁵ c ⁶ c ⁷ c ⁸ c ⁹ c ¹⁰ c ¹¹ c ¹² c ¹³ c ¹⁴ c ¹⁵ c ¹⁶ c ¹⁷ c ¹ is absent or an aliphatic amino acid residue; c ² is absent or a polar hydrophilic or aromatic hydrophobic amino acid residue; c ³ and c ⁴ Are independently absent or are polar hydrophilic, aromatic hydrophobic or aliphatic amino acid residues; c ⁵ is absent or a polar hydrophilic, aliphatic or aromatic amino acid residue group; c ⁶ is absent or a polar hydrophilic or aliphatic amino acid residue; c ⁷ is a polar hydrophilic or aliphatic amino acid residue; c ⁸ is a polar hydrophilic, hydrophobic or aromatic amino acid residue; c ⁹ is a polar hydrophilic, aliphatic or aromatic hydrophobic amino acid residue; c ¹⁰ is a polar hydrophilic, aromatic hydrophobic or aliphatic hydrophobic amino acid residue; c ¹¹ -c ¹³ are independently polar hydrophilic or aromatic hydrophobic amino acid residues; c ¹⁴ is an aliphatic or aromatic hydrophobic amino acid residue; c ¹⁵ is a polar hydrophilic or neutral hydrophobic amino acid residue; c ¹⁶ is absent or a polar hydrophilic amino acid residue; c ¹⁷ is an aromatic hydrophobic or aliphatic hydrophobic amino acid residue.

In one aspect, a ¹ is a polar hydrophilic amino acid residue; a ² is an aromatic hydrophobic amino acid residue; a ³ is an aliphatic hydrophobic amino acid residue; a ⁴ is a neutral hydrophobic amino acid residue; a ⁵ is a polar hydrophilic amino acid residue; b ¹ is an aliphatic or aromatic amino acid residue; b ² is Ile; b ³ is a polar hydrophilic amino acid residue; b ⁴ is a polar hydrophilic or aromatic amino acid residue; b ⁵ -b ⁹ are independently polar hydrophilic or aliphatic amino acid residues; b ¹⁰ is an aliphatic amino acid residue; b ¹¹ is Tyr; b ¹² is an aliphatic hydrophobic amino acid residue; b ¹³ is an aliphatic or polar hydrophilic amino acid residue; b ¹⁴ and b ¹⁶ are independently polar hydrophilic amino acid residues; b ¹⁵ is an aliphatic hydrophobic amino acid residue; b ¹⁷ is an aliphatic acidic amino acid residue; c ¹ is absent or an aliphatic amino acid residue; c ² is absent or a polar hydrophilic or aromatic hydrophobic amino acid residue; c ³ and c ⁴ are not present independently or are polar hydrophilic, aromatic hydrophobic or aliphatic amino acid residues; c ⁵ is absent or a polar hydrophilic amino acid residue; c ⁶ is absent or a polar hydrophilic or aliphatic amino acid residue; c ⁷ is a polar hydrophilic or aliphatic amino acid residue; c ⁸ is a polar hydrophilic, hydrophobic or aromatic amino acid residue; c ⁹ is a polar hydrophilic, aliphatic or aromatic hydrophobic amino acid residue; c ¹⁰ is a polar hydrophilic, aromatic hydrophobic or aliphatic hydrophobic amino acid residue; c ¹¹ -c ¹³ are independently polar hydrophilic or aromatic hydrophobic amino acid residues; c ¹⁴ is an aliphatic or aromatic hydrophobic amino acid residue; c ¹⁵ is a polar hydrophilic or neutral hydrophobic amino acid residue; c ¹⁶ is absent or a polar hydrophilic amino acid residue; c ¹⁷ is an aromatic hydrophobic or aliphatic hydrophobic amino acid residue.

In certain aspects, a ¹ is Ser, Asp or Thr; a ² is Tyr; a ³ is Ala, Ser, Trp or Gly; a ⁴ is Met or Ile; a ⁵ is His, Gly or Asn; b ¹ is Tyr, Gly, Ile or Asp; b ² is Ile; b ³ is Ser, Thr, Tyr or Asn; b ⁴ is Trp, Arg or Pro; b ⁵ is Ser, Asn or Gly; b ⁶ is Ser, Arg, Asp or Gly; b ⁷ is Ser, His or Gly; b ⁸ is Ser, Ile, Asp or Thr; b ⁹ is Leu, Ile or Thr; b ¹⁰ is Gly, Lys or Phe; b ¹¹ is Tyr; b ¹² is Ala or Ser; b ¹³ is Asp, Gly or Pro; b ¹⁴ is Ser; b ¹⁵ is Val or Phe; b ¹⁶ is Lys or Gln; b ¹⁷ is Gly; c ¹ is absent or an aliphatic amino acid residue; c ² is absent or Tyr; c ³ and c ⁴ are not present independently or are Tyr, Asn, Val or Glu; c ⁵ is absent or is Ser, Gly or Trp; c ⁶ is absent or is Ser, Gly, Glu or Leu; c ⁷ is Gly, Arg or Asp; c ⁸ is Trp, Pro, Ser or Thr; c ⁹ is His, Gly or Tyr; c ¹⁰ is Val, Tyr or Arg; c ¹¹ -c ¹³ are independently Ser, Phe, Tyr, Asp or Asn; c ¹⁴ is Phe, Val or Gly; c ¹⁵ is Met or Asp; c ¹⁶ is absent or is Asp or Asn; c ¹⁷ is Tyr or Val.

In another particular aspect, a ¹ is Ser or Asp; a ² is Tyr; a ³ is Ala or Ser; a ⁴ is Met or Ile; a ⁵ is His or Asn; b ¹ is Tyr or Gly; b ² is Ile; b ³ is Ser, Thr, Tyr or Asn; b ⁴ is Trp, Arg or Pro; b ⁵ is Ser or Asn; b ⁶ is Ser or Arg; b ⁷ is His or Gly; b ⁸ is Ile or Thr; b ⁹ is Leu, Ile or Thr; b ¹⁰ is Gly or Phe; b ¹¹ is Tyr; b ¹² is Ala or Ser; b ¹³ is Asp or Gly; b ¹⁴ is Ser; b ¹⁵ is Val or Phe; b ¹⁶ is Lys or Gln; b ¹⁷ is Gly; c ¹ is absent or Gly; c ² is absent or Tyr; c ³ and c ⁴ are not present independently or are Tyr, Gly or Val; c ⁵ is absent or Ser; c ⁶ is Ser or Gly; c ⁷ is Gly or Arg; c ⁸ is Trp or Pro; c ⁹ is His, Gly or Tyr; c ¹⁰ is Val or Tyr; c ¹¹ -c ¹³ are independently Ser, Tyr, Phe or Asp; c ¹⁴ is Phe or Val; c ¹⁵ is Met or Asp; c ¹⁶ is absent or Asp; c ¹⁷ is Tyr or Val.

Another specific aspect is the following (a), (b), (c), (d), (e) or (f): (a) heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 22 CDR2 has the amino acid sequence set forth in SEQ ID NO: 18, and heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 14; (b) heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 92, heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 93, and heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 94; (c) heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 98, heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 99, and heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 100; (d) the heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 104, the heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 105, and the heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 106; (e) heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 110, heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 111, and heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 112; Or (f) heavy chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 116, heavy chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 117, and heavy chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 118.

The present invention also provides isolated human antibodies, or antigen-binding fragments thereof, or immunologically functional immunoglobulin fragments that specifically bind to NGF, wherein the antibody or fragment comprises (a), (b) and (c) the light chain comprises a light chain variable region, containing approximately 1 × 10 ^-9 or less are separated from the human NGF polypeptide with K _D of about 1 × 10 ^-8 M, or other than a living body with IC ₅₀ standard Neutralizes human NGF bioactivity in the assay: (a) CDR1 region a ¹ a ² a ³ a ⁴ a ⁵ a ⁶ a ⁷ a ⁸ a ⁹ a ¹⁰ a ¹¹ a ¹² a ¹ is a polar hydrophilic amino acid residue; a ² , a ¹¹ and a ¹² are independently aliphatic or hydrophobic amino acid residues; a ³ , a ⁵ , a ⁷ and a ⁸ are independently aliphatic, polar hydrophilic or hydrophobic amino acid residues; a ⁴ is a polar hydrophilic amino acid residue; a ⁶ is an aliphatic or hydrophobic amino acid residue; a ⁹ is absent or an aliphatic or polar hydrophilic amino acid residue; a ¹⁰ is an aliphatic, aromatic or hydrophobic amino acid residue; (b) a CDR2 region b ¹ b ² b ³ b ⁴ b ⁵ b ⁶ b ⁷ comprising amino acid residues of the formula: wherein b ¹ is an aliphatic, polar hydrophobic or hydrophobic amino acid residue; b ² is an aliphatic or hydrophobic amino acid residue; b ³ and b ⁴ are independently polar hydrophilic, aliphatic or hydrophobic amino acid residues; b ⁵ is a polar hydrophilic or aliphatic hydrophobic amino acid residue; b ⁶ is a polar hydrophilic or aliphatic hydrophobic amino acid residue; b ⁷ is a polar hydrophilic amino acid residue; (c) CDR3 region c ¹ c ² c ³ c ⁴ c ⁵ c ⁶ c ⁷ c ⁸ c ⁹ c ¹⁰ c ¹¹ c ¹² c ¹³ c ¹⁴ c ¹⁵ c ¹⁶ c ¹⁷ c ¹ and c ² are independently polar hydrophilic amino acid residues; c ³ is a polar hydrophilic, aliphatic or hydrophobic amino acid residue; c ⁴ , c ⁵ and c ⁶ are independently aliphatic, polar hydrophilic or hydrophobic amino acid residues; c ⁷ is absent or a polar hydrophilic or aliphatic hydrophobic amino acid residue; c ⁸ is a polar hydrophilic or hydrophobic amino acid residue; c ⁹ is a polar hydrophilic amino acid residue.

In one aspect, a ¹ , a ³ , a ⁴ , a ⁷ and a ⁸ are independently polar hydrophilic amino acid residues; a ² , a ⁶ , a ¹¹ and a ¹² are independently aliphatic hydrophobic amino acid residues; a ⁵ is a polar hydrophilic or aliphatic amino acid residue; a ⁹ is absent or an aliphatic or polar hydrophilic amino acid residue; a ¹⁰ is an aliphatic or aromatic amino acid residue; b ¹ is an aliphatic, polar hydrophobic or hydrophobic amino acid residue; b ² is an aliphatic hydrophobic amino acid residue; b ³ , b ⁴ and b ⁷ are independently polar hydrophilic amino acid residues; b ⁵ and b ⁶ are independently polar hydrophilic or aliphatic hydrophobic amino acid residues; c ¹ and c ² are independently polar hydrophilic amino acid residues; c ³ is a polar hydrophilic, aliphatic or hydrophobic amino acid residue; c ⁴ , c ⁵ and c ⁶ are independently aliphatic, polar hydrophilic or hydrophobic amino acid residues; c ⁷ is absent or an aliphatic hydrophobic amino acid residue; c ⁸ is a hydrophobic amino acid residue; c ⁹ is a polar hydrophilic amino acid residue.

In certain aspects, a ¹ , a ³ , a ⁴ and a ⁷ are Arg, Ser, Gln and Ser, respectively; a ² is Ala; a ⁵ is Gly or Ser; a ⁸ is Ser or Ile; a ⁹ is absent or is Ser or Gly; a ¹⁰ is Ala, Tyr, Trp or Phe; b ¹ is Asp, Gly, Ala or Val; b ² and b ³ are Ala and Ser, respectively; b ⁴ is Ser or Asn; b ⁵ is Leu or Arg; b ⁶ is Glu, Ala or Gln; b ⁷ is Ser or Thr; c ¹ and c ² are Gln; c ³ is Phe, Tyr, Arg or Ala; c ⁴ is Asn, Gly or Ser; c ⁵ is Ser or Asn; c ⁶ is Tyr, Ser, Trp or Phe; c ⁷ is absent or is Pro or His; c ⁸ is Leu, Trp, Tyr or Arg; c ⁹ is Thr.

In another specific aspect, a ¹ , a ² , a ³ , a ⁴ and a ⁷ are Arg, Ala, Ser, Gln and Ser, respectively; a ⁵ is Gly or Ser; a ⁸ is Ser or Ile; a ⁹ is absent or is Ser or Gly; a ¹⁰ is Ala or Tyr; b ¹ is Asp or Gly; b ² and b ³ are Ala and Ser, respectively; b ⁴ is Ser or Asn; b ⁵ is Leu or Arg; b ⁶ is Glu, Ala or Gln; b ⁷ is Ser or Thr; c ¹ And c ² is Gln; c ³ is Phe, Tyr, Arg or Ala; c ⁴ is Asn, Gly or Ser; c ⁵ is Ser or Asn; c ⁶ is Tyr, Ser, Trp or Phe; c ⁷ is absent or is Pro or His; c ⁸ is Leu, Trp, Tyr or Arg; c ⁹ is Thr.

Another particular aspect is the following (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j): (a) Light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 24, light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 20, and light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 16; (b) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 95, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 96, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 97; (c) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 101, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 102, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 103; (d) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 107, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 108, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 109; (e) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 113, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 114, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 115; (f) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 119, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 120, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 121; (g) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 122, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 123, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 124; (h) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 125, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 126, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 127; (i) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 128, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 129, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 130; Or (j) the light chain CDR1 has the amino acid sequence set forth in SEQ ID NO: 132, the light chain CDR2 has the amino acid sequence set forth in SEQ ID NO: 133, and the light chain CDR3 has the amino acid sequence set forth in SEQ ID NO: 134.

The invention also inserts a polynucleotide sequence encoding a novel anti-human NGF human antibody, a vector comprising said polynucleotide sequence encoding an anti-human NGF human antibody, a polynucleotide encoding an anti-human NGF human antibody To a host cell transformed with the vector, a formulation comprising an anti-human NGF human antibody, and a method of making and using the same.

The present invention also provides a method for detecting NGF levels in a biological sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof of the invention. Anti-NGF antibodies of the invention are competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays and enzyme-linked immunosorbent assays (ELISAs) for detecting and quantifying NGF (Sola, 1987, Monoclonal Antibodies: A Manual of Techniques , pp. 147-158, CRC Press, Inc.). The antibody can bind NGF with an affinity suitable for the assay method to be used.

The invention also includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising at least one antibody of the invention, or an antigen-binding fragment thereof, or an immunologically functional immunoglobulin fragment as needed. It provides a method of treating diseases associated with increased production of NGF or increased sensitivity to NGF.

Certain preferred embodiments of the present invention will become apparent as described in more detail in the following specific preferred embodiments and claims.

1 depicts a graph showing that NGF activity is neutralized upon DRG neuronal basal neutralization biological assay by 4D4 monoclonal antibody purified from hybridoma conditioned media.

FIG. 2 depicts a graph showing neutralization of VR1 expression and NGF activity stimulated by human NGF activity in DRG neuronal basal neutralization biological assay by anti-NGF monoclonal antibody (4D4) purified from hybridoma conditioned media. will be.

Figure 3 shows the recombinant anti-NGF 4D4 monoclonal antibody when the recombinant anti-NGF 4D4 monoclonal antibody is expressed as IgG1 or IgG2 or in cells grown in a rotator culture (R) or stirred flask (S). A graph showing that NGF activity is neutralized upon DRG neuronal basal neutralization biological assay by transient expression of raw antibodies.

4 shows an arrangement of neurotrophin sequences. Numbers and secondary structural elements on the sequence refer to mature human NGF. Conserved residues are marked with * and regions with low sequence homology are darkened. Human NGF is SEQ ID NO: 135; Mouse NGF is SEQ ID NO: 136; BDNF is SEQ ID NO: 137; NT3 is SEQ ID NO: 138.

5 shows 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) shows anti-NGF CDR1 heavy chain sequence and% identity to antibodies.

6 shows 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) Anti-NGF CDR2 heavy chain sequence and% identity to antibodies are shown.

7 shows 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) shows anti-NGF CDR3 heavy chain sequence and% identity to antibodies.

8 shows 14D10 (SEQ ID NO: 119), 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) antibody (4G6a is shown as 20031028340 in the figure). Anti-NGF CDR1 for 4G6b is described as 20031028351 in the drawings; 4G6c is described as 20031071526 in the drawings; 4G6d is described as 20031028344 in the drawings; 4G6e is described as 20031000528 in the drawings). Light chain arrangement and percent identity are shown.

9 shows 14D10 (SEQ ID NO: 123), 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) antibody (4G6a is 20031028340 in the figure) Anti-NGF CDR2; 4G6b is described as 20031028351 in the drawings; 4G6c is described as 20031071526 in the drawings; 4G6d is described as 20031028344 in the drawings; 4G6e is described as 20031000528 in the drawings). Light chain arrangement and percent identity are shown.

10 shows 14D10 (SEQ ID NO: 123), 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) antibody (4G6a is shown as 20031028340 in the figure). Anti-NGF CDR3; 4G6b is described as 20031028351 in the drawing; 4G6c is described as 20031071526 in the drawing; 4G6d is described as 20031028344 in the drawing; 4G6e is described as 20031000528 in the drawing). Light chain arrangement and percent identity are shown.

11 shows 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) antibody (4G6a is 20031028340 in the figure) 4G6b is described as 20031028351 in the drawing; 4G6c is described as 20031071526 in the drawing; 4G6d is described as 20031028344 in the drawing; 4G6e is described as 20031000528 in the drawing). The arrangement and percent identity are shown.

Figure 12 shows 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) Anti-NGF heavy chain sequence and identity (%) for antibody.

The headings of sections used herein are for illustrative purposes only and are not intended to be limiting to the headings described. All references disclosed herein are expressly incorporated herein by reference for all purposes.

Justice

Conventional techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally performed in the art or as described herein. The following techniques and methods can be performed generally as described in the methods well known in the art and in the various general, more specific references described and discussed throughout this specification. See, eg, 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. See also. Except where specific meaning is intended, the nomenclature used in connection with the analytical chemistry, synthetic organic chemistry and pharmaceutical chemistry and pharmaceutical chemistry described herein and laboratory methods and techniques in the chemistry field will be appreciated by those skilled in the art. It is well known and commonly used. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and transportation, and treatment of patients.

Except as otherwise indicated, as used herein, the following terms are understood to have the following meanings: The terms "biological characteristics", "biological characteristics" and "activity" in connection with the antibodies of the invention As used herein interchangeably, the term refers to epitope affinity and specificity (eg, anti-human NGF human antibodies that bind human NGF), the ability to antagonize the activity of a targeting polypeptide (eg, NGF Activity), in vivo stability of the antibody and immunogenic properties of the antibody. Other biological properties or characteristics of the antibodies that can be recognized in the art include, for example, cross-reactivity (ie, cross-reactivity with non-human homologs or other proteins or tissues of a generally targeted polypeptide) and mammalian cells. The ability to preserve the high expression level of the protein. The above-described properties or characteristics are different, including ELISAs, competitive ELISAs, surface plasmon resonance assays, in vitro and in vivo neutralization assays (see, eg, Example 2), and humans, primates or any other circles as needed. Observation or measurement can be made using techniques known in the art, including but not limited to immunohistochemistry with tissue sections obtained from the original. Specific activity and biological properties of anti-human NGF human antibodies are described in more detail in the Examples below.

As used herein, the term “isolated polynucleotide” means a polynucleotide of genome, cDNA or synthetic origin, or some combination thereof, wherein polynucleotides separated according to their origin are (1) the entirety of polynucleotides. Or unrelated to some, the "isolated polynucleotide" is found in nature, (2) binds to a polynucleotide that is not bound in nature, or (3) as part of a long sequence It is not naturally occurring.

As used herein, the term “isolated protein” refers to (1) a title protein in which at least some other proteins are generally found with the title protein, and (2) other proteins of the same origin, e.g. Title protein liberated by (3) the title protein expressed in cells of different species, (4) the title protein isolated from at least about 50% of polynucleotides, lipids, carbohydrates or other substances that bind in nature, (5) Title proteins that are not bound in nature by covalent or non-covalent interactions with portions of the protein that bind to "isolated proteins," (6) covalent or non-covalent interactions with polynucleotides that do not bind in nature. The title protein that is actionably bound, or (7) natural foot The title refers to a protein that is not enemy. Such isolated proteins are encoded by genomic DNA, cDNA, mRNA or other RNA, or combinations thereof of synthetic origin. Preferably, the isolated protein is substantially free of proteins or polypeptides or other contaminants found in the natural environment that would violate its use (therapeutic, diagnostic, prophylactic, research, or otherwise). It is advantageous.

An “isolated” antibody is an antibody that is identical to and separated from and / or recovered from a component of its natural environment. Contaminating components of the antibody's natural environment are materials that violate the diagnostic or therapeutic use of the antibody, which includes enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) purified to at least 95% by weight, most preferably at least 99% by weight of the antibody as determined by the Lowry method, or (2) an N-terminal or internal amino acid using a rotary cup sequencing machine. At least 15 residues of the sequence are purified to a suitable degree or (3) purified for homogeneity by SDS-PAGE under reduced or non-reduced conditions using Coomassie blue or preferably silver dyes. Since at least one component of the antibody's natural environment is not present, the isolated antibody includes the antibody in situ within recombinant cells.

The term “polypeptide” or “protein” is one of a natural protein, ie, a molecule having a sequence of naturally occurring and specifically non-recombinant cells, or a protein produced by a genetically engineered cell or recombinant cell, or a natural sequence Or a molecule having more than one amino acid deleted, added and / or substituted, and the term also encompasses molecules having the amino acid sequence of a natural protein. The terms "polypeptide" and "protein" specifically include sequences in which one or more amino acids of an anti-NGF antibody, or anti-NGF antibody, have been deleted, added and / or substituted.

The term "polypeptide fragment" means a polypeptide having an amino-terminal deletion, a carboxy-terminal deletion and / or an internal deletion. In certain embodiments, the fragment is at least 5 to about 500 amino acids in length. In certain embodiments, it is apparent that the fragment has 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 that include a binding domain. In the case of an anti-NGF antibody, useful fragments include, but are not limited to, CDR regions, variable domains of heavy or light chains, portions of antibody chains or variable regions thereof comprising two CDRs, and the like.

The term “specific binder” refers to a natural or non-natural 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 that binds to NGF" means a specific binding agent that specifically binds to all parts of NGF. In certain embodiments, the specific binding agent that binds to NGF is an antibody that specifically binds to NGF.

The term “immunologically functional immunoglobulin fragment” as used herein means a polypeptide fragment comprising at least the CDRs of an immunoglobulin heavy and light chain. The immunologically functional immunoglobulin fragments of the invention can bind antigens. In a preferred embodiment, the antigen is a ligand that specifically binds to a receptor. In such embodiments, the binding of the immunologically functional immunoglobulin fragments of the present invention interferes with the binding of the ligand to its receptor and the biological response by the ligand binding to the receptor. Preferably, the immunologically functional immunoglobulin fragments of the invention specifically bind to NGF. Most preferably, the fragment specifically binds to human NGF.

The term "naturally occurring" as used herein and applied to an object means that the thing can be found naturally. For example, a polypeptide or polynucleotide sequence that is isolated from a natural source and is present in an organism (including viruses) that is not intentionally modified by humans, is naturally occurring.

The term "practically combined" means associated components that enable them to perform their inherent functions under suitable conditions. For example, a regulatory sequence "practically linked" to a protein coding sequence is linked to express the protein coding sequence under conditions suitable for the transcriptional activity of the regulatory sequence.

The term "regulatory sequence" as used herein refers to a polynucleotide sequence that can affect the expression, processing or intracellular localization of the coding sequence to which the regulatory sequence is bound. The nature of these regulatory sequences depends on the host organism. In certain embodiments, the regulatory sequences of the prokaryotes comprise a promoter, a ribosomal binding site and a transcription termination sequence. In other specific embodiments, the regulatory sequences of the eukaryote include a promoter, a transcriptional enhancer sequence, a transcription termination sequence, and a polyadenylation sequence comprising one or multiple recognition sites for a transcription factor. In certain embodiments, a "regulatory sequence" comprises a leader sequence and / or a fusion partner sequence.

The term "polynucleotide" as referred to herein means a single-stranded or double-stranded nucleic acid polymer consisting of at least 10 nucleotides in length. In certain embodiments, nucleotides, including polynucleotides, are ribonucleotides or deoxyribonucleotides or modified forms of nucleotides of either type. Such modifications include ribose modifications such as bromouridine, arabinosides and 2 ', 3'-dideoxyribose, and phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodicele Internucleotide binding modifications such as noate, phosphoroanilothioate, phosphoroaniladeate and phosphoroamideate. The term “polynucleotide” specifically includes DNA in single stranded and double stranded forms.

The term “oligonucleotide” as referred to herein includes naturally occurring, mutated nucleotides bound together by naturally occurring / naturally occurring or non-naturally occurring oligonucleotide bonds. Oligonucleotides are generally a subset of polynucleotides that are single-stranded and comprise components having 200 nucleotides or more in length. In certain embodiments, the oligonucleotides are 10 to 60 nucleotides in length. In certain embodiments, oligonucleotides have 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides in length. Oligonucleotides are, for example, single stranded or double stranded to construct genetic mutants. Oligonucleotides of the invention are sense or antisense oligonucleotides associated with protein-coding sequences.

The term “naturally occurring nucleotide” includes deoxyribonucleotides and ribonucleotides. The term "mutated nucleotide" includes nucleotides having a mutated or substituted pull and the like. The term "oligonucleotide bond" refers to such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanilideate, phosphoroamideate and the like. Oligonucleotide bonds. See, eg, LaPlanche et al., 1986, Nucl., Incorporated herein by reference for all purposes. Acids Res., 14: 9081; Stec et al. 1984, J. Am. Chem. Soc., 106: 6077; Stein et al. 1988, Nucl. Acids Res., 16: 3209; Zon et al. 1991, Anti-Cancer Drug Design, 6: 539; Zon et al. 1991, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; US Patent No. 5,151,510 to Stec et al .; See Uhlmann and Peyman, 1990, Chemical Reviews, 90: 543. Oligonucleotides include detectable labels that can detect oligonucleotides or hybrids thereof.

The term "vector" includes nucleic acid molecules capable of carrying other nucleic acids bound to nucleic acid molecules. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be bound. Another type of vector is a viral vector, where additional DNA fragments can bind into the viral gene. Certain vectors can be autonomously replicated, for example, in host cells introduced into bacterial vectors and episomal mammalian vectors having bacterial initiation points. Other vectors (eg, non-episomal mammalian vectors), when introduced into a host cell, can be inserted into the host cell genome and replicate along the host genome. In addition, certain vectors are capable of expressing genes to which they are practically linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, recombinant vectors used in recombinant DNA techniques are often in the form of plasmids. In the context of the present invention, plasmids are most commonly used in the form of vectors, so "plasmid" and "vector" can be used interchangeably. However, the present invention includes other types of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) that can perform the same function.

The term "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector is introduced. Those skilled in the art will understand that these terms are intended to mean not only specific title cells but also descendants of such cells. Since certain variations can occur in successive generations by mutations or environmental influences, in fact such offspring are not identical to parent cells but are still included within the term “host cell” category as used herein. A wide variety of host expression systems include bacteria, yeast, baculovirus and mammalian expression systems (as well as phage display expression systems) and can be used to express the antibodies of the invention. An example of a suitable bacterial expression vector is pUC19. To recombinantly express the antibody, the host cell was transfected with one or more recombinant expression vectors carrying the DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, whereby the light and heavy chains were transferred to the host cell. It is expressed in, preferably the host cells are secreted into the cultured medium and the antibody can be recovered from the medium. 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories, Ausubel, F.M. Standard recombinant DNA methodology as described in US Protocol No. 4,816,397 to Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), and Boss et al. Yields antibody heavy and light chain genes and This gene is used to insert this gene into an expression vector and to introduce the vector into a host cell.

The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence, thereby expressing a selected gene of interest. The term includes, as long as the selected gene is present, a descendant of the parent cell, whether or not the descendant is morphologically or genetically identical to the parent cell.

The term “transduction” means that the gene is generally transferred from one bacterium to another by phage. "Transduction" also refers to the acquisition and delivery of eukaryotic cell sequences by retroviruses.

The term "transfection" means that the foreign DNA or exogenous DNA is taken up by the cell, and the cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art and described herein. See, eg, Graham et al. 1973, Virology 52: 456; 2001, MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratories of Sambrook et al .; Davis et al. 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; And Chu et al., 1981, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA components into suitable host cells.

As used herein, the term “transformation” means that the genetic properties of the cell are changed, and the cell is transformed when the cell is modified to contain new DNA. For example, a cell is transformed when the cell is genetically modified from its natural state. After transfection or transduction, the transforming DNA can be physically synthesized into the cell's chromosome to be temporarily retained as an episomal component without being recombined or replicated with the cell's DNA or independently replicated as a polamide. . Cells are considered to be "stable transformed" when DNA replicates during cell division.

When used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, the term "naturally occurring" or "natural" refers to materials that are found in nature and are not manufactured by humans. Similarly, "non-naturally occurring" or "non-natural" as used herein means a material that is not found in nature and that is structurally modified or synthesized by humans.

The term “antigen” refers to a molecule or portion of a molecule that can be used in an animal to produce a molecule that can be bound by an optional binder such as an antibody and additionally an antibody that can bind to the epitope of the antigen. An antigen has one or more epitopes.

As is known in the art, the term “identity” refers to a relationship determined by comparing the sequence of two or more polypeptide molecules or a sequence between two or more nucleic acid molecules. As used herein, "identity" also means the degree of sequence relationship between a polypeptide or nucleic acid molecule, and optionally determines the match between strings of two or more nucleotides or two or more amino acid sequences. It is. "Identity" is a measure of the same percentage of match between gap arrangements recorded in a particular mathematical model or computer program (ie, "algorithm") that exists at any number and less than or equal to two sequences.

The term "similarity" is used in this field in a related concept that is not in contrast to "identity", but "similarity" means a measure of relevance, including identical matches and conservative substitution matches. If both polypeptide sequences have, for example, 10/20 identical amino acids and the rest are both non-conservative substitutions, the percentage of identity and similarity are both 50%. In the same embodiment, if there are five or more positions where conservative substitutions are present, the percentage of identity is 50%, but the percentage of similarity is 75% (15/20). Thus, if conservative substitutions are present, the percent similarity between the two polypeptides will be higher than the percent identity between the two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in the literature: COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A.M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D.W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A.M. and Griffin, H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al. 1988, SIAM J. Applied Math. 48: 1073; And 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press.

Preferred methods for determining identity were designed so that the sequences tested matched one another. Methods of determining identity are described as publicly available computer programs. Preferred computer program methods for determining identity between two sequences are described in the GCG program package including GAP (see Deevere et al., 1984, Nucl. Acid.Res. 12: 387; Genetics Computer Group, University of Wisconsin, Madison, Wisconsin, USA). Available), BLASTP, BLASTN and FASTA (see, 1990, J. Mol. Biol. 215: 403-410 to Altschul et al.). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (NCB / NLM / NIH Bethesda, Blast Manual, Altschul et al., MD 20894; 1990, Altschul et al., Supra). The well known Smith Waterman algorithm can also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences can only match short regions of two sequences, and even though there is no significant association between the two sequences in the full length, such small regions have very high sequence identity. Thus, in certain embodiments, the alignment method chosen (GAP program) spans at least 50 consecutive amino acids of the target polypeptide.

For example, using the computer algorithm GAP (obtained from the University of Wisconsin, Madison, Wisconsin, USA), two polypeptides to determine percent sequence identity were aligned so that their respective amino acids were optimally matched (algorithm). "Matched length" as determined by). In a particular embodiment, the gap opening penalty (calculated as 3 X average diagonal) as well as the control matrix such as PAM250 or BLOSUM62; "average diagonal" is the average of the diagonals of the control matrix used; "diagonal" is Score or number for each perfect amino acid match by a particular control matrix) and gap extension penalty (generally 1/10 times the gap opening penalty) can be used in conjunction with the algorithm. In certain embodiments, standard control matrices [Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5: 345-352 on PAM 250 control matrices; Henikoff et al. 1992, Proc. Natl. Acad. Sci USA 89: 10915-10919 may also be used in the algorithm.

In certain embodiments, the parameters for polypeptide sequence comparison include: Algorithm: Needleman et al., 1970, J. Mol. Biol. See 48: 443-453; Control matrix: Henikoff et al., 1992, BLOSUM 62, supra; Gap Penalty: 12 Gap Stretch Penalty: 4 Threshold of Similarity: 0. The GAP program is effective to perform with the above parameters. In certain embodiments, the aforementioned parameters are default parameters for polypeptide comparison (no penalty for terminal gap) using the GAP algorithm.

The term “homology” refers to the degree of similarity between protein or nucleic acid sequences. Homology information is useful for understanding the genetic relationship of a particular protein or nucleic acid species. Homology can be determined by alignment and comparison of sequences. In general, to determine amino acid homology, protein sequences were compared to databases of known protein sequences. Homologous sequences share a common functional identity along these sequences. A high degree of similarity or identity usually indicates homology, but a low degree of similarity or identity does not necessarily indicate a lack of homology.

Several methods can be used to compare amino acids from one sequence to amino acids of another sequence to determine homology. In general, the methods fall into two categories: (1) comparison of physical properties such as polarity, charge, and van der Waals volumes to generate a similarity matrix; And (2) comparison of possible substitutions of amino acids with all other amino acids in a sequence based on the observation of many protein sequences from known homologous proteins to generate a Point Accepted Mutation Matrix (PAM).

Program needles (EMBOSS packages) or stretchers, such as modules in the Vector NTI 9.0.0 software package, with omission parameters (for example, GAP penalty 5, GAP opening penalty 15, GAP extension penalty 6.6). (%) Can also be calculated using (EMBOSS package) or program alignment X.

As used herein, twenty conventional amino acids and their abbreviations are in accordance with conventional practice. See IMMUNOLOGY--A SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates: Sunderland, MA, 1991, which is hereby incorporated by reference for all purposes. Stereoisomers of twenty conventional amino acids (e.g., D-amino acids), non-natural amino acids such as α-, α-unsubstituted amino acids, N-alkyl amino acids, lactic acid and other nonconventional amino acids are also disclosed herein. Is a suitable component for the polypeptide. Examples of unusual amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoseline, N-acetylserine , N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used herein, according to standard conventions and conventions, the left direction is the amino terminal direction and the right direction is the carboxy terminal direction.

Naturally occurring residues can be classified into the following classes according to common side chain properties: 1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; 2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; 3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro; 4) aliphatic hydrophobicity: Ala, Ile, Leu, Val, Pro; 5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 6) acidic: Asp, Glu; 7) basic: His, Lys, Arg; 8) residues affecting chain origin: Gly, Pro; 9) aromatic: His, Trp, Tyr, Phe; And 10) aromatic hydrophobicity: Phe, Trp, Tyr.

Conservative amino acid substitutions include the replacement of a member of one of the classes with another member of the same class. Conservative amino acid substitutions include non-naturally occurring amino acid residues, which generally include those by chemical peptide synthesis rather than those synthesized in the biological world. Such amino acids include peptidomide and other reverse phase amino acid components.

Non-conservative substitutions include the replacement of one member of a class with a member of another class. Such substituted residues may be introduced into regions of a human antibody that are homologous to non-human antibodies or into non-homologous regions of a molecule.

In substitutions according to certain embodiments, hydropathic indexes of amino acids are contemplated. Each amino acid is determined hydrotherapy values based on its hydrophobicity and charge characteristics. This figure is as follows: 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); Glutamate (-3.5); Glutamine (-3.5); Aspartic acid salts (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The importance of amino acid hydrotherapy values that confer interactive biological function on proteins is understood in the art (see, eg, Kyte et al. 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids may be substituted with other amino acids having similar hydrotherapy values or scores and have similar biological activity. In certain embodiments, substitutions based on hydrotherapy values include substitution of amino acids with hydrotherapy values within ± 2. In certain embodiments, those having numerical values within ± 1 are included, and in certain embodiments, those within ± 0.5 are also included.

It is understood in the art that analogous amino acids are effectively substituted on the basis of hydrophilicity, in particular the biologically functional proteins or peptides resulting from such substitutions are used in immunological embodiments as described herein. In certain embodiments, the very local mean hydrophilicity of a protein regulated by the hydrophilicity of adjacent amino acids is related to its immunogenicity and antigenicity, ie the biological properties of the protein.

Hydrophilicity values of amino acid residues are shown below: arginine (+3.0); Lysine (+3.0); Asparagine salts (+3.0 ± 1); Glutamate (+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). Substitution based on similar hydrophilicity values includes, in certain embodiments, substitutions of amino acids with hydrophilicity values within ± 2 and substitutions of amino acids with hydrophilicity values within ± 1 and amino acids within ± 0.5. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity values. Such regions are referred to as "epitope core regions".

Typical amino acid substitutions are shown in Table 1.

Those skilled in the art can determine suitable variants of the polypeptides as described herein using well known techniques. In certain embodiments, those skilled in the art can identify appropriate regions of the molecule that are substituted with target regions that do not disrupt the activity but do not significantly affect the activity. In other embodiments, the skilled person can identify residues and portions of molecules that are conserved among analogous polypeptides. In further embodiments, even regions that have a significant effect on biological activity or structure can be made conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

In addition, those skilled in the art can review structure-acting studies that identify residues of similar polypeptides that are important for activity or structure. Through this comparison, the skilled person can predict the importance of amino acid residues in a protein corresponding to amino acid residues that are important for the activity or structure of the analogous protein. Those skilled in the art can substitute chemically similar amino acids for amino acid residues predicted to be important amino acid residues.

Those skilled in the art can also analyze amino acid sequences and three-dimensional structures regarding the structure of analogous polypeptides. Those skilled in the art can use this information to predict the arrangement of amino acid residues of an antibody with respect to the three-dimensional structure of the antibody. In certain embodiments, such residues relate to important interactions with other molecules, so those skilled in the art can choose not to cause radical changes to amino acid residues expected at the surface of the protein. In addition, those skilled in the art can make experimental variants in which a single amino acid is substituted at each preferred amino acid residue. This variant can then be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about suitable variants. For example, if a change in a particular amino acid residue results in loss of activity, undesirably reduced or inadequate, then variants with this variation are preferred. That is, one of ordinary skill in the art can readily determine the amino acids so that additional variations do not occur alone or in combination with other mutations, based on information gathered from general experiments.

There have been many scientific presentations predicting secondary structure. Moult, 1996, Curr. Op. in Biotech. 7: 422-427; Chou et al. 1974, Biochemistry 13: 222-245; Chou et al. 1974, Biochemistry 113: 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, Biophys. See J. 26: 367-384. In addition, computer programs that are helpful in predicting secondary structure are generally available. One way of estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins with at least 30% sequence identity or at least 40% similarity often have similar structural topologies. Recent developments in protein structure databases (PDBs) have increased the predictability of secondary structures, including the number of possible folds in polypeptide or protein structures. Holm et al. 1999, Nucl. Acid. Res. See 27: 244-247. There is a limited number of folds in a particular polypeptide or protein and it is suggested that once the critical number of structures is established, the prediction of the structure will be more accurate (Brenner et al. 1997, Curr. Op. Struct. Biol. 7: 369-376).

Additional methods for estimating secondary structure are described in "threading" (Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87; see Sippl et al., 1996, Structure, 4: 15-19), "profile analysis." (See 1991, Science 253: 164-170 to Bowie et al., 1990, Meth. Enzym. 183: 146-159 to Gribskov et al., 1987, Proc. Nat. Acad. Sci. 84: 4355-4358 to Gribskov et al.) And "evolutionary linkage" (Holm 1999, supra; and Brenner 1997, supra).

In certain embodiments, antibody variants include glycosylation variants, wherein the number and / or type of glycosylation sites is mutated as compared to the amino acid sequence of the morphopeptide. In certain embodiments, protein variants comprise more or less N-linked glycosylation sites than natural proteins. The N-linked glycosylation site is characterized by the following sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residues represented by X are all amino acid residues except proline. Substitution of the amino acid residues that produce such sequences provides a possible new site to which N-linked carbohydrate chains can be added. Optionally, substitutions to remove these sequences eliminated existing N-linked carbohydrate chains. In addition, one or more N-linked glycosylation sites (generally naturally occurring sites) are removed to provide rearrangement of the N-linked carbohydrate chains resulting in one or more new N-binding sites. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted or substituted with other amino acids (eg, serine) corresponding to the parent amino acid sequence. Cysteine variants are useful when the antibody is folded back into a biologically active conformation after separating insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than natural proteins and generally have enough cysteine residues to minimize interactions due to unpaired cysteine.

In additional embodiments, antibody variants may comprise an antibody comprising a modified Fc fragment or a modified heavy chain constant region. Fc fragments or heavy chain constant regions representing “crystallized fragments” can be mutated to confer changed binding properties antibodies. See, eg, Burton and Woof, 1992, Advances in Immunology 51: 1-84; Ravetch and Bolland, 2001, Annu. Rev. Immunol. 19: 275-90; Shields et al., 2001, Journal of Biol. Chem 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100: 245-251; Medesan et al., 1998, Eur. J. Immunol. 28: 2092-2100, all of which are incorporated herein by reference. Such mutations may include substitutions, additions, deletions or any combination thereof, and are generally site specific using one or more mutagenic oligonucleotides according to methods known herein, as well as methods described herein. Produced by mutagenesis (see, eg, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001, Cold Spring Harbor, NY and Berger and Kimmel, METHODS IN ENZYMOLOGY). , Volume 152, Guide to Molecular Cloning Techniques, 1987, obtained from Academic Press, Inc., San Diego, California).

In certain embodiments, amino acid substitutions reduce (1) susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity to form protein complexes, and (4) Change binding affinity and / or (5) confer or modify physicochemical or functional properties on such polypeptides. In certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in naturally occurring sequences (in certain embodiments, part of a polypeptide outer domain that forms an intermolecular bond). In a preferred embodiment, conservative amino acid substitutions generally do not substantially alter the structural characteristics of the sequence (e.g., replacement amino acids do not destroy the helix that occurs in the sequence or other types of features characterized by the sequence). Does not collapse the secondary structure). Examples of polypeptide secondary structures and tertiary structures recognized in the art are, respectively, PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 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), Nature 354: 105.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of template peptides. Non-peptide compounds of this type are termed "peptide analogs" or "peptide analogs". For example, Fauchere, 1986, Adv., Incorporated herein by reference for all purposes. Drug Res. 15:29; Veber & Freidinger, 1985, TINS p. 392; And Evans et al., 1987, J. Med. Chem. See 30: 1229. Such compounds are often shaped with the help of computer molecular modeling. Peptide analogs structurally similar to therapeutically useful peptides exhibit similar therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to typical polypeptides such as human antibodies (i.e., those having biochemical or pharmacological activity), but are well known in the art and may be represented by -CH ₂ -NH-,- Optionally substituted with a bond selected from CH ₂ -S-, -CH ₂ -CH _2- , -CH = CH- (cis and trans), -COCH _2- , -CH (OH) CH ₂ -and -CH ₂ SO- One or more peptide bonds. In certain embodiments, systematic substitution of one or more amino acids of a consensus sequence having the same type of D-amino acid (eg, D-lysine instead of L-lysine) may be used to produce more stable peptides. Can be. In addition, methods known in the art (see Rizo & Gierasch, 1992, Ann. Rev. Biochem. 61: 387, incorporated herein by reference for all purposes), for example, to make the peptide cyclic By adding internal cysteine residues that can form intermolecular disulfide bonds, one can generate limited peptides that comprise a co-sequence or substantially identical co-sequence variants.

"Antibody" or "antibody peptide (class)" means a complete antibody or a binding fragment thereof that competes with a specific antibody that specifically binds. In certain embodiments, the binding fragments were prepared by recombinant DNA technology. In additional embodiments, binding fragments were prepared by enzymatic or chemical cleavage of the complete antibody. Binding fragments include, but are not limited to, F (ab), F (ab '), F (ab') ₂ , Fv, and single chain antibodies.

The term “heavy chain” includes all immunoglobulin polypeptides having variable region sequences sufficient to confer specificity for NGF. The term “light chain” includes all immunoglobulin polypeptides having variable region sequences sufficient to confer specificity for NGF. The full-length heavy chain contains the variable region domain V _H and three constant region domains C _H 1, C _H 2 and C _H 3. The V _H domain is at the amino-terminus of the polypeptide and the C _H 3 domain is at the carboxy-terminus. The term “heavy chain” as used herein includes the full length heavy chain and fragments thereof. The full length light chain comprises the variable region domain V _L and the constant region domain C _L. Like the heavy chain, the variable region domain of the light chain is at the amino-terminus of the polypeptide. The term "light chain" as used herein includes the full length light chain and fragments thereof. The F (ab) fragment comprises the variable regions of one light chain and C _H 1 and one heavy chain. The heavy chain of the F (ab) molecule cannot form disulfide bonds with other heavy chain molecules. F (ab ') fragments comprise one light chain and one heavy chain comprising both additional constant regions, C _H 1 and C _H 2 domains, and disulfide bonds between the chains forming the F (ab') ₂ molecule It can be formed between these two heavy chains. The Fv region includes the variable regions of the heavy and light chains but does not include the constant regions. Single chain antibodies are Fv molecules in which heavy and light chain variable regions are linked by a flexible linker to form a single chain polypeptide that forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication Nos. WO 88/01649 and US Pat. Nos. 4,946,778 and 5,260,203.

In certain embodiments, it is understood that "bispecific" or "bivalent" antibodies that differ from "multispecific" or "multifunctional" antibodies include binding sites with the same antigenic specificity.

In evaluating the antibody binding and specificity of the present invention, the amount of ligand that binds to the receptor to which the excess antibody binds is at least about 20%, 40%, 60%, 80%, 85% or more (among others, in vivo). When measured using a competitive binding assay), the antibody substantially inhibits binding of the ligand to the receptor.

"Neutralizing antibody" means an antibody molecule capable of substantially blocking or reducing the effector function of the target antigen to which the antibody binds. Thus, "neutralizing" anti-NGF antibodies can substantially reduce or block effector functions such as receptor binding of NGF and / or induction of cellular responses. “Substantially reduced” results in at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80% effector function of a target antigen (eg, human NGF), Even more preferably at least about 85%, most preferably at least about 90%.

The term "epitope" includes all determinants, preferably polypeptide determinants, capable of specifically binding an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, epitope determinants are specific three-dimensional structural properties and / or Has specific charge characteristics. Epitopes are regions of antigen to which antibodies bind. In certain embodiments, the antibody specifically binds to the antigen when the antibody selectively recognizes the target antigen in a complex mixture of proteins and / or polymers. In a preferred embodiment, when the equilibrium dissociation constant is ≤ 10 ^-8 M, more preferably the equilibrium dissociation constant is ≤ 10 ^-9 M and most preferably the dissociation constant is ≤ 10 ^-10 M, the antibody is specific for the antigen. As an enemy.

When two antibodies recognize the same or steric overlapping epitopes, the antibody binds to the "essentially identical epitope" as a control antibody. The most widely used and fast method for determining whether two antibodies bind to the same or steric overlapping epitopes is the competition assay, which can be formed in several different formats using labeled antigens or labeled antibodies. . In general, the ability of unlabeled antibodies to immobilize antigen to substrate and block binding of labeled antibodies was measured using radioactive or enzymatic labels.

As used herein, the term “formulation” means a chemical compound, a mixture of chemical compounds, a biological macromolecule or an extract made of a biological substance.

As used herein, the term “label” or “labeled” refers to, for example, radiolabeled amino acids or labeled avidin (eg, fluorescent markers, chemiluminescent, preferably detectable by optical or colorimetric methods). Binding to a detectable marker by binding an attachment to a polypeptide of a biotin component detectable with a marker or streptavidin comprising a detectable marker such as enzymatic activity. In certain embodiments, the label can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be conveniently used with the methods described herein. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ^{99 m} Tc, ¹¹¹ In, ¹²⁵ I , ¹³¹ I), fluorescent labels (eg, fluorescent isothiocyanates or FITC, rhodamine or lanthanide), enzymatic labels (eg, horseradish peroxidase, β-galactosidase, lucifer Recognition by heptane labels such as lase, alkaline phosphatase), chemiluminescent labels, biotinyl groups and secondary receptors (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains or epitope labels) Already determined polypeptide epitopes. In certain embodiments, the label is bound to spacer arms of various lengths (eg, (CH ₂ ) _n when n <about 20) to reduce potential steric hindrance.

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

As used herein, the term “pharmaceutical agent or drug” means a chemical compound or composition that can induce the desired therapeutic effect when properly administered to a patient. A "pharmaceutical effective amount", an expression for a pharmaceutical composition comprising one or multiple antibodies of the present invention, is a sensitivity threshold for external stimuli such that the sensitivity threshold of the patient considered to be comparable to the level observed in a healthy subject is restored. It will be understood to mean the amount of the pharmaceutical composition which can extinguish the reduction of.

"Disorder" is any condition that can be treated in accordance with the present invention. "Disorders" and "symptoms" can be used interchangeably herein, including chronic and acute NGF-mediated disorders or NGF-mediated disorders, including such pathological symptoms that make a mammal susceptible to a disorder in question.

The terms "NGF-mediated disease" and "NGF-mediated symptoms" include all medical symptoms or disorders associated with increased levels of NGF or increased susceptibility to NGF, including but not limited to: acute pain, Toothache, trauma-induced pain, surgical pain, pain caused by amputation or abscess, burning pain, dehydration disease, trigeminal neuralgia, cancer, chronic alcoholism, seizures, hypothalamic syndrome, diabetes, acquired immunodeficiency syndrome ("AIDS") , Toxins and chemotherapy, general headaches, migraines, cluster headaches, mixed-vascular syndromes and non-vascular syndromes, tension headaches, general inflammation, arthritis, rheumatic diseases, lupus, osteoarthritis, fibromyalgia, Inflammatory bowel disease, irritable bowel syndrome, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, inflammatory components Skin lesions, sunburn, cardiitis, dermatitis, myositis, neuritis, collagen vascular disease, chronic inflammatory symptoms, inflammatory pain and related hyperalgesia and allodynia, neuropathic pain and related hyperalgesia and allodynia, diabetic neuropathic pain , Burning pain, pain maintained by sympathetic nerves, afferent syndrome, asthma, epithelial tissue damage or dysfunction, herpes simplex, visceral dyskinesia in the respiratory, genitourinary, gastrointestinal or vascular areas, wounds, burns, allergic skin Reactions, pruritus, vitiligo, general gastrointestinal disorders, colitis, gastrointestinal ulceration, duodenal ulcers, vasomotor or allergic rhinitis, or bronchial disorders, dysmenorrhea, dyspepsia, esophageal reflux, pancreatitis, and visceral pain.

As used herein, when used with an excipient- or pharmaceutical composition comprising one or more anti-human NGF human antibodies, the terms "effective amount" and "therapeutically effective amount" indicate the desired result ( That is, the result required when treated with an excipient- or anti-human NGF human antibody of the present invention is a required decrease in inflammation and / or pain, for example) or one or more biological activity levels of NGF. It means a sufficient amount or dosage to reduce. More specifically, a therapeutically effective amount is sufficient to inhibit, for some time, an anti-human that inhibits one or more clinically defined pathological processes associated with symptoms, eg, inflammation or pain, in a subject treated with the agent in vivo. Amount of NGF human antibody. In the present invention, an "effective amount" of anti-NGF antibodies can prevent, stop, control or reduce the recognition of pain associated with all painful medical symptoms. In the method of the present invention, the term "modulation" and its grammatical modifications mean the prevention, partial or complete inhibition, reduction, delay or slowing down of unwanted consequences such as, for example, pain. The effective amount will vary depending on the specific excipient- or anti-human NGF human antibody selected, and also depends on the severity of the various factors and symptoms and disorders associated with the subject to be treated. For example, if excipient- or anti-human NGF human antibodies are administered in vivo, factors such as the patient's age, weight and health as well as the dose response curve and toxicity data obtained in preclinical animal experiments are among the factors to be considered. . If the agent is contacted with ex vivo cells, various preclinical ex vivo studies should also be designed to assess parameters such as absorption, half-life, dosage, toxicity, and the like. Determination of an effective amount or therapeutically effective amount for a given agent is within the ability of those skilled in the art.

As used herein, the terms “nerve growth factor” and “NGF” are limited to all mammalian species of native sequence NGF, including recombinant human NGF 1-120, as shown in SEQ ID NO: 30.

As used herein, the term "substantially pure" or "substantially purified" means a compound or species that is the predominant species present in the species (ie, is present in abundance on a molar basis than other individual species in the composition). . In certain embodiments, the substantially purified fraction is a composition comprising at least about 50% (based on moles) of all polymeric species present in the species. In certain embodiments, the substantially pure composition will comprise at least about 80%, 85%, 90%, 95% or 99% of the polymeric species present in the composition. In certain embodiments, the species is purified to substantially homogeneity (contaminated species cannot be detected in the composition by conventional detection methods), wherein the composition consists substantially of a single polymeric species.

The term "patient" includes human and animal subjects.

"Treat" or "treat" means both therapeutic treatment and prophylactic methods. Those in need of treatment include those who have a tendency to have a disorder or who have been prevented as well as those who have already developed the disorder.

Except as otherwise required in the context, singular terms shall include the plural and plural terms shall include the singular.

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

In one aspect, the present invention provides fully human monoclonal antibodies having biological and immunological specificities that are increased against human NGF and bind to human NGF. In another aspect, the invention provides nucleic acids comprising nucleotide sequences encoding amino acid sequences for heavy and light chain immunoglobulin molecules, in particular sequences corresponding to their variable regions. Particular embodiments of this aspect of the invention are sequences corresponding to the complementarity determining regions (CDRs), specifically CDR1 to CDR3, of the heavy and light chains provided by the invention. In another aspect, the invention provides hybridoma cells and cell lines expressing immunoglobulin molecules and antibodies, preferably monoclonal antibodies of the invention. The present invention also provides biologically and immunologically purified methods for the preparation of antibodies, preferably monoclonal antibodies having biological and immunological specificities that are increased against human NGF and bind to human NGF.

The ability to clone and reconstruct the million-sized human locus of artificial yeast chromosomes (YACs) and to introduce artificial yeast chromosomes into the mouse gonads not only creates useful models of human disease but also maps to very large or native maps. Provides an advantageous way to uncover the functional components of the left. Moreover, the use of techniques to replace the mouse lobe with human equivalents provides unique insight into the expression and regulation of human gene products during development, interaction with other systems, and their relevance in disease development and progression.

An important practical method of this strategy is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which endogenous Ig genes are inactivated provides an opportunity to study the mechanisms underlying the programmed expression and synthesis of antibodies as well as the role of antibodies in B-cell development. do. This strategy also provides a source for generating 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 have been produced in non-human mammals, including but not limited to rodents such as mice and rats, and lagomorphs such as rabbits. In certain embodiments, human antibodies were produced in hybridoma cells. In certain embodiments, human antibodies have been produced recombinantly.

The term “recombinant” with respect to an antibody includes antibodies produced, expressed, created or isolated by recombinant means. Representative examples include antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant, combinatorial human antibody libraries, and animals isolated from human immunoglobulin genes (eg, mice) Antibodies (e.g., Taylor, LD, et al., Nucl. Acids Res. 20: 6287-6295 (1992); or by any means including splicing of human immunoglobulin gene sequences to other DNA sequences Antibodies prepared, expressed, created or isolated Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.

Human antibodies have at least three advantages over non-human and chimeric antibodies for use in human therapy: 1) Because the effector portion of the antibody is the human body, it can interact more with other parts of the human immune system [ Destroying target cells more efficiently, eg, by complement-dependent cytopathy (CDC) or antibody-dependent cellular cytopathy (ADCC); 2) The human immune system cannot recognize human antibodies as heterologous, so that the antibody response to these injected antibodies may be less expressed in fully heterologous non-human antigens or partially heterologous chimeric antibodies; 3) Injected non-human antibodies have been reported to have shorter half-lives than human antibodies in human circulation. Injected human antibodies have essentially the same half-life as naturally occurring human antibodies which allow for lower doses and fewer times of administration than a given dose.

Thus, fully human antibodies are expected to minimize the intrinsic immunogenic and allergic responses to mice or mouse-induced MAbs, thereby increasing the efficacy and stability of the administered antibody. Thus, the fully human antibodies of the invention can be used in the treatment of chronic and repetitive pain, in those treatments where repeated antibody administration is required. Thus, a particular advantage of the anti-NGF antibodies of the present invention is that the antibodies are fully human antibodies and in a non-acute manner while minimizing the general side effects associated with human anti-mouse antibodies or incomplete human antibodies from non-human species described above. It can be administered to a patient.

To generate human antibodies without generating mouse antibodies, those skilled in the art can genetically engineer mouse strains having large fragments of the human Ig locus from which no mouse antibodies are produced. Large human Ig fragments can not only moderate antibody production and expression, but also preserve a variety of large gene diversity. By using mice for antibody diversification and selection and lack of immunological resistance to human proteins, human antibodies regenerated from these mouse strains are highly affinity antibodies that bind to all antigens of interest, including human antigens. Can be calculated. Hybridoma technology can be used to generate and select antigen-specific human MAbs with desirable specificity.

Transgenic animals (eg mice) can be used that can produce a complete repertoire of fully human antibodies without immunization of endogenous immunoglobulins in immunization. Metastasis of the human germline immunoglobulin gene sequence in such germline mutant mice will produce human antibodies at two subsequent antigenic stimuli (eg, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555, (1993); Nature of Jakobovits et al., 362: 255-258, (1993); Year in Immun, 7:33 (1993); Bruggmann et al., Nature 148: 1547-1553 ( 1994), Nature Biotechnology 14: 826 (1996); Gross, JA, et al., Nature, 404: 995-999 (2000); And US Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, and 5,545,806 (all purposes). Each of which is hereby incorporated by reference in its entirety). Human antibodies can also be produced in phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1992); Marks et al., J. Mol. Biol., 222: 581 (1991). Cole et al. And Boerner et al. Are also available for the production of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therap, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86-95 (1991).

Recombinant human antibodies can also be ex vivo mutagenesis (or somatic mutagenesis in vivo when using animal transgenics to human Ig sequences), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are human germline VH and VL. A sequence derived from a sequence associated with a sequence, which does not naturally exist in the human antibody germline repertoire in vivo.

In certain embodiments, those skilled in the art can use constant regions from species other than human in combination with human variable regions in said mice to produce chimeric antibodies.

Naturally occurring antibody structure

Naturally occurring antibody structural units generally include tetramers. Each tetramer generally comprises two identical pairs of polypeptide chains, each pair having one full length "light" chain (typically having a molecular weight of about 25 kDa) and one full length "heavy" chain (Typically having a molecular weight of about 50-70 kDa). The amino-terminal portions of each light and heavy chain generally comprise a variable region of about 100 to 110 or more amino acids, which recognizes the antigen. The carboxy-terminal portion of each chain is specified as a constant region that can function as an effector. Human light chains are generally classified into κ and λ light chains. Heavy chains are generally classified as μ, σ, γ, α or ε, each of which is defined as antibody isotypes such as IgM, IgD, IgG, IgA and IgE. IgG has a variety of subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly classified into subclasses including, but not limited to, IgA1 and IgA2. Generally in the full length light and heavy chains, the constant and variable regions are linked by a "J" region of about 12 or more amino acids, and also comprise a "D" region of about 10 or more amino acids. Connected with heavy chains. For example, FUNDAMENTAL IMMUNOLOGY Ch. 7, 2 ^nd ed., (Paul, W., ed.), 1989, Raven Press, NY, which is incorporated herein by reference. The variable region of each light / heavy chain pair generally forms an antigen-binding site.

The variable regions represent the same general structure of the relatively conserved outline region (FR) that binds to three hypervariable regions, commonly referred to as complementarity determining regions or CDRs. The CDRs of each of the two chains of each pair are combined into outline regions and are capable of binding to specific epitopes. From the N-terminus to the C-terminus, the light and heavy chain variable regions generally comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid sequence for each domain is generally described by Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.) Or Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917; as defined by Nature 342: 878-883 to Chothia et al.

Bispecific or bifunctional antibodies

Bispecific or bifunctional antibodies are generally artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced in a variety of ways, including but not limited to, fusion of hybridomas or binding F (ab ') fragments. For example, Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol. See 148: 1547-1553.

Preparation of Antibodies

The present invention provides antibodies that bind to human NGF. Such antibodies can be produced by immunization with full length NGF or fragments thereof. Antibodies of the invention may be polyclonal or monoclonal, and / or may be recombinant antibodies. In a preferred embodiment, the antibody of the invention is a human antibody produced by immunization of a transgenic animal, for example, capable of producing a human antibody (see, eg, International Patent Application Publication No. WO 93/12227). do it).

The complementarity determining regions (CDRs) of the light and heavy chain variable regions of the anti-NGF antibodies of the invention can be implanted into the same or different species of structural regions (FRs). In certain embodiments, CDRs of the light and heavy chain variable regions of the anti-NGF antibody can be transplanted into common human FRs. To generate consensus human FRs, FRs from several human heavy or light chain amino acid sequences were arranged to identify consensus amino acid sequences. Anti-NGF antibody heavy or light chain FRs may be replaced with FRs of different heavy or light chains. Trace amino acids of the heavy and light chain FRs of the anti-NGF antibody were not substituted, but the remaining FR amino acids may be substituted. Trace amino acids are specific amino acids at positions not normally found in FRs. The implanted variable regions in the anti-NGF antibodies of the invention can be used with constant regions that are different from the constant regions of the anti-NGF antibodies. Optionally, the implanted variable region is part of a single chain Fv antibody. CDR transplantation is described, for example, in US Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference.

Antibodies of the invention are preferably prepared using transgenic mice that have substantial portions of the locus that produce human antibodies inserted into cells that produce antibodies of the mouse, and that have been further engineered to produce no antibodies of endogenous mice. Such mice have the ability to produce human immunoglobulin molecules and antibodies and produce or do not produce substantially reduced amounts of immunoglobulin molecules and antibodies. The techniques used to obtain these results are described in patents, applications, and references described herein. In a preferred embodiment, the methods used by those skilled in the art are described in International Patent Application Publication No. WO 98/24893, incorporated herein by reference. See Mendez et al., 1997, Nature Genetics 15 : 146-156, incorporated herein by reference.

The monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques including general monoclonal antibody methodology, such as standard somatic cell hybridization technique of Kohler and Milstein (1975, Nature 256 : 495). have. It is preferred to use somatic hybridization methods, but generally other techniques for producing monoclonal antibodies (e.g., transformation of B-lymphocyte virus or tumor gene transformation) can be used.

Preferred animal systems for making hybridomas are mice. Hybridoma production in mice is widely established and techniques for isolation of immunized splenocytes for fusion are well known in the art. Fusion partners (eg, mouse myeloma cells) and fusion preparation methods are also well known.

In a preferred embodiment, transgenic mice comprising portions of the human immune system rather than the mouse system can be used to produce human monoclonal antibodies directed against NGF. Transgenic mice referred to herein as “HuMab” mice are endogenous μ And human immunoglobulin gene minilocus, which encodes unrearranged human heavy chains (μ and γ) and κ light chain immunoglobulin sequences, along with target mutations that inactivate the κ chain locus (Lonberg et al., 1994). , Nature 368: 856-859). Thus, mice will exhibit reduced expression of mouse IgM or κ and, in the immunization response, class switch and somatic mutations in human heavy and light chain transgenes introduced to produce human IgG κ monoclonal antibodies with high affinity. (See Lonberg et al., Supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13 : 65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764 : 536-546). . The preparation of HuMab mice is described in Taylor et al., 1992, Nucleic Acids Res. 20 : 6287-6295; Chen et al., 1993, International Immunology 5 : 647-656; Tuaillon et al., 1994, J. Immunol. 152 : 2912-2920; Lonberg et al., 1994, Nature 368 : 856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113 : 49-101; Taylor et al., 1994, International Immunology 6 : 579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13 : 65-93; Harding & Lonberg, 1995, Ann. NY Acad. Sci 764 : 536-546; Fishwild et al., 1996, Nature Biotechnology 14 : 845-851. See also US Pat. No. 5,545,806, which is further incorporated herein by reference; No. 5,569,825; 5,625,126; 5,625,126; 5,633,425; 5,633,425; 5,789,650; 5,789,650; 5,877,397; 5,877,397; No. 5,661,016; 5,814,318; 5,874,299; 5,874,299; And 5,770,429; In US Pat. No. 5,545,807 to Surani et al., As well as all references to Lonberg and Kay et al .; International Patent Application Publication No. WO 93/1227 published June 24, 1993; WO 92/22646 published December 23, 1992; And WO 92/03918, published March 19, 1992. Alternatively, the HCo7, HCo12 and KM transgenic mouse strains described in the examples below can be used to produce human anti-NGF antibodies.

The present invention provides human monoclonal antibodies specific for and neutralizing bioactive human NGF polypeptides. The present invention also provides antibody heavy and light chain amino acid sequences that are highly specific for NGF polypeptides and neutralize the NGF polypeptide when the antibody binds to the NGF polypeptide. This high specificity allows anti-human NGF human antibodies and human monoclonal antibodies with similar specificity to be used as effective immunotherapy for NGF related diseases.

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

In one aspect, the invention provides an isolated human antibody comprising at least one amino acid sequence shown in SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22, 24 and 79-130, which is highly Affinity can bind to NGF polypeptide epitopes and neutralize NGF polypeptide activity. Preferably, such antibodies bind to the same epitope or essentially the same epitope as the 4D4 antibody provided herein.

In a preferred embodiment, the isolated human antibody is 1 × 10 ^-9 M or bonded to NGF polypeptide with a lower dissociation constant (K _D) of the 1 × 10 ^-7 M or in vitro neutralization with IC ₅₀ of less black Inhibits NGF-induced survival. In a more preferred embodiment, the isolated human antibody is 1 × 10 ^-10 M or bonded to NGF polypeptide with a lower dissociation constant (K _D) and in vitro neutralization with 1 × 10 ^-8 M or IC ₅₀ of less Inhibits NGF-induced survival in the assay. In even more preferred embodiments, the isolated human antibody binds to human NGF polypeptide having a dissociation constant (K _D ) of 1 × 10 ⁻¹¹ M or less and with an IC ₅₀ of 1 × 10 ⁻⁹ M or less. Inhibits NGF-induced survival in in vitro assays. Examples of anti-human NGF human antibodies meeting the binding and neutralization criteria mentioned above are provided herein.

Most preferred anti-human NGF human antibodies of the invention are referred to herein as 4D4 and have the VL and VH polypeptide sequences as shown in SEQ ID NO: 12 and SEQ ID NO: 10, respectively. The polynucleotide sequences encoding VL and VH of 4D4 are shown in SEQ ID NO: 11 and SEQ ID NO: 9, respectively. The properties of the anti-human NGF human antibodies of the invention are shown specifically in the Examples. Of particular note is the high ability to neutralize the NGF polypeptide activity described herein and the high affinity for the NGF polypeptide.

The dissociation constant (K _D ) of anti-human NGF human antibody can be measured by surface plasmon resonance, as generally described in Example 9. In general, surface plasmon resonance analysis is performed using a BIAcore system (available from Pharmacia Biosensor, Piscataway, NJ) with surface plasmon resonance (SPR) ligands (recombinant NGF polypeptides immunized in biosensor matrices) and analytes. Real-time binding interactions between (antibodies in solution) were measured. Surface plasmon analysis can be performed by immunizing the analyte (antibody in the biosensor matrix) and displaying the ligand (recombinant V in solution). The dissociation constant (K _D ) of anti-human NGF human antibody can also be measured using KinExA methodology. In certain embodiments of the invention, the antibody binds to NGF with a K _D between approximately 10 ⁻⁸ M and 10 ⁻¹² M. The term "K _D " as used herein refers to the dissociation constant of a particular antibody-antigen interaction. K _D was measured as shown in Example 9 for all purposes of the present invention.

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

In certain embodiments, conservative modifications of the heavy and light chains of the anti-NGF antibody (and corresponding modifications to the coding nucleotides) exhibit functional and chemical properties similar to those of the heavy and light chains of the anti-NGF antibodies described herein. Anti-NGF antibodies can be produced. In contrast, substantial modifications in the functional and / or chemical properties of the anti-NGF antibody may be indicated by substitutions selected from the amino acid sequences of the heavy and light chains, wherein the selected substitutions of the amino acid sequences of the heavy and light chains are (a) To maintain the structure of the molecular backbone (eg, sheet or helical conformation) in the substitution region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains; These effects are very different.

For example, “conservative amino acid substitutions” include substitution of natural amino acid residues with non-natural residues that have little or no effect on the polarity or charge of the amino acid residues at that position. In addition, all natural residues in the polypeptide may also be substituted with alanine, which is already described in detail in "alanine scanning mutagenesis".

If such substitutions are desired, the required amino acid substitutions (regardless of conservative or non-conservative substitutions) can be determined by one skilled in the art. In certain embodiments, amino acid substitutions can be used to identify important residues of the anti-NGF antibodies described herein or to increase or decrease the affinity of the anti-NGF antibodies.

As is well known, small changes in amino acid sequences such as deletions, additions or substitutions of one, some or several amino acids can induce alleles of the original protein having substantially 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 the gene can be readily accomplished by various well known techniques such as site specific mutagenesis. Thus, the present invention can predict “variant” or “mutant” anti-NGF human antibodies that have substantially similar properties to the anti-NGF human antibodies described herein (eg, each referenced herein). See WO 00/56772, incorporated by reference). Thus, with respect to anti-NGF human antibodies, the term “variant” or “mutant” means all binding molecules (molecule X), which means (i) the hypervariable regions CDR1, CDR2 and CDR3 of the heavy chain as a whole, or the seconds of the light chain. The variable regions CDR1, CDR2 and CDR3 are at least 80% homologous, preferably at least 90% homologous, more preferably with the hypervariable regions set forth in SEQ ID NOS: 14, 18 and 22 or SEQ ID NOS: 16, 20 and 24, respectively. To be at least 95% homologous and (ii) the variant or mutant can inhibit the activity of human NGF to the same extent as the control anti-NGF human antibody having the same conformation region as that of the molecule X.

Usually, the anti-NGF human antibody variant is at least about 80% amino acid sequence identity, preferably at least about, with the amino acid sequence set forth in SEQ ID NOS: 14, 18 and 22 and / or SEQ ID NOS: 16, 20 and 24, respectively, as a whole 85% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, more preferably at least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably at least about 98% sequence identity, more preferably at least about Light and / or heavy chains having 99% amino acid sequence identity Will have CDRs.

More preferably, the anti-NGF human antibody variant is at least about 80% amino acid sequence identity, even more preferred, with the amino acid sequence set forth in SEQ ID NOS: 12, 80, 82, 84, 86, 88, 89, 90 or 91, respectively, as a whole; Preferably at least about 81% sequence identity, more preferably at least about 82% sequence identity, more preferably at least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, more preferably Preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably at least about 88% sequence identity, more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably At least about 91% sequence identity, more preferably At least about 92% sequence identity, more preferably at least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably SEQ ID NOS: 10, 81, 83, 85 or as a light chain variable region and / or a whole having at least about 97% sequence identity, more preferably at least about 98% sequence identity, more preferably at least about 99% amino acid sequence identity, respectively At least about 70% amino acid sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% sequence identity, more preferably at least about 81% sequence identity, more preferably at least about 82% with the amino acid sequence described in 87 Sequence identity, more preferably At least about 83% sequence identity, more preferably at least about 84% sequence identity, more preferably at least about 85% sequence identity, more preferably at least about 86% sequence identity, more preferably at least about 87% sequence identity, more preferably At least about 88% sequence identity, more preferably at least about 89% sequence identity, more preferably at least about 90% sequence identity, more preferably at least about 91% sequence identity, more preferably at least about 92% sequence identity, more preferably At least about 93% sequence identity, more preferably at least about 94% sequence identity, more preferably at least about 95% sequence identity, more preferably at least about 96% sequence identity, more preferably at least about 97% sequence identity, more preferably Write it down It will have about 98% sequence identity, more preferably at least a heavy chain variable region having about 99% amino acid sequence identity.

A “variant” with respect to a polynucleotide means a nucleic acid molecule having at least about 75% nucleic acid sequence identity with a polynucleotide sequence of the invention. Usually, polynucleotide variants have at least about 75% nucleic acid sequence identity, more preferably at least about 80% nucleic acid sequence identity, more preferably at least about 81% nucleic acid sequence identity, more preferably at least with the novel nucleic acid sequences described herein About 82% nucleic acid sequence identity, more preferably at least about 83% nucleic acid sequence identity, more preferably at least about 84% nucleic acid sequence identity, more preferably at least about 85% nucleic acid sequence identity, more preferably at least about 86% nucleic acid sequence identity , More preferably at least about 87% nucleic acid sequence identity, more preferably at least about 88% nucleic acid sequence identity, more preferably at least about 89% nucleic acid sequence identity, more preferably at least about 90% nucleic acid sequence identity, more preferably At least about 91% nucleic acid sequence identity, more preferably at least about 92% nucleic acid sequence identity, more preferably at least about 93% nucleic acid sequence identity, more preferably at least about 94% nucleic acid sequence identity, more preferably at least about 95% nucleic acid sequence Identity, more preferably at least about 96% nucleic acid sequence identity, more preferably at least about 97% nucleic acid sequence identity, more preferably at least about 98% nucleic acid sequence identity, more preferably at least about 99% nucleic acid sequence identity.

In certain embodiments, the present invention provides an antibody having a percent identity with an antibody of the invention, or the heavy chain variable region, light chain variable region, CDR1 of the invention as described in Examples 10 and 5-10 of this application. , An antibody comprising a heavy chain variable region, a light chain variable region, a CDR1, CDR2 or CDR3 region having a percent identity with a CDR2 or CDR3 region.

In certain embodiments, the present invention provides isolated human antibodies that specifically bind to nerve growth factors and include heavy and light chains, wherein the heavy chains comprise heavy chain variable regions comprising the amino acid sequences described below. An amino acid sequence that is at least 70% or 75% identical to the amino acid sequence set forth in SEQ ID NO: 10, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; Amino acid sequences at least 70%, 80%, 85% or 95% homologous to the amino acid sequence set forth in SEQ ID NO: 81, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 80%, 85% or 95% identical to the amino acid sequence set forth in SEQ ID NO: 83, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 80% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 85, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 75% or 80% identical to the amino acid sequence set forth in SEQ ID NO: 87, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 56% identical to the amino acid sequence set forth in SEQ ID NO: 79, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In certain embodiments, the present invention provides isolated human antibodies that specifically bind to nerve growth factors and comprise heavy and light chains, wherein the light chains comprise light chain variable regions comprising the amino acid sequences described below. : An amino acid sequence at least 70%, 75%, 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 12, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 85% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 80, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 74%, 90% or 94% identical to the amino acid sequence set forth in SEQ ID NO: 88, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 80%, 85% or 87% identical to the amino acid sequence set forth in SEQ ID NO: 89, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 85%, 90% or 94% identical to the amino acid sequence set forth in SEQ ID NO: 90, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 85%, 90%, 95% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 91, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 80%, 90%, 95% or 96% identical to the amino acid sequence set forth in SEQ ID NO: 82, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 84, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 86, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In certain other embodiments, the invention provides an isolated human antibody that specifically binds to a nerve growth factor and comprises human heavy chain CDR1, wherein the heavy chain CDR1 is the amino acid sequence set forth below: SEQ ID NO: 98 At least 40% or 60% identical to the amino acid sequence set forth in SEQ ID NO: 105, SEQ ID NO: 110 or SEQ ID NO: 22, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In another embodiment, the invention provides an isolated human antibody that specifically binds to a nerve growth factor and comprises human heavy chain CDR2, wherein the heavy chain CDR2 is the amino acid sequence set forth below: SEQ ID NO: 99 An amino acid sequence at least 70%, 82% or 94% identical to the described amino acid sequence, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 76% identical to the amino acid sequence set forth in SEQ ID NO: 106, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 59% identical to the amino acid sequence set forth in SEQ ID NO: 18, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 117, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; Or an amino acid sequence at least 70%, 75% or 80% identical to the amino acid sequence set forth in SEQ ID NO: 111, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In another embodiment, the present invention provides an isolated human antibody that specifically binds to a nerve growth factor and comprises human light chain CDR1, wherein the CDR1 is the amino acid sequence described below: SEQ ID NO: 101 An amino acid sequence at least 70% or 80% identical to an amino acid sequence, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70%, 75%, 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 95, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 75%, 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 119, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 75%, 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 122, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 125, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 75%, 80% or 90% identical to the amino acid sequence set forth in SEQ ID NO: 24, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 80% identical to the amino acid sequence set forth in SEQ ID NO: 107, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; Or an amino acid sequence that is at least 70% or 80% identical to the amino acid sequence set forth in SEQ ID NO: 113, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In an additional embodiment, the invention provides an isolated human antibody that specifically binds to a nerve growth factor and comprises human light chain CDR2, wherein said CDR2 is the amino acid sequence set forth below: SEQ ID NO: 102 An amino acid sequence that is at least 70% or 85% identical to the described amino acid sequence, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 96, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 120, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 123, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 126, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 129, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 20, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 108, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 133, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 114, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In another embodiment, the present invention provides an isolated human antibody that specifically binds to a nerve growth factor and comprises human light chain CDR3, wherein said CDR3 is the amino acid sequence set forth below: SEQ ID NO: 103 An amino acid sequence at least 70% or 85% identical to the amino acid sequence, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 97, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% or 78% identical to the amino acid sequence set forth in SEQ ID NO: 121, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 70% or 78% identical to the amino acid sequence set forth in SEQ ID NO: 127, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 78% identical to the amino acid sequence set forth in SEQ ID NO: 130, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 78% identical to the amino acid sequence set forth in SEQ ID NO: 16, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence that is at least 70% or 85% identical to the amino acid sequence set forth in SEQ ID NO: 109, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 78% identical to the amino acid sequence set forth in SEQ ID NO: 134, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof; An amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 115, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

The sequences of the 4D4 antibody heavy and light chain variable regions are described in SEQ ID NOS: 10 and 12, respectively. However, even if many potential CDR-contacting residues are substituted with other amino acids, the antibody still retains substantial affinity for the antigen. In addition, many outline residues that do not contact CDRs in the heavy and light chains are amino acids or other mouse antibodies from the corresponding positions of other human antibodies by human common amino acids without significant loss of affinity or non-immunogenicity of the human antibodies. It can be substituted from amino acid from. The selection of various selective amino acids can be used to produce modifications of anti-NGF antibodies and fragments thereof having various combinations of affinity, specificity, non-immunogenicity, ease of manufacture, and other desired features.

In alternative embodiments, the antibodies of the invention may be expressed in cell lines other than hybridoma cell lines. In such embodiments, sequences encoding particular antibodies can be used for transformation of appropriate mammalian host cells. In accordance with this embodiment, for example, as illustrated in US Pat. Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (all documents incorporated herein by reference for all purposes), for example, viruses Known methods or transductions known in the art for polynucleotide introduction into host cells, including polynucleotide packaging within (or into viral vectors) and host cell transduction with viruses (or vectors) Transformation can be done using the specification method. In general, the transformation method used depends on the host to be transformed. Methods of introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to: dextran-mediated transfection, potassium phosphate precipitation, polybrene-mediated transfection, protoplast fusion , Electroporation, polynucleotide encapsulation in liposomes and direct microinjection of DNA into the nucleus.

Nucleic acid molecules encoding amino acid sequences of the heavy chain constant region, heavy chain variable region, light chain constant region or light chain variable region of the NGF antibody of the present invention were inserted into an appropriate expression vector using standard ligation techniques. In a preferred embodiment, the anti-NGF antibody heavy or light chain constant region was added to the C-terminus of the appropriate variable region and bound to the expression vector. Vectors were selected to be functional in the particular host cell generally used (ie, the vector may be compatible with host cell machinery such as amplification of genes and / or expression of genes). For a review of expression vectors see METH. ENZ. 185 (Goeddel, ed.), 1990, Academic Press. See also.

In general, expression vectors used in all host cells will include sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. In certain embodiments, such sequences, referred to collectively as "flanking sequences" will generally include one or more nucleotide sequences, such as: a promoter, one or more enhancer sequences, a replication start point, a transcription termination sequence, A complete intron sequence comprising donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a liposome 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 comprises a "marker" -coding sequence (ie, an oligonucleotide molecule located at the 5 'or 3' end of the anti-NGF antibody polypeptide coding sequence); The oligonucleotide sequence encodes another marker such as polyHis (eg, hexaHis) or FLAG, HA (hemagglutinin influenza virus), or myc present in commercially available antibodies. This marker is generally fused to the polypeptide depending on the expression of the polypeptide and can be used as an affinity purification or as a means of detecting NGF antibodies from host cells. Affinity purification can be performed using, for example, column chromatography using an antibody against a label as an affinity matrix. Optionally, the label can be removed from the purified anti-NGF antibody polypeptide by various means, such as using specific peptidase for cleavage.

The flanking sequences can be homologous (ie, from the same species and / or lineage as the host cell), heterologous (ie from species or lineages other than the host cell), hybrid (ie flanking from one or more sources) Combination of sequences], synthetic or natural. If the flanking sequence is functional and can be activated in host cell tissue, the source of the flanking sequence can be obtained from all prokaryotes or eukaryotes, all vertebrate or invertebrates, or all plants.

Flanking sequences useful in the vectors of the invention can be obtained by a variety of methods well known in the art. In general, flanking sequences useful herein can be identified by mapping and / or restriction endonuclease cleavage and thus can be isolated from appropriate tissue using appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence is known. Here, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Genome library using polymerase chain reaction (PCR) and / or using appropriate probes such as oligonucleotides and / or flanking sequence fragments from the same species or other species, if only all or part of the flanking sequence is known Can be obtained by screening. If the flanking sequence is unknown, a fragment of DNA comprising the flanking sequence can be separated from, for example, a large piece of DNA comprising a coding sequence or other gene or genes. Cleavage with restriction endonuclease to produce appropriate DNA fragments followed by separation using agarose gel purification, Qiagen ^® column chromatography (available from Chatsworth, Calif.) Or other methods well known to those skilled in the art. The selection of appropriate enzymes for use in this method will be apparent to those skilled in the art.

Initiation of replication is generally part of a commercially available prokaryotic expression vector, which aids in the amplification of the vector in a host cell. If the selected vector does not include an origin of replication, it can be chemically synthesized based on known sequences and can be linked to the vector. For example, the replication initiation point from plasmid pBR322 (obtained from New England Biolabs, Beverly, Mass.) Is suitable as the initiation point for most Gram-negative bacteria and various viral initiation points (eg, SV40, Folio). Hemp, adenovirus, vesicular stomatitus virus (VSV) or papilloma virus such as HPV or BPV] are useful as cloning vectors in mammalian cells. In general, the starting point of the replication moiety does not require a mammalian expression vector (eg, the SV40 starting point is often used because it includes the viral early promoter).

The transcription terminal sequence is generally located at terminal 3 'of the polypeptide coding region and serves to terminate transcription. In general, the transcription termination sequence in prokaryotic cells is a poly-T sequence followed by a G-C rich fragment. Such sequences can be easily cloned from a library or commercially available portions of a vector, while can be readily synthesized using nucleic acid synthesis methods such as those described herein.

Selectable marker genes encode proteins essential for host cell growth and survival in selective culture media. Common selection marker genes encode the following proteins: (a) proteins that are resistant to antibiotics or other toxins (eg, ampicillin, tetracycline or kanamycin) in prokaryotic host cells; (b) proteins that compensate for nutrient deficiencies in cells; Or (c) a protein that supplies important nutrients that are not available from complete or specific media. Preferred selectable markers are kanamycin resistance gene, ampicillin resistance gene and tetracycline resistance gene. Conveniently, the neomycin resistance gene can also be selected and used in both prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene to be expressed. Amplification is a process in which genes that require production of proteins important for cell survival or growth are repeated in series in the chromosome to be continuously generated in recombinant cells. Examples of suitable selectable markers in mammalian cells include dihydrofolate reductase (DHFR) and promoter deficient thymidine kinase genes. Mammalian cell transformants were placed under selection pressure, where the transformants were only adapted and survived by selectable genes present in the vector. The selection pressure induces amplification of both the selectable gene and the DNA encoding another gene, such as an antibody that binds the NGF polypeptide, under conditions that continuously increase the concentration of the selection agent in the medium. do. As a result, increased quantities of polypeptides, such as anti-NGF antibodies, were synthesized from amplified DNA.

Ribosome-binding sites are generally essential for initiation of translation of mRNA and are characterized by shine-dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). This element is generally located 3 'to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

In some cases, glycosylation is required in the eukaryotic host cell expression system, which can manipulate various pre- or prosequences to increase glycosylation or yield. For example, a peptidase cleavage site of a particular single peptide that may affect glycosylation can be altered or presequences added. The final protein product may have one or more additional amino acids that are likely to be expressed that are not entirely removed at one position (the first amino acid of the mature protein). For example, the final protein product may have one or two amino acid residues at the peptidase cleavage site attached to the amino-terminus. Optionally, in the case of cleavage using enzymes in these regions within a mature polypeptide, the use of several enzyme cleavage sites results in truncated forms of the desired polypeptide.

Expression and cloning vectors of the invention generally include a promoter that is recognizable by the host organism and capable of binding to a molecule encoding an anti-NGF antibody. The promoter is an untranscribed sequence (typically within about 100 bp to 1000 bp) located upstream of the start codon of the structural gene (ie, 5 ') that regulates the transcription of the structural gene. Promoters are generally included in one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under the control of inducible promoters in response to changes in culture conditions, such as the presence or absence of nutrients, or changes in temperature. Constitutive promoters, on the other hand, are genes that bind and are uniformly transcribed with little or no regulation in gene expression. Many promoters recognized by a variety of potential host cells are well known. Suitable promoters may be bound to DNA encoding the heavy or light chain comprising an anti-NGF antibody of the invention by removing the promoter from the original DNA by restriction digestion and inserting the required promoter sequence into the vector.

Suitable promoters for use in yeast hosts are also well known in the art. Yeast enhancers can be used advantageously with yeast promoters. Suitable promoters for use in mammalian host cells are well known and include, but are not limited to, those obtained from the genome of the following viruses: polyoma virus, chicken pox virus, adenovirus (eg, adenovirus 2). ), Bovine papilloma virus, chicken sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably Simian virus 40 (SV 40). Other suitable mammalian promoters include heterologous mammalian promoters (eg, heat shock promoters and actin promoters).

Additional promoters include, but are not limited to: the SV 40 early promoter region (Bernoist and Chambon, 1981, Nature 290 : 304-10); CMV promoter (Thomsen et al., 1984, Proc. Natl, Acad. USA 81: 659-663); Promoters involved in the 3 ′ long terminal repeat of the Raus sarcoma virus (Yamamoto et al., 1980, Cell 22 : 787-97); Herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78 : 1444-45); Promoter and regulatory sequences from the metalthionine gene (Brinster et al., 1982, Nature 296 : 39-42); And prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75 : 3727-31); Or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80 : 21-25). It also has tissue specificity and has the following animal transcriptional regulatory regions that can be used in transgenic animals: elastase I gene regulatory regions 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, Hepatology 7 : 425-515); Insulin gene regulatory regions active in pancreatic beta cells (Hanahan, 1985, Nature 315 : 115-22); Immunoglobulin gene regulatory regions active in lymphoid cells (Grosschedl et al., 1984, Cell 38 : 647-58; Adames et al., 1985, Nature 318 : 533-38; Alexander et al., 1987, Mol. Cell. Biol. 7 : 1436-44); Tumor virus regulatory regions of mouse breasts active in testicular cells, breast cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45 : 485-95); Albumin gene regulatory regions active in the liver (Pinkert et al., 1987, Genes and Devel. 1 : 268-76); Α-fetoprotein gene regulatory regions active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5 : 1639-48; Hammer et al., 1987, Science 235 : 53-58); Α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel 1 : 161-71); Β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315 : 338-40; Kollias et al., 1986, Cell 46 : 89-94); Myelin basic protein gene regulatory regions active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48 : 703-12); Myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314 : 283-86); And gonadotropin free gene regulatory regions active in the hypothalamus (Mason et al., 1986, Science 234 : 1372-78).

Enhancer sequences can be inserted into the vector to increase the transcription of the DNA encoding the light or heavy chain comprising the anti-NGF antibody of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA about 10-300 bp long and act on the promoter to increase transcription. Enhancers found at the positions of both 5 'and 3' of the transcription unit are relatively orientation- and position-independent. Several enhancer sequences are available from mammalian genes (ie globin, elastase, albumin, α-feto-protein and insulin). However, in general, enhancers from viruses are used. SV 40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers and adenovirus enhancers known in the art are typical facilitating factors for activation of promoters in eukaryotes. Enhancers can be positioned as vectors at 5 'or 3' relative to the coding sequence, which is generally located at the 5 'site of the promoter.

Expression vectors of the invention can be constructed from initiation vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. If one or more flanking sequences described herein are not present in the vector, they can be obtained separately and bound to the vector. The method used for obtaining each flanking sequence is well known by those skilled in the art.

After constructing the vector and inserting the light chain containing the anti-NGF antibody or the nucleic acid molecule encoding the heavy or light chain and the heavy chain into an appropriate site of the vector, the completed vector is inserted into an appropriate host cell for amplification and / or polypeptide expression. can do. Transformation of expression vectors for anti-NGF antibodies into selected host cells can be performed by well known methods such as: transfection, injection, calcium phosphate co-precipitation, electroporation, microinjection, Lipofection, DEAE-dextran-mediated transfection or other known technique. The method chosen depends on the type of host cell to be used. Such and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., Supra.

When cultured under appropriate conditions, the host cell synthesized an anti-NGF antibody and can be collected from the culture medium if the host cell secretes the antibody into the medium, or directly from the host cell producing it if not secreted. Can be obtained. Selection of an appropriate host cell will depend on several factors, such as the level of expression required, polypeptide modifications desired or essential for activity (eg glycosylation or phosphorylation) and ease of folding into biologically active molecules.

Mammalian cell lines as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including: Chinese Hamster Ovary (CHO) Cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2) and many other cell lines. In certain embodiments, the cell line can be selected by measuring whether the cell line produces antibodies with high expression levels and constitutively carries NGF binding properties. In another embodiment, cell lines from a B cell lineage that are unable to produce antibodies but have the ability to produce and produce heterologous íforms can be selected.

Antibodies of the invention are useful for detecting NGF in biological samples and identifying cells or tissues that produce NGF protein. Antibodies of the invention that specifically bind to NGF may be useful for the treatment of NGF mediated diseases. The antibody can be used in a binding assay to detect NGF and inhibit NGF from complexing with the NGF receptor. The antibodies that bind to NGF and block interaction with other binding compounds have therapeutic uses to alleviate NGF mediated diseases. In a preferred embodiment, the antibody against NGF can cause disruption of the NGF induced signaling cascade to block NGF binding to the NGF receptor.

The invention also relates to one or more of the invention in the manufacture of a medicament for the treatment of a painful disorder or condition caused by increased expression of NGF or increased sensitivity to NGF in a patient, such as any disorder or condition described herein. It relates to the use of the antibody.

In a preferred embodiment, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies of the invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant do. Preferably, the acceptable formulation material is nontoxic at the dosages and concentrations used for the recipient. In a preferred embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an anti-NGF antibody.

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

In certain embodiments, the pharmaceutical composition modifies, maintains or preserves, for example, the pH, osmolarity, viscosity, purity, color, isotonicity, flavor, sterilization, stability, dissolution or release rate, adsorption or permeation of the composition. Formulation materials for In such embodiments, suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfate or sodium hydrogen sulfide); Buffer solutions (eg, borate, bicarbonate, tris-HCl, citrate, phosphate or other organic acids); Volume control agents (eg mannitol or glycine); Chelating agents (eg ethylenediamine tetraacetic acid (EDTA); complexing agents (eg caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; Disaccharides; and other carbohydrates (e.g. glucose, mannose, or dextrins); proteins (e.g. serum albumin, gelatin or immunoglobulins); pigments, anti-microbial and diluents; emulsifiers; hydrophilic polymers (e.g. poly Vinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g. sodium); preservatives (e.g. benzalkonium chloride, benzoic acid, salicylic acid, chimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine Sorbic acid or hydrogen peroxide); solvents (e.g. glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g. mannitol or sorbitol); suspending agents; surfactants or Wetting agents (e.g., polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol and tiloxapal); stability enhancers ( For example sucrose or sorbitol); enteric enhancers (e.g., alkali metal halide compounds, preferably sodium chloride, potassium chloride, mannitol sorbitol); transport excipients; diluents; prosthetics; and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18 ^th Edition, (AR Gennaro, ed.), 1990, obtained from Mack Publishing Company, which is incorporated by reference.

In certain embodiments, the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, according to the intended route of administration, mode of transport and dosage required. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES above. In certain embodiments, the composition affects the rate of in vivo clearance, rate of in vivo release, stability, and physical state of the antibodies of the invention.

In certain embodiments, the primary excipient or carrier in the pharmaceutical composition is aqueous or non-aqueous in nature. For example, a suitable excipient or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, which is generally capable of supplementing other substances in the composition for parenteral administration. Neutral buffered saline or physiological saline mixed with serum albumin is further typical excipients. In a preferred embodiment, the pharmaceutical composition of the present invention comprises tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which further comprises sorbitol, sucrose, Tween-20 and / or Suitable substituents thereof. In certain embodiments of the invention, the anti-NGF antibody composition is mixed with a selection composition having the desired purity and an optimal formulation formulation (see REMINGTON'S PHARMACEUTICAL SCIENCES, cited above), in the form of a lyophilized cake or aqueous solution. Prepared for storage. Moreover, in certain embodiments, anti-NGF antibody products are formulated as lyophilisates using suitable prostheses such as sucrose.

The pharmaceutical compositions of the invention can be selected for parenteral administration. Optionally, the composition may be selected for transport through the digestive tract, such as oral or for inhalation. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation component is preferably present at an acceptable concentration at the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological or slightly lower pH, generally in the range of about pH 5 to about pH 8.

When intended for parenteral administration, the therapeutic compositions used in the present invention will provide a form of a pyrogen-free parenterally acceptable aqueous solution comprising the required anti-NGF antibody dissolved in a pharmaceutically acceptable excipient. Can be. Particularly suitable excipients for parenteral injection are sterile distilled water which formulates the anti-NGF antibody as a sterile isotonic solution, which is suitably preserved. In certain embodiments, the formulations are injectable microparticles, bio-corrosive particles, high molecular compounds (eg, polylactic acid and polyglycolic acid) that provide controlled release and sustained release products that can be transported via depot injection. Formulations of the molecule as needed, together with agents such as beads or liposomes. In certain embodiments, hyaluronic acid can also be used, which has the effect of increasing the duration in circulation. In certain embodiments, an implantable drug transporter can be used to inject the desired antibody molecule.

The pharmaceutical compositions of the present invention may be formulated for inhalation. In this embodiment, the anti-NGF antibody is formulated in a dry, inhalable powder for convenient inhalation. In a preferred embodiment, the anti-NGF antibody inhalation solution can also be formulated with a propellant for aerosol transport. In certain embodiments, the solution may be sprayed. Thus, methods for administering and formulating lungs are further described in International Patent Publication No. PCT / US94 / 001875, which describes lung transport of chemically modified proteins, which is incorporated herein by reference.

The formulations can also be administered orally. Anti-NGF antibodies administered in this manner can be formulated with or without carriers commonly used in the preparation of solid pharmaceutical formulations such as tablets and capsules. In certain embodiments, the capsule may be designed to release the effective portion of the formulation at a gastrointestinal tract location where the bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate uptake of anti-NGF antibodies. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

Pharmaceutical compositions of the invention are preferably provided comprising an effective amount of one or multiple anti-NGF antibodies in admixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable excipients, solutions may be prepared in unit pharmaceutical formulations. Suitable excipients include, but are not limited to: inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions comprising formulations comprising anti-NGF antibodies in sustained or controlled release formulations will be apparent to those skilled in the art. Formulation techniques of various other sustained or controlled release vehicles such as liposome carriers, bio-corrosive microparticles or porous beads and depot injections are also known to those skilled in the art. See International Patent Publication No. PCT / US93 / 00829, which is described by reference herein, for the controlled release of microparticles of porous polymers for the transport of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped drugs (eg, films or microcapsules). Sustained release release matrices may include: polyesters, hydrogels, polylactides (described in US Pat. No. 3,773,919 and EP 058481, respectively, incorporated herein by reference), Copolymers of L-glutamic acid and γ ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22 : 545-556), copolymers of poly (2-hydroxyethyl-methylacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15 : 167-277 and Langer, 1982, Chem. Tech. 12 : 98-105), ethylene vinyl acetate (Langer et al., Supra) or poly-D (-) 3-hydroxy Butyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes, which may be prepared by various methods known in the art. See, eg, Eppstein et al., 1985, Proc. Natl. Acad. Sci, USA 82 : 3688-3692; European Patent Application Publication No. EP 036,676; See EP 088,046 and EP 143,949.

Pharmaceutical compositions used for administration in vivo are generally provided as sterile preparations. It can be sterilized by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using the method before or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or as a solvent. Parenteral compositions are generally stored in containers with sterile access ports, such as vials or intravenous solution bags with stoppers that can be inserted into subcutaneous needles.

Once the pharmaceutical composition has been formulated, it is stored in sterile vials in the form of solvents, suspensions, gels, emulsions, solids or dehydrogenated or lyophilized powders. Such formulations are stored in an easy-to-use form or in a form that requires reconstitution (eg, lyophilized) prior to administration.

The invention also provides kits for providing single-dose dosage units. Kits of the invention each comprise both a primary container containing dried protein and a secondary container containing an aqueous formulation. In certain embodiments of the present invention, the kits provide single and multi-threaded prefield cyclers (eg, liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition containing a therapeutically used anti-NGF antibody depends, for example, on the therapeutic situation and the subject. Those skilled in the art will appreciate that dosage levels suitable for treatment may be partially determined by the transport molecule, signs of the use of anti-NGF antibodies, route and size of administration (weight, body surface area or organ size) and / or symptoms of the patient (age and overall health). Will vary depending on the condition. In certain embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In a preferred embodiment, the dosage range is from 0.1 μg / kg to about 30 mg / kg; More preferably from 1 μg / kg to about 30 mg / kg; Even more preferably from 5 μg / kg to about 30 mg / kg.

The number of administrations will depend on the pharmacokinetic parameters of the particular anti-NGF antibody of the formulation used. In general, the clinician has administered the composition until the dosage achieves the desired effect. Therefore, the compositions were administered by a single administration or by two or more administrations (with or without the same amount of the desired molecule) or by continuous infusion via an insertion device or catheter over time. Moreover, refining at a suitable dosage is an area of common work typically made by a person skilled in the art and done by a person skilled in the art. Appropriate doses were identified using appropriate dose-response data. In certain embodiments, antibodies of the invention can be administered to a patient for an extended period of time. Routine administration of the antibodies of the present invention minimized immune adverse reactions or allergic reactions generally associated with antibodies to human antigens, such as non-complete human antibodies produced in non-human species, in non-human animals.

The route of administration of the pharmaceutical composition is according to known methods, including, for example: oral; Injection by intravenous, intraperitoneal, intracranial (brain parenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes; Sustained release system; Or injection using an insertion device. In certain embodiments, the composition may be administered by injection or infusion or infusion device continuously.

The composition can be administered topically through the insertion of a membrane, sponge or other suitable material in which the required molecules are absorbed or encapsulated. In certain embodiments, when using an insertion device, the device is inserted into an appropriate tissue or organ and the required molecules are transported by diffusion, sustained-release concentrates or continuous administration.

It would also be desirable to use the anti-NGF antibody pharmaceutical compositions of the invention ex vivo. In this example, cells, tissues or organs removed from the patient were transplanted back to the patient after exposure to the anti-NGF antibody pharmaceutical composition.

In particular, anti-NGF antibodies can be transplanted and transported into specific cells genetically engineered for polypeptide expression and secretion using the methods described herein. In certain embodiments, such cells are animal or human cells and may be autologous, heterologous, or heterologous. In certain embodiments, the cells can be immortalized. In another embodiment, to reduce the chance of an immune response, encapsulation can be avoided to avoid infiltration of tissue surrounding the cells. In further embodiments, the encapsulated material is generally a biocompatible semi-permeable polymer enclosure or membrane for releasing a protein product, but this prevents destruction of cells by other harmful elements from the patient's immune system or surrounding tissue. It can prevent.

The following examples, including performing experiments and obtaining results, are provided for illustrative purposes only and are not intended to limit the invention.

Example 1

Production of human NGF protein from E. coli cells

Cloning of rHu-NGF (1-120)

The nucleotide sequence encoding human NGF was amplified from cDNA using oligonucleotide primers having the sequences set forth in SEQ ID NO: 27 and SEQ ID NO: 28 and standard PCR techniques. The 5 'primer produces a methionine initiation codon that is present just before the NdeI restriction site and codon 1 (serine) of the mature sequence. The 3 ′ primer creates a BamH1 restriction site that is present immediately after the stop codon. The resulting PCR product was gel purified, digested with restriction enzymes NdeI and BamH1 and then linked into vector pCFM1656 (ATCC Accession No. 69576) digested with NdeI and BamH1. The linked DNA was transformed into appropriate host cells of E. coli strain 657. Clones were screened to generate recombinant protein products and to see if they had a plasmid with the correct nucleotide sequence (ie SEQ ID NO: 29). The amino acid sequence of recombinant human NGF 1-120 is shown in SEQ ID NO: 30.

The expression vector pCFM1656 (ATCC Accession No. 69576) is derived from the expression vector system described in US Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the pCFM836 plasmid (US Pat. No. 4,710,473) described by the following procedure: (a) Endogenous to two sites by end filling with T4 polymerase followed by blunt end bonding. Destroys NdeI restriction sites; (b) replacing the DNA sequence between a single AatII and ClaI restriction site comprising a synthetic P _L promoter with a similar fragment obtained from pCFM636 (US Pat. No. 4,710,473) comprising a PL promoter; And (c) then replacing the small DNA sequence between the single ClaI and KpnI restriction sites with an oligonucleotide obtained by annealing two probes having the nucleotide sequence shown in SEQ ID NO: 31 and SEQ ID NO: 32.

E. coli K12 host strain (Amgen strain 657) is a derivative of E. coli W1485 (K12 strain) obtained from E. coli Genetic Stock Center, New Haven Yale University, Connecticut (CGSC strain 6159).

Expression of rHu-NGF (1-120)

E. coli cells containing NGF expressing constructs (as described above) were fermented in a fed-culture in rich media. The cells were grown at 30 ° C. until the OD at 600 nm became 49 and then the temperature was raised to 42 ° C. The cells were harvested by centrifugation 4 hours after the temperature was raised. The final OD is 75. Expression yield was measured at approximately 0.15 g / L.

Refolding and Purification of rHu-NGF (1-120)

The cell paste was lysed in a Microfluidizer and centrifuged at 10,000 Xg for 30 minutes, then the pellet was washed with 1% deoxycholic acid, centrifuged as above and the resulting pellet was washed with cold water and refilled. -Centrifuged. The resulting pellet (WIBs-washed inclusion body) was resuspended in 8 M guanidine HCl and 50 mM Tris (pH 8.5) containing denaturing 10 mM DTT, dissolved for 1 hour at room temperature and centrifuged at 10,000 xg for 30 minutes. After separation, the supernatant was carefully decanted and diluted 25-fold with an aqueous buffer solution containing redox pairs at 4 ° C. for 5 days. The resulting refolded solution was adjusted to pH 3.0 and filtered through a 0.45 uM filter. The refold solution was purified using a Sp-Sepharose fast flow column using standard NaCl gradients. The pool from the cation exchange column was then concentrated and the partition was cooled to -80 ° C. The purity of the protein was measured by SDS-polyacrylamide gel electrophoresis (SES-PAGE) and analyzed by Coomassie Blue staining. By this method the purified protein shows a major band of at least 90%.

Example 2

Production of human monoclonal antibodies against nerve growth factor (NGF)

Transgenic HuMab and KM Mice

Fully human monoclonal antibodies against NGF were prepared using HCo7, HCo12, HCo7 + HCo12 and KM strains of transgenic mice expressing human antibody genes, respectively. In each of these mouse strains, the endogenous mouse kappa light chain gene was homozygous cleaved as described in Chen et al. (1993, EMBO J. 12: 811-820) and the endogenous mouse heavy chain gene was assigned to International Patent Application Publication No. Homozygous cleavage as described in Example 1 of WO 01/09187 (incorporated by reference). Each mouse strain contains KCo5, a human kappa light chain transgene, as described in Fishwild et al. (1996, Nature Biotechnology 14: 845-851). HCo7 strains contain the HCo7 human heavy chain transgene as described in US Pat. Nos. 5,545,806, 5,625,825 and 5,545,807, incorporated by reference. The HCo12 strain contains the HCo12 human heavy chain transgene as described in Example 2 of International Patent Application Publication No. WO 01/09187 (incorporated by reference). The HCo7 + HCo12 strain contains both HCo7 and HCo12 heavy chain trans genes and is semi-conjugated for each transgene. KM mice contain the SC20 heavy chain transgene as described in Tomizuka et al. (1997, Nature Genet. 16, 133-143 and 2000, Proc. Natl. Acad. Sci, 97, 722-727). This transgene is not inserted into the mouse chromosome but propagates as an independent chromosome fragment. The fragment comprises approximately 15 MB of human chromosome 14. This includes the entire human heavy chain locus, including all VH, D and JH gene segments and all heavy chain constant region isotypes. All such strains are referred to herein as HuMab mice.

HuMab immunization

To generate fully human monoclonal antibodies against NGF, HuMab mice were immunized with purified recombinant NGF derived from E. coli cells as antigens (Example 1). General immunization schemes for HuMab mice are described in Lonberg et al. (1994, Nature 368: 856-859; supra from Fishwild et al .; and International Patent Application Publication No. WO 98/24884, each of which theories are cited by reference). Is described in the above). 6-16 week old mice were first injected with antibodies. HuMab mice were immunized with purified recombinant preparations of NGF antigen (25-100 μg) injected intraperitoneally (IP) or subcutaneously (SC).

HuMab transgenic mice were immunized with complete Freund's adjuvant and antigens dissolved in both injections, followed by IP injection of antigen dissolved in incomplete Freund's adjuvant (up to 9 total immunizations). ). Dozens of 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 response was confirmed by posterior bleeding.

To select HuMab mice that produce antibodies that bind human NGF, the sera of immunized mice were tested by ELISA as described in Fishwild et al., Supra. Briefly, purified recombinant NGF (Example 1) from E. coli cells were dissolved in PBS at 1-2 μg / mL, coated in microplates at 50 μL / well and incubated overnight at 4 ° C., followed by PBS / Twin ( Blocking with 200 μL / well of 5% chicken serum dissolved in 0.05%). Plasma dilutions of NGF-immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS / Twin and then incubated for 1 hour at room temperature with goat-anti-human IgG Fc-specific polyclonal reagent bound to horseradish peroxidase (HRP). Plates were washed with PBS / Twin and incubated for 1 hour at room temperature with goat anti-human IgG Fc-specific polyclonal reagent bound to horseradish peroxidase (HRP). After washing, the plates were developed with ABTS substrate (Sigma Chemical Co., St. Louis, Missouri, Cat. No. A-1888, 0.22 mg / mL) and had optical density at a wavelength of 415-495 nm. OD) was analyzed spectrophotometrically. Mice with sufficient titers of anti-NGF human immunoglobulin were used to generate monoclonal antibodies as described below.

Production of Hybridomas Producing Human Monoclonal Antibodies to NGF

Two days before the sacrifice of mice, antigens were further antigenically stimulated to produce monoclonal antibodies, which were then sacrificed and spleens removed. Mouse splenocytes were isolated from HuMab mice and fused to mouse myeloma cell lines with PEG using standard protocols. In general, 10-20 fusions were generated for each antigen.

Briefly, single cell suspensions of splenic lymphocytes from immunized mice were administered in P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC Accession No. CRL 1580) with 50% PEG (available from Sigma). Fusion with / 4. The cells were plated on a wide bottom microplate at approximately 1 × 10 ⁵ / well, followed by DMEM supplemented with 10% fetal bovine serum, 10% P388D1 (ATCC Accession No. CRL TIB-63) -adjusted medium, 5 mM HEPES. Obtained from Mediatech, catalog number CRL 10013, 3-5% origen (origen, available from IGEN) dissolved in high glucose, L-glutamine and sodium pyruvate, 0.055 mM 2-mercaptoethanol, 50 mg / Incubated for about 2 weeks in selection medium containing mL of gentamicin and 1 × HAT (available from Sigma, catalog number CRL P-7185). After 1-2 weeks, cells were cultured in exclusion in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production of antigen-specific antibodies. Each well was screened by ELISA (described above) against human anti-NGF monoclonal IgG antibodies. When growing hybridomas on a large scale, medium was generally identified after 10-14 days. Anti-NGF monoclonal antibodies were subcloned at least 2 times with limiting dilution if antibody secreting hybridomas were replated and screened again and still positive for human IgG. Stable subclones were then in vitro cultured in tissue culture medium for characterization to produce small amounts of antibodies.

Screening for Human Monoclonal Antibodies That Bind to NGF

The ELISA assay described above was used to screen hybridomas that showed positive reactivity with NGF immunogens. Hybridomas secreting monoclonal antibodies that bind NGF with high binding activity were subcloned and further characterized. One clone from each hybridoma maintaining parental reactivity was selected to create 5-10 vial cell banks stored in liquid nitrogen.

Isotype-specific ELISAs were performed to determine the isotype of the monoclonal antibodies produced as described herein. In this experiment, 1 μg / mL of mouse-anti-human kappa light chain solution dissolved in PBS was coated in 50 μL / well in microplate wells and incubated at 4 ° C. overnight. After blocking with 5% chicken serum, the plates were reacted with supernatants from each test monoclonal antibody and purified isotype control. The plate was incubated for 1-2 hours at ambient temperature. Wells were then reacted with human IgG-specific horseradish peroxidase-bound goat anti-human polyclonal antiserum and plates developed and analyzed as follows.

Monoclonal antibodies purified from hybridoma supernatants that bind well to NGF as detected by ELISA were further tested for biological activity using various bioassays as described below.

Example  3

potent NGF   Anti-neutralizing activity NGF   Screening of antibodies and Cloning

The efficacy of the antibody initially identified in Example 2 as an inhibitor of NGF activity (ie, NGF “neutralization”) is assessed by measuring the ability of each modified peptide to block NGF induction of vanilloid receptor-1 (VR1) expression. It was.

Dorsal Root Ganglion Neuronal Cell Culture

All 19-day-old fetus (E19) rats that were surgically removed from the uterus after anesthesia was finally anesthetized with time-pregnant Sprague-Doleley rats (obtained from Charles River, Wilmington, Mass.). The dorsal root ganglions (DRG) were dissected one by one in a sterile state from the vertebral section. DRG was collected into ice-cold L-15 medium (obtained from GibcoBRL) containing 5% heat inactive horse serum (obtained from GibcoBRL, Grand Island, NY) and loosely coupled tissues and blood vessels were removed. DRG was washed twice with Dulbecco's phosphate buffered saline (DPBS, available from GibcoBRL) without Ca ²⁺ and Mg ²⁺ at pH 7.4. The DRG was then dissociated into a single cell suspension using a papain dissociation system (obtained from Worthington Biochemical Corp., New Jersey Freehold). Briefly, DRG was incubated in digestion solution containing 20 U / ml papain dissolved in Egg's balanced salt solution (EBSS) for 50 minutes at 37 ° C. The cells were heat-polished in dissociation medium consisting of MEM / Ham's F12 1: 1, 1 mg / ml of ovomucoid inhibitor and 1 mg / ml of ovalbumin and 0.005% deoxyribonuclease I (DNase). -polished) dissociated by crushing through Pasteur pipette.

Dissociated cells were pelleted at 200 × g for 5 minutes and resuspended in EBSS containing 1 mg / ml ovomucoid inhibitor, 1 mg / ml ovalbumin and 0.05% DNase. Cell suspensions were centrifuged at 200 xg for 6 minutes with a gradient solution containing 10 mg / ml ovomucoid inhibitor, 10 mg / ml ovalbumin to remove cell debris and then 88-μm nylon mesh (Pittsburg, Penn.) Coagulum) to remove clots. Cell counts were measured with a hemocytometer and cells were cultured in 100 μg / ml of poly-ornithine (obtained from Sigma, St. Louis, MT) and 1 μg / ml of mouse laminin (obtained from GibcoBRL) -coated 96-well plates Seeded at 10 × 10 ³ cells / well. Complete media consists of minimal essential media (MEM) and Ham's F12 1: 1, penicillin (100 U / ml), streptomycin (100 μg / ml) and 10% heat inactive horse serum (available from GibcoBRL). The medium was maintained at 37 ° C., 5% CO ₂ and 100% humidity. To regulate the growth of non-neuronal cells, 5-fluoro-2'-dioxyuridine (75 μM) and uridine (180 μM) were included in the medium.

Treatment of NGF and Anti-NGF

Two hours after plate, cells were treated with recombinant human β-NGF (obtained from Amgen) or recombinant rat β-NGF (obtained from R & D Systems, Minneapolis, Minnesota) at a concentration of 10 ng / ml (0.38 nM). It was. Positive controls containing series-diluted anti-NGF antibodies (obtained from R & D Systems) were fed to each culture plate. Test antibodies were added at 10 concentrations using 3.16-fold serial dilutions. All samples were diluted in complete medium before addition to the medium. It was incubated for 40 hours before measuring VR1 expression.

Measurement of VR1 Expression in DRG Neurons

The medium was fixed for 15 minutes in 4% paraformaldehyde dissolved in Hank's balanced saline solution and blocked with Superblock (obtained from Pierce, Rockford, Ill.) And then in Tris-HCl (obtained from Sigma) -buffered saline (TBS). Permeated 0.25% Nonidet P-40 (available from Sigma) for 1 hour at room temperature. The medium was washed once with TBS containing 0.1% Tween 20 (available from Sigma) and incubated with rabbit anti-VR1 IgG for 1 hour and a half at room temperature followed by Eu-labeled anti-rabbit secondary antibody (Turkue, Finland). Obtained from Wallac Oy) at room temperature for 1 hour. Each antibody was incubated and washed with TBS (3 times with gentle shaking for 5 minutes). Enhancer solution (150 μl / well, available from Wallac Oy) was added to the medium. Fluorescence signals were measured with a time-resolved fluorometer (available from Wallac Oy). VR1 expression in samples treated with mutated peptides was determined by comparing the NGF titer standard curve from 0-1000 ng / ml. The inhibition rate (compared to the maximal possible inhibition) in the effect of NGF on VR1 expression in DRG neurons was measured in comparison to the control without NGF treatment. The results are shown in Table 2 and Table 5.

Cell lines were named # 110- # 129. Antibodies from cell lines # 119, # 124 and # 125 demonstrated very useful NGF neutralizing activity (FIG. 1). The # 124 cell line is the parent cell line and is also named 4D4. The # 119 and # 125 cell lines were subcloned from the 4D4 parent cell line. Additional samples were grown from raw vials containing hybridoma # 124 (4D4) and named # 167 (4D4).

Antibodies produced by hybridoma # 167 (4D4) were subjected to the same DRG neuronal basal NGF neutralization assay as in the sample. It was demonstrated that antibody # 167 (4D4) exhibited strong anti-NGF activity with an IC ₅₀ of 0.50 nM consistent with the activities of samples # 119, # 124 and # 125 (FIG. 2). The activity of the four samples is shown in Table 2.

N-terminal sequencing and mass spectrometry

Purified anti-NGF hybridoma antibody samples were prepared for protein sequencing and LC / MS analysis. The antibody was purified from the conditioned medium by concentrating the medium until the volume was 15 mL or less using Amicon centriprep-30. The batch of rProA (available from Pharmacia) resin was washed four times with PBS and 50% slurry was made in PBS after the last wash. Appropriate amount of rProA resin (approximately 5 ug antibody / ul resin but not used at 50 ul or less) was added to the antibody sample and incubated overnight at 4 ° C. The Ab-resin mixture was centrifuged and the unbound fractions collected. 0.5 ml of PBS was added and transferred to a 0.45 um Spin-X (obtained from CoStar) tube and the sample was centrifuged at 10000 rpm for 3 minutes. The resin was washed at least three times with 0.5 ml of PBS, then 0.1 M glycine (pH 2.7) was added in a 1.5 × volume of resin, incubated for 10 minutes at room temperature and centrifuged again at 10000 rpm for 3 minutes to obtain a supernatant. This elution step was repeated two more times and then the mixed supernatant was neutralized with 1/25 volume of 1.0 M Tris (pH 9.2).

After the final filtration step through a new Spin-x tube (0.2 um), the antibody was quantified using a standard Bradford assay using human IgG as a standard, or absorbance at 280 for large samples. . The gel was also developed with 2 ug of human IgG1, k (obtained from Sigma) using 2 ug of each sample. In mass spectrometry, 4 μg of samples were deglycolated, reduced and loaded online on HPLC (HP1090) connected with a Finingan 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.

Both the N-terminal sequences of the light and heavy chains of the anti-NGF # 167 (4D4) antibody sample are consistent with the two N-terminal sequences of the anti-NGF # 119 (4D4) antibody sample. In addition, the measured mass of the antibody indicates that the isolated antibodies from # 167 and # 119 hybridomas are identical. The measured deconvoluted mass 23096 of the anti-NGF # 167 light chain is consistent with the measured mass 23096 of the anti NGF Ab # 119 light chain.

Cloning of Anti-NGF Antibody Heavy and Light Chains

The hybridoma expressing the monoclonal antibody in the 4D4.D7 potent NGF binding monoclonal antibody was used as raw material to isolate total RNA using TRIzol ^® reagent (obtained from Invitrogen). The first template cDNA was synthesized using random primers with an extended linker (5'-GGC CGG ATA GGC CTC CAN NNN NNT-3 ') (SEQ ID NO: 33) and preliminary 5' RACE (acute amplification of cDNA ends) The assay was performed using the GeneRacer ™ kit (obtained from Invitrogen) according to the manufacturer's instructions. In the preparation of the full light chain encoding the cDNA, the forward primer is GeneRacer ™ nested primer and the reverse primer is 5'-GGG GTC AGG CTG GAA CTG AGG-3 '(SEQ ID NO: 34). In the preparation of cDNA encoding the variable region of the heavy chain, the forward primer is GeneRacer ™ nested primer and the reverse primer is 5′-TGA GGA CGC TGA CCA CAC G-3 ′ (SEQ ID NO: 35). RACE products were cloned into pCR4-TOPO (available from Invitrogen) and sequences were determined. The consensus sequence was used to design primers for full length antibody chain PCR amplification.

In the preparation of cDNA encoding the anti-NGF 4D4.D7 kappa light chain, the 5 'PCR primers encode the amino terminus of the signal sequence, the XbaI restriction site and the optimized Kozak sequence (5'-CAG CAG AAG CTT CTA GAC CAC CAC). CAT GGA CAT GAG GGT GCC CGC TCA GCT CCT GGG-3 '; SEQ ID NO: 36). 3 'primers encode carboxy terminal and termination codons as well as SalI restriction sites (5'-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3'; SEQ ID NO: 37). The resulting PCR product fragment was purified, digested with XbaI and SalI, gel isolated and bound to mammalian expression vector pDSRα20 (see International Patent Application Publication No. WO 90/14363, incorporated herein by reference). I was. pDSRα20 is prepared by replacing “guanosine” of nucleotide 2563 with “adenosine” by site specific mutagenesis in pDSRα19.

In the preparation of cDNA encoding the anti-NGF 4D4.D7 heavy chain, the 5 'PCR primer encodes the amino terminus of the signal sequence, the XbaI restriction site and the optimized Kozak sequence (5'-CAG CAG AAG CTT CTA GAC CAC CAT GGA GTT GGG GCT GTG CTG GGT TTT CCT TGT T-3 '; SEQ ID NO: 38). 3 'primers encode carboxy terminal and termination codons as well as SalI restriction sites (5'-GCA TGT CGA CTC ATT TAC CCG GAG ACA GGG AGA G-3'; SEQ ID NO: 39). The resulting product was purified, digested with XbaI and SalI, gel separated and bound to pDSRα20 vector.

The calculated mass (23099) of the DNA sequence of the anti-HGF Ab 4D4 clone light chain determined by translating the nucleotide to the expected amino acid and adding together the molecular weight of the amino acid is consistent with the measured mass determined by mass spectrometry. The measured nonlinear mass of the anti-NGF # 167 heavy chain (49479) is consistent with the measured mass of the anti NGF Ab # 119 heavy chain (49484) and also the DNA of the heavy chain of the anti-NGF Ab 4D4 clone (Table 3) within the mechanical deviation. It is also consistent with the theoretical mass of the sequence.

The data of the N-terminal protein sequence and LC / MS confirm that hybridoma # 119 expresses the same antibody as hybridoma # 167. In addition, the calculated mass of the antibody based on the sequence further corroborates this finding.

Example 4

Expression of Anti-NGF Antibody in Chinese Hamster Ovary (CHO) Cells

Calcium phosphate method was used to transform 4D4-heavy / pDSRα19 IgG2 or 4D4-heavy / pDSRα19 IgG1 and NGF-kappa / pDSRα19 plasmid into dihydrofolate reductase deficiency (DHFR ⁻ ) serum-matched Chinese hamster ovary (CHO) cells. Co-transfection stably expressed the 4D4 anti-NGF mAb. Transfected cells were selected in medium containing diluted serum but not hydroxyxanthine-thymidine for growing cells expressing the DHFR enzyme. Transfected clones were screened using an assay such as ELISA to detect expression of 4D4 anti-NGF mAb in conditioned media. For the most expressed clones, the concentration of methotrexate (MTX) for DHFR amplification was increased. MTX amplified clones were screened using assays such as ELISA to detect more expression of 4D4 anti-NGF mAb in conditioned media. The most expressing clones were subcloned for generation of homologous populations and cell banks.

Recombinant anti-NGF antibodies of the invention can be generated in DHFR deficient Chinese hamster ovary cells using a protocol as described above for anti-NGF monoclonal antibodies. DNA sequences encoding the complete heavy or light chain of each anti-NGF antibody of the invention were cloned into expression vectors. Expression vectors capable of expressing the full heavy chain of the appropriate anti-NGF antibody and expression vectors expressing the complete light chain were co-transfected into CHOd ⁻ cells. For example, to generate an anti-NGF antibody, a vector capable of expressing a full heavy chain comprising an amino acid sequence as described in SEQ ID NO: 40 and a full light chain comprising the amino acid sequence as set out in SEQ ID NO: 44 Vectors capable of expressing were co-transfected into cells. The full heavy and complete light chains of the 4D4 antibody with various IgG heavy chain constant regions are shown in Table 4.

Example 5

term- NGF   4 D4  Characterization of Antibody Activity

Neutralizing NGF in a DRG neuronal basal NGF neutralizing bioassay, in which transiently expressed anti-NGF 4D4 antibodies generated in cells grown under spinner (S) or roller (R) conditions were run as described above (Example 3). Were tested to confirm the ability of the antibody to react.

NGF antibody was transiently expressed in 293T cells suitable for serum-free suspension. Transfection was performed in either 500 mL or 1 L medium. Briefly, conditioned media was removed by centrifuging the cell inoculum (5.0 × 10 ⁵ cells / mL × media volume) at 2,500 RPM for 10 minutes at 4 ° C. The cells were resuspended in serum free DMEM and centrifuged again at 2,500 RPM for 10 minutes at 4 ° C. After aspirating the wash solution, cells were harvested in growth medium [DMEM / F12 (3: 1) + 1X Insulin-Transferrin-Selenium Supplement + 1X Pen Strep Glut + 2 mM L-Glutamine + in 1 L or 3 L spinner flask medium. 20 mM HEPES + 0.01% Pluronic F68]. Spinner flask medium was maintained at 125 RPM in a magnetic stir plate in a wet thermostat maintained at 37 ° C. and 5% CO ₂ . Plasmid DNA was bound with transfection reagent in 50 mL conical tubes. DNA-transfection reagent complexes were prepared at 5% of the final medium volume in serum-free DMEM. 1 μg plasmid DNA / mL medium was first added to serum-free DMEM followed by 1 μL X-TremeGene RO-1539 / mL medium. The complex was incubated at room temperature for approximately 30 minutes and then added to the cells in the spinner flask. Transfection / expression was carried out for 7 days and then the conditioned medium was obtained by centrifugation at 4,000 RPM for 60 minutes at 4 ° C.

In rotator transient transfection, grown 293T adherent cells were used and maintained in DMEM supplemented with 5% FBS + 1X non-essential amino acids + 1X Pen Strep Glut + 1X sodium pyruvate. Approximately 4-5 × 10 ⁷ 293T cells were inoculated overnight in a 850 cm 2 rotary bottle. The inoculated cells were then transfected the next day using FuGene6 transfection reagent. DNA-transfection reagent mixtures were prepared approximately in 6.75 mL serum-free DMEM. 675 μl of FuGene6 transfection reagent was added first followed by 112.5 μg of plasmid DNA. The composite was incubated for 30 minutes at room temperature. Then the whole mixture was added to the bottle. The rotary bottle was fed with a 5% CO ₂ gas mixture, tightly closed and placed in a 37 ° C. thermostat on a roller rack rotating at 0.35 RPM. The medium was replaced with 100 mL DMEM + 1X Insulin-Transferrin-Selenium Supplement + 1X Pen Strep Glu + 1X Non-Essential Amino Acid + 1X Sodium Pyruvate and transfection was performed for 24 hours. In general, two 100 ml 48 hour harvests were obtained from each carousel. Harvested serum-free media was pooled together and centrifuged at 4,000 RPM for 30 minutes at 4 ° C.

Both 4D4.IgG1 and 4D4.IgG2 exhibit potent activity with IC ₅₀ values of about 0.14 nM to about 0.2 nM against human NGF (FIG. 2). The results of the activity assay are shown in Table 5. The antibody showed little activity against rat NGF (FIG. 3). The results were similar to the activity of the antibodies tested directly from the hybridomas described above.

Example 6

term- NGF   Production of antibodies

Anti-NGF antibodies were produced by expression in clone lines of CHO cells. In each production process, cells from a single vial were lysed into serum-free cell culture medium. Cells were first grown in T-flask and then transferred to spinner flasks and then grown in a stainless steel reactor scaled up to a 2000 L bioreactor. Nutrients containing the concentrated media components were produced in a 2000 L bioreactor using fed-batch culture, which was added to maintain cell growth and culture viability. Production continued for approximately two weeks, which is the time at which the antibody was structurally produced in the cells and secreted into the cell culture medium.

Predetermined pH, temperature and dissolved oxygen levels were adjusted in the production reactor: pH was adjusted by addition of carbon dioxide gas and sodium carbonate; Dissolved oxygen was controlled by air, nitrogen and oxygen gas flows.

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

Example 7

Cross-reactivity with other neurotropins

Cross-reactivity of 4D4 antibodies against human NT3 or human BDNF was tested with different bioassays including DRG neuron survival assays for human NT3 and DA uptake assays in cultured DA neurons against human BDNF.

Treating DRG Media with NT3, Anti-NT3 and Anti-NGF Antibodies

Two hours after plating, DRG cells (separation described in Example 3) were treated with 100 ng / ml of recombinant hNT-3 (3.8 nM). A series of diluted anti-hNT3 antibodies (obtained from R & D) were used as positive controls. An unknown sample (anti-NGF Ab sample) of varying concentrations was added at 10 points 3.16 fold series diluted. All samples were diluted in complete medium before addition to the medium.

Measurement of MAP2 Expression in DRG Neurons

The medium was fixed for 4 minutes in 4% paraformaldehyde dissolved in Hank's balanced saline solution, blocked with Superblock (obtained from Pierce) and then 0.25% Nonidet P dissolved in Tris-HCl (obtained from Sigma) -buffered saline (TBS). Permeation was performed at −40 (from Sigma) at room temperature (PT) for 1 hour. The medium was washed once with TBS containing 0.1% Tween 20 (obtained from Sigma) and incubated with mouse anti-MAP2 IgG (obtained from Chemicon, Temecula, Calif.) At room temperature for 1 and a half hours followed by Eu-labeling. Was incubated at room temperature for 1 hour with anti-mouse secondary antibody (obtained from Wallac Oy, Turku, Finland). Each antibody was incubated and washed with TBS (3 min 5 min shaking). Enhancer solution (150 μg / well, available from Wallac Oy) was added to the medium and the fluorescence signal was measured with a time-resolved fluorometer (obtained from Wallac Oy).

Culture of Fetal Midbrain

A 19 day old fetus (E19) Sprag-Doleley (obtained from Jackson Labs) was used. The ventral midbrain tissue rich in dopamine functional neurons was removed and transferred to chilled Dulbecco's phosphate buffered saline (DPBS) at pH 7.4 without Ca ⁺⁺ and Mg ⁺⁺ . Tissue fragments were dissociated into single cell suspensions using a papain dissociation system (obtained from Worthington Biochemical Corp., Freehold, NJ). Briefly, tissue fragments were incubated in digestive solution containing 20 units / ml of papain dissolved in Egg's Balanced Salt Solution (EBSS) for 50 minutes at 37 ° C. Pasteurized heat-polished cells in dissociation medium consisting of MEM / Ham's F12 1: 1, 1 mg / ml of ovomucoid inhibitor and 1 mg / ml of ovalbumin and 0.005% deoxyribonuclease I (DNase) Dissociate by grinding through the pet. Dissociated cells were pelleted at 200 × g for 5 minutes and resuspended in EBSS containing 1 mg / ml ovomucoid inhibitor, 1 mg / ml ovalbumin and 0.005% DNase. Cell suspensions were centrifuged at 200 xg for 6 minutes through a gradient solution containing 10 mg / ml ovomucoid inhibitor, 10 mg / ml ovalbumin to remove cell debris and then obtained from 25 μg Nitex nylon mesh (Tetko, Inc.) The coagulum was removed by filtration through). Dissociated cells were plated on tissue culture plates at a density of 100,000 / cm 2. Plates were previously described (J. Pharmacol. Exp. Ther. 1992; 262: 1274-1283 by Louis JC et al.) 100 μg / ml of poly-ornithine (available from Sigma) and 1 μg / ml of mouse laminin (from GibcoBRL). Available). The culture medium consists of minimum essential medium (MEM) / Ham's F12 1: 1, 12% horse serum (obtained from Gibco), 100 μg / ml transferrin and 2.5 μg / ml insulin (obtained from Sigma). The medium was maintained at 37 ° C., 5% CO ₂ and 100% humidity for 6 days.

Midbrain media treatment with BDNF and anti-BDNF or anti-NGF

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

DA influx in the midbrain neurons

Dopamine uptake assays were performed as previously described (Friedman, L. and Mytilineou, C., Neuroscience Letters 1987; 79: 65-72). On day 6, the medium was washed once with pre-incubated Crebes-ringer phosphate buffer (pH 7.4) containing 5.6 mM glucose, 1.3 mM EDTA and 0.5 mM pargiline, monoamine oxidase inhibitor. The medium was incubated at 37 ° C. for 60 minutes in an influx buffer containing 50 nM [ ³ H] DA (NEN). The influx buffer was removed to stop influx and the medium was washed three times with Crebes-Ringer phosphate buffer. Cells were lysed by adding an optical phase supermix (obtained from Wallac), a liquid scintillation cocktail, to release [ ³ H] DA directly into the medium. Cell lysates were then counted for radioactivity with a microbeta-plus liquid scintillation counter (obtained from Wallac, Inc.). Low affinity DA influx was assessed by adding 0.5 mM GBR12909, a specific inhibitor of the high affinity DA influx site (Heikkila RE and Mazino L, European Journal of Pharmacology 1984; 103: 241-8), to the influx buffer. The low affinity DA inflow was subtracted from the total inflow to obtain the Mars DA inflow.

Example 8

Identification of Epitopes on Anti-NGF Antibodies

Epitope Mapping by Limited Proteolysis

5 μg of NGF was incubated for 30 min at 4 ° C. with 4D4 (11 μg) in 0.1 M Tris buffer at pH 7.5. The complex was then digested with 1 μg of protease (subtilisin) at 37 ° C. for 1 and 2 hours. HPLC peptide maps were compared with each other to find peptides protected by 4D4 antibodies. Limited proteolysis of NGF indicates that several major peptides are released early from NGF. Of particular interest were the peptides S18.3, S18.5 and S34.4, which were protected by antibodies from proteolysis. Other peaks were not formed large or protected. Peptides protected from 2 experiments (1 hour and 2 hours digest) are shown in Table 7.

Protection rates were calculated from peptide peak heights. S18.5 comprises two peptides but only one peptide (SSSHPIFHR; SEQ ID NO: 46) is protected by the 4D4 antibody, as the rest of the peptide peak (HWNSY; SEQ ID NO: 47) is detected at 280 nm absorbance. This is because it is not changed by the addition of 4D4 antibody. Peptide S18.3 is the C-terminal portion of S34.4 both derived from the same loop region. N-terminal and central loop regions are also possible epitopes.

Microcon Isolation of Digested Peptides

Subtilisin-digested materials (3 μg each) were incubated at 4 ° C. for 30 minutes with active 4D4 antibody and inactive monoclonal antibody (# 162) (8 μg) in 0.1 M Tris buffer at pH 7.5. The bound / unbound peptides were separated by Microcon 10 (obtained from Millipore Corp., Bedford, Mass.) And the two fractions (bound and unbound) were analyzed by HPLC to detect antibodies bound to the antibody. Two reduced peaks were confirmed by HPLC comparison of unbound fractions after treatment with 4D4 antibody and # 162 and Microcon separation showing antibody binding peptides was recovered. 4D4 binding peptides are as follows:

S1 (4.4) ---- SRKAVRR (113-119) (SEQ ID NO: 49), C-terminus; And

S2 (28.3) ---- EVMVL (35-39) (SEQ ID NO: 50), loop area

NGF samples were selectively digested with Lys-C (K) for 24 hours. Cysteine residues were reduced and carboxymethylated without denaturing agent. Samples were incubated with monoclonal antibodies 4D4 and AMG162 before microcon 100 separation. Was executed. Bound and unbound fractions were analyzed by reverse phase HPLC. Only two peptides were identified as antibodies that bind K-peptides as described below. The calculated mass of the peptide was determined by sequencing and the mass spectrometry of the peptide was consistent. Peptides as shown below were mapped to the N-terminal and C-terminal regions.

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

        Calculated mass = 2821; Measured mass = 2828.2; N-terminal

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

         Calculated mass = 2452; Measured mass = 2459.5; C-terminal

The epitope mapping experiments described above indicate that at least three regions, including the N-terminal (1-9), internal to sequence (46-57), and C-terminal (96-98) regions, are possible epitopes for 4D4 antibodies. In addition, AspN digestion indicates that the peptide fragment consisting of --- SSHPIFHRGEFSVC --- (SEQ ID NO: 53) is protected by 4D4 antibody while trypsin digestion is --- SSHPIFHR --- (SEQ ID NO: 54) Peptide fragments shown are not protected by the 4D4 antibody. Therefore, at the N-terminus, the sequence of GEFSVC (SEQ ID NO: 55) is the most important for binding with 4D4 antibodies.

To more clearly define epitopes for anti-NGF antibody 4D4.IgG1, a total of 23 peptides were synthesized using standard techniques based on the whole human mature NGF (hNGF) sequence (Table 8). The peptide was 15 amino acids long with 10 amino acids overlapped and linked cysteine at the C-terminus for binding to the matrix. Human anti-hNGF Ab 4D4.IgG1 described above was used in the mapping experiments.

Human NGF peptide fragments were diluted in PBS with 5% DMSO and 1 mM EDTA at pH 6.23. Final peptide concentrations were normalized to the same molar concentration (about 100 μg / ml) at 55 μM. Peptides were incubated in Reacti-Bind Maleimide activated 96 well microplates (obtained from Pierce, Cat. No. 15150) at 100 μl / well for 2 hours at room temperature followed by stirring at 4 ° C. overnight. Human NGF (100 μg / ml) was used as a positive control. Plates were washed with wash buffer (KPL) and blocked with 0.2% fat-free dry milk (dissolved in PBS-EDTA buffer at pH 6.23) for 2 hours at room temperature and then further blocked with 5% BSA for 1 hour. . Plates were then incubated with human anti-NGF antibodies at various concentrations (0, 3, 10, 30 μg / ml) and then for 2 hours with goat anti-hFc Ab-HRP (KPL). The signal was shown as TMB substrate and read at 450 nm after addition of stop solution (KPL).

In 23 human NGF peptides, at least four major peaks showing 4D4 binding were observed. 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 peptide # 18-21 (SEQ ID NO: 73-SEQ ID NO: 76), TTTHT --- LSRKC (100-115).

Four binding peaks of 4D4 were mapped to N-terminal, C-terminal and internal domains as well as loops L2 and L4 of NGF as described in Weismann et al. (1999, Nature 401: 184-8). These results are shown in Table 9.

Wiesmann et al. Addressed the crystal structure of hNGF binding to trkA receptors, indicating that the N-terminus (residues 2-9) are important for receptor binding (Weismann et al. 1999, Nature 401: 184-8). The residues in these segments of NGF also play an important role for specificity for trkA rather than trkB or trkC receptors. Antibody 4D4 is selective for human NGF rather than BDNF and NT-3 as well as mouse / rat NGF due to the N-terminal difference between human NGF and other neurotropins.

Antibody 4D4 was assigned to peptide # 10 (SEQ ID NO: 65) (NSVFK ---, 46-60) and peptide # 17 (SEQ ID NO: 72) (TTTHTFVKALTMDGKC, 81-95) corresponding to loops L2 and L4, respectively. It represents two of seven distinct regions with higher diversity than the average sequence diversity among neurotropins. Swapping experiments between NGF and BDNF in these seven regions indicate that L2 and L4 are important for the biological activity of NGF. In addition, substitution of five NT3 residues in loops L2 and L4 with these NGF biological activities also induced NGF-like activity while maintaining NT3 activity. Therefore, L2 and L4 are suitable regions for antibody 4D4 to bind NGF more selectively than BDNF or NT-3.

Antibody 4D4 also binds to peptide # 16 (SEQ ID NO: 71) (WNSYATTTHTFVKAL, 76-90), which matches the internal domain of the NGF crystal structure. This region shows 100% homology between human NGF and mouse NGF but differs from other neurotropins. 4D4 shows much weaker activity against rat / mouse NGF compared to activity against human NGF. Therefore, binding of this portion of NGF does not play an important role in species specificity but is important for selectivity in neurotrophin.

Antibody 4D4 also has a C-terminal region of NGF [peptide # 19-21 (SEQ ID NO: 74-SEQ ID NO: 76) that is one of the regions of human NGF that distinguishes NGF from other neurotropins (BDNF and NT3). TMDGK --- LSRKC, 91-115. Binding to these regions helps explain why 4D4 is not active on other neurotropins. In addition, there is a single amino acid difference between human NGF and mouse NGF at the C-terminus, similar to the N-terminus where species differences are found, which is why these single amino acids are selective for human NGF over 4D4 rat / mouse NGF. Is one of.

Finally, 4D4 also interacts with an internal domain as described by peptide # 10 (SEQ ID NO: 65) (--- KARDC, 50-60) of human NGF, which is characterized by NGF being higher than trkB or trkC. It is an important region that preferably binds to trkA, which further explains the selective neutralizing activity of 4D4 on human NGF.

Example 9

Affinity measurement of monoclonal antibodies by KinExA

Binding of Ab 4D4 (38859-80) to huNGF (29714-91) was tested on KinExA. Briefly, Reacti-Gel 6x (available from Pierce) was pre-coated with huNGF and blocked with BSA. 10 pM and 30 pM Ab 4D4 samples were incubated for 8 hours at room temperature with varying concentrations of huNGF (available from Amgen) before developing through huNGF-coated beads. The amount of bead-binding antibody was quantified with fluorescent (Cy5) labeled goat anti-human IgG antibody (obtained from Jackson Immuno Research). The binding signal is proportional to the concentration of free antibody at equilibrium. Dissociation equilibrium constants (K _D ) were obtained from nonlinear regression analysis of competition curves using a dual-curve one-site homogeneous binding model (KinEx ™ software). K _D is about 4 pM for Ab 4D4 binding to huNGF.

Example 10

Identification of Additional Anti-NGF Antibodies

Additional anti-NGF antibodies (designated 14D10, 6G9, 7H2, 14F11 and 4G6), generated and identified as described in Examples 2 and 3 above, were selected for further study. Briefly, conditioned media was tested for binding activity. Antibodies from the medium were purified and sequenced. The expected mass was compared with the mass spectrometry data of the antibodies from the conditioned medium. The antibody was cloned. Two of the clones were expressed in CHO cells and tested for activity as described above. The results are shown in Table 10.

The sequences of the light and heavy chain variable regions of these antibodies were then compared with the other sequences as well as with the 4D4 antibody sequence (FIGS. 5 and 6). The percent homology of the heavy chain variable regions identified from this comparison 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.

It is to be understood that the above specification highlights specific embodiments of the invention and all such variations or alternative equivalents are included within the spirit and scope of the invention as set forth in the appended claims.

                         SEQUENCE LISTING <110> Kenneth, Wild       Treanor, James       Huang, Haichun       Inoue, Heather       Zhang, Tie J.       Martin, Frank <120> Human anti-NGF Neutralizing Antibodies as Selective NGF Pathway Inhibitors <130> 02-1240 <150> US 60 / 487,431 <151> 2003-07-15 <160> 138 <170> PatentIn version 3.0 <210> 1 <211> 990 <212> DNA <213> Homo sapiens <400> 1 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 2 <211> 330 <212> PRT <213> Homo sapiens <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 3 <211> 978 <212> DNA <213> Homo sapiens <400> 3 gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300 aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960 tccctgtctc cgggtaaa 978 <210> 4 <211> 326 <212> PRT <213> Homo sapiens <400> 4 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 5 <211> 981 <212> DNA <213> Homo sapiens <400> 5 gccagcacca aggggccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300 aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 360 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840 gacggctcct tcttcctcta cagcaggcta accgtgraca agagcaggtg gcaggagggg 900 aatgtcttct catgctccgt gakgcatgag gctctgcaca accactacac acagaagagc 960 ctctccctgt ctctgggtaa a 981 <210> 6 <211> 327 <212> PRT <213> Homo sapiens <400> 6 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 7 <211> 321 <212> DNA <213> Homo sapiens <400> 7 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 8 <211> 107 <212> PRT <213> Homo sapiens <400> 8 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 9 <211> 369 <212> DNA <213> Homo sapiens <400> 9 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggctt caccttaaga agttatagca tgaactgggt tcgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtcgta gtagtcatac catattctac 180 gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ttcactgtat 240 ctgcaaatgg acagcctgag agacgaggac acggctatgt attactgtgc gagagtatat 300 agcagtggct ggcacgtctc tgattatttt gactactggg gccagggaat cctggtcacc 360 gtttcctca 369 <210> 10 <211> 123 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser         115 120 <210> 11 <211> 321 <212> DNA <213> Homo sapiens <400> 11 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaacca 120 gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 12 <211> 107 <212> PRT <213> Homo sapiens <400> 12 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 13 <211> 42 <212> DNA <213> Homo sapiens <400> 13 gtatatagca gtggctggca cgtctctgat tattttgact ac 42 <210> 14 <211> 14 <212> PRT <213> Homo sapiens <400> 14 Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr 1 5 10 <210> 15 <211> 27 <212> DNA <213> Homo sapiens <400> 15 caacagttta atagttaccc gctcact 27 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 17 <211> 51 <212> DNA <213> Homo sapiens <400> 17 tacattagtc gtagtagtca taccatattc tacgcagact ctgtgaaggg c 51 <210> 18 <211> 17 <212> PRT <213> Homo sapiens <400> 18 Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 19 <211> 21 <212> DNA <213> Homo sapiens <400> 19 gatgcctcca gtttggaaag t 21 <210> 20 <211> 7 <212> PRT <213> Homo sapiens <400> 20 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 21 <211> 15 <212> DNA <213> Homo sapiens <400> 21 agttatagca tgaac 15 <210> 22 <211> 5 <212> PRT <213> Homo sapiens <400> 22 Ser Tyr Ser Met Asn 1 5 <210> 23 <211> 33 <212> DNA <213> Homo sapiens <400> 23 cgggcaagtc agggcattag cagtgcttta gcc 33 <210> 24 <211> 11 <212> PRT <213> Homo sapiens <400> 24 Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 <210> 25 <211> 1131 <212> DNA <213> Homo sapiens <400> 25 gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacacctgca acgtgaatca caagcccagc aacaccaagg tggacaagag agttgagctc 300 aaaaccccac ttggtgacac aactcacaca tgcccacggt gcccagagcc caaatcttgt 360 gacacacctc ccccgtgccc acggtgccca gagcccaaat cttgtgacac acctcccccg 420 tgcccacggt gcccagagcc caaatcttgt gacacacctc ccccatgccc acggtgccca 480 gcacctgaac tcctgggagg accgtcagtc ttcctcttcc ccccaaaacc caaggatacc 540 cttatgattt cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac 600 cccgaggtcc agttcaagtg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 660 ccgcgggagg agcagttcaa cagcacgttc cgtgtggtca gcgtcctcac cgtcctgcac 720 caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 780 cccatcgaga aaaccatctc caaaaccaaa ggacagcccc gagaaccaca ggtgtacacc 840 ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 900 ggcttctacc ccagcgacat cgccgtggag tgggagagca gcgggcagcc ggagaacaac 960 tacaacacca cgcctcccat gctggactcc gacggctcct tcttcctcta cagcaagctc 1020 accgtggaca agagcaggtg gcagcagggg aacatcttct catgctccgt gatgcatgag 1080 gctctgcaca accgcttcac gcagaagagc ctctccctgt ctccgggtaa a 1131 <210> 26 <211> 376 <212> PRT <213> Homo sapiens <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro             100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg         115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys     130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val             180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr         195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu     210 215 220 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys                 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln             260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met         275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro     290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu                 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile             340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln         355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly     370 375 <210> 27 <211> 29 <212> DNA <213> Artificial <220> PCR primer for human NGF <400> 27 acaccacata tgtcatcatc ccatcccat 29 <210> 28 <211> 40 <212> DNA <213> Artificial <220> <223> PCR Primer for human NGF <400> 28 accacaggat cctccttatg cacgacgcac agctttacgg 40 <210> 29 <211> 375 <212> DNA <213> Artificial <220> <223> Nucleotide sequence encoding recombinant human met-NGF <400> 29 catatgtcat catcccatcc catcttccac aggggcgaat tctcggtgtg tgacagtgtc 60 agcgtgtggg ttggggataa gaccaccgcc acagacatca agggcaagga ggtgatggtg 120 ttgggagagg tgaacattaa caacagtgta ttcaaacagt acttttttga gaccaagtgc 180 cgggacccaa atcccgttga cagcgggtgc cggggcattg actcaaagca ctggaactca 240 tattgtacca cgactcacac ctttgtcaag gcgctgacca tggatggcaa gcaggctgcc 300 tggcggttta tccggataga tacggcctgt gtgtgtgtgc tcagccgtaa agctgtgcgt 360 cgtgcataag gatcc 375 <210> 30 <211> 121 <212> PRT <213> Artificial <220> <223> amino acid sequence of recombinant human met-NGF (1-120) <400> 30 Met Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile             20 25 30 Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser         35 40 45 Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro     50 55 60 Val Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr 65 70 75 80 Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys                 85 90 95 Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val             100 105 110 Leu Ser Arg Lys Ala Val Arg Arg Ala         115 120 <210> 31 <211> 55 <212> DNA <213> Artificial <220> <223> 5 'primer sequence to generate expression vector pCFM1656 (ATCC #        69576 <400> 31 cgatttgatt ctagaaggag gaataacata tggttaacgc gttggaattc ggtac 55 <210> 32 <211> 49 <212> DNA <213> Artificial <220> <223> 3 'primer sequence to generate expression vector pCFM1656 (ATCC #        69576 <400> 32 taaactaaga tcttcctcct tattgtatac caattgcgca accttaagc 49 <210> 33 <211> 24 <212> DNA <213> Artificial <220> PCR primers <220> <221> Unsure (222) (18) .. (23) N is a, c, t, or g <400> 33 ggccggatag gcctccannn nnnt 24 <210> 34 <211> 21 <212> DNA <213> Artificial <220> PCR primers <400> 34 ggggtcaggc tggaactgag g 21 <210> 35 <211> 19 <212> DNA <213> Artificial <220> PCR primers <400> 35 tgaggacgct gaccacacg 19 <210> 36 <211> 54 <212> DNA <213> Artificial <220> PCR primers <400> 36 cagcagaagc ttctagacca ccatggacat gagggtgccc gctcagctcc tggg 54 <210> 37 <211> 34 <212> DNA <213> Artificial <220> PCR primers <400> 37 cttgtcgact caacactctc ccctgttgaa gctc 34 <210> 38 <211> 55 <212> DNA <213> Artificial <220> PCR primers <400> 38 cagcagaagc ttctagacca ccatggagtt ggggctgtgc tgggttttcc ttgtt 55 <210> 39 <211> 34 <212> DNA <213> Artificial <220> PCR primers <400> 39 gcatgtcgac tcatttaccc ggagacaggg agag 34 <210> 40 <211> 449 <212> PRT <213> Homo sapiens <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser     130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val         195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys     210 215 220 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser                 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp             260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn         275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val     290 295 300 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys                 325 330 335 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr             340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr         355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu     370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys                 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu             420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly         435 440 445 Lys      <210> 41 <211> 453 <212> PRT <213> Homo sapiens <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly     130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val         195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys     210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val             260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val         275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser     290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala                 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro             340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln         355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala     370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu                 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser             420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser         435 440 445 Leu Ser Pro Gly Lys     450 <210> 42 <211> 450 <212> PRT <213> Homo sapiens <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser     130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val         195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys     210 215 220 Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu             260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu         355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu         435 440 445 Gly lys     450 <210> 43 <211> 499 <212> PRT <213> Homo sapiens <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Arg Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Arg Ser Ser His Thr Ile Phe Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Asp Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Val Tyr Ser Ser Gly Trp His Val Ser Asp Tyr Phe Asp Tyr             100 105 110 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly     130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val         195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Leu Lys     210 215 220 Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro 225 230 235 240 Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys                 245 250 255 Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser             260 265 270 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu         275 280 285 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu     290 295 300 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser 305 310 315 320 His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu                 325 330 335 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr             340 345 350 Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn         355 360 365 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro     370 375 380 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 385 390 395 400 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val                 405 410 415 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val             420 425 430 Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro         435 440 445 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr     450 455 460 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val 465 470 475 480 Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu                 485 490 495 Ser Pro Gly              <210> 44 <211> 214 <212> PRT <213> Homo sapiens <400> 44 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 45 <211> 5 <212> PRT <213> Homo sapiens <400> 45 Phe Phe Glu Thr Lys 1 5 <210> 46 <211> 9 <212> PRT <213> Homo sapiens <400> 46 Ser Ser Ser His Pro Ile Phe His Arg 1 5 <210> 47 <211> 5 <212> PRT <213> Homo sapiens <400> 47 His Trp Asn Ser Tyr 1 5 <210> 48 <211> 12 <212> PRT <213> Homo sapiens <400> 48 Asn Ser Val Glu Lys Gln Tyr Phe Phe Glu Thr Lys 1 5 10 <210> 49 <211> 7 <212> PRT <213> Homo sapiens <400> 49 Ser Arg Lys Ala Val Arg Arg 1 5 <210> 50 <211> 5 <212> PRT <213> Homo sapiens <400> 50 Glu Val Met Val Leu 1 5 <210> 51 <211> 25 <212> PRT <213> Homo sapiens <400> 51 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys             20 25 <210> 52 <211> 20 <212> PRT <213> Homo sapiens <400> 52 Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val 1 5 10 15 Leu Ser Arg Lys             20 <210> 53 <211> 14 <212> PRT <213> Homo sapiens <400> 53 Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 <210> 54 <211> 8 <212> PRT <213> Homo sapiens <400> 54 Ser Ser His Pro Ile Phe His Arg 1 5 <210> 55 <211> 6 <212> PRT <213> Homo sapiens <400> 55 Gly Glu Phe Ser Val Cys 1 5 <210> 56 <211> 15 <212> PRT <213> Homo sapiens <400> 56 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys 1 5 10 15 <210> 57 <211> 16 <212> PRT <213> Homo sapiens <400> 57 Ile Phe His Arg Gly Glu Phe Ser Val Ala Asp Ser Val Ser Val Cys 1 5 10 15 <210> 58 <211> 16 <212> PRT <213> Homo sapiens <400> 58 Glu Phe Ser Val Ala Asp Ser Val Ser Val Trp Val Gly Asp Lys Cys 1 5 10 15 <210> 59 <211> 16 <212> PRT <213> Homo sapiens <400> 59 Asp Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Cys 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Homo sapiens <400> 60 Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Cys 1 5 10 15 <210> 61 <211> 16 <212> PRT <213> Homo sapiens <400> 61 Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Cys 1 5 10 15 <210> 62 <211> 15 <212> PRT <213> Homo sapiens <400> 62 Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn 1 5 10 15 <210> 63 <211> 16 <212> PRT <213> Homo sapiens <400> 63 Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Cys 1 5 10 15 <210> 64 <211> 16 <212> PRT <213> Homo sapiens <400> 64 Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Cys 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> Homo sapiens <400> 65 Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Ala Arg Asp Cys 1 5 10 15 <210> 66 <211> 16 <212> PRT <213> Homo sapiens <400> 66 Gln Tyr Phe Phe Glu Thr Lys Ala Arg Asp Pro Asn Pro Val Asp Cys 1 5 10 15 <210> 67 <211> 16 <212> PRT <213> Homo sapiens <400> 67 Thr Lys Ala Arg Asp Pro Asn Pro Val Asp Ser Gly Ala Arg Asp Cys 1 5 10 15 <210> 68 <211> 16 <212> PRT <213> Homo sapiens <400> 68 Pro Asn Pro Val Asp Ser Gly Ala Arg Asp Ile Asp Ser Lys His Cys 1 5 10 15 <210> 69 <211> 15 <212> PRT <213> Homo sapiens <400> 69 Ser Gly Ala Arg Asp Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Homo sapiens <400> 70 Ile Asp Ser Lys His Trp Asn Ser Tyr Ala Thr Thr Thr His Thr Cys 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Homo sapiens <400> 71 Trp Asn Ser Tyr Ala Thr Thr Thr His Thr Phe Val Lys Ala Leu Cys 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Homo sapiens <400> 72 Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Cys 1 5 10 15 <210> 73 <211> 16 <212> PRT <213> Homo sapiens <400> 73 Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Cys 1 5 10 15 <210> 74 <211> 16 <212> PRT <213> Homo sapiens <400> 74 Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Cys 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> Homo sapiens <400> 75 Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Ala Val Cys 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Homo sapiens <400> 76 Phe Ile Arg Ile Asp Thr Ala Ala Val Ala Val Leu Ser Arg Lys Cys 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Homo sapiens <400> 77 Thr Ala Ala Val Ala Val Leu Ser Arg Lys Ala Val Arg Arg Ala Cys 1 5 10 15 <210> 78 <211> 15 <212> PRT <213> Homo sapiens <400> 78 Cys Ala Ala Val Ala Val Leu Ser Arg Lys Ala Val Arg Arg Ala 1 5 10 15 <210> 79 <211> 125 <212> PRT <213> homo sapien <400> 79 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Thr Thr Tyr             20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met         35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe     50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Ala Arg Asn Tyr Tyr Gly Ser Gly Thr Tyr Tyr Tyr Tyr Tyr Gly Met             100 105 110 Asn Val Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser Ser         115 120 125 <210> 80 <211> 107 <212> PRT <213> homo sapien <400> 80 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ile Trp             20 25 30 Leu Ala Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 81 <211> 127 <212> PRT <213> homo sapien <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Glu Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Ser Trp Asn Arg Gly Ile Ile Gly Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Lys Glu Gly Tyr Tyr Gly Ser Gly Arg Pro Gly Tyr Phe Tyr Tyr             100 105 110 Val Met Asp Val Trp Gly Gln Gly Thr Thr Val Val Thr Val Ser Ser         115 120 125 <210> 82 <211> 108 <212> PRT <213> homo sapien <400> 82 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Gly             20 25 30 Phe Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Ala Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 83 <211> 127 <212> PRT <213> homo sapien <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Ser Trp Asn Arg Gly Ile Ile Gly Tyr Ala Gly Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Val Lys Glu Gly Tyr Tyr Gly Ser Gly Arg Pro Gly Tyr Phe Tyr Tyr             100 105 110 Val Met Asp Val Trp Gly Gln Gly Thr Thr Val Val Thr Val Ser Ser         115 120 125 <210> 84 <211> 108 <212> PRT <213> homo sapien <400> 84 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Val Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 85 <211> 121 <212> PRT <213> homo sapien <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Thr Trp Asn Ser Gly Ile Leu Gly Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Lys Glu Glu Gly Ser Gly Arg Tyr Tyr Asn Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 86 <211> 107 <212> PRT <213> homo sapien <400> 86 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 87 <211> 124 <212> PRT <213> homo sapien <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr             20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Asp Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gln Trp Leu Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp             100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 88 <211> 107 <212> PRT <213> homo sapien <400> 88 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 89 <211> 107 <212> PRT <213> homo sapien <400> 89 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp His Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 90 <211> 108 <212> PRT <213> homo sapien <400> 90 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro                 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 91 <211> 107 <212> PRT <213> homo sapien <400> 91 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 92 <211> 5 <212> PRT <213> homo sapien <400> 92 Thr Tyr Trp Ile Gly 1 5 <210> 93 <211> 17 <212> PRT <213> homo sapien <400> 93 Ile Ile Tyr Pro Gly Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly      <210> 94 <211> 16 <212> PRT <213> homo sapien <400> 94 Asn Tyr Tyr Gly Ser Gly Thr Tyr Tyr Tyr Tyr Tyr Gly Met Asn Val 1 5 10 15 <210> 95 <211> 11 <212> PRT <213> homo sapien <400> 95 Arg Ala Ser Gln Gly Ile Ser Ile Trp Leu Ala 1 5 10 <210> 96 <211> 7 <212> PRT <213> homo sapien <400> 96 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 97 <211> 9 <212> PRT <213> homo sapien <400> 97 Gln Gln Ala Asn Ser Phe Pro Trp Thr 1 5 <210> 98 <211> 5 <212> PRT <213> homo sapien <400> 98 Asp Tyr Ala Met His 1 5 <210> 99 <211> 17 <212> PRT <213> homo sapien <400> 99 Gly Ile Ser Trp Asn Arg Gly Ile Ile Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 100 <211> 17 <212> PRT <213> homo sapien <400> 100 Gly Tyr Tyr Gly Ser Gly Arg Pro Gly Tyr Phe Tyr Tyr Val Met Asp 1 5 10 15 Val      <210> 101 <211> 12 <212> PRT <213> homo sapien <400> 101 Arg Ala Ser Gln Ser Val Ser Ser Gly Phe Leu Ala 1 5 10 <210> 102 <211> 7 <212> PRT <213> homo sapien <400> 102 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 103 <211> 9 <212> PRT <213> homo sapien <400> 103 Gln Gln Tyr Gly Ser Ser Pro Tyr Thr 1 5 <210> 104 <211> 5 <212> PRT <213> homo sapien <400> 104 Asp Tyr Ala Met His 1 5 <210> 105 <211> 17 <212> PRT <213> homo sapien <400> 105 Gly Ile Ser Trp Asn Arg Gly Ile Ile Gly Tyr Ala Gly Ser Val Lys 1 5 10 15 Gly      <210> 106 <211> 17 <212> PRT <213> homo sapien <400> 106 Gly Tyr Tyr Gly Ser Gly Arg Pro Gly Tyr Phe Tyr Tyr Val Met Asp 1 5 10 15 Val      <210> 107 <211> 12 <212> PRT <213> homo sapien <400> 107 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 108 <211> 7 <212> PRT <213> homo sapien <400> 108 Val Ala Ser Ser Arg Ala Thr 1 5 <210> 109 <211> 9 <212> PRT <213> homo sapien <400> 109 Gln Gln Tyr Gly Ser Ser Pro Tyr Thr 1 5 <210> 110 <211> 5 <212> PRT <213> homo sapien <400> 110 Asp Tyr Ala Met His 1 5 <210> 111 <211> 17 <212> PRT <213> homo sapien <400> 111 Gly Ile Thr Trp Asn Ser Gly Ile Leu Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 112 <211> 11 <212> PRT <213> homo sapien <400> 112 Glu Gly Ser Gly Arg Tyr Tyr Asn Phe Asp Tyr 1 5 10 <210> 113 <211> 12 <212> PRT <213> homo sapien <400> 113 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 114 <211> 7 <212> PRT <213> homo sapien <400> 114 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 115 <211> 8 <212> PRT <213> homo sapien <400> 115 Gln Gln Tyr Gly Ser Ser Tyr Thr 1 5 <210> 116 <211> 5 <212> PRT <213> homo sapien <400> 116 Asp Tyr Gly Met Asn 1 5 <210> 117 <211> 17 <212> PRT <213> homo sapien <400> 117 Asp Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 118 <211> 15 <212> PRT <213> homo sapien <400> 118 Glu Gln Trp Leu Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val 1 5 10 15 <210> 119 <211> 11 <212> PRT <213> homo sapien <400> 119 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 120 <211> 7 <212> PRT <213> homo sapien <400> 120 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 121 <211> 9 <212> PRT <213> homo sapien <400> 121 Gln Gln Tyr Asn Ser Tyr Pro Trp Thr 1 5 <210> 122 <211> 11 <212> PRT <213> homo sapien <400> 122 Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu Ala 1 5 10 <210> 123 <211> 7 <212> PRT <213> homo sapien <400> 123 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 124 <211> 9 <212> PRT <213> homo sapien <400> 124 Gln Gln Arg Ser Asn Trp His Arg Thr 1 5 <210> 125 <211> 12 <212> PRT <213> homo sapien <400> 125 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 126 <211> 7 <212> PRT <213> homo sapien <400> 126 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 127 <211> 9 <212> PRT <213> homo sapien <400> 127 Gln Gln Tyr Asn Ser Tyr Pro Trp Thr 1 5 <210> 128 <211> 11 <212> PRT <213> homo sapien <400> 128 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 129 <211> 7 <212> PRT <213> homo sapien <400> 129 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 130 <211> 9 <212> PRT <213> homo sapien <400> 130 Gln Gln Arg Ser Asn Trp Pro Trp Thr 1 5 <210> 131 <211> 108 <212> PRT <213> homo sapien <400> 131 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 132 <211> 11 <212> PRT <213> homo sapien <400> 132 Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu Ala 1 5 10 <210> 133 <211> 7 <212> PRT <213> homo sapien <133> 133 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 134 <211> 9 <212> PRT <213> homo sapien <400> 134 Gln Gln Tyr Gly Ser Ser Pro Tyr Thr 1 5 <210> 135 <211> 120 <212> PRT <213> homo sapien <400> 135 Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys             20 25 30 Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val         35 40 45 Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val     50 55 60 Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80 Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln                 85 90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu             100 105 110 Ser Arg Lys Ala Val Arg Arg Ala         115 120 <210> 136 <211> 120 <212> PRT <213> mus musculus <400> 136 Ser Ser Thr His Pro Val Phe His Met Gly Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys             20 25 30 Gly Lys Glu Val Thr Val Leu Ala Glu Val Asn Ile Asn Asn Ser Val         35 40 45 Phe Arg Gln Tyr Phe Phe Glu Thr Lys Cys Arg Ala Ser Asn Pro Val     50 55 60 Glu Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80 Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Thr Asp Glu Lys Gln                 85 90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu             100 105 110 Ser Arg Lys Ala Thr Arg Arg Ala         115 120 <210> 137 <211> 126 <212> PRT <213> homo sapien <400> 137 His Ser Asp Pro Ala Arg Arg His Ser Asp Pro Ala Arg Arg Gly Glu 1 5 10 15 Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala Asp Lys             20 25 30 Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu Glu Lys         35 40 45 Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys     50 55 60 Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp Lys 65 70 75 80 Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg Ala                 85 90 95 Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile Arg Ile             100 105 110 Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg         115 120 125 <210> 138 <211> 119 <212> PRT <213> homo sapien <400> 138 Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp Ser 1 5 10 15 Glu Ser Leu Trp Val Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg Gly             20 25 30 His Gln Val Thr Val Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro Val         35 40 45 Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys     50 55 60 Asn Gly Cys Arg Gly Ile Asp Asp Lys His Trp Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu                 85 90 95 Val Gly Trp Arg Trp Ile Arg Ile Asp Thr Ser Cys Val Cys Ala Leu             100 105 110 Ser Arg Lys Ile Gly Arg Thr         115

Claims (95)

An isolated specifically binding to nerve growth factor (NGF) having a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12 Human antibodies. delete delete delete delete delete delete delete delete delete delete delete The antibody according to claim 1, which has the following (a) and (b) (a) a human heavy chain having the amino acid sequence set forth in SEQ ID NO: 22 CDR1, human body having the amino acid sequence set forth in SEQ ID NO: 18 Heavy chain CDR2 and having the amino acid sequence set forth in SEQ ID NO: 14 Human heavy chain CDR3; (b) human light chain having the amino acid sequence set forth in SEQ ID NO: 24     CDR1, human body having the amino acid sequence set forth in SEQ ID NO: 20     Light chain CDR2 and having the amino acid sequence set forth in SEQ ID NO: 16     Human light chain CDR3. delete delete The antibody of claim 13, wherein the antibody is isolated from human NGF polypeptide with a K _D of 1 × 10 ⁻¹¹ to 1 × 10 ⁻⁹ and an in vitro standard with an IC ₅₀ of 0.2 × 10 ⁻⁹ M to 1 × 10 ⁻⁸ M An antibody characterized by neutralizing human NGF bioactivity in an assay. delete delete The antibody of claim 1, wherein the heavy and light chains are linked by a flexible linker to form a single chain antibody. The antibody of claim 19, wherein the antibody is a single chain Fv antibody. The antibody of claim 1, wherein the antibody is a Fab antibody. The antibody of claim 1, wherein the antibody is a Fab 'antibody. The antibody of claim 1, wherein the antibody is a (Fab ′) ₂ antibody. The antibody of claim 1, wherein the antibody is a fully human antibody. The antibody of claim 1, wherein the antibody inhibits NGF signaling. delete delete A method for treating pain associated with a pain disorder or condition caused by increased expression of NGF or increased sensitivity to NGF selected from the following, comprising a therapeutically effective amount of the antibody of claim 25 and a pharmaceutically acceptable carrier. Pharmaceutical composition Acute pain, toothache, trauma, trauma, amputation Pain, burning pain, dehydration diseases, trigeminal neuralgia, cancer caused by abscesses, Chronic alcoholism, seizures, thalamic syndrome, diabetes, acquired immunodeficiency Syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster Headache, mixed-vascular syndrome or non-vascular syndrome, Tension headache, general inflammation, arthritis, rheumatic diseases, lupus, Osteoarthritis, Fibromyalgia, Inflammatory Bowel Disease, Irritable Bowel Syndrome, Inflammatory Eye disorders, inflammatory or unstable bladder disorders, psoriasis, with inflammatory components Skin lesions, sunburn, heartitis, dermatitis, myositis, neuritis, collagen Vascular disease, chronic inflammatory symptoms, inflammatory pain and related hyperalgesia, and Allodynia, neuropathic pain and related hyperalgesia or allodynia, Diabetic neuropathic pain, pain maintained by sympathetic nerves, Afferent syndrome, asthma, epithelial tissue damage or dysfunction, Visceral herpes, respiratory, genitourinary, gastrointestinal or vascular areas Motility disorders, wounds, burns, allergic skin reactions, pruritus, vitiligo, General gastrointestinal disorders, colitis, gastrointestinal ulceration, duodenal ulcers, Vasomotor nerve or allergic rhinitis, or bronchial disorder, dysmenorrhea, Indigestion, esophageal reflux, pancreatitis, or visceral pain. delete A method for detecting NGF in biological samples other than humans, comprising the following steps: (a) under the condition that the antibody can bind to NGF,  Contacting the sample; And (b) measuring the level of bound antibody in the sample. An isolated human antibody that specifically binds to a nerve growth factor having the following heavy and light chain variable regions, wherein the heavy chain variable region is SEQ ID NO: 10, SEQ ID NO: 79, SEQ ID NO: 81, Having an amino acid sequence as set forth in any one of SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87, wherein the light chain variable region is SEQ ID NO: 12, SEQ ID NO: 80, SEQ ID NO: 82, Having the amino acid sequence set forth in any one of SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 131 Isolated human antibody. delete delete delete delete An isolated human antibody that specifically binds to nerve growth factor (NGF) having the following heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3 The heavy chain CDR1, CDR2 and CDR3 are selected from the following (a) to (f) (a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 22,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 18,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; (b) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 92,     A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 93,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; (c) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 98,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 99,     A heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 100; (d) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 104,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 105,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 106; (e) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 110,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 111,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 112; (f) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 116,     A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 117,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 118; The light chain CDR1, CDR2 and CDR3 are selected from the following (g) to (p) (g) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 24,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 20,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 16; (h) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 95,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 96,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 97; (i) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 101,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 102,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 103; (j) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 107,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 108,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 109; (k) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 113,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 114,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 115; (l) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 119,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 120,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 121; (m) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 122,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 123,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 124; (n) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 125,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 126,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 127; (o) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 128,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 129,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 130; (p) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 132,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 133,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 134. delete delete delete delete delete delete 37. The antibody of claim 31 or 36, wherein the heavy and light chains are linked by a flexible linker to form a single chain antibody. The antibody of claim 43, wherein the antibody is a single chain Fv antibody. 37. The antibody of claim 31 or 36, wherein the antibody is a Fab antibody. 37. The antibody of claim 31 or 36, wherein the antibody is a Fab 'antibody. 37. The antibody of claim 31 or 36, which is a (Fab ') ₂ antibody. 37. The antibody of claim 31 or 36, wherein the antibody is a fully human antibody. 37. The antibody of claim 31 or 36, wherein the antibody inhibits NGF signaling. delete delete 50. A method for the treatment of pain associated with a painful disorder or condition caused by increased expression of NGF or increased sensitivity to NGF selected from the following, comprising a therapeutically effective amount of the antibody of claim 49 and a pharmaceutically acceptable carrier. Pharmaceutical composition Acute pain, toothache, trauma, trauma, amputation Pain, burning pain, dehydration diseases, trigeminal neuralgia, cancer caused by abscesses, Chronic alcoholism, seizures, thalamic syndrome, diabetes, acquired immunodeficiency Syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster Headache, mixed-vascular syndrome or non-vascular syndrome, Tension headache, general inflammation, arthritis, rheumatic diseases, lupus, Osteoarthritis, Fibromyalgia, Inflammatory Bowel Disease, Irritable Bowel Syndrome, Inflammatory Eye disorders, inflammatory or unstable bladder disorders, psoriasis, with inflammatory components Skin lesions, sunburn, heartitis, dermatitis, myositis, neuritis, collagen Vascular disease, chronic inflammatory symptoms, inflammatory pain and related hyperalgesia, and Allodynia, neuropathic pain and related hyperalgesia or allodynia, Diabetic neuropathic pain, pain maintained by sympathetic nerves, Afferent syndrome, asthma, epithelial tissue damage or dysfunction, Visceral herpes, respiratory, genitourinary, gastrointestinal or vascular areas Motility disorders, wounds, burns, allergic skin reactions, pruritus, vitiligo, General gastrointestinal disorders, colitis, gastrointestinal ulceration, duodenal ulcers, Vasomotor nerve or allergic rhinitis, or bronchial disorder, dysmenorrhea, Indigestion, esophageal reflux, pancreatitis, or visceral pain. delete A method for detecting NGF in biological samples other than humans, comprising the following steps: (a) Under conditions in which the antibody is capable of binding NGF, to claim 31 or 36.     Contacting the sample with an antibody of the sample; And (b) measuring the level of bound antibody in the sample. A nucleic acid molecule encoding the antibody of claim 31 or 36. A host cell, except for human cells, comprising the nucleic acid of claim 55. An isolated cell line producing the antibody of claim 31 or 36. NGF specific binding agent capable of binding to NGF having the following heavy and light chain variable regions The heavy chain variable region has an amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, and SEQ ID NO: 87; The light chain variable region is 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 and SEQ ID NO: 131 having the amino acid sequence of any one. 59. A method for treating pain associated with a pain disorder or condition caused by increased expression of NGF or increased sensitivity to NGF, selected from the following comprising a therapeutically effective amount of the binding agent of claim 58 and a pharmaceutically acceptable carrier. Pharmaceutical composition Acute pain, toothache, trauma, trauma, amputation Pain, burning pain, dehydration diseases, trigeminal neuralgia, cancer caused by abscesses, Chronic alcoholism, seizures, thalamic syndrome, diabetes, acquired immunodeficiency Syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster Headache, mixed-vascular syndrome or non-vascular syndrome, Tension headache, general inflammation, arthritis, rheumatic diseases, lupus, Osteoarthritis, Fibromyalgia, Inflammatory Bowel Disease, Irritable Bowel Syndrome, Inflammatory Eye disorders, inflammatory or unstable bladder disorders, psoriasis, with inflammatory components Skin lesions, sunburn, heartitis, dermatitis, myositis, neuritis, collagen Vascular disease, chronic inflammatory symptoms, inflammatory pain and related hyperalgesia, and Allodynia, neuropathic pain and related hyperalgesia or allodynia, Diabetic neuropathic pain, pain maintained by sympathetic nerves, Afferent syndrome, asthma, epithelial tissue damage or dysfunction, Visceral herpes, respiratory, genitourinary, gastrointestinal or vascular areas Motility disorders, wounds, burns, allergic skin reactions, pruritus, vitiligo, General gastrointestinal disorders, colitis, gastrointestinal ulceration, duodenal ulcers, Vasomotor nerve or allergic rhinitis, or bronchial disorder, dysmenorrhea, Indigestion, esophageal reflux, pancreatitis, or visceral pain. delete 59. The binder according to claim 58, which is a protein. A nucleic acid molecule encoding the binding agent of claim 61. A host cell, except for human cells, comprising the nucleic acid of claim 62. An isolated cell line producing the binder of claim 58. delete delete A method for detecting NGF in biological samples other than humans, comprising the following steps: (a) under the conditions in which the binder can bind to NGF, Contacting the sample; And (b) measuring the level of bound antibody in the sample. 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, SEQ ID NO: 24, SEQ ID The nucleotide sequence encoding the amino acid set forth in any one of: NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 or SEQ ID NOS: 79-134. The isolated nucleic acid molecule. An isolated human antibody that specifically binds to nerve growth factor (NGF) having the following heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3 The heavy chain CDR1, CDR2 and CDR3 are selected from the following (a) to (f) (a) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 22,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 18,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 14; (b) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 92,     A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 93,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 94; (c) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 98,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 99,     A heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 100; (d) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 104,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 105,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 106; (e) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 110,     Heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 111,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 112; (f) a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 116,     A heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 117,     Heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 118; The light chain CDR1, CDR2 and CDR3 are selected from the following (g) to (p) (g) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 24,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 20,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 16; (h) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 95,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 96,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 97; (i) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 101,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 102,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 103; (j) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 107,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 108,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 109; (k) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 113,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 114,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 115; (l) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 119,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 120,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 121; (m) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 122,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 123,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 124; (n) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 125,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 126,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 127; (o) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 128,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 129,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 130; (p) light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 132,     Light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 133,     Light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 134, Wherein the antibody is isolated from human NGF polypeptide with a K _D of 1 × 10 ⁻¹¹ to 1 × 10 ^-9 and in an in vitro standard assay with an IC ₅₀ of 0.2 × 10 ⁻⁹ M to 1 × 10 ⁻⁸ M Neutralizes human NGF bioactivity. delete delete delete delete delete delete delete delete delete 70. A polynucleotide encoding the antibody of claim 69. An expression vector comprising the polynucleotide of claim 79. A host cell, except for human cells, comprising the expression vector of claim 80. 82. The host cell of claim 81, wherein the cell is a eukaryotic cell. 83. The host cell of claim 82, wherein the cell is a CHO cell. Including the pharmaceutically effective amount of the monoclonal antibody or pharmaceutically acceptable salts thereof of claim 1, and increased sensitivity to NGF or increased expression of NGF selected from the following, including a pharmaceutically acceptable carrier, diluent or excipient Drugs for treating pain associated with related pain disorders or symptoms Acute pain, toothache, trauma, trauma, amputation Pain, burning pain, dehydration diseases, trigeminal neuralgia, cancer caused by abscesses, Chronic alcoholism, seizures, thalamic syndrome, diabetes, acquired immunodeficiency Syndrome ("AIDS"), toxins and chemotherapy, general headache, migraine, cluster Headache, mixed-vascular syndrome or non-vascular syndrome, Tension headache, general inflammation, arthritis, rheumatic diseases, lupus, Osteoarthritis, Fibromyalgia, Inflammatory Bowel Disease, Irritable Bowel Syndrome, Inflammatory Eye disorders, inflammatory or unstable bladder disorders, psoriasis, with inflammatory components Skin lesions, sunburn, heartitis, dermatitis, myositis, neuritis, collagen Vascular disease, chronic inflammatory symptoms, inflammatory pain and related hyperalgesia, and Allodynia, neuropathic pain and related hyperalgesia or allodynia, Diabetic neuropathic pain, pain maintained by sympathetic nerves, Afferent syndrome, asthma, epithelial tissue damage or dysfunction, Visceral herpes, respiratory, genitourinary, gastrointestinal or vascular areas Motility disorders, wounds, burns, allergic skin reactions, pruritus, vitiligo, General gastrointestinal disorders, colitis, gastrointestinal ulceration, duodenal ulcers, Vasomotor nerve or allergic rhinitis, or bronchial disorder, dysmenorrhea, Indigestion, esophageal reflux, pancreatitis, or visceral pain. delete delete 85. The antibody of claim 84, wherein the monoclonal antibody is isolated from human NGF polypeptide with a K _D of 1 × 10 ⁻¹¹ to 1 × 10 ⁻⁹ and an IC ₅₀ of 0.2 × 10 ⁻⁹ M to 1 × 10 ⁻⁸ M A pharmaceutical agent characterized in that it neutralizes human NGF bioactivity in an in vitro standard assay. delete delete delete delete delete delete delete delete

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