Anti-nerve growth factor (ngf) antibodies possessing high stability in vivo

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.

EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.

16 cl, 7 dwg, 13 tbl, 8 ex

 

The level of technology

Nerve growth factor (NGF) is a secreted protein that was discovered over 50 years ago as a molecule that promotes survival and differentiation of sensitive and sympathetic neurons. The beta chain of NGF is fully responsible for the activity of NGF, stimulating the growth of nerves. Beta chain forms homodimer and is part of a larger protein complex. NGF is a member of a family of neurotrophic factors known as the neurotrophins. NGF with high affinity binds to the kinase receptor tropomyosin-called TrkA. NEF is also able to bind to a receptor, known as p75NTRa member of the superfamily of receptors of tumor necrosis factor, which also interacts with other neurotrophins. Structure and function of NGF considers, for example, Sofroniew, M. V. et al. (2001) Annu. Rev. Neurosci. 24:1217-1281; Weismann, S. and de Vos, A. M. (2001) Cell. Mol. Life Sci. 58:748-759; Fahnestock, M. (1991) Curr. Top.Microbiol. Immunol. 165:1-26.

Although initially NGF was identified by its ability to ensure the survival and differentiation of neurons, there is increasing evidence that the impact on development is only one aspect of the biology of NGF. In particular, it has been shown the involvement of NGF in the transmission and maintenance of persistent or chronic pain. For example, it is demonstrated that both local and systemic introduction�of NGF causes hyperalgesia and allodynia (Lewin, G. R. et al. (1994). J. Neurosci, 6:1903-1912). Intravenous infusion of NGF to the person causing myalgia throughout the body, while local injection causes hyperalgesia and allodynia in the area of injection, in addition to systemic effects (Apfel, S. C. et al. (1998) Neurology 51_:695-702). In addition, some forms of cancer excess NGF promotes the growth and infiltration of nerve fibers with the induction of cancer pain (Zhu, Z. et al. (1999) J. din. Oncol. 17:241-228).

The involvement of NGF in the mechanisms of chronic pain has led to considerable interest in therapeutic approaches based on inhibition effects of NGF (see, e.g., Saragovi, H. U. and Gehring, K. (2000) Trends Pharmacol. Sci. 21:93-98). For example, a soluble form of the receptor TrkA was used to block NGF activity that has been shown to significantly suppress the formation of neuromas, responsible for neuropathic pain, without damaging the bodies of neuronal cells with pathological changes (Kryger, G. S. et al, (2001) J. Hand Surg. (Am.) 26:635-644).

Another method for neutralizing NGF activity is the use of antibodies against NGF, examples of which have been described (see, e.g., PCT publication Nos. WO 2001/78698, WO 2001/64247, WO 2002/096458, WO 2004/032870, WO 2005/061540, WO 2006/131951, WO 2006/110883, U.S. patent No. 7 449 616, the publication U.S. No. US 20050074821, US 20080033157, US 20080182978 and US 20090041717). In models of neuropathic pain in animals (for example, ligation of a nerve or spinal nerve�) systemic injection of neutralizing antibodies against NGF prevents allodynia, and hyperalgesia (Ramer, M. S. and Bisby, M. A. (1999) Eur. J. Neurosci. 11:837-846; Ro, L. S. et al. (1999) Pain 79:265-274). Moreover, treatment with a neutralizing antibody against NGF causes a significant decrease in pain in models of pain associated with cancer in mice (Sevcik, M. A. et al. (2005) Pain 115:128-141).

Thus, in the light of the foregoing, additional antagonists of NGF.

Summary of the invention

The present invention is directed to the creation of antibodies against NGF, with a higher stability in vivo. In particular, the present invention is directed to the creation of antibodies against NGF containing conservative region of human IgG4, wherein the conservative region of human IgG4 contains the mutation is preferably a mutation in a hinge region, and wherein the antibody demonstrates an unexpectedly long terminal elimination half-life, such as a terminal elimination half-life in cynomolgus monkey of at least 15 days and typically in the range from about 15 to about 22 days (or in the range from 15 days to 22 days), or in the range from about 15 days to 28 days (or in the range from 15 days to 28 days), or in the range from about 21 days to about 28 days (or in the range from 21 to 28 days). This stable antibody against NGF (for example, with stabilized antibody hinge region) also shows a terminal periodprivate in rats for at least 8 days in the typical case is in the range from about 8 to about 9 days (or in the range of 8 to 9 days). In other embodiments, the embodiment of a stabilized antibody against NGF (for example, with stabilized antibody hinge region) may have an average terminal half-life in humans of at least 10-30 days, or at least 10 days, at least 15 days at least 20 days, at least 25 days, at least 30 days, or in the range from about 10 days to about 40 days, or in the range from about 15 days to about 30 days (or in the range from 10 to 40 days or in range from 15 to 30 days). In other embodiments, the embodiment of a stabilized antibody against NGF (for example, with stabilized antibody hinge region) may have an average pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Preferably, the mutation in a conservative region of IgG4 is a mutation in the hinge region. Even more pre�respectfully, the mutation in the hinge region within the conservative region of IgG4 contains a serine mutation at amino acid position corresponding to amino acid position 108 in sequence ID no: 9 (which shows the amino acid sequence of wild-type conservative area of human IgG4). Accordingly, the present invention is aimed at creating antibodies against nerve growth factor (NGF), comprising a conservative region of human IgG4, namely conservative region of human IgG4, which includes a mutation in a hinge region comprising the mutation of serine at amino acid position corresponding to amino acid position 108 in sequence ID no: 9. More preferably, the series in the amino acid position corresponding to amino acid position 108 in sequence ID no: 9 is replaced by Proline. In a preferred embodiment, a conservative region of human IgG4 composed of antibodies against NGF contains the amino acid sequence of SEQ. ID no: 10. Alternatively, this document describes other possible mutations that stabilize IgG4.

The preferred antibody against NGF according to the invention is the PG110 antibody, the amino acid sequence of the heavy chain which is presented in SEQ. ID no: 13 and amino acid sequence of the light chain of which is presented in SEQ. ID No. 16. In line with this. the present invention is directed to the creation of antibodies against NGF containing conservative region of human IgG4, wherein the antibody contains a heavy chain comprising the amino acid sequence of SEQ. ID no: 13 and a light chain comprising the amino acid sequence of SEQ. ID no: 16. In another embodiment, the present invention is directed to the creation of antibodies against NGF containing conservative region of human IgG4, wherein the antibody contains a heavy chain encoded by a nucleotide sequence of SEQ. ID no: 11 and a light chain encoded by the nucleotide sequence of SEQ. ID no: 14. In another embodiment, the present invention is directed to the creation of antibodies against NGF containing a heavy chain comprising the amino acid sequence of SEQ. ID no: 13, wherein the antibody has a terminal elimination half-life in cynomolgus monkey of at least 15 days (and typically is in the range from about 15 to about 22 days, or in the range from 15 days to 22 days, or in the range from about 15 days to 28 days, or in the range from 15 to 28 days, or in the range from about 21 days to about 28 days, or in the range from 21 to 28 days and/or has a terminal elimination half-life in humans of at least 10-30 days (or at least 10 days, or at least 15 days, or at least 20 days or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days, or in the range from about 15 days to about 30 days, or in the range from 10 to 40 days or in range from 15 to 30 days). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). Preferably, the heavy chain encodes the nucleotide sequence of SEQ. ID no: 11. Preferably, the antibody contains a light chain comprising the amino acid sequence of SEQ. ID no: 16. Preferably, the light chain encodes the nucleotide sequence of SEQ. ID no:14.

In another embodiment, the antibody against NGF contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1 (which shows the variable region of the heavy chain of the antibody PG110). In another embodiment, the antibody against NGF stereotaxically region light chain, comprising the amino acid sequence of SEQ. ID no: 2 (which shows a variable region light chain antibody PG110). In another embodiment, the antibody against NGF contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2. In another embodiment, the antibody against NGF competes for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

In another embodiment, the antibody against NGF contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively (where LAST. ID nos: 3, 4 and 5 demonstrate the CDR 1, 2 and 3, respectively, in the composition of the variable region of the heavy chain of the antibody PG110), IN other embodiments, the antibody against NGF contains a variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively (where LAST. ID nos: 6, 7 and 8 show the CDR 1, 2 and 3, respectively, comprising a variable region light chain antibody PG110). In another Varian�e embodiment, the antibody against NGF contains the variable region of the heavy chain, comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and contains a variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively.

Preferably, the antibody against NGF possess one or more of the following functional properties:

a) binds to human NGF but does not bind to neurotrophic factor brain (BDNF) man neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) persons;

b) binds to human NGF or rats with KDof 100 PM or less;

b) inhibits binding of NGF to TrkA or p75NTR;

d) inhibits NGF-dependent proliferation of cells TF-1;

e) inhibits NGF-dependent survival of the dorsal root ganglia of the spinal cord of the chick;

(e) inhibits NGF-dependent proliferation of axons of cells PC 12.

In another embodiment, the antibody against NGF according to the invention does not show the syndrome of a ricochet when administered to a subject. For example, the size of the dosage and frequency of doses for antibodies can be selected so that the antibody does not show the syndrome of a ricochet when administered to a subject.

In another embodiment, the antibody against NGF according to the invention is able to alleviate pain in a subject over a long period of time, for example, for a period of at least about one week, or at least about two weeks, or at least about four weeks, or at least about eight weeks, or at least about twelve weeks, or at least from about one week to about twelve weeks, or at least about four weeks to about twelve weeks, or at least about eight weeks to about twelve weeks, or for at least one week, or at least two weeks or at least four weeks, or at least eight weeks, or at least twelve weeks, or at least from one to twelve weeks, or at least four to twelve weeks, or at least eight to twelve weeks after administration of a single dose of the antibody against NGF entity.

In particular, in a preferred embodiment of the present invention is directed to the creation of antibodies against NGF, which has combined the useful features of the prolonged terminal half-life and prolonged period of pain relief. Accordingly, the present invention also aim to develop antibodies against NGF containing conservative region of human IgG4, wherein the conservative region of IgG4 brow�ESA contains a mutation (preferably a mutation in a hinge region), wherein the antibody has a terminal elimination half-life in humans of at least 10-30 days, or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days, or in the range from about 15 days to about 30 days (or in the range of 10-40 days or in the range 15-30 days), and wherein the antibody alleviates pain for a period of from at least about one week to about twelve weeks, or at least from one week to twelve weeks, or at least about four weeks to about twelve weeks, or at least from four weeks to twelve weeks after administration of a single dose of the antibody to man (or at least one week, or at least two weeks, or at least four weeks, or at least eight weeks, or at least twelve weeks, or from one to twelve weeks, or four to twelve weeks, or eight to twelve weeks after administration of a single dose of the antibody to the human being). Preferably, the mutation in the hinge region contains a serine mutation at amino acid position corresponding to amino acid position 108 in sequence ID no: 9, preferably, the replacement of serine by Proline at position amino acids�, corresponding to amino acid position 108 in sequence ID no: 9. More preferably, conservative region of human IgG4 comprises the amino acid sequence of SEQ. ID no: 10. In various embodiments embodiment, the antibody may exhibit one or more of the functional properties described in this document. In preferred embodiments, the antibody competes for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

In another embodiment, the present invention is directed to the creation of antibodies against nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, and wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less. Preferably, the antibody has an average terminal half-life in humans of at least 10-30 days, or at least 10 days, or at IU�e 15 days or at least 20 days, or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days, or in the range from about 15 days to about 30 days (or in the range from 10 to 40 days or in range from 15 to 30 days). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). The antibody may further exhibit one or more additional functional properties such as binding to human NGF, but the lack of binding neurotrophic factor brain (BDNF) man neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) persons; the inhibition of NGF-dependent survival of the dorsal root ganglia of the spinal cord of the chick; and/or the inhibition of NGF-dependent proliferation of axons of cells PC 12. Preferably, the antibody alleviates pain in a subject for at least approximately one� weeks to about twelve weeks, or at least from about four weeks to about twelve weeks, or at least about eight weeks to about twelve weeks, or at least from one week to twelve weeks, or at least from four weeks to twelve weeks, or at least eight to twelve weeks (or at least one week, or at least four bulldog, or at least eight weeks, or at least twelve weeks, or from one to twelve weeks, or four to twelve weeks, or from eight to twelve weeks) after administration of a single dose of the antibody against NGF entity. Preferably, the antibody contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, or an antibody contains a variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, or an antibody contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and variable area light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively. Preferably, the antibody contains a variable region heavy C�PI comprising the amino acid sequence of SEQ. ID no: 1, or the antibody contains a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2 or an antibody contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, or the antibody competes for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

In various preferred embodiments the embodiment of the present invention is directed to the creation of antibodies against NGF, which has the following features:

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody has a medium term�iny the half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in range�not four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region of human IgG4, comprising a mutation in a hinge region, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) t�individual region of the heavy chain, comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative region of human IgG4, comprising a mutation in a hinge region, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region comprising a mutation in a hinge region, wherein the antibody has a mean terminal p�the period half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative region comprising a mutation in a hinge region, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four �about seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody binds to human NGF or rats with a kd of 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less, and wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising CDR 1, 2 and 3 having the amino�istotnie sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising (i) variable region of the heavy chain, �comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region of human IgG4, comprising a mutation in a hinge region, wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative field TgG4 person, including a mutation in a hinge region, wherein the antibody binds to human NGF or rats with a kd of 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3 and 4 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region comprising a mutation in a hinge region, wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising (i) variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, (ii) variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, and (iii) conservative region comprising a mutation in a hinge region, wherein the antibody binds to human NGF or rats with KDof 100 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less.

Antibody against the nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 comprises a mutation, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-0 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

Antibody against the nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 comprises a mutation, wherein the antibody has a terminal elimination half-life in cynomolgus monkey of at least 15 days. Additionally, or in accordance with another variant antibody may possess intermediate pharmacological period probiogen the person at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks).

In various embodiments embodiment, the antibody against NGF according to the invention can be, for example, chimeric, humanized antibody or human antibody, or an antibody, which were removed potential epitopes of T cells.

Another feature is that the present invention is directed to a pharmaceutical composition containing the antibody against NGF according to the invention and a pharmaceutically acceptable carrier.

Another feature is that the present invention is directed to a method of weakening or inhibition associated with NGF disease or condition in a subject, namely, the method comprising administering to the subject antibodies against NGF according to the invention. Non-limiting examples associated with NGF diseases and conditions include pain with inflammation, postoperative pain, neuropathic pain, pain in fractures, pain in the joints in gout, post-herpetic neuralgia, pain in cancer, pain in osteoarthritis or rheumatoid arthritis, sciatica, pain associated with crisis sickle-cell anemia, headache, dysmenorrhea, endometriosis, musculoskeletal pain, chronic pain in the lower back, fibromyalgia, sprains, visceral pain, ovarian cysts, prostatitis, cystitis, interstitial cystitis, pain in the area on�reoperations scar, migraine, trigeminal neuralgia, pain from burns and/or wounds, pain associated with trauma, pain associated with diseases of the musculoskeletal system, ankylosing spondylitis, macroglossinae pathology, the pain caused by bone metastases, pain, caused by HIV, erythromelalgia or pain caused by pancreatitis or kidney stones. Other examples associated with NGF diseases and conditions include malignant melanoma, xeroderma and asthma, such as uncontrolled asthma with severe hypersensitivity of the respiratory tract, and chronic cough. Particularly preferred diseases and conditions for treatment in accordance with the methods of the invention include pain during inflammation (in particular, pain caused by osteoarthritis or rheumatoid arthritis), musculoskeletal pain (particularly chronic pain in the lower back), neuropathic pain (including diabetic neuropathy), pain in cancer and the pain caused by bone metastases, interstitial cystitis/ painful syndrome of the bladder, pain associated with chronic non-bacterial prostatitis, pain associated with endometriosis and/or uterine fibroids and post-operative pain.

The antibody can be administered, e.g., intravenously, subcutaneously (e.g., using a syringe-R�chki or subcutaneous implant), intramuscular or intra-articular, however, other suitable methods of administration described in this document. Preferably, the antibody is administered in a dose ranging from 0.1 mg/kg to 3 mg/kg or at a dose in the range from 0.1 mg/kg to 30 mg/kg Antibody may be administered, for example, at a dose in the range from about 3 μg/kg to about 3000 μg/kg, with a preferred dosage range from 100 μg/kg to 300 μg/kg. In other embodiments embodiment, the antibody is administered in a dose ranging from 0.1 mg/kg to 30 mg/kg, or in the range from 0.1 mg/kg to 20 mg/kg, or between 0.1 mg/kg to 10 mg/kg, or between 1 mg/kg to 30 mg/kg, or between 1 mg/kg to 20 mg/kg, or between 1 mg/kg to 10 mg/kg, however, other suitable ranges of dosages and doses described in this document. Additionally, we can apply the formulation of an antibody with a fixed dose.

The antibody may be administered alone or in combination with one or more additional pharmaceutical agents. For example, the second pharmaceutical agent, such as NSAIDs, analgesics (e.g., opioid analgesic), a local anesthetic, block anesthesia, phenolic block, therapeutic antibody, anticonvulsant, antidepressant, local capsaicin, a steroid or antiviral agent can be administered in combination with the antibodies�ohms against NGF according to the invention. In particular, the second preferred pharmaceutical agents for combined therapy, in conjunction with the antibody of the invention include opioid analgesics, such as morphine, etc. Other preferred second pharmaceutical agents for combined therapy include TrkA inhibitors and inhibitors of protein kinase C (PKC).

In a preferred embodiment, the present invention is directed to a method of mitigating or inhibiting pain in a subject, namely, the method comprising administering to the subject antibodies against nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody alleviates pain in a subject for at least four to twelve weeks (or at least from one to twelve weeks, or at least eight to twelve weeks, or four to twelve weeks, or from one to twelve weeks, or eight to twelve weeks, or for at least one week, or at least four weeks, or at least eight weeks, or at least twelve weeks) after administration of a single dose of the antibody against NGF entity. Preferably, the antibody against NGF contains a variable region �agelou chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and variable area light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively. Preferably, the antibody against NGF contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2. Preferably, the type of pain selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain in cancer, pain from bone metastases, interstitial cystitis, painful syndrome of the bladder, pain associated with chronic non-bacterial prostatitis, pain associated with endometriosis, pain associated with uterine fibroids and post-operative pain. Preferably, the antibody against NGF is administered in a dose ranging from 0.1 to 3 mg/kg or at a dose in the range from 0.1 mg/kg to 30 mg/kg. Preferably, the antibody is administered intravenously or subcutaneously. Preferably, the antibody has a terminal elimination half-life in humans of at least 10-30 days (or at least 10 days, or at least 15 days, or according to møn�Shea least 20 days or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days, or in the range of about 15 to about 30 days, or in the range from 10 to 40 days or in range from 15 to 30 days).

In another preferred embodiment, the present invention is directed to a method of weakening or inhibition associated with nerve growth factor (NGF) disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet, namely, the method comprising administering to the subject antibodies against NGF containing conservative region of human IgG4, wherein the conservative region of human IgG4 comprises a mutation in a hinge region, and wherein the antibody has a terminal elimination half-life in humans of at least 10-30 days (or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, IZH at least 30 days, or in the range from about 10 days to about 40 days, or in the range of about 15 to about 30 days, or in the range from 10 to 40 days or in range from 15 to 30 days), and wherein the antibody is administered in such dosage and frequency to avoid the subject of emergence of a syndrome of a ricochet. Additionally, or in accordance with another variant antibody may possess environments�them pharmacological half-life in humans of at least 30 days or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). Preferably, conservative region of human IgG4 contains a mutation at the amino acid position corresponding to amino acid position 108 in SEQ. ID no: 9. Preferably, the series in the amino acid position corresponding to amino acid position 108 in SEQ. ID no: 9 is replaced by Proline. Preferably, conservative region of human IgG4 comprises the amino acid sequence of SEQ. ID no: 10. Preferably, the antibody against NGF contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and variable area light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively. Preferably, the antibody against NGF contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2. Preferably,�titulo competes for binding to NGF with an antibody, containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2. Preferably, associated with NGF the disease or condition is pain selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain in cancer, pain from bone metastases, interstitial cystitis, painful syndrome of the bladder, pain associated with chronic non-bacterial prostatitis, pain associated with endometriosis, pain associated with uterine fibroids and post-operative pain. Preferably, the antibody against NGF is administered in a dose ranging from 0.1 to 3 mg/kg or in a range from 0.1 mg/kg to 30 mg/kg. Preferably, the antibody is administered intravenously or subcutaneously.

Another feature is that the invention is directed to a use of the antibody against NGF according to the invention for obtaining a medicinal product for use for the purpose of weakening or inhibition associated with NGF disease or condition in a subject. Non-limiting examples associated with NGF diseases and conditions include those listed above. Preferred associated with �PH disease or condition is pain. Preferably, the type of pain selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain in cancer, pain from bone metastases, interstitial cystitis, painful syndrome of the bladder, pain associated with chronic non-bacterial prostatitis, pain associated with endometriosis, pain associated with uterine fibroids and post-operative pain.

Another feature is that the present invention is directed to the use of antibodies against NGF according to the invention for obtaining a medicinal product used for the purpose of weakening or inhibiting pain in a subject so that the facilitating or inhibiting pain in a subject lasted for at least from about one week to about twelve weeks, or at least about four weeks to about twelve weeks, or at least about eight weeks to about twelve weeks (or within one to twelve weeks, or four to twelve weeks, or from eight to twelve weeks, or at least one week, or at least two weeks, or at least four weeks, or at least eight weeks, or at least twelve weeks) poslesvecheniya single dose of the antibody against NGF entity.

Another feature is that the invention is directed to a use of the antibody against NGF according to the invention for obtaining a medicinal product used for the purpose of weakening or inhibition associated with NGF disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet. In particular, the antibody is administered in such dosage and frequency, to avoid the subject of the syndrome of a ricochet.

Another feature is that the present invention is directed to the creation of nucleic acid molecules encoding the heavy chain and/or light chains of antibodies against NGF according to the invention, and vectors (e.g., expression vectors) containing such vectors, host cells containing such vectors and methods for expression of antibodies against NGF with the use of host cells according to the invention.

Another feature is that the present invention is directed to a method of weakening or inhibition associated with nerve growth factor (NGF) disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet. The method comprises administering to the subject antibodies against NGF containing conservative region of human IgG4, wherein the conservative region of human IgG4 included�AET mutation (preferably, a mutation in a hinge region), and wherein the antibody has a terminal elimination half-life in cynomolgus monkey of at least 15 days, more preferably at least 21 days, and wherein the antibody is administered in such dosage and frequency, to avoid the subject of the syndrome of a ricochet. In another embodiment of the present invention, the antibody has a terminal elimination half-life in cynomolgus macaque in the range from about 15 days to about 22 days (or 15-22 days), or in the range from about 15 days to about 28 days (15-28 days), or in the range from about 21 days to about 28 days (or 21-28 days). In another embodiment, the antibody has a terminal elimination half-life in rats of at least 8 days. In yet another embodiment of the present invention, the antibody has an average terminal half-life in humans of at least 10-30 days (or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days, or in the range of about 15 to about 30 days, or in the range from 10 to 40 days or in range from 15 to 30 days). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological period �of olivedale the person at least 30 days or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). Preferred mutations include those described above. Preferred antibodies include those that have sequences and/or additional functional properties as described above. Non-limiting examples associated with NGF diseases and conditions include those listed above.

The invention also is directed to a use of the antibody prosthesis NGF according to the invention for obtaining a medicinal product for use for the purpose of weakening or inhibition associated with NGF disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet.

Kits containing the antibody against NGF according to the invention, also described in this document. For example, the kit can contain an antibody against NGF and guidance on the application of antibodies for the treatment associated with NGF disease or condition.

Brief description of graphic materials

Figure 1 presents a graph showing �wezwanie PG110 with nerve growth factor (NGF) human rights and the absence of binding neurotrophic factor brain (BDNF) person, neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) of a person, according to ELISA.

Figure 2A presents a graph showing the inhibition of binding of NGF with TrkA receptor antibody PG110, according to the experiment on the binding of radioactively labeled ligand.

Figure 2B presents a graph showing the inhibition of binding of NGF to its receptor p75NTRthe PG110 antibody, according to the experiment on the binding of radioactively labeled ligand.

Figure 3A presents a graph showing the inhibitory effect of PG110 antibody on cell proliferation, TF-1, NGF stimulated person.

Figure 3B presents a graph showing the inhibitory effect of PG110 antibody on cell proliferation, TF-1, NGF stimulated rats.

Figure 3C presents a graph showing the inhibitory effect of PG110 antibody on cell proliferation, TF-1, NGF stimulated mouse.

Figure 4 presents a graph showing the effect of treatment with antibody PG110 on the skin damage in rats.

Detailed description of the invention

The invention relates to antibodies against nerve growth factor, which have increased stability in vivo, which is obvious, for example, from an unexpectedly long terminal elimination half-life in cynomolgus macaques. Antibodies of the invention include modificat�Yu conservative area of human IgG4 in the composition of the antibody by introducing mutations in a conservative region of IgG4, preferably in the hinge region of a conservative field.

To facilitate understanding of the present invention first provides the definitions of certain terms. Additional definitions are set out in the detailed text description.

I. Definitions

The terms "nerve growth factor" or "NGF" are used in this document interchangeably, and include variants, isoforms, homologues, orthologs, and paralogy. For example, an antibody specific to human NGF, may in some cases demonstrate cross-reactivity with NGF species, distinct from humans. In other embodiments, embodiments, the antibody is specific to human NGF, may be completely specific for human NGF, and may not exhibit species-specific or other type of cross-reactivity. The term "human NGF" refers to the sequence of human NGF, such as containing the amino acid sequence of the chain of NGF-R human form precursor which has the access number in Genbank database NP_002497, and is encoded by a nucleotide sequence with access number in the database Genbank NM 002506.

The sequence circuit NGF-R may be different from that of NGF-R in Genbank with an access number NP_002497, for example, the presence of conservative substitutions or non-conservative substitutions in regions of NGF-R man has a living�significant extent the same biological function, that and NGF-R man from Genbank with an access number NP_002497. The term "rat NGF" refers to the sequence of rat NGF, such as containing the amino acid sequence of the chain of NGF-R rats, form precursor which has the access number in Genbank database XP_227525, and is encoded by a nucleotide sequence with access number in Genbank database XP_227525. The term "mouse NGF" refers to the sequence of rat NGF, such as containing the amino acid sequence of the chain of NGF-R, mouse, form precursor which has the access number in Genbank database NP_038637, and is encoded by a nucleotide sequence with access number in Genbank database NM_013609.

The term "TrkA receptor" as used in this document means the receptor for NGF, known to specialists in this field as tropomyosin receptor kinase A and Neurocrine receptor tyrosinekinase type 1 (NTRK1). Given as non-limiting examples of sequence receptor TrkA person include the amino acid sequence shown in Genbank database with access numbers NP_0010123 31 (isoform 1), NP_002520 (isoform 2) and NP_001007793 (isoform 3).

The term "receptor p75NTR"as used in this document means the receptor of neurotrophin with a molecular mass of approximately 75 kDa that binds to NGF and other neurotrophins, receptors, which are described, for example in the work of Bothwell, M. (1996) Science 272:506-507. Specified as a non-limiting example, the sequence of the receptor p75NTRman is the amino acid sequence shown in Genbank database with an access number NP_002498 encoded by the nucleotide sequence provided in Genbank database with an access number NM_002507.

The term "terminal half-life period" used in this document in respect of the antibody against NGF is the amount of time required for the concentration of antibodies measured in serum of the subject, which was introduced to the antibody is decreased by half assuming complete absorption and distribution of antibodies. When using a group of subjects, as a measure of terminal half-life of the antibody can be used an indicator of the geometric mean terminal half-life of the subjects.

The term "pharmacological half-life" as used in this document in respect of antibodies against NGF means the average amount of time maintain the effect of the drug in vivo (average retention time for the effect of the drug). It can be calculated as the ratio of the area under the efficiency curve at the initial time, adjusted relative to the baseline (AUMEC) to the square dependence of the accumulated and correction�x relative to the baseline effect of the drug versus time (area under the curve of efficiency, AUBC), using the following formula:

When using a group of subjects, as a measure of the pharmacological half-life of the antibody can be used an indicator of the geometric mean pharmacologic half-life in the regions.

The term "mutation in a hinge region" as used in this document means a mutation such as point mutation, substitution, insertion or deletion in a hinge region conservative domain of immuno globulin.

The term "inhibition" as used in this document means a statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can mean reducing a biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

The terms "antibody" or "immunoglobulin" as used in this document interchangeably include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains that keep increased stability in vivo, as described in this document. "Antibody" comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises variable region heavy chain (denoted in this document with POM�using abbreviations V Hand conservative region of the heavy chain. Conservative region of the heavy chain consists of three domains, CH1, CH2 and CH3, Each light chain comprises a variable region light chain (denoted in this document by using the abbreviation VLand conservative region light chain. The conservative region of the light chain consists of one domain, CL. Region VHand VLcan be further subdivided into hypervariable region, called regions, complementarity determining (CDR), interspersed with the more conservative areas, called frame regions (FR). Each region of VHand VLis composed of three CDR and four FR located in the direction from N-to C-Terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Conservative region of the antibodies may mediate the binding of the immunoglobulin with the tissues or factors body = host, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "monoclonal antibody" as used in this document refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population, are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are vysokospetsifichnymi, as are produced against a single antigenic site. Additionally, unlike conventional drugs (polyclonal) antibodies, which typically contain different antibodies generated against different determinants (epitopes), each monoclonal antibody generated against a single determinant on the antigen. Monoclonal antibodies can be prepared using methods known to experts in this field, for example, hybrid method, as described by Kohler et al. (1975) Nature, 256:495, using transgenic animals, as described, e.g., Lonberg, et al. (1994) Nature 368(6474); 856-859), recombinant DNA methods (see, e.g., U.S. patent No. 4, 816 567) or with the use of phage libraries of antibodies and methods described, for example, in the work of Clarkson et al., Nature, 352:624-628 (1991) and Marks et al,J. Mol. Biol, 222:581-597 (1991). Monoclonal antibodies include chimeric antibodies, human antibodies and humanized antibodies, and may be natural or recombinant methods.

The term "recombinant antibody" refers to antibodies that are prepared, expressed, created or selected with the use of recombinant human�on the Internet ways such as (a) antibodies isolated from an animal (e.g., mouse) which is transgenic or transpromotional in respect of immunoglobulin genes (e.g., genes of human immunoglobulins), or from hybridomas obtained from such an animal, (b) antibodies isolated from a host cell transformed to Express the antibody, e.g., from transfection, (b) antibodies isolated from a recombinant combinatorial library of antibodies (e.g., containing the sequence of human antibodies) using phage display, and (d) antibodies prepared, expressed created or selected by any other means, including the splicing of sequences of immunoglobulin genes (e.g., genes of human immunoglobulins) to other DNA sequences. Such recombinant antibodies may contain a variable and conservative region, derived from sequences of human immunoglobulins geneticheskogo type. However, in some embodiments, such recombinant human antibodies can be subjected to mutagenesis in vitro, and consequently the amino acid sequence of the regions VHand VLrecombinant antibodies are sequences that, though descended from geneticheskih sequences VHand VLperson, may not exist in PR�kind in the kit geneticheskih human antibodies in vivo.

The term "chimeric immunoglobulin" or " antibody means an immunoglobulin or antibody whose variable regions are derived from a first species, and whose conservative region are derived from a second biological species. Chimeric immunoglobulins or antibodies can be constructed, for example, by genetic engineering, from segments of immunoglobulin genes belonging to different biological species.

The term "humanized antibody" or "humanized immunoglobulin" means an antibody or immunoglobulin, which contains at least one humanisierung chain of the immunoglobulin or antibody (i.e., at least one humanisierung light or heavy chain). The term "chain of humanized antibody" or "chain of a humanized immunoglobulin" (i.e., "light chain of a humanized immunoglobulin" or "heavy chain of a humanized immunoglobulin" means a chain of the immunoglobulin or antibody (i.e., light or heavy chain, respectively), which includes a variable region that contains a variable frame region, substantially derived from an immunoglobulin or antibodies, and region, complementarity determining (CDR) (e.g., at least one CDR, preferably two CDRs, more preferably Tr� CDR), substantially derived from an immunoglobulin or antibody belonging to the mind, distinct from the person, and additionally includes conservative region (for example, at least one conservative region or part of it, in the case of light chain, and preferably three conservative region in the case of the heavy chain). The term "humanitariannet variable region" (e.g., "humanitariannet variable region light chain" or "humanitariannet variable region of the heavy chain") refers to a variable region that includes a variable frame region, substantially derived from an immunoglobulin or antibodies, and region, complementarity determining (CDR), substantially derived from an immunoglobulin or antibody belonging to the mind, distinct from the person.

It is assumed that the term "human antibody", as used in this document includes antibodies having variable regions in which a frame region, region CDR sequences originate from immunoglobulin geneticheskogo type of person, as described, for example, in Kabat et al. (Cm. Kabat, et al. (1991) Sequences of pro ferns of Immmological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Furthermore, if the antibody contains a conservative area of a conservative region also comes from continuous�of linota immunoglobulins geneticheskogo type of person. Antibodies may include amino acid residues not encoded by the sequences of immunoglobulins geneticheskogo type of person (e.g., mutations introduced with the use of random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, it is understood that the term "human antibody", as used in this document does not include antibodies in which CDR sequences derived from antibodies geneticheskogo type belonging to a different mammal species, such as mouse were embedded in a frame sequence of a human antibody.

A selected antibody" when used in this document means an antibody, substantially free of other antibodies having different antigenic specificnosti (e.g., the selected antibody that is specific binds to NGF, substantially free of antibodies that are specific contact with antigens, distinct from NGF). In addition, the selected antibody is typically substantially free of other cellular material and/or chemical compounds.

When used in this document, the terms "specific binding", "specific associated", "selective binding" and "selectively binds" means that an antibody or its antigen-binding part of the exhibit �substantial affinity to a particular antigen or epitope and, usually, do not exhibit significant cross reactivity against other antigens and epitopes. "Substantial" or preferred binding includes binding with an affinity of at least 106, 107, 108, 109M-1or 1010M-1. More preferred are affinity more than 107M-1preferably more than 108M-1. It is assumed that the values, which is an intermediate in relation to those specified in this document, are within the scope of the present invention and a preferred binding affinity can be indicated as a range of affinely, for example, from 106up to 1010M-1preferably from 107up to 1010M-1, more preferably from 108up to 1010M-1. An antibody that "does not show substantial cross-reactivity" is an antibody which does not substantially contact with an undesirable entity (e.g., unwanted protein object). Specific or selective binding can be determined using any of a recognized in the field of the method for determining such binding, including, for example, the analysis of graphs of Scatchard and/or analysis of competitive binding.

It is assumed that the term "KD"used in �that document means the equilibrium dissociation constant of the specific interaction of antibody-antigen or affinity of the antibody to the antigen. In one embodiment, the antibody of the present invention binds to the antigen (e.g., NGF) with an affinity (KD) equal to about 100 PM or less (i.e., or better) (e.g., about 90 PM, or about 80 PM, or about 70 PM, or about 60 PM, or about 50 PM, or about 40 PM, or about 30 PM), according to measurements carried out using the analysis method of surface plasma resonance or analysis of cell binding. In preferred embodiments, the antibody binds to NGF with an affinity (KD), which is in the range of about 25-35 PM.

It is assumed that the term "Kass"as used in this document means the rate constant of the Association for the Association of the antibody in the complex of the antibody/antigen.

It is assumed that the term "Kdiss"as used in this document means the rate constant of dissociation for the dissociation of antibodies from complex antibody/antigen.

The term "IC50"as used in this document means the concentration of antibody which inhibits the answer, as in the analysis of in vitro and in vivo analysis, to a level equal to 50% of the maximal inhibitory response, i.e., the average between the maximum inhibitory response and the response in the absence of treatment.

It is assumed that the term "nucleic acid molecule", as used in �that document include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. It is assumed that the term "selected nucleic acid molecule" as used in this document in relation to nucleic acids encoding antibodies or parts of antibodies (e.g., VHVL, CDR3) that binds to NGF, means a nucleic acid molecule in which the nucleotide sequence that encodes the antibody or part of an antibody that is free of other nucleotide sequences encoding antibodies that bind to antigens that are different from NGF, while these other sequences may naturally planirovati this nucleotide sequence in the genomic DNA of a person.

The term "functionally connected" means that a nucleic acid sequence positioned so that there is a functional relationship of this sequence to another nucleic acid sequence. For example, DNA for a signal sequence or secretory leader sequence functionally attached to DNA for a polypeptide if it is expressed in the form of a pre-protein that participates in the secretion of the polypeptide;

the promoter or enhancer functionally joined to a coding sequence if it has �lianie on the transcription of the sequence;

or the binding site of the ribosome is functionally joined to a coding sequence if it is positioned in such a way as to facilitate translation. In General, a "functionally attached to" means that the United DNA sequences are contiguous and, in the case of a secretory leader sequences, contiguous and in reading frame. However, enhancers do not have to be adjoining. The joining is achieved by ligation by standard restriction sites. If such sites are not available, use synthetic oligonucleotide adapters or linkers in accordance with standard methods. Nucleic acid is "functionally connected" when it is arranged in such a way that there is a functional relationship of this sequence to another nucleic acid sequence. For example, a promoter or enhancer is functionally joined to a coding sequence if it affects the transcription of the sequence. In relation to the sequences that regulate transcription, functionally attached means that the United DNA sequences are contiguous and, if you want to join two protein coding region, they are adjacent and are in the same reading frame. Relative�rise to sequences of switching areas, the term functionally attached indicates that the sequence is able to influence recombination in the cutover.

It is assumed that the term "vector" as used in this document means a nucleic acid molecule capable of another nucleic acid to which it was attached. One type of vector is a "plasmid", which means a closed double-stranded DNA loop, which can be legirovanyh additional segments of DNA. Another type of vector is a viral vector, wherein additional DNA segments can be legirovanyh in the viral genome. Some vectors are capable of Autonomous replication in the host cell into which they were introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal vectors mammals). Other vectors (e.g., episomal vectors mammals) can be integrated into the genome of the host cell after introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of driving the expression of genes to which they were functionally connected. Such vectors are in this document referred to as "recombinant expression vectors" (or simply "expression vectors"). In General, gene expression in�story, useful for use in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably. However, it is understood that the present invention includes such other forms of expression vectors, viral vectors (e.g., replication defective retroviruses, adenoviruses and adrenosterone viruses), which serve equivalent functions.

It is understood that the term "recombinant host" (or simply "host"), used in this document means a cell into which has been introduced the recombinant expression vector. It should be understood that it is assumed that such terms refer not only to the cell but to the progeny of such cells. Due to the fact that certain modifications may occur in succeeding generations as a result of mutations or the influence of environmental factors, such descendants may actually not be identical to the parent cell, but nevertheless remain included in the scope of the term "host" when used in this document.

The terms "treating", "treatment" and "treat" as used in this document refer to therapeutic or prophylactic methods described in this document. The methods of "treatment" include an introduction to the subject the antibody according to the present invented�Yu, for example, the entity associated with the NEF disease or condition, prevention, cure, delay, reduce the severity or reduce the intensity of one or more symptoms of a disease or condition.

The term "associated with NGF disease or condition" as used in this document means a disease or condition in which activity of NGF is involved in the occurrence of one or more symptoms of a disease or condition associated with, or mediates, or contributes to them.

Used in this document, the term "subject" includes any human or any other species of animal distinct from man. In one of the embodiments the subject is a human. The term "animal, non-human," includes all vertebrates, e.g., mammals or mammals, such as all primates, except humans, sheep, dog, cow, chickens, amphibians, reptiles, etc. have

Used in this document the term "syndrome of a ricochet" means a decreased efficiency NGF binding agents such as an antibody against NGF observed in the subject after an initial period of effectiveness after single or repeated administration. For example, treatment with antibody against NGF may initially ease the pain, for example, resulting from inflammation or nerve damage Il� etiology followed by a period of reduced analgesic effectiveness, during which the pain becomes almost as intense or more intense than before treatment. In another example, the antibody against NGF can demonstrate initial effectiveness of the entity for some period of time after single or repeated administration, such as a period length of one week after administration (e.g., days 1-7), followed by a period of reduced efficiency, such as a period from 1 to 2 weeks after administration (e.g., 7-14 days after administration). Over the period of the "syndrome of a ricochet" can be followed by a recovery period of effectiveness of the antibody against NGF. For example, there may be a biphasic profile of analgesia after single or repeated injection of antibodies against NGF, with an intermediate period of low efficiency or even excessively increased pain. The syndrome of a ricochet can be assessed, for example, in clinical trials, a pain in experimental models of pain and/or other models of the efficiency of suppression of NGF. The syndrome of a ricochet can be associated, for example, with increased pain in a subject and/or more severe adverse events (such as abnormal sensitivity, range from the degree to dysesthesia, paresthesia and Hyper - yl� hypoesthesia) during the period of the syndrome of a ricochet. Not limited to any one mechanism, the syndrome of a ricochet can be caused by changes in the expression of NGF, changes in the expression or transmission of a signal receptor TrkA or P75 or any other mechanism which leads to transient decrease in efficiency after single or repeated administration of the antibody against NGF after an initial period of effectiveness.

The various features of the present invention is described in more detail in the following subsections.

II. Antibodies of the invention

A. Increased stability in vivo

Antibodies against NGF according to the invention are characterized by enhanced stability in vivo, which is obvious from a long terminal half-life observed in vivo. He limited to any one mechanism, it is believed that prolonged terminal half-life of antibodies is the result of a reduced rate of clearance of the antibody and not increase the volume of distribution of the antibody. Antibodies of the invention include the conserved region of human IgG4, which contains a mutation. A preferred mutation is a mutation in a hinge region. Preferably, the mutation in the hinge region contains a serine mutation at amino acid position 108 in sequence ID no: 9 (SEQ. ID no: 9 shows the amino acid sequence of wild-type conservative�active region human IgG4). More preferably, the mutation in the hinge region contains a mutation replacing serine at amino acid position 108 in sequence ID no: 9 by Proline. In a preferred embodiment, a conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10.

Antibody against NGF according to the invention shows unexpectedly long terminal elimination half-life, such as a terminal elimination half-life in cynomolgus monkey of at least 15 days and typically in the range from about 15 to about 22 days (or in the range from 15 to 22 days), or in the range from about 15 days to 28 days (or in the range from 15 to 28 days), or in the range from about 21 days to about 28 days (or in the range from 21 to 28 days). This stable antibody against NGF also shows a terminal elimination half-life in rats of at least 8 days, typically in the range from about 8 to about 9 days (or in the range of 8 to 9 days). As described in more detail in example 4, PG110, the antibody against NGF according to the invention, shows the average terminal period of half-excretion in cynomolgus rhesus at least 15 days and, in the typical case, even longer. For example, in a study of cynomolgus rhesus observed average terminal elimination half-life in the range of about 15 to �rimero 22 days. In another study in cynomolgus rhesus observed average terminal elimination half-life in the range from about 21 to about 28 days. Additionally, PG110 shows the average terminal elimination half-life in rats from about 8 to about 9 days. Additionally, as those skilled in the art knows that the terminal half-life of IgG in humans is approximately two times greater than that of monkeys, predicted that antibodies against NGF according to the invention, such as PG110, will have a terminal half-life in humans of at least 10-30 days, or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, or, more preferably, at least 30 days or at least 40 days or in the range from about 10 days to about 40 days (or in the range of 10-40 days), or in the range of about 15 to about 30 days (or in the range 15-30 days). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range of four�x to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). As described in more detail in example 8, it was shown that the antibody against NGF according to the invention has a mean pharmacologic half-life in humans that fall in the above ranges.

Terminal half-life of PG110 in cynomolgus macaques significantly longer than the half-life reported by the experts in this field for other IgG4 antibodies in cynomolgus macaques. For example, it was reported the half-life of about 40-90 hours (approximately 1.6-3.8 days) in cynomolgus macaques for CDP571, IgG4 antibodies against TNF (see Stephens, S. et al. (1995) Immunol. 85:668-674). Similarly, it was reported the half-life of approximately 3 days in cynomolgus macaques for natalizumab, IgG4 antibodies against the integrin (see Refusal CHMP Assessment Report for Natalizumab, European Medicines Agency, London, 15 November 2007, Doc. Ref. EMEA/CHMP/8203/2008).

A preferred mutation in the hinge region used in the invention is the replacement of serine by Proline at position 108 in SEQ. ID no: 9. This mutation was previously described by experts in this field (see Angal, S. et al. (1993) Mol. Immunol. 30:105-108), and, as reported, it makes it impossible heterogeneity of IgG4 molecules, in particular, the formation of polymolecular antibodies containing one heavy chain and one light chain. Accordingly, the replacement of amino acids�, other than Proline, at position 108 of SEQ. ID no: 9 also included in the scope of the present invention, wherein the substitution causes the same effect as the substitution of Ser for Pro, in the prevention of heterogeneity of IgG4 molecules (for example, education of polymolecular antibodies). The ability of mutations at position 108 to prevent the heterogeneity of IgG4 molecules can be estimated, as described in Angal et al. (1993), see above.

Additionally or alternatively, was described modification at position 108 of SEQ. ID no: 9, distinct from mutations in the hinge region of IgG, which increases the affinity of the interaction of FcRn-IgG, leading to increased half-life of the modified IgG. Examples of such additional go alternative modifications include mutations at one or more residues of the conservative regions of IgG, relevant: Thr250, Met252, Ser254, Thr256, Thr307, Glu308, Met428, His433 and/or Asn434 (as further described in Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604; Petkova, S. B. ef al. (2006) Int. Immunol. 18:17591769; Hinton, P. R. et al. (2004)./. Biol. Chem. 279:6213-6216; Kamei, D. T. et al. (2005) Biotechnol. Bioeng. 92:748-760; Vaccaro, C. et al. (2005) Nature Biotechnol. 23:1283-1288; Hinton, P. R. et al. (2006) J. Immunol. 176:346-356).

Additionally, alternatively, the mutations in the hinge region, have been described by other stabilizing modifications of the conservative region of lgG4. For example, in other embodiments embodiment, the mutation is a conservative area TgG4 person includes replacement �area from CH3 lgG4 on the CH3 region of IgGI, replacement regions CH2 and CH3 from lgG4 on region CH2 and CH3 from 1 IgG or replacement of arginine at position 409 of the conservative region of lgG4 (according to Kabat numbering) for lysine, as further described in the publication of U.S. patent 20080063635. In other embodiments embodiment, the mutation is a conservative area TgG4 person includes replacement Arg409, Phe405 or Lys370 (according to Kabat numbering), such as replacement Arg409 on Lys, Ala, Thr, Met or Leu, or replacement Phe405 on Ala, Val, Gly or Leu, as further described in PCT publication WO 2008/145142.

The desired mutation can be introduced into the domain of the conservative region of human lgG4 using standard recombinant DNA techniques such as site-directed mutagenesis or mediated PCR mutagenesis of nucleic acid encoding the conservative region of human lgG4. Optionally, DNA encoding a variable region of the heavy chain of the antibody can be inserted into expression vector encoding mutant conservative region of human lgG4, so that the variable region and conserved region were functionally related, resulting in a vector that encodes a full-sized heavy chain of immunoglobulin, in which conservative field is conservative mutant region of human lgG4. Thereafter, the expression vector can be used for expression panoramer�th heavy chain immunoglobulin using standard methods of recombinant expression of proteins. For example, the antibody against NGF according to the invention can be constructed, as further described in example 1.

Terminal half-life of antibodies can be determined using standard methods known to experts in this field. For example, after administration of the antibody to a subject (e.g., cynomolgus rhesus monkey, rat line sprag-dawli), can be obtained blood samples at different time points after injection and the concentration of antibody in serum from blood samples with the use of well-known specialists in this field of methods for determining the concentration of antibodies (such as ELISA). Calculation of terminal half-life of antibodies can be performed using known pharmacokinetic methods, for example, by using computer systems and software, developed for calculation of pharmacokinetic parameters (non-limiting example of which is the system for pharmacokinetic analysis company SNBL USA with the software WinNonlin).

V. Variable regions of the antibody

Preferred variable regions of antibodies for use in antibody against NGF according to the invention are variable regions of the heavy and light chains of the antibody PG110. The variable region of the heavy chain PG110 shown in the LAST. ID no: 1 and variable region�there is a light chain PG110 shown in the LAST. ID no: 2. Accordingly, in one of the embodiments the antibody against NGF according to the invention contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1. In another embodiment, the antibody against NGF according to the invention contains a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2. In another embodiment, the antibody against NGF according to the invention contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

A full-sized amino acid sequence of the heavy chain PG110 (variable and conservative regions) shown in the LAST. ID no: 13. This heavy chain may be prepared from a precursor of the heavy chain, which contains a leader or signal sequence, such as amino acid sequence shown in SEQ. ID no: 12. The predecessor of the heavy chain with SEQ. ID no: 12 is encoded by the nucleotide sequence shown in SEQ. TH no:11.

A full-sized amino acid sequence of the light chain PG110 (variable and conservative regions) shown in the LAST. ID no: 16. This light chain can be prepared from precursor light chain, which contains �ederney or signal sequence, such as the amino acid sequence shown in SEQ. ID no: 15. Predecessor light chain with SEQ. ID no: 15 is encoded by the nucleotide sequence shown in SEQ. ID no: 14.

Accordingly, in another embodiment, the present invention is directed to the creation of antibodies against NGF containing a heavy chain comprising the amino acid sequence of SEQ. ID no: 13, wherein the antibody has a half-life of serum from cynomolgus monkey of at least 15 days. In another embodiment of the present invention the half-life of serum from cynomolgus macaque can be in the range from about 15 days to about 22 days (or in the range of 15-22 days). In another embodiment, the half-life of serum in rats may be at least 8 days, or be in the range of from about 8 days to about 9 days (or in the range of 8-9 days). In yet another embodiment, the half-life of the serum in humans may be at least 10-30 days, or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, or at least 30 days, or at least 40 days, or to be in the range from about 10 days to about 40 days (or in the range of 10-40 days), or in the range from 15 to 30 days (or in the range of 15-30 d�her). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). Preferably, encodes a heavy chain nucleotide sequence SEQ. ID no: 11. Preferably, the light chain of the antibody comprises the amino acid sequence of SEQ. ID no: 16. Preferably, the light chain encodes the nucleotide sequence of SEQ. ID no: 14.

In another embodiment, the present invention is directed to the creation of antibodies against NGF containing a heavy chain comprising the amino acid sequence of SEQ. ID no: 13 and a light chain comprising the amino acid sequence of SEQ. ID no: 16.

In another embodiment, the present invention is directed to the creation of antibodies against NGF containing a heavy chain encoded by a nucleotide sequence of SEQ. ID no: 11 and a light chain encoded by the nucleotide sequence of SEQ. ID no: 14./p>

Given the fact that the specificity of binding of PG110 provide region, complementarity determining (CDR) of the variable domain, in other embodiments, the antibody against NGF according to the invention contains a region CDR of the heavy chain PG110, light chain PG110 or both circuits. CDR 1, 2 and 3 of the heavy chain PG110 shown in the LAST. ID. Nos. 3, 4 and 5, respectively. CDR 1, 2 and 3 light chain PG110 shown in the LAST. ID nos: 6, 7 and 8, respectively. Accordingly, in one of the embodiments the antibody against NGF according to the invention contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively. In another embodiment, the antibody against NGF according to the invention contains variableyou region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively. In another embodiment, the antibody against NGF according to the invention contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and contains a variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively.

In another embodiment, the antibody against NGF according to the invention may contain variable regions of heavy and light C�drink, comprising the amino acid sequence homologous to the variable regions of heavy and/or light chains PG110, and wherein the antibodies retain the increased stability in vivo, demonstrated PG110. For example, the variable region of the heavy chain of the antibody against NGF may contain amino acid sequence that is at least 90% homologous, more preferably at least 95% homologous, more preferably at least 97% homologous, or more preferably at least 99% homologous to the amino acid sequence SEQ. ID no: 1. Variable region light chain antibodies against NGF can contain amino acid sequence that is at least 90% homologous, more preferably at least 95% homologous, more preferably at least 97% homologous, or more preferably at least 99% homologous to the amino acid sequence SEQ. ID no: 2.

When used in this document, the percent homology between two amino acid sequences is equivalent to the percent identity between two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences (i.e., % homology = number �gentechnik positions/total number of positions ×100), considering the number of admissions and length of each pass required for optimal alignment of two sequences. Comparison of sequences and determination of percent identity between two sequences can be performed using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which was used in the ALIGN program (version 2.0), using a matrix comparison of the amino acid residues RAM, the fine for the length of the pass is $ 12, and the fine for the length of the pass is $ 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm proposed by Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is used in the GAP program in the structure of the software package GCG (available at http://www.gcg.com), using the matrix Blossum 62 matrix RAM, penalty for pass equal 16, 14, 12, 10, 8, 6 or 4 and the fine for the length equal to 1, 2, 3, 4, 5 or 6.

In another embodiment, the antibody against NGF according to the invention may contain variable regions of the heavy and light chains comprising the amino acid sequence of variable regions of heavy and/or light chains G110, but in the sequence(s) were made by one or more conservative substitutions, although the antibody maintains enhanced stability in vivo, demonstrated PG110. For example, the variable region of the heavy chain of the antibody against NGF may contain amino acid sequence that is identical to the amino acid sequence SEQ. ID no: 1, except 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to SEQ. ID no: 1. Variable region light chain antibodies against NGF can contain amino acid sequence that is identical to the amino acid sequence SEQ. ID no: 2, except 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to SEQ. ID no: 2.

It is assumed that when using this document, the term "conservative amino acid substitution" means amino acid modifications that do not affect or do not change significantly the parameters of the binding or stability of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibody according to the present disclosure using standard methods known to experts in this field, such as site-directed mutagenesis and indirect pnrm mutagenesis. Conservative Amin�acid substitutions include amino acid substitutions, when this amino acid residue is replaced with an amino acid residue having a similar side chain. Family of amino acid residues having similar side chain, defined in the relevant field of knowledge. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, series, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine), branched at the beta carbon atom side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, ritalin). Thus, one or more amino acid residues consisting of variable regions of PG110 can be replaced by other amino acid residues from the same family, were identified based on the properties of the side chain, and the altered antibody can be tested for retention of function by applying methods of functional analysis described in this document.

In another embodiment, the antibody against NGF according to the invention contains an antigen-binding region (i.e., variable regions), cat�that bind to the same epitope, the PG110 antibody, or a crosshair compete with PG110 for binding to NGF. Accordingly, in one of the embodiments the antibody against NGF according to the invention compete for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

Such cross-competing antibodies can be identified based on their ability to cross-compete with PG110 in the standard analysis of binding to NGF. For example, you can use the standard ELISA in which recombinant NEF protein (e.g., NGF-R person) immobilized on the tablet, one of the antibodies is fluorescently labeled, and evaluates the ability of unlabeled antibodies competitively inhibit the binding of labeled antibodies. Additionally, or in accordance with another option to assess the ability of antibodies to cross-competition can be applied BIAcore analysis. Suitable methods for analysis of binding that can be used to test the ability of the antibody to compete for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino�isatou sequence SEQ. ID no: 2, is further described in detail in example 2.

In other embodiments embodiment, the antibody against NGF according to the invention shows one or more functional properties of the antibody PG110. For example, the antibody against NGF according to the invention may exhibit one or more of the following functional properties:

a) binds to human NGF but does not bind to neurotrophic factor brain (BDNF) man neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) persons;

b) binds to human NGF or rats with a kd of 100 PM or less;

b) inhibits binding of NGF to TrkA or p75NTR;

d) inhibits NGF-dependent proliferation of cells TF-1;

e) inhibits NGF-dependent survival of the dorsal root ganglia of the spinal cord of the chick;

(e) inhibits NGF-dependent proliferation of axons of cells PC 12.

These functional properties can be evaluated using in vitro assays, as detailed in examples 2 and 3. In relation to the specific binding of an antibody to NGF person when used in this document, the term "not associated with neurotrophic factor brain (BDNF), neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) of a person" means that the observed quantitative measure of the binding of an antibody to BDNF, NT-3 or NT-4 in the standard analysis of binding (e.g., ELISA �any other suitable method of analysis in vitro as described in the examples) is comparable with the background level of binding (for example, for the control antibody), for example, exceeding the background level is not more than 2 times, or binding to BDNF, NT-3 or NT-4 is less than 5% when compared with the binding to human NGF (the level of binding to human NGF is set as 100% binding).

In another embodiment, the present invention is directed to the creation of antibodies against nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10 (or in this conservative region of human IgG4 contains a serine mutation at amino acid position 108 in SEQ. ID no: 9, preferably a substitution of serine for Proline at position 108), and wherein the antibody binds to human NGF or rats with KDof 100 PM or less (or, according to another variant, with KDequal to 300 PM or less, 200 PM or less, 150 PM or less, 75 PM or less or 50 PM or less, inhibits binding of NGF to TrkA or p75NTRwith IC50equal to 250 PM or less (or, in accordance with another option, with IC50equal to 500 PM or less, 400 PM or less, 300 PM or less, or 200 PM or less, and inhibits NGF-dependent proliferation of cells TF-1 with IC50equal to 50 ng/ml or less (or, in the solicitation�Wii with another option with IC50equal to 150 ng/ml or less, 100 ng/ml or less, 75 ng/ml or less, or 40 ng/ml or less), Preferably, the antibody has an average terminal half-life in humans of at least 10-30 days, or at least 10 days, or at least 15 days, or at least 20 days, or at least 25 days, or at least 30 days, or in the range from about 10 days to about 40 days (or in the range of 10-40 days)or in the range from about 15 days to about 30 days (or in the range 15-30 days). Additionally, or in accordance with another variant antibody may possess intermediate pharmacological half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks (or in the range of four to six weeks), or in the range of at least four to seven weeks (or in the range from four to seven weeks), or in the range of at least four to eight weeks (or in the range of four to eight weeks). Additionally, or in accordance with another variant antibody may exhibit an average terminal half-life in cynomolgus monkey of at least 15 days and typically in the range from about 15 to about 22 days (or in the range from 15 to 22 days), or in the range of about� about 15 days to 28 days (or in the range from 15 to 28 days), or in the range from about 21 days to about 28 days (or in the range from 21 to 28 days). Additionally, or in accordance with another variant antibody may exhibit a terminal elimination half-life in rats of at least 8 days, typically in the range from about 8 to about 9 days (or in the range of 8 to 9 days). The antibody may further exhibit one or more additional functional properties such as binding to human NGF, but the lack of binding neurotrophic factor brain (BDNF) man neurotrophin 3 (NT-3) the person or neurotrophin 4 (NT-4) persons;

inhibition of NGF-dependent survival of the dorsal root ganglia of the spinal cord of the chick; and/or the inhibition of NGF-dependent proliferation of axons of cells PC 12. Preferably, the antibody alleviates pain for a period of at least from about one week to about twelve weeks after administration of a single dose of the antibody against NGF entity. Preferably, the antibody contains a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and the antibody contains a variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and the antibody contains� variable region of the heavy chain, comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, and variable area light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID. nos: 6, 7 and 8, respectively. Preferably, the antibody contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, or the antibody contains a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2 or an antibody contains a variable region heavy chain comprising the amino acid sequence of SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2, or the antibody competes for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID no: 2.

In another embodiment, the antibody against NGF according to the invention does not cause the syndrome of a ricochet when administered to a subject (for example, the antibody is administered in such dosage and frequency, to avoid the subject of the syndrome of a ricochet). The syndrome of a ricochet, in which the antibody against NGF demonstrates a decreased efficiency in a subject after the initial�CSOs efficiency time after single or repeated administration, it was reported in the as studies on animal model and clinical studies of other antibodies against NGF. For example, this effect, called "rebound phenomenon", was reported for antibodies against NGF rats in a model of chronic constriction (CCI) in rats (Ro, L-S. et al. (1999) Pain 79:265-274). Additionally, it was reported on clinical studies of pain with the use of antibodies against NGF tanezumab (also known as RN624, E3, CAS registry no.88026657-9), in which the period of increased manifestations of adverse events, such as sensitivity to touch and a sense of tingling, was observed after the initial pain period (see the report presentation Hefti, Franz F., Rinat Neuroscience, LSUHSC, Shreveport, Louisiana, made on 26 September 2006) is Not limited to any one mechanism, it is believed that the prolonged terminal half-life of antibodies against NGF according to the invention allows them to avoid induction of the syndrome of a ricochet. Thus, other advantages of antibodies against NGF according to the invention include a more stable and long-lasting activity in vivo, compared with other prototype antibodies against NGF. Taking into account the prolonged terminal half-life of antibodies against NGF according to the invention, it is possible to apply a lower dosage (compared with other antibodies against NGF), the antibody can prima�ive at more frequent intervals, if it is necessary, therefore, dosages and regimens can be chosen so that the subject does not occur the syndrome of a ricochet.

In another embodiment, the antibody against NGF according to the invention is able to alleviate pain in a subject over a long period, for example, the antibody is able to ease the pain for a period of at least from about one week to about twelve weeks (or for from one week to twelve weeks) after administration of a single dose of the antibody against NGF entity. In one embodiment, the antibody alleviates pain for a period of at least about one week (or at least one week) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about two weeks (or at least two weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about four weeks (or at least four weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about eight weeks (or at least eight weeks) after administration of a single dose �ntitle against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about twelve weeks (or for at least twelve weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least from about four weeks to about twelve weeks (or four weeks to twelve weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least from about eight weeks to about twelve weeks (or eight weeks to twelve weeks) after administration of a single dose of the antibody against NGF entity.

The ability of the antibody to alleviate pain in a subject can be assessed using the methods of analysis adopted in this area. Suitable animal model to estimate the duration of pain relief antibodies against NGF is described for example in PCT publication Nos. WO 2006/131951 and published U.S. patent 20080182978. Non-limiting examples of such animal models include a model of neuropathic pain induced by chronic compression of the sciatic nerve, a model of postoperative pain, including dissection of the front paw model of pain in rheumatoid arthritis, including arthritis, induzirovanny� full of adjuvant freind (CFA), and models of pain with the cancer, such as those described in the works Halvorson, K. G. et al. (2005) Cancer Res. 65:9426-9435 and Sevcik, M. A. et al. (2005) Pain 115:128-141. Additionally, pain relief can be assessed clinically in humans, and duration of pain relief can be determined on the basis of patient reports(s) receiving treatment with antibody against NGF, about the degree of pain.

In other embodiments embodiment, the antibody against NGF according to the invention can contain a variable region of the heavy chain and/or variable region light chain antibodies against NGF, described by experts in this field. For example, the variable region of the heavy chain and/or variable region light chain antibodies against NGF, described in PCT publication no WO 2001/78698, PCT publication no WO 2001/64247, PCT publication no WO 2002/096458, PCT publication no WO 2004/032870, PCT PCT publication no WO 2004/058184, PCT publication no WO 2005/061540, PCT publication no WO 2005/019266, PCT publication no WO 2006/077441, PCT publication no WO 2006/131951, PCT publication no WO 2006/110883, PCT publication no WO 2009/023540, U.S. patent No. 7 449 616;

the publication of U.S. patent No. US 20050074821, the publication of U.S. patent No. US 20080033157, the publication of U.S. patent No. US 20080182978 or publication of U.S. patent No. US 20090041717, may be applied to the antibody against NGF according to the invention.

In other embodiments embodiment, the antibody against NGF according to the invention can contain a variable region heavy C�PI and/or variable region light chain antibodies against NGF, which is prepared with the use of standard, well-known specialists in this field, the method of obtaining monoclonal antibodies, such as the standard method of hybridization of somatic cells, described by Kohler and Milstein (1975) Nature 256:495 to generate monoclonal antibodies that are not owned by the person (antibodies, which can then be humanized), as well as ways of using libraries in phage display format or methods with the use of transgenic animals expressing genes of human immunoglobulins. Methods with the use of libraries in phage display format for the selection of antibodies is described, for example, McCafferty et al, Nature, 348:552-554 (1990), Clarkson et al. Nature, 352:624-628 (1991), Marks et al, J. MolBiol, 222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23, 344-348; U.S. patents №№5 223 409; 5 403 484 and 5 571 698 owned by Ladner et al; U.S. patents №№5 427 908 and 5 580 717 owned by Dower et al; U.S. patents №№5 969 108 and 6 172 197 owned by McCafferty et al; and U.S. patents№№5 885 793; 6 521 404; 6 544 731; 6 555 313; 6 582 915 and 6 081 593 owned by et al. The methods of using the transgenic animals expressing genes of human immunoglobulin, to obtain the antibodies are described, for example, Lonberg, et al. (1994) Nature 368(6474):856-859; Lonberg, N. and Hussar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY. Acad. Sci. 764:536-546; U.S. patents№№5 545 806; 5 569 825; 5 625 126; 5 633 425; 5 789 650; 5 877 397; 5 661 016; 5 814 318; 5 874 299 and 5 770 429; all Prina�lie Lonberg and Kay; U.S. patent No. 5, 545 807 owned by Surani et al, PCT publications Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all belong to Lonberg and Kay; PCT publication WO 02/43478 owned by Ishida et al, U.S. patents№№5 939 598; 6 075 181; 6 114 598; 6 150 584 and 6 162 963 owned by Kucherlapati et al.

In various embodiments embodiment, the antibody against NGF according to the invention may be a chimeric antibody, humanized antibody or human antibody. Additionally, the antibody may be an antibody, which were removed potential epitopes of T cells. Ways to remove potential epitopes for T-cells to reduce potential immunogenicity of the antibody have been described in the art (see, for example, the publication of U.S. patent No. 20030153043 owned by Can et al).

The antibody or part of an antibody according to the invention may be derived from another functional molecule, or be connected with it (e.g., another peptide or protein). Accordingly, it is assumed that the antibodies or parts of antibodies according to the invention include derivatives and otherwise modified forms of antibodies PG110 described in this document. For example, the antibody or part of an antibody according to the invention can be functionally connected (by chemical coupling, genetic fusion, noncovalent Association or otherwise) to one or more other molecules, such as other antic�lo (for example, bispecific antibody or diatel), detected agent, cytotoxic agent, a pharmaceutical agent and/or a protein or peptide that can mediate Association of the antibody or part of an antibody with another molecule (such as the korowai area streptavidin or polyhistidine label).

One type derivatif antibodies receive a transverse cross-linking two or more antibodies (of the same type or different types, for example, to create bispecific antibodies). Suitable agents for crosslinking include those, which heterobifunctional having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidomethyl-N-hydroxysuccinimidyl ether), or homobifunctional (for example, disuccinimidyl suberate). Such linkers are available for purchase in the company Pierce Chemical Company, Rockford, Illinois, USA.

Useful the detected agents can be obtained from derivatives of the antibodies or parts of antibodies according to the invention include fluorescent compounds. Examples of the fluorescent detectable agents include fluorescein, the fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrine, etc. can Also be obtained from the derivative of the antibody with detektirovanie enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase, etc. When the antibody is obtained as a derivative to detect the enzyme, it is detected by adding additional reagents that are used by the enzyme with the formation of the detected reaction product. For example, when there is detected the agent is horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine leads to the manifestation of a colored reaction, which can be used to detect. The antibody can also be derived with Biotin, and detected through indirect measurement of the level of binding of the avidin or streptavidin.

III. Antibodies

Another feature of the present disclosure relates to nucleic acid molecules that encode the antibodies of the disclosure. The nucleic acid may be present in a whole cell, cell lysate, or in a partially purified or substantially purified form. Nucleic acid is "isolated" or "substantially purified" when it is purified from other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard methods, including treatment with alkali/LTOs, zonal centrifugation in CsCl solution, column chromatography, agarose gel electrophoresis and others well known to specialists in this field ways. Cm. F. Ausubel,et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid according to the present disclosure may be, for example, DNA or RNA, and may contain or not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule. Nucleic acids according to the present disclosure may be obtained using standard techniques of molecular biology.

A preferred nucleic acid molecule according to the invention comprises the nucleotide sequence of SEQ. ID no: 11. Another preferred nucleic acid molecule according to the invention comprises the nucleotide sequence of SEQ. ID no: 14.

After receiving the DNA fragments encoding the segments of the VHand VLwith these DNA fragments can be produced additional manipulation using standard techniques of recombinant DNA, for example, to transform the genes of variable regions of genes in a full-sized antibody chains so that the variable region is functionally linked to the conservative sphere (see e.g., example 1). It is assumed that the term "functionally linked" when used in this context means that two fragments of DNA are joined such that the amino acid sequence encoded by the two DNA fragments are in the same R�the MCA reading.

Antibodies can be produced in the host cell using methods known to those skilled in the art (e.g., Morrison, S. (1985) Science 229:1202). For example, for expression of the antibody, DNA encoding the heavy and light chains can be inserted into expression vectors such that the genes were functionally linked to sequences controlling transcription and translation. It is assumed that in this context, the term "functionally linked" means that the antibody gene Legerova into the vector in such a way that sequences controlling transcription and translation and located within the vector, perform the functions for which they were intended, on the regulation of the transcription and translation of the gene of the antibody. Investigating the expression vector and sequences that control expression, selected so that they were compatible with the expression used in the host cell. Gene light chain antibody gene and the heavy chain of the antibody can be inserted into different vectors, or, more typically, both genes are inserted into the same expression vector. The genes of the antibody is inserted into the expression vector using standard methods (for example, subsidized prices of complementary restriction sites on the fragment of the antibody gene and the vector, or ligation of blunt ends restriction sites no�Ute). Additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the chains of the antibody from the host cell. Gene chain antibodies can be cloned into the vector such that the signal peptide was attached in one frame read from N-Terminus of the gene chain antibodies. The signal peptide may be the signal peptide of the immunoglobulin or a heterologous signal peptide (i.e., a signal peptide of a protein other than an immunoglobulin).

In addition to genes chain antibodies, recombinant expression vectors typically carry regulatory sequences that control the expression of genes chains of the antibody in the host cell. It is assumed that the term "regulatory sequence" includes promoters, enhancers and other elements that control expression (e.g., polyadenylation signals) that control the transcription or translation of genes chain antibodies. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). The person skilled in the art it is clear that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of host cell for transformation, the desired level of protein expression, etc. Preferred regulatory sequences for expression in the cells of the host, belonging to mammals, include viral elements that provide high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), aqualicious simian virus 40 (SV40), adenovirus (such as adenovirus major late promoter (AdMLP) and polyomavirus. In accordance with another option, can be used non-viral regulatory sequences, such as promoter of ubiquitin gene or the promoter of the gene of β-globin gene. Further, regulatory elements composed of sequences from other sources, such as the promoter of the SRa system, which contains sequences from the early SV40 promoter and the long terminal repeat of the virus T-cell leukemia type 1 (Takebe, Y. et al. (1988) Mol, Cell. Biol. 8:466-472).

In addition to genes chains of antibodies and regulatory sequences, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in the cells of the host (for example, oridzhiny replication), genes and selectable markers. Gene selectable marker facilitates the selection of host cells into which has been introduced a vector (see, e.g., U.S. patent Nos. 4 399 216, 4 634 665 and 5 017 179, all owned by Axel et al.). For example, in the typical case, the gene selectable marker p�idet resistance to medicinal substances, such as G418, hygromycin or methotrexate, the cell host, which was introduced vector. Preferred genes of selectable markers include the dihydrofolate reductase gene (DHFR) (for use in dhfr - cells with the selection/amplification in the presence of methotrexate) and the gene peo (for selection in the presence of G418).

For expression of the light and heavy chain expression vector(s) encoding the heavy and light chains, transferout in a host cell using standard methods. Means that various forms of the term "transfection" includes a wide variety of ways, commonly used for introducing exogenous DNA into a prokaryotic or a eukaryotic host cell, e.g., electroporation, calcium phosphate precipitation, and transfection with the use ؤ]❊]-dextran, etc. Although it is theoretically possible to Express the antibodies in prokaryotic and eukaryotic cells-hosts, expression of antibodies in eukaryotic cells, and most preferably in the cells of the host mammal belonging, is the most preferred because such eukaryotic cells and in particular mammalian cells, more likely than prokaryotic cells, will provide the Assembly and secretion of properly coagulated and immunologically active antibody. It was reported, Thu� prokaryotic expression of antibody genes ineffective to obtain a high yield of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred the host cell belonging to the mammal for expression of recombinant antibodies according to the present disclosure include cells Chinese hamster ovary (Cho cells) (including dhfr cells-CHO is described in Uriaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a selectable marker DHFR, for example, as described in R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), cells myeloma NSO, COS cells and SP2 cells. Another preferred expression system is the system is the GS gene expression described in WO 87/04462 (owned by Wilson), WO 89/01036 (owned by Bebbington) and EP 338 841 (owned by Bebbington). When recombinant expression vectors encoding antibody genes, introduced into the host cell belonging to mammals, antibodies get, cultivating the host cell within a period of time sufficient for receiving expression of the antibody in the cells of the host, or, more preferably, secretion of the antibody into the culture medium in which they grow the host cell. The antibodies can be extracted from the culture medium using standard methods purification of proteins.

In a preferred embodiment, the present invention is directed to a expression vector that encodes the antibody against NGF, wherein the vector contains a nucleotide pic�egovernance LAST. ID no: 11 encoding a heavy chain of the antibody and the nucleotide sequence of SEQ. ID no: 14, encoding light chain of the antibody. A preferred expression vector of the invention contains the gene for GS (glutamylcysteine). In yet another preferred embodiment of the present invention is directed to a host cell containing the expression vector according to the invention. A preferred host cell of the invention is a cell Cho (Chinese hamster ovary). In yet another preferred embodiment of the present invention is directed to a method of expression of antibodies against NGF, comprising culturing the host cell contains the expression vector containing the nucleotide sequence of SEQ. ID no: 11 (encoding the heavy chain of the antibody and the nucleotide sequence of SEQ. ID no: 14 (encoding light chain antibodies), so that the expressed antibody against NGF containing a heavy chain encoded by SEQ. ID no: 11 and a light chain encoded by SEQ. ID no: 14.

Another feature is that the invention relates to a method of producing antibodies against NGF, which carries a mutation in a conservative region of the antibody (e.g., a mutation in a hinge region), the method comprising introducing appropriate mutations in a conservative region, e.g.�, using standard techniques of recombinant DNA. For example, the present invention is directed to a method of producing antibodies against NGF, wherein the antibody comprises (i) a variable region heavy chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 3, 4 and 5, respectively, (ii) variable region light chain comprising CDR 1, 2 and 3 having the amino acid sequence SEQ. ID nos: 6, 7 and 8, respectively, and (iii) conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10 (and, for example, wherein the antibody has a mean terminal elimination half-life in humans of at least 10-30 days), or, additionally or in accordance with another option, has a mean pharmacologic half-life in humans of at least 30 days, or at least 35 days, or at least 40 days, or in the range of at least four to six weeks, or in the range of four to six weeks, or in the range of at least four to seven weeks or in the range from four to seven weeks, or in the range of at least four to eight weeks, or in the range of four to eight weeks), the method of producing the antibody comprises introducing a mutation replacing serine at position AMI�of ocelote 108 in the LAST. ID no: 9 by Proline with the creation of the conservative region of human IgG4, containing the amino acid sequence of SEQ. ID no: 10. Preferably, the variable region of the heavy chain comprises the amino acid sequence of SEQ. ID no: 1. Preferably, the variable region of the light chain comprises the amino acid sequence of SEQ. ID no: 2. Preferably, the heavy chain comprises the amino acid sequence of SEQ. ID no: 13. Preferably, the light chain includes the amino acid sequence of SEQ. ID no: 16.

IV. The pharmaceutical composition

Another feature is that the present invention is directed to a compositions, e.g., pharmaceutical compositions containing the antibody according to the invention in admixture with a pharmaceutically acceptable carrier. In the preferred embodiments of pharmaceutical compositions suitable for administration intravenously, subcutaneously (e.g., with a syringe-pen) or intra-articular, however, other suitable methods of administration described in this document. In one of the embodiments the composition can include a combination of multiple (e.g., two or more antibodies according to the invention, for example, antibodies that bind to different epitopes on NGF.

When used in this document, "pharmaceutically acceptable carrier" includes any and �CoE solvents, salt, dispersing medium, the agents for coating, antibacterial and antifungal agents, isotonic and attracive absorption agents, etc., which are physiologically compatible. Depending on the method of administration, the active substance may be covered with material that protects the active substance from the action of acids and other natural conditions that may inactivate the substance.

"Pharmaceutically acceptable salt" means a salt that retains the desired biological activity of the parent compound and does not cause any undesired Toxicological effects (see, e.g., Berge, S. M., et al. (1977)./. Pharm. Sci. 66:1-19). Examples of such salts include salts formed by the addition of acid, and salts formed by the addition of the base. Salts formed by the addition of acids, include salts that are derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, Hydrobromic, itestosterone, phosphorous and the like, and nontoxic organic acids such as aliphatic mono - and dicarboxylic acids, phenyl-substituted carboxylic acids, hydroxycarbonate acids, aromatic acids, aliphatic and aromatic sulfonic acid, etc. Salts formed by the addition of bases include salts, derived alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, and nontoxic organic amines, such as N,N'-dibenziletilendiaminom, N-methylglucamine, chloroprocaine, choline, diethanolamine, Ethylenediamine, procaine, etc.

Pharmaceutical compositions may be administered alone or in combination therapy, i.e. in combination with other agents. For example, combination therapy can include a composition of the present invention with at least one or more additional pharmaceutical agents. For example, at least one or more additional pharmaceutical agents may be administered separately or may also be included in the composition. In preferred embodiments, the antibody against NGF according to the invention is administered in combination with a second pharmaceutical agent, wherein the second pharmaceutical agent selected from the group consisting of NSAIDs, analgesics (including opioid analgesics and atypical analgesics, local anesthetics, drugs for conductive anesthesia, drugs for phenol blockade, therapeutic antibodies, steroids, anticonvulsants, antidepressants, local capsaicin and antiviral agents. In particular, the preferred second class of pharmaceutical agents for the prima�need to alleviate pain are the opioid analgesics. Additionally, or in accordance with another variant, the second mode of treatment may be combined with the use of the antibodies according to the invention, for example, for pain relief. Examples of such second modes of treatment include radiation therapy (for example, in the case of pain in cancer), surgical procedures (e.g., ablation (using a needle) procedures on Gusarova ganglia and the area located behind the trigeminal node, trigeminal neuralgia), hypnosis and acupuncture.

Examples of NSAIDs include acetylated salicylates, including aspirin; aacetaminophen salicylates, including salsalate, diflunisal; acetic acid derivatives, including etodolac, diclofenac, indomethacin, Ketorolac, nabumetone; propionic acid derivatives, including fenoprofen, flurbiprofen, ibuprofen, Ketoprofen, naproxen, naproxen sodium, oxaprozin; fenamate, including meclofenamic acid, mefenamic acid, phenylbutazone, piroxicam; inhibitors of COX-2, including celecoxib, etoricoxib, valdecoxib, rofecoxib, lumiracoxib. Examples of analgesics include paracetamol (acetaminophen), tramadol, capsaicin (local), opioid analgesics and atypical analgesics. Examples of opioid analgesics include morphine, codeine, thebaine, hydromorphone, hydrocodone, oxycodone, Oxymorphone desomorphine, diacetylmorphine, Nicomorphine, dipropanoylmorphine, benzylmorphine, Ethylmorphine, fentanyl, pethidine, methadone, tramadol and propoxyphene. Examples of atypical analgesics include tricyclic antidepressants, carbazepine, gabapentin, pregabalin, DULOXETINE and caffeine. Examples of steroids include intra-articular corticosteroids (LAC) and prednisolone. Examples of therapeutic antibodies include antibodies against TNF, such as Remicade® and Humira®, and antibodies against CD20, such as Rituxan® and Arzerra™. Examples of antiviral agents include acyclovir and oseltamivir phosphate (Tamiflu®).

In a preferred embodiment, the combination therapy can include an antibody against NGF according to the present invention with at least one or more TrkA inhibitors (e.g., compounds that are antagonists of TrkA activity). The TrkA inhibitors may act, for example, by extracellular interaction with the TrkA receptor, or by intracellular interaction with components of signal transduction from TrkA (for example, by inhibiting the kinase activity of TrkA). Non-limiting examples of extracellular TrkA inhibitors include antibodies against TrkA (such as humanized antibodies against TrkA, described in the publication of U.S. patent No. 20090208490 and the publication of U.S. patent No. 20090300780) and peptide mimetics of NGF, which �antagonistami TrkA (such as those described in the work Debeir, T. et al. (1999) Proc. Natl. Acad. Sci. USA 96:4067-4072). Non-limiting examples of intracellular TrkA inhibitors include able to penetrate into the cell peptides are antagonists of TrkA function (e.g., as described in Hirose, M. et al. (2008) J. Pharmacol. Sci. 106:107-113; Ueda, K. et al. (2010) J. Pharmacol. Sci., March 30, 2010 issue) inhibitors in the form of small molecules, such as inhibitors of TrkA kinase (e.g. as described by Wood, E. R. et al. (2004) Bioorg. Mod. Chem. Lett. 14:953-957; Tripathy, R. et al. (2008) Bioorg. Med. Chem. Lett. 18:3551-3555). Other non-limiting examples of TrkA inhibitors include ARRY-470 and ARRY-872 (Array Biopharma).

In yet another preferred embodiment, the combination therapy can include an antibody against NGF according to the present invention with at least one or more inhibitors of protein kinase C (PKC) (e.g., compounds that are antagonists of PKC activity).

The composition of the present invention may be administered using various methods known to experts in this field. The person skilled in the art would understand that the path and/or the method of administration will vary depending on the desired results. Preferably, the carrier is suitable for intravenous, intraarticular, subcutaneous, intramuscular, parenteral, intratumoral, intranasal, intravesicular, intra-articular, oral, mucosal, podjasek�wow, spinal or epidermal management or installation in the body cavity (e.g., peritoneal cavity, pleural cavity, paranasal cavities of the nose) or on the surface of the eye, or the introduction into the lungs by inhalation. For specific routes of administration can be selected by applying suitable products for delivery. For example, for subcutaneous or intramuscular injection can be used the autoinjector (e.g., one that allows to carry out the introduction). These pens, also called injectors, known to specialists in this field, including those that incorporate a liquid dose of the antibody (e.g., an autoinjector for single use, used for the introduction of Humira®), and, more preferably, those that contain a dose of the antibody in the form of dry matter, which is reduced in liquid form immediately before injection. Also for subcutaneous administration can be used subcutaneous implants. Additionally, transdermal delivery can be achieved using topical transdermal (percutaneous) of the plaster (e.g. plaster). Transdermal delivery can also be achieved with the use of injections dry powder (such as injection using injectors available for purchase from the company Glide Pharma). Additionally, for delivery to the lungs (for example, in the treatment�AI asthma or chronic cough), can be applied with spray solution in the nebulizer.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as the formulation of controlled release, including implants, transdermal patches, and microencapsulated delivery systems. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycol acid, collagen, polyarteritis and polylactic acid. Many methods for preparing such formulations are patented or generally known to specialists in this field. See, for example. Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

For the introduction of compounds according to the invention with the use of certain routes of administration may require a coating compound sheath, or joint compound introduction with a material to prevent its inactivation. For example, the compound can be administered to a subject in an appropriate carrier, for example, in liposomes, or a diluent. Pharmaceutically acceptable diluents include sodium chloride solution and aqueous buffer solutions. Liposomes include emulsions of gum from corn fibers of the type water-in-oil and oil-in-water, and standard liposomes (Strejan et al. (1984) J. Neuroimmunol, 7:27).

Pharmaceutically acceptable� carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate admission. The use of such media and agents for pharmaceutically active substances known to specialists in this field. Except in cases where any standard medium or agent is incompatible with the active compound, their use in pharmaceutical compositions according to the invention is regarded as coming within the scope of the invention. Auxiliary active compounds can also be introduced into the composition.

Therapeutic compositions typically must be sterile and stable under conditions of manufacture and storage. Compositions can be prepared in the form of a solution, microemulsion, liposome, or other ordered structure suitable for high concentration of the drug. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, etc.), and their suitable mixtures. The desired fluidity can be maintained, for example, through the use of the shell, for example, lecithin, maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases it will be preferable to include in the composition isotonic agents, for example, sugars, property, such as mannitol, sorbitol, or sodium chloride. Prolonged absorption injects�innovative compositions can be achieved by the inclusion in the composition of the agent, which delay absorption, for example monostearate salts and gelatin.

Sterile injectable solutions can be prepared by adding the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization with the use of microfiltration. Generally, dispersions are prepared by adding the active compound into a sterile medium containing a basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which produce a powder of the active substance in a mixture with additional desired ingredient from their solution, previously resterilizing filtering.

Dosing regimens adjusted to provide the optimum desired response (e.g., therapeutic response). For example, can be entered one-time loading dose, several divided doses may be administered within a certain time or the dose may be proportionally reduced or increased, in accordance with the requirements of a particular therapeutic situation. Typical �Azova dose (which may be entered in accordance with the mode of dosing as described further below) may be in the range from about 0.1 μg/kg to 1 mg/kg or 3 mg/kg or 30 mg/kg or 300 μg/kg or 3000 µg/kg (3 mg/kg) or 30 mg/kg or 100 mg/kg or more, depending on the factors described in this document. For example, the antibody against NGF can be administered at a dose of about 1 μg/kg, about 10 μg/kg, about 20 μg/kg, about 50 μg/kg, about 100 μg/kg, about 200 μg/kg, about 300 μg, about 400 μg/kg, about 500 μg/kg, about 1 mg/kg, about 2 mg/kg or about 3 mg/kg. In preferred embodiments, the antibody against NGF is administered in a dose within the range of from about 3 μg/kg to about 3000 µg/kg. In another preferred embodiment, the antibody against NGF is administered in a dose of 100 µg/kg. In another preferred embodiment, the antibody against NGF is administered at a dose of 200 µg/kg. In another preferred embodiment, the antibody against NGF is administered at a dose of 300 mcg/kg. In another preferred embodiment, the antibody against NGF is administered in a dose of 400 µg/kg.

For repeated administrations over several days, weeks, or months, or longer, depending on the condition, the treatment lasts until a desired suppression of symptoms or until sufficient therapeutic levels (e.g., pain). Cited as an example of a dosing regimen comprises administering an initial dose in the range of from about 3 μg/kg to 500 μg/kg, with subsequent monthly support�her a dose of about 3 mg/kg to 500 μg/kg of the antibody against NGF. In another embodiment of the present invention, the dose is about 200 μg/kg, administered once every month. In another embodiment of the present invention, the dose is about 400 mg/kg, administered once every two months. However, it may be useful to other dosing regimens, depending on the nature of pharmacokinetic decay, which wants to achieve a medical practitioner. For example, in some embodiments of the incarnation involves the introduction of doses one to four times a week. However, given the long duration of the pain relief antibodies against NGF can be used less frequent administration of doses. In some embodiments embodiment, the antibody against NGF is injected once every week, once every 2 weeks once every 3 weeks once every 4 weeks, once every 5 weeks, once every 6 weeks once every 7 weeks once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 15 weeks, once every 20 weeks, once every 25 weeks, once every 26 weeks or longer. In some embodiments embodiment, the antibody against NGF is injected once every 1 month, once every 2 months once every 3 months once every 4 months, once every 5 months, once every 6 month� or longer.

As is discussed further in example 6, in a preferred embodiment, the antibody against NGF according to the invention is administered (e.g., human) intravenously at a dose ranging from 0.1 mg/kg to 0.2 mg/kg, preferably 0.15 mg/kg, once every 12 weeks. In another preferred embodiment, the antibody against NGF according to the invention is administered (e.g., person) subcutaneously in a dose ranging from 0.2 mg/kg to 0.4 mg/kg, preferably 0.3 mg/kg, once every twelve weeks. In other embodiments embodiment, the antibody against NGF according to the invention is administered in a dose ranging from 0.1 mg/kg to 3 mg/kg, or between 0.1 mg/kg to 30 mg/kg, or between 0.1 mg/kg to 20 mg/kg, or between 0.1 mg/kg to 10 mg/kg, or between 1 mg/kg to 30 mg/kg, or between 1 mg/kg to 20 mg/kg, or in the range from 1 mg/kg to 10 mg/kg.

A special advantage is the formulation development of parenteral compositions in the form of dosage units for ease of administration and uniformity of dosage. The shape of the dosing unit when used in this document means physically separated units, prepared as single dosages for the subjects receiving treatment; each unit contains a predetermined quantity of active compound calculated to produce the required terapevticheskoj� effect in Association with the required pharmaceutical carrier. Specification for forms of dosage units according to the invention is attributable to and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the field of preparation of such active compounds for the treatment of sensitivity in patients. For example, non-limiting examples of forms of dosage units comprise from 0.2 mg (corresponding to a dose of 3 mg/kg for a patient weighing about 70 kg), 2 mg (corresponding to a dose of 30 µg/kg for a patient weighing about 70 kg) and 7 mg (corresponds to a dose of 100 µg/kg for a patient weighing about 70 kg).

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) fat-soluble antioxidants, such as ascorbyl palmitate, trouble soothing (VNA), stabilizer (VIT), lecithin, propylgallate, alpha-tocopherol, etc.; and (3) metal-chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

In one embodiment, the formulations can be presented in the form of dosage units and may be a prig�are made in any way, known to experts in the field of pharmacy. The shape of the dosing unit when used in this document means physically separated units, prepared as single dosages for the subjects receiving treatment; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in Association with the required pharmaceutical carrier. The amount of active substance that can be combined with a carrier material to obtain a single dosage form will vary depending upon the subject treated and the particular route of administration. The amount of active substance that can be combined with a carrier material to obtain a single dosage form, in the General case will be equal to the amount of the composition that provides a therapeutic effect. In the General case, on the basis of one hundred percent, this amount will range from about 0.001 per cent to about ninety percent of active substance, preferably from about 0.005 percent to about 70 percent, most preferably from about 0.01 percent to about 30 percent.

The phrases "parenteral administration" and "introduced parenterally when used in this document means the methods introduced�I, than entirelynew and local administration, usually with the use of injection, and includes, in particular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, vnutriglaznogo, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Examples of suitable aqueous and nonaqueous carriers that may be used in pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable mixtures, vegetable oils, such as olive oil, and injectable organic esters, such as ethyloleate. The desired fluidity can be maintained, for example, through the use of the materials for the shell, for example, lecithin, maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. In particular, examples of adjuvants which are well known to specialists in this field include, for example, inorganic adjuvants (such as aluminum salts, for example, Alu�the action of the phosphate and aluminium hydroxide), organic adjuvants (such as squalene), adjuvants based on oil, virosomes (e.g. virosome that contain membrane-associated hemagglutinin and neuraminidase derived from the influenza virus).

Prevention of presence of microorganisms may be ensured both sterilization methods, see above, and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. may Also be desirable to include in the composition isotonic agents such as sugars, sodium chloride, etc. in addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

When compounds of the present invention are administered as pharmaceuticals to humans or animals, they can be let separately or as part of a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably from 0.005 to 70%, for example, from 0.01 to 30%) of the active substance in combination with a pharmaceutically acceptable carrier.

Regardless of the chosen route of administration, the compounds of the present invention that can be used in a suitable hydrated form, and/or pharmaceutical compositions of the present invention is prepared�I formulations in pharmaceutically acceptable dosage forms by standard methods, known to specialists in this field.

The actual levels of dosages of active ingredients in the pharmaceutical compositions of the present invention can be modified so as to obtain an amount of active substance that is effective to achieve the desired therapeutic response with respect to a particular patient, composition and method of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions used according to the present invention, or the ester, salt or amide, route of administration, time of administration, rate of excretion of the specific compound, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions used, age, gender, body weight, condition, General health and prior medical history of the patient receiving treatment, as well as factors known to experts in the field of medicine. A physician or veterinarian having a standard expertise that can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, the physician or veterinarian could start doses of the compounds according to the invention, Prim�employed in pharmaceutical compositions located on a level lower than required to achieve the desired therapeutic effect and gradually increase the dosage to achieve the desired effect. In General, suitable daily dose of the composition according to the invention will be equal to the number of connections, which is the lowest dose effective to obtain a therapeutic effect. Such an effective dose in the General case will depend on the factors described above.

Therapeutic compositions may be administered with the use of medical devices known to specialists in this field. For example, in a preferred embodiment therapeutic composition according to the invention may be administered with the use of needle-free products for subcutaneous administration, such as products disclosed in U.S. patents№№5 399 163, 5 383 851, 5 312 335, 5 064 413, 4 941 880,4 790 824 or 4 596 556. Examples of well known implants and modules useful in the present invention include: U.S. Patent No. 4 487 603, which describes an implantable microinfusion pump for dispensing of drugs at a controlled rate; U.S. patent No. 4, 486 194, which describes therapeutic product for the introduction of drugs through the skin; U.S. patent No. 4, 447 233, which describes a medical infusion pump for the delivery of drugs at a certain with�oresti infusion; U.S. patent No. 4, 447 224, which describes an implantable infusion apparatus with adjustable flow for continuous drug delivery; U.S. patent No. 4 439 196, which describes the osmotic delivery system of medicines with a multi-chamber compartments; and U.S. patent No. 4 475 196, which describes the osmotic delivery system of medicines. Many other implants, delivery systems, and modules known to specialists in this field,

In some embodiments the monoclonal antibodies of the invention can be included in the formulation, ensuring the required distribution in vivo. For example, the blood-brain barrier (BBB) does not miss many highly hydrophilic compounds. To ensure that therapeutic compounds of the invention across the BBB (if required) may be developed compound, where they are placed, for example, in liposomes. Information on methods of obtaining liposomes, see, e.g., U.S. patents 4 522 811; 5 374 548 and 5 399 331. Liposomes can contain one or more residues, which are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, e.g., V. V. Ranade (1989) J. din. Pharmacol. 29:685). Examples of guiding residues include folate and Biotin (see, e.g., U.S. patent 5416016 owned by Low et al.); Mann�side (Umezawae/o/., (19SS) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBSLett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); the receptor for surfactant protein A (Briscoe et al, (1995) Am. J. Physiol. 1233:134), different species of which may include formulations according to the invention, as well as components of molecules according to the invention; R120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

V. Methods of using antibodies of the invention

Another feature is that the present invention is directed to a method for the treatment of, for example, attenuation or inhibition associated with NGF disease or condition in a subject, namely, the method comprising administering to the subject antibodies against NGF according to the invention. Preferably, the antibody against NGF is used to reduce or alleviate pain, e.g., pain associated with the disease or condition in which the development or preservation of pain is mediated, at least partially, NEF. Non-limiting examples associated with NGF diseases and conditions include pain with inflammation, pain after surgery, post-operative pain (including dental pain), neuropathic pain, peripheral neuropathy, diabetic neuropathy, pain in fractures, pain in the joints in gout, post-herpetic neuralgia, pain in oncological diseases�aniah, pain in osteoarthritis or rheumatoid arthritis, sciatica, pain associated with crisis sickle-cell anemia, headaches (e.g., migraine, tension headaches, headaches), pain during dysmenorrhea, endometriosis, uterine fibroids, musculoskeletal pain, chronic pain in the lower back, fibromyalgia, sprains, visceral pain, ovarian cysts, prostatitis, chronic pelvic pain, cystitis, interstitial cystitis, a painful syndrome of the bladder and/or painful bladder syndrome, pain associated with chronic non-bacterial prostatitis, pain in the postoperative scar, migraine, trigeminal neuralgia, pain from burns and/or wounds, pain associated with trauma, pain associated with diseases of the musculoskeletal system, ankylosing spondylitis, around-articular pathology, the pain caused by bone metastases, pain, caused by HIV, erythromelalgia or pain caused by pancreatitis or kidney stones, malignant melanoma, xeroderma, asthma (e.g., uncontrolled asthma, severe hypersensitivity of the respiratory tract), and chronic cough, demyelinating diseases, chronic alcoholism, stroke, thalamic pain syndrome, the pain caused by the toxins, the pain caused by chemotherap�th, fibromyalgia, inflammatory bowel disorders, irritable bowel syndrome, inflammatory eye disorders, disorders associated with inflammatory or unstable bladder, psoriasis, skin diseases with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen (vascular) disease, a condition of chronic inflammation, pain during inflammation and associated hyperalgesia or allodynia, neuropathic pain associated with hyperalgesia or allodynia, diabetic neuropathic pain, causalgia, simpaticeskii supported the pain, syndromes of deafferentation, damage or dysfunction of epithelial tissues, disorders of motility of the internal organs of the respiratory, urogenital, gastrointestinal or vascular systems, allergic skin reactions, pruritus, vitiligo, General gastrointestinal disorders, colitis, stomach ulcers, duodenal ulcers, vasomotor or allergic rhinitis, bronchial disorders, dyspepsia, gastroesophageal reflux, pancreatitis and visceralgia.

Additionally, identified the involvement of NGF in the progression of cancers, such as prostate cancer, thyroid cancer, lung cancer, prolactinoma and melanoma. Accordingly, in another embodiment associated with NGF disease or condition, which� can cure with the use of antibodies against NGF according to the invention, is cancer, preferably prostate cancer, thyroid cancer, lung cancer, prolactinoma or melanoma. Thus, in another embodiment, the present invention is also directed to a method of treating cancer in a subject, preferably prostate cancer, thyroid cancer, lung cancer, prolactinoma or melanoma, comprising administering antibodies against NGF according to the invention to a subject.

Additionally, in another embodiment associated with NGF disease or condition may be HIV/AIDS. Blocking NGF with the use of antibodies against NGF according to the invention can block HIV-infected macrophages, thereby providing treatment for HIV/AIDS. Accordingly, in another embodiment, the present invention is also directed to a method for the treatment of HIV/AIDS in a subject, comprising administering antibodies against NGF according to the invention to a subject.

Particularly preferred diseases and conditions for treatment in accordance with the methods of the invention include pain during inflammation (in particular, pain caused by osteoarthritis or rheumatoid arthritis), musculoskeletal pain (particularly chronic pain in the lower back), pain in cancer, neuropathic pain (particularly pain with diabetes�cal neuropathy), the pain caused by bone metastases, interstitial cystitis/ painful syndrome of the bladder, pain associated with chronic non-bacterial prostatitis, the pain caused by endometriosis and/or fibroids of the uterus, and postoperative pain.

Pain and/or symptoms associated with endometriosis and/or uterine fibroids may include dysmenorrhoea; chronic memenstrual pelvic pain; dyspareunia; dichasia; hypermenorrhea; pain in the lower abdomen or back; the failure infertility and reproductive functions; dysuria; bloating and pain when urinating; nausea, vomiting and/or diarrhea. Symptoms can also include symptoms associated with endometrial damage and fibroids that are located outside of the peritoneal cavity, including, for example, such symptoms of thoracic endometriosis as haemoptysis, pneumothorax or hemothorax, and such manifestations lamisa lungs as dyspnoea and blackening in the lung.

In particular, in a preferred embodiment, the antibody against NGF according to the invention is used to treat pain. Preferably, the type of pain being treated, selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain cancer and pain with endometriosis and/or uterine fibroids. Suitable�NGOs, in a preferred embodiment, the present invention is directed to a method for treating pain in a subject, comprising administering antibodies against NGF according to the invention to a subject in such a way that the pain of the subject treated. Preferably the pain is selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain cancer and pain with endometriosis and/or uterine fibroids. Accordingly, in one embodiment embodiment of the present invention is directed to a method of treatment of pain in osteoarthritis in a subject, comprising administering antibodies against NGF according to the invention is thus that the pain in osteoarthritis in a subject treated. In another embodiment, the present invention is directed to a method for the treatment of chronic pain in the lower back in a subject, comprising administering antibodies against NGF according to the invention in such a way that chronic pain in the lower back of the subject treated. In another embodiment, the present invention is directed to a method of treatment of pain in diabetic neuropathy in a subject, comprising administering antibodies against NGF according to the invention is thus that the pain of diabetic neuropathy in a subject subjected�is treatment. In another embodiment, the present invention is directed to a method for treating pain in cancer disease in a subject, comprising administering antibodies against NGF according to the invention is thus that the pain has cancer in a subject treated. In another embodiment, the present invention is directed to a method of treatment of pain with endometriosis and/or uterine fibroids in a subject, comprising administering antibodies against NGF according to the invention is thus that the pain with endometriosis and/or uterine fibroids in a subject is cured.

In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment associated with NGF disease. Non-limiting examples associated with NGF diseases or conditions include those listed above. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain in Oncology Zab�disease and pain with endometriosis and/or uterine fibroids. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain in osteoarthritis. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of chronic pain in the lower back. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain in diabetic neuropathy. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain in cancer. In another embodiment, the present invention is directed to the creation of antibodies against NGF, as described in this document, for the treatment of pain with endometriosis and/or uterine fibroids.

In some embodiments embodiment the present invention is directed to a method of mitigating or inhibiting pain in a subject, namely, the method comprising administering to the subject antibodies against nerve growth factor (NGF), comprising a conservative region of human IgG4, wherein the conservative region of human IgG4 contains the amino acid sequence of SEQ. ID no: 10, wherein the antibody alleviates pain in a subject for IU�Isha least from about four to about twelve weeks (or at least from four to twelve weeks or for at least four weeks, or at least eight weeks, or at least twelve weeks, or from one to twelve weeks, or four to twelve weeks, or eight to twelve weeks) after administration of a single dose of the antibody against NGF entity. Preferably the pain is selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain cancer and pain with endometriosis and/or pain with uterine fibroids. Preferably, the antibody against NGF is administered in a dose ranging from about 0.1 mg/kg to 3 mg/kg, or 0.1 mg/kg to 3 mg/kg, or from about 0.1 mg/kg to about 30 mg/kg, or 0.1 mg/kg to 30 mg/kg or in one of the other ranges of the dosages described in this document.

In some embodiments embodiment the present invention is directed to a method of weakening or inhibition associated with nerve growth factor (NGF) disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet, the method comprising administering to the subject antibodies against NGF containing conservative region of human IgG4, wherein the conservative region of human IgG4 comprises a mutation in a hinge region at position 108 amino acids of SEQ. ID no: 9, wherein �ntitle has a terminal elimination half-life in humans of at least 10-30 days (or at least 10 days or at least 15 days, or at least 20 days, or at least 25 days, or at least 30 days, or at least 40 days, or in the range from about 10 days to about 40 days, or in the range of 10-40 days, or in the range from about 15 days to about 30 days, or in the range 15-30 days), wherein the antibody is administered to the subject in such dosage and frequency, to avoid the subject of the syndrome of a ricochet. Preferably, the series at amino acid position 108 in SEQ. ID no: 9 is replaced by Proline. Preferably, conservative region of human IgG4 comprises the amino acid sequence of SEQ. ID no: 10. Preferably, the antibody competes for binding to NGF with an antibody containing the variable region of a heavy chain including amino acid sequence SEQ. ID no: 1, and a variable region light chain comprising the amino acid sequence of SEQ. ID No.:

2. Preferably, associated with NGF the disease or condition is selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain cancer and pain with endometriosis and/or pain with uterine fibroids. Preferably, the antibody against NGF is administered in a dose ranging from about 0.001 mg/kg to about 30 mg/kg, more preferably from 1 mg/kg to 3 mg/kg, or one of the other ranges of the dosages described in this document. More preferably, in order to avoid the syndrome of a ricochet, the antibody is administered in the lower range of dosages, for example, in the range of 0.001 mg/kg to 1 mg/kg, or in the range from 0.001 mg/kg to 1 mg/kg or in a range from 0.001 mg/kg to 0.5 mg/kg or in a range from 0.001 mg/kg to 0.3 mg/kg, or in the range of 0.01 mg/kg to 1 mg/kg or in a range from 0.01 mg/kg to 0.5 mg/kg or 0.01 mg/kg to 0.3 mg/kg. More preferably, in order to avoid the syndrome of a ricochet, the antibody is administered in the lower range of dosages (as indicated above) and with more frequent intervals, e.g. once a week or once every two weeks, or once every four weeks.

The invention also is directed to a use of the antibody against NGF according to the invention for obtaining a medicinal product for use for the purpose of weakening or inhibition associated with NGF disease or condition in a subject. Non-limiting examples associated with NGF diseases or conditions include those listed above. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of pain. In another embodiment, the present invention is directed to a antibody n�of otiv NGF according to the invention for obtaining a medicinal product for the treatment of pain, selected from the group consisting of pain in osteoarthritis, chronic pain in lower back, pain in diabetic neuropathy, pain cancer and pain with endometriosis and/or uterine fibroids. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of pain in osteoarthritis. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of chronic pain in the lower back. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of pain in diabetic neuropathy. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of pain in cancer. In another embodiment, the present invention is directed to the creation of antibodies against NGF according to the invention for obtaining a medicinal product for the treatment of pain with endometriosis and/or uterine fibroids. In preferred embodiments, the antibody against NGF is administered at a dose within a range of elprimero 3 mg/kg to about 3000 μg/kg, or in a dose of 100 μg/kg or at a dose of 300 mcg/kg. In another preferred embodiment, the antibody against NGF according to the invention is administered (e.g., human) intravenously at a dose ranging from 0.1 mg/kg to 0.2 mg/kg, preferably 0.15 mg/kg, once every 12 weeks. In another preferred embodiment, the antibody against NGF according to the invention is administered (e.g., person) subcutaneously in a dose ranging from 0.2 mg/kg to 0.4 mg/kg, preferably 0.3 mg/kg, once every twelve weeks. In other embodiments embodiment, the antibody against NGF according to the invention is administered in a dose ranging from 0.1 mg/kg to 3 mg/kg, or between 0.1 mg/kg to 30 mg/kg, or between 0.1 mg/kg to 20 mg/kg, or between 0.1 mg/kg to 10 mg/kg, or between 1 mg/kg to 30 mg/kg, or between 1 mg/kg to 20 mg/kg, or in the range from 1 mg/kg to 10 mg/kg. However, other suitable ranges of doses and the dose indicated above in the section relating to pharmaceutical compositions.

In preferred embodiments, the antibody against NGF is administered intravenously. In another preferred embodiment, the antibody against NGF is administered subcutaneously or intra-articular. However, other suitable routes of administration described above in the section relating to pharmaceutical compositions.

In preferred embodiments, the antibody against NGF on subramaniyapuram pain in a subject which antibody is administered for a long period. For example, in one embodiments, the antibody alleviates pain for a period of at least from about one week to about twelve weeks (or at least one week to twelve weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about one week (or at least one week) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about two weeks (or at least two weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about four weeks (or at least four weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about eight weeks (or at least eight weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least about twelve weeks (or at IU�e twelve weeks) after administration of a single dose of the antibody against NGF entity. In one embodiment, the antibody alleviates pain for a period of at least from about four weeks to about twelve weeks (or four weeks to twelve weeks) after administration of a single dose of the antibody against NGF entity. In another embodiment, the antibody alleviates pain for a period of at least from about eight weeks to about twelve weeks (or eight weeks to twelve weeks) after administration of a single dose of the antibody against NGF entity.

In another embodiment, the antibody against NGF is administered together with a second pharmaceutical agent or the second mode of treatment. The antibody and another agent or antibody and the second mode of treatment may be introduced or conducted simultaneously or in accordance with another option, may be administered first antibody, followed by a second pharmaceutical agent or the second mode, or may be introduced or administered a second pharmaceutical agent or mode, followed by the antibody. Non-limiting examples of suitable second pharmaceutical agents and second modes of treatment mentioned above in the section relating to pharmaceutical compositions. In particular, we are considering the second pharmaceutical agents for use in combination with the antibody according to the invention are an opioid�ligetti. Other preferred second pharmaceutical agents for use in combination with an antibody of the invention are inhibitors of TrkA (for example, extracellular inhibitors or intracellular TrkA TrkA inhibitors, as described in detail in the section relating to pharmaceutical compositions) and inhibitors of protein kinase C (PKC).

Another feature is that the present invention is directed to a method of weakening or inhibition associated with nerve growth factor (NGF) disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet, the method comprising administering to the subject antibodies against NGF according to the invention, such as an antibody against NGF containing conservative region of human IgG4, wherein the conservative region of human IgG4 includes a mutation (preferably, a mutation in a hinge region), and wherein the antibody has a terminal elimination half-life in cynomolgus monkey of at least 15 days. In another embodiment, the antibody may have a terminal half-life in cynomolgus macaque in the range of about 15 to about 22 days (or in the range from 15 to 22 days), or in the range from about 15 days to about 28 days (or in the range from 15 to 28 days), or in the range from about 21 days to about 28 days (or in range�not from 21 to 28 days). In another embodiment, the antibody has a terminal elimination half-life in rats of at least 8 days. In another embodiment, the antibody has an average terminal half-life in humans of at least 10-30 days (or at least 10 days, at least 15 days at least 20 days, at least 25 days, at least 30 days, at least 40 days, or in the range from about 10 days to about 40 days, or in the range of 10-40 days, or in the range from about 15 days to about 30 days, or in the range 15-30 days). Preferred mutations include mutation, described in detail in this document previously. Preferred antibodies include antibodies against NGF sequences and/or having the functional properties described in detail in this document previously. Non-limiting examples associated with NGF diseases or conditions include those described in detail in this document previously. The invention also provides the use of antibodies against NGF according to the invention for obtaining a medicinal product for use for the purpose of weakening or inhibition associated with NGF disease or condition in a subject, therefore, to avoid the subject of the syndrome of a ricochet (for example, the antibody is administered in such dosage and frequency to avoid appearance uniform illumination, wide�incurred the subject of the syndrome of a ricochet).

VI. Products

Also in the scope of the present invention includes kits containing the antibodies of the invention that do not necessarily include application guidance for the treatment associated with NGF disease or condition. These sets can contain a label indicating the intended use of the package contents. The term label includes any inscription, marketing materials or written materials delivered to or set, or otherwise associated with the set.

For example, the present invention also aims at creating a packaged pharmaceutical composition, wherein the PG110 antibody containing a heavy chain as shown in SEQ. ID no: 13 and a light chain as shown in SEQ. ID no: 16), or its derivatives, as described in this document, Packed in the kit or product. The kit or article of the invention contains materials useful for the treatment, including prevention, treatment and/or diagnosis associated with NGF disease or condition in a subject. In the preferred embodiments associated with NGF the disease or condition is pain, inflammation (in particular, pain caused by osteoarthritis or rheumatoid arthritis), musculoskeletal pain (particularly chronic pain in the lower back), neuropathic pain (particularly pain in diabetic neuropathy), pain at Onco�logicheskih diseases (in particular, pain caused by bone metastases), pain associated with endometriosis and/or fibroids of the uterus, and postoperative pain. The kit or product comprises a container and a label or leaflet leaflet or printed material attached to the container or accompanying it, which reports information relating to the application of the PG110 antibody for the treatment associated with NGF disease or condition described in this document.

The kit or article means a packaged product comprising components with which administered the PG110 antibody for the treatment associated with NGF disease or condition. The kit preferably contains a box or container in which is placed the components in this kit, and can also include a Protocol for the introduction of the PG110 antibody and/or a leaflet-liner". In a box or container placed components according to the invention, which preferably are of plastic, polyethylene, polypropylene, ethylene or propylene vessels. For example, suitable containers for the PG110 antibody include, among other things, bottles, vials, syringes, pens, etc.

The term "leaflet-liner" when used in this document refers to the instructions that are usually included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, introduction, contraindications and/�whether the warnings related to the use of such therapeutic products. In one of the embodiments flyer-insert of the invention informs a reader, including a subject, for example, a buyer who will enter PG110 to ensure that the PG110 antibody is intended for the treatment associated with NGF disease or condition described in this document. In one of the embodiments leaflet leaflet describes specific therapeutic benefits the PG110 antibody, including pain relief. In another embodiment, the leaflet-liner may include a description of the dosage of PG110 antibody. In another embodiment, the leaflet-liner may include a description of the route and frequency of administration of the PG110 antibody. In another embodiment, the leaflet-liner of the invention may also be provided to the subject that will receive the antibody PG110, information regarding the combined applications for the purposes of both safety and efficiency. For example, in some embodiments, the kit further comprises a second pharmaceutical composition comprising an additional therapeutic agent, are packaged together or promoted in the market together with guidelines for the establishment of both agents for the treatment associated with NGF disease or condition. Particularly preferred diseases and conditions for treatment with application�m sets according to the invention include pain during inflammation (in particular, the pain caused by osteoarthritis or rheumatoid arthritis), musculoskeletal pain (particularly chronic pain in the lower back), neuropathic pain (including diabetic neuropathy), pain in cancer and the pain caused by bone metastases, pain associated with endometriosis and/or uterine fibroids and post-operative pain.

Other embodiments of the present invention are described in the following examples. The present invention is additionally illustrated by the following examples, which should not be construed as further limiting. The contents of the lists of sequences, figures and all references, patents and published patent applications cited in this application are specifically incorporated in this document by reference.

Examples

Example 1 Construction of antibodies against NGF, PG110, with a mutant hinge region of IgG4

In this example, the mutant form humanized antibody against NGF created with the introduction of the replacement of serine by Proline at hinge region of the conservative region of IgG4.

Were applied variable region of the heavy chain and variable region light chain of a humanized antibody against NGF alphaD11. A humanized antibody alphaD11 described in detail in PCT publications WO 2005/061540 and WO 2006/131951. Amino acid sequentially�th variable regions of the heavy chain alphaD11 (Hu-alphaD11 V Hshown in SEQ. ID no: 1. Amino acid sequence of variable region light chain alphaD11 (Hu-alphαD11 VLshown in SEQ. ID no: 2. Region CDR1, 2 and 3 the Hu-alphaD11 VHshown in the LAST. ID nos: 3, 4 and 5, respectively. Region CDR1, 2 and 3 consisting HualphaD11 VLshown in the LAST. ID nos: 6, 7 and 8, respectively.

A nucleic acid sequence encoding the Hu-alphaD11 VHwas attached to the 3'-end of the conservative region of IgG4-Pro (Lonza Biologic) (which encodes a mutation corresponding to the substitution of amino acid residue 108 in a conservative sequence with serine to Proline). The signal sequence derived from mouse IgGI, was attached to the 5'-end to obtain the complete cDNA sequence for the heavy chain of Hu-alphaD11. Amino acid sequence of wild-type conservative area of human IgG4 are shown in the LAST. ID no: 9 and amino acid sequence of a mutant of the conservative region of human IgG4 are shown in the LAST. ID no: 10. In the LAST part. ID nos: 9 and 10, an amino acid that is replaced with serine (SEQ. ID no: 9) by Proline (in the LAST. ID no: 10), is located at amino acid positions 108.

A nucleic acid sequence encoding the Hu-alphaD11 VLwas attached to the 3'-end of the conservative region of the human Kappa (supplied by Lonza Biologies), and signal sequence, receiving�tion of mouse IgGI, was attached to the 5'-end to obtain the full sequence that encodes a variable region light chain.

Since the antibody was expressed in the cells of the Chinese hamster ovary (Cho cells), held codon optimization (Geneart; with application software GeneOptimizer™), which included the adaptation of sequences of the antibodies to the deviation from uniformity in use. codons, genes characteristic of Cricetulus griseus (Chinese hamster). Additionally, avoid areas of very high (>80%) or very low (<30%) GC content, where possible. During the optimization process avoided the CIS-acting sequences of the following motifs: nutrigenie TATA-boxes, chi-sites and also joining the ribosome; the areas of at-rich or GC-rich sequences; sequence elements ARE, INS, CRS (involved in replication of the vector in bacteria); repeated sequences and secondary structures of the RNA; (cryptic) donor and acceptor splicing sites and points of branching; set vnutriennikh recognition sites for restriction enzymes (Eco RI, Hind III, Pvu I and Not I). Sequence optimization of antibody genes, including optimizing the use of codons, and expression of antibody genes in cells SNO further described in detail in PCT application WO 2006/122822, when�alejada company Lonza Biologies PLC.

Optimized sequences of the heavy and light chains (including signal sequence) was cloned in GS-vector REE.4 and REE.4, respectively (supplied by Lonza Biologies), obtaining two vectors, each containing one gene (SGV). Then constructed a vector containing two genes (DGV), by ligating the complete expression cassette from the vector of the heavy chain vector light chain with the creation of a single vector expressing the full genes of both heavy and light chains and gene GS (glutamylcysteine).

The resulting mutant antibody is called PG110. The nucleotide sequence of the complete heavy chain of PG110 (including the signal sequence, variable region and mutant conservative region IgG4) are shown in the LAST. ID no: 11. Amino acid sequence of the complete heavy chain of PG110 (including the signal sequence, variable region and mutant conservative region IgG4) are shown in the LAST. ID no: 12, wherein amino acid residues 1-19 comprise the signal sequence and amino acid residues 20-141 comprise a variable region. Amino acid sequence of the Mature heavy chain PG110, without the signal sequence (including the variable region and mutant conservative region IgG4) are shown in the LAST. ID no: 13.

U�echidna the sequence of the entire light chain PG 110 (including the signal sequence, variable region and a conservative region of the Kappa) is shown in the LAST. ID no: 14. The amino acid sequence of the entire light chain PG110 (including the signal sequence, variable region and a conservative region of the Kappa) is shown in the LAST. ID no: 15, amino acid residues 1 -20 constitute the signal sequence and amino acid residues 21-127 comprise a variable region. Amino acid sequence of the Mature light chain PG110, without the signal sequence (comprising a variable region and a conservative region of the Kappa) is shown in the LAST. ID no: 16.

To check the expression of the antibody PG110 vector DGV, encoding heavy and light chain antibody PG110, temporarily transfusional cells SNOC SV (supplied by Lonza Biologies). Cells (0,125×106viable cells per well) were sown in 24-well plates in the medium based on DMEM with addition of 10% serum of fetuses of cattle and 6 mm L-glutamine and incubated overnight at 37°C (incubator with 10% CO2). Before transfection the medium for planting were replaced with 800 μl of fresh medium and cells were incubated for 1 h at 37°C.

For each transfection, 5 μl PG110 DGV were resuspended in 100 μl of medium for transfection (OptiMEM, Invitrogen). As a positive control was used a vector encoding a different antibody IgG4/world, and in kacestvennogo control buffer used without a vector. For each 5 ál of transfection reagent Lipofectamine-2000 (Invitrogen) was diluted in 100 μl of medium for transfection. After 5 min incubation at room temperature the DNA and diluted Lipofectamine reagent were combined, stirred and left at ambient temperature for 20 min then the mixture volume of 205 ál was added into the hole 24-hole tablet containing cells. Cells were incubated for 68-72 h at 37°C. the Collected supernatant of the culture and clarified by centrifugation before making a quantitative determination of the presence of antibodies.

Quantification in the medium from transfected cells was performed using standard ELISA method for IgG collected. It includes bondage samples and standards in a 96-well plate, coated with antibody against human IgG Fc. The bound analyte was detected using the antibody against Kappa chain of a human, conjugated with horseradish peroxidase and a chromogenic substrate tetramethylbenzidine. The color development was proportional to the number of assembled antibodies present in the sample. Standard samples were prepared from commercially purchased stock solution of antibody IgG4/Kappa. The results demonstrated that DGV encoding heavy and light chain PG110, was capable of expressing the collected antibody.

Example 2: Parameters of binding of the mutant anti�ate PG110 against NGF

In this example investigated the binding specificity and kinetics of binding of PG110, mutant antibodies against NGF, prepared as described in example 1.

A. the binding Specificity

Profile the selectivity of binding of PG110 with human neurotrophins were determined using enzyme-linked immunosorbent assay binding (ELISA). Tablets for ELISA coated 100 ng/well human NGF (R&D Systems, cat. No. 256-GF), neurotrophic factor brain (BDNF) (R&D Systems, cat. No. 248-BD), neurotrophin 3 (NT3) (R&D Systems, cat. No. 267-N3) or neurotrophin 4 (NT4) (R&D Systems, cat. No. 268-N4). PG110 added in covered neurotrophins wells in concentrations ranging from 3 PM to 3 nm. After washing (PBS 0.5% (by volume) Tween 20, pH 7.3), the binding of PG110 were detected using biotinylating antibodies against human IgG (Rockland Immunochemical Inc., cat. No. 609-1602), conjugated with streptavidin-linked alkaline phosphatase (Sigma Aldrich, cat. No. S2890), followed by a color development reaction by adding 4-methylumbelliferone (Sigma Aldrich, cat. No. m). The reaction product was quantitatively assessed using a fluorimeter (excitation at 360 nm; emission at 440 nm).

The results are summarized in the chart in figure 1. Binding PG110 covered in human NGF wells showed a concentration dependence, with a concentration corresponding to half of the max�maximum binding, equal 726 PM (obtained by defining three times on one plate for analysis). In contrast, there was no visible binding PG110 analyzed with holes, covered with BDNF, NT3 or NT4 person who otherwise gave detected response when used as a positive control antibodies specific to these neurotrophins. These results demonstrate that PG 110, when tested in concentrations up to 3 nm in vitro that is specific binds to human NGF and does not show cross-reactivity to related neurotrophins.

B. Kinetics of binding

BIAcore analysis was used to assess the kinetics of binding in the interaction between PG110 and either recombinant rat NGF (rrNGF), or recombinant human NGF (rhNGF).

Recombinant human beta-nerve growth factor (rhNGF) (R&D Systems, cat. no.256 GF/CF) or recombinant human beta-nerve growth factor rat (rrNGF) (R&D Systems, cat.No. 556 GF/CF) was covalently immobilizovana on a sensor chip CM 5 (GE Healthcare, formerly Biacore AB, Uppsala, Sweden) via primary amino groups using a set of aminoscience (GE Healthcare), with HBSEP (10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mm NaCl, 3 mm ethylenediaminetetraacetic acid (EDTA) and 0.005% Tween® 20, pH 7.4) as the electrode buffer. The surface of the sensor chip was activated with the introduction of�art 400 mm N-(3-dimethylaminopropyl)-M'-ethylcarbodiimide (EDC) and 100 mm N-hydroxysuccinimide (NHS) (1:1, by volume) for 7 min at a flow rate of 10 µl/min. R-FST (recombinant human or recombinant rat) was diluted to 200 ng/ml in 10 mm sodium acetate, pH 4.0, and the diluted solution was added to the activated surface and maintained for different periods of time to obtain different density surfaces. For quantitative analysis of interactions the density of the surface 60 EN prepared by introducing 50 µl of the diluted P-NGF (contact time 5 min). Unreacted NHS ester groups were decontaminated using 70 μl (the contact time of 7 min) solution of ethanolamine (1 M, pH 8.5).

The parameters of the experiments conducted with immobilized NGF, were as follows: electrode buffer: HBSEP containing 100 µg/ml bovine serum albumin; flow rate: 25 µl/min; temperature: 37°C; ligand density: 60 EN/EN 60 (rhNGF/rrNGF); analyte: concentration PG110: 2 nm, 4 nm, 8 nm, 17 nm, 33 nm and 66 nm in electrode buffer; contact time: 240 h; dissociation time: 600 h; regeneration: 2×1 min, in 10 mm glycine, pH of 1.5.

Data evaluation and kinetics was performed using the software GraphPad Prism (version 5.01, GraphPad Software Inc., San Diego, CA, USA) and BIAevaluation software (version 4.0.1, GE Healthcare), writing data to the model of binding Langmuir 1:1.

The results are summarized below in table 1 (where KassOZNA�AET rate constant of the Association, Kdissmeans the rate constant of dissociation and KDmeans the equilibrium dissociation constant). Data are shown as mean ± (standard error of the mean) for 3 separate determinations.

Table 1
Kinetic constants of the interaction of PG110 with immobilized NGF
AnalytLigandKass(L. mol-1with-1)Kdiss(-1)KD
PG110rhNGFof 1.6×106±3,5×1031, 2 ×10-5±3,0×10-672±2 PM
PG110rrNGFof 1.7×105±2,0×103of 1.5×10-5±5,6×10-692±34 PM

The results of the study showed that interaction with PG110 NGF was characterized by high affinity binding, and there were no significant differences in affinity (KDfor both species-specific homologs (owned by the man and the rat).

Further care.�th analysis of the kinetics of binding was performed using material PG110 Fab, the obtained fragmentation on PG110 papain-sepharose (Thermo Scientific). The immobilization of rhNGF was performed using HBSEP as the electrode buffer at 25°C. the surface of the sensor chip was activated by the introduction of a mixture of 200 mm EDC and 50 mm NHS (1:1, by volume) for 7 min at a flow rate of 10 µl/min. rhNGF was diluted to 500 ng/ml in 10 mm sodium acetate, pH 4.0, and 20 µl (contact time 2 min) of the diluted solution was added to the activated surface of each of the three cells for receiving the immobilization levels 93,1,100,3 and 88.7 EN. Unreacted NHS ester groups were decontaminated using 70 μl (the contact time of 7 min) of ethanolamine (1 M, pH 8.5).

A series of concentrations of PG110 Fab(0,39, 0,78, 1,56, 3,13, 6,25,12,5 and 25 nm), 250 µl is passed over the freshly-immobilized rhNGF surfaces at a flow rate of 50 ál/min, 37°C in an electrode buffer HPSEP containing 100 µg/ml bovine serum albumin. For the dissociation was monitored for 30 min, and after completion of the analysis of the interaction was achieved regeneration using a single injection (30 µl) of 10 mm glycine, pH of 1.5. Additionally, for a more precise definition is very slow (kdisswatched the dissociation of a single high concentration (100 nm) PG110 Fab for 8 h on each surface with rhNGF and used the data to quantify the rate constants of dissociation (kdiss). Data were entered in a fashion�ü linking the Langmuir 1:1 with the use of k dissdefined by indications of long term measurements of dissociation. To assess the reproducibility of the kinetic constants analysis was performed in three replicates on three freshly-immobilized surfaces. The calculated kinetic constants are summarized in table 2.

Table 2:
Kinetic constants of the interaction of PG110 Fab with immobilized recombinant human NGF
AnalytThe level of immobilizationKass(L. mol-1with-1)Kdiss(-1)KD
PG110 Fab93,13,6×105of 1.1×10-531,3 PM
100,33,6×1051, 2 ×10-532,7 PM
88,74,4×1051, 2 ×10-527,8 PM
Average3,8×10 5of ±0.5×105of 1.1×10-5±0,06×10-530,6±0,25 PM

Example 3: Funktsionaalsusgarantii mutant PG110 antibody against NGF

In this example, the various functional characteristics of PG110, mutant antibodies against NGF, prepared as described in example 1, were studied by means of in vitro assays.

A. Inhibition of binding of NGF to receptors TrkA and p75NTRStudies on the binding of radioactively labeled ligands was performed to compare the inhibitory effect of PG110 on the binding of NGF receptors TrkA and p75NTRperson. Cells NEK expressing a full-sized human receptors - either TrkA or p75NTRwere incubated with 2 nm125I-NGF in the presence of PG110 in a final concentration of 0.01-100 nm. Unlabeled NGF (with different concentrations from 0 to 1 μm) was also introduced into the reaction mixture. Reactions were carried out in flat-bottomed high disposable vials RT (Thermo Life Sciences). At the first stage of radioactively labeled NGF, unlabeled NGF and antibody and PG110 mixed and incubated in test tubes for 10 min at room temperature, with shaking, in the buffer for binding (1 × PBS, 0.9 mm CaCl2, 0.5 mm MgCl2, 0,1% BSA fraction V, 0.1% of (wt./about.) glucose). Then added 200 μl of the prepared cells (diluted to 5×105cells/�l). After additional 30 min incubation at room temperature with vigorous shaking, each reaction was divided into three plastic tubes (plastic microtube capacity 0.4 ml, Sarstedt 72.700), adding to each tube 100 µl of the reaction mixture. Each microtube already contained 200 µl of 150 mm sucrose solution in buffer for binding. Then these tubes were centrifuged at 20 000 × g at 4°C for 30 seconds to precipitate the cells. Sucrose provides a density gradient, which separates superseded by any radioactively labeled NGF. After that, the tube was frozen in a bath of dry ice in ethanol. Then pointed pieces of those frozen tubes were placed in separate plastic vials (Naiad Ltd) for counting using a gamma counter LKB Wallac for the purpose of quantification of125I-NGF associated with the cell.

The results are illustrated by graphs in figures 2A and 2B, the effect of PG110 on the binding of NGF with TrkA is shown in figure 2A, and the effect of PG110 on the binding of NGF with p75NTRshown in figure 2B. Inhibition of PG110 binding125I-NGF receptors TrkA and p75NTRhad the character of concentration dependence, with geometric mean (95% confidence interval) values of IC50equal to 170 (88-331) PM and 206 (86-491) PM, respectively (in both sluchayah=3). The control antibody with the same isotype not inhibited binding125I-NGF to any of the receptors. These results demonstrate that PG110 effectively blocks the interaction of binding of human NGF with both its receptors in vitro.

B. quantification of cell proliferation of TF-1

TF-1 is a cell line èritromieloza people who Express TrkA person and proliferate in response to NGF. In these experiments cells TF-1 was cultured the presence of 10 ng/ml recombinant human NGF, rat or mouse, with increasing concentrations of antibody PG110, and quantitatively evaluated the proliferation of cells after 40 h with the use of the colorimetric method of analysis based on metabolic recovery of the yellow tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to purple formazan.

Before use in this analysis, cells TF-1 was cultured for 1 week in RPMI-1640 medium containing 10% serum fruits of bovine (FBS) with 2 ng/ml GM-CSF (R&D Systems, cat. No. 215-GM-50). Cells were washed, were resuspended in RPMI-1640+10% FBS to a concentration of 300,000 cells/ml and re-seeded on 96-well plates (15,000 cells/well in 50 μl). Not less than 60 min after the addition of 96-well plates for analysis of cells treated with recombinant human NGF, rat or washed 10 ng/ml) in RPMI-1640 medium, containing 10% FBS (50 µl/well) containing antibody PG110. The medium containing NGF and the test antibody, prepared as 2-fold solution relative to the concentration in the test sample with at least 30 min prior to adding it to the pre-seeded cells. The test antibody was used for analysis in the concentration range from 0.6 ng/ml and 24 μg/ml were included In the analysis of the control wells that contained either the medium or the cells TF-1 in the absence of NGF ("single cell test"). Each treatment was performed three times. After 40 h incubation period (37°C, 5% CO2), conducted a quantitative assessment of cell proliferation with the use of the kit for determination of cell proliferation using MTT (ATSS cat. No. 30-C). 10 µl of MTT reagent was added before further incubation for 4 h at 37°C. thereafter, the wells were incubated with a reagent containing a detergent (100 μl/well; gentle stirring) overnight at room temperature in the dark. After that he registered the absorbance at 570 n. a. Final average OD values for the three repetitions of the measurement was calculated by subtracting the mean value for the blank sample cell.

The incubation of cells TF-1 with NGF in the presence of PG110 (0,6 ng/ml 24 µg/ml) led to dependent on the concentration of the inhibition of cell proliferation. The results are illustrated by graphs in Fig�Oh 3A-3C, where figure 3A shows the treatment effect of PG110 on cell proliferation, TF-1, NGF stimulated human figure 3C shows a treatment effect of PG110 on cell proliferation, TF-1, NGF stimulated rats, and figure 3C shows the effect of processing on PG110 cell proliferation, TF-1, NGF stimulated mouse. PG110 demonstrated similar efficacy in the inhibition of all three homologues tested NGF, and the values of IC50approximately 30 ng/ml. the values of IC50summarized below in table 3 (values of IC50expressed in ng/ml):

Table 3:
Summary by the values of IC50for PG110 in measuring the cell proliferation of TF-1
Recombinant human NGFRecombinant rat NGFRecombinant mouse NGF
Zh. average95% confidencenZh. average95% confidencenZh. average95% confidencen
intervalintervalinterval
29,426,5-32,7527,824,5-31,6530,827,4-34,53

Quantification of cell proliferation of TF-1 demonstrates that PG110 with equal efficiency neutralizes activation of TrkA receptors human factors NGF both human and rodents.

V. Inhibition of NGF-dependent survival of the dorsal root ganglia of the spinal cord of chicken

To test the ability of PG110 to inhibit the effect of NGF on sensory neurons used in vitro assays using primary cultures of cells of the dorsal root ganglion (DRG) from a chicken embryo on day 8. Under these conditions, the survival of chick DRG depends on the presence of exogenous NGF added to the culture medium.

On the dorsal root ganglia were isolated from chicken embryos on day 8 and collected in Falcon�stuffing tubes 50 ml (in 5 ml nutrient mixture ham F-12+Glutamax I: GIBCO 31765027). In each experiment the total number of allocated ganglia was approximately 400, which is approximately 20 embryos. Further DRGs was trypsinization for 5 min at 37°C (trypsin-EDTA Euroclone, ECB3052D) and dissociatively with a syringe of 10 ml (with the "yellow" needle 20G) 4-5 times before centrifuging for 3 min at 800 rpm.min.

After careful removal of containing trypsin medium the cells were resuspended in 10 ml fresh medium (nutrient mixture ham F-12+Glutamax I), then the procedure was repeated dissociation and brought the volume of the medium to achieve a seeding concentration of 100 000-400 000 cells/ml. Cells were sown without treatment, half of the final volume of medium containing 20% horse serum (Euroclone ECS0091L) using 24-well plates (Falcon 353047) that were previously coated with poly-b-lysine (100x=1 mg/ml solution of poly-b-lysine hydrobromide; Sigma P2636) dissolved in distilled water (30 min under UV, after 30 min of drying in a sterile fume hood).

After attachment of cells to the walls of the plate wells (30 min in 5% CO2incubator, 37°C), prepared solutions of either NGF or NGF/antibody against NGF in 2-fold concentration in the half of the final volume and added to each well to achieve the desired final concentration (i.e., end - % content of the serum of the horse=10%; final NGF concentration=5 ng/ml).Were included in the analysis of the control wells which contained the cells of the DRG in the absence of NGF. Each condition was tested in two replicates. After adding mixtures of either NGF or NGF/antibody against NGF the plates returned to the incubator (5% CO2, 37°C). Counting the number of cells was carried out after 48 h by counting the cells of the DRG, observed along the vertical diameter of each hole (microscope Nikon TMS, 10X magnification). In the calculation included only those cells of the DRG, which are easily identified by their morphological characteristics, i.e., round, bright, svetoprelomlyayuschimi cells with long axons.

The effectiveness of the PG110 antibody in neutralizing NGF was tested in a concentration range from 10 ng/ml to 25 μg/ml for neutralization of recombinant human NGF (rhNGF), recombinant rat NGF (rrNGF) or recombinant mouse NGF (rmNGF). In these experiments, 100% inhibition of survival was equivalent to the number of cells counted by culturing cells without NGF, in the absence of antibodies against NGF. Conversely, 0% inhibition was equivalent to the number of cells counted in the case when the cells were treated with 5 ng/ml of the corresponding isoforms of NGF, in the absence of antibodies against NGF.

Incubation of cells in the presence of PG110 (10 ng/ml - 25 μg/ml) resulted in dependent on the concentration of reduced survival of cells with values of IC50from 10 to 50 ng/ml for all species specific gomal�GOV NGF (n=1). These data demonstrate that PG110 inhibits the activity against NGF-sensitive neurons.

G. Inhibition of growth of axons of cells PC-12

PC 12 is a cell line of rat pheochromocytoma (a tumor derived from chromaffin cells) that Express receptors of the rat TrkA and p75NTR. When cultured on coated with collagen tablets in the presence of NGF cells PC 12 differentiate into neurons, similar sympathetic, becoming flat and showing significant growth (axons). Inhibition of NGF-mediated growth of axons was used as a semiquantitative method for measuring in vitro the ability of PG110 to inhibit the interaction of NGF to receptors of the rat TrkA and p75NTR.

Cells PC 12 (ECAAC 88022401) subjected to the initial impact (i.e., pre-treated NGF) by washing 100 000 cells serum-free medium (RPMI-1640 with Glutamax I, Gibco-Invitrogen 61870-010) and re-seeding on plastic coated with collagen flasks (collagen type I, BD 35-4236, used in the form of a working solution of 0.5 mg/ml) in RPMI-1640 medium with Glutamax-I containing 10% FBS and recombinant rat NGF (R&D Systems, cat. no.556-NG-100) (100 ng/ml). Before seeding the cells gently missed at least 5 times through a needle 21G for the disaggregation of clumps of cells. The medium containing NGF was removed and replaced twice during the period�and pre-treatment duration of 1 week.

At the end of this period the pre-treated cells PC 12 were washed with serum-free medium and trypsinization (trypsin-EDTA Euroclone, ECB3052D) for 2-3 min in a humid incubator (5% CO2, 37°C). Trypsin was blocked by the addition containing serum medium, and the cells were centrifuged, washed with serum-free medium and were resuspended in RPMI-1640 medium with Glutamax-I containing 10% FBS. The cells gently missed at least 5 times through a needle 21G for the disaggregation of clumps of cells before re-sowing coated with collagen on Petri dishes at a density of 50 000 cells/ml. Prepared three cups for each of the analyzed conditions. For testing antibodies prepared 2 the incubation mixture (recombinant rat NGF+antibody against NGF) for 1 h before adding to the pre-seeded cells. The final concentration of NGF was 20 ng/ml, and the antibody against NGF were tested at four dilutions:

20 μg/ml, 2 μg/ml, 200 ng/ml and 20 ng/ml.

Assessment of NGF induced proliferation of axons were performed after 72 h. this time point was removed the medium and washed the cells not containing calcium and magnesium PBS (GIBCO, 10010) and fixed for 30 min with a solution of 4% formaldehyde in PBS. Microscopic images (20X magnification) were obtained using microscope Nikon Eclipse TE2000-E microscope and software Leica IM1000 Image Manager. Estimated� inhibition of growth of axons, and evaluation (++;+/-; -) was assigned on the basis of the number of cells showing the phenotype of undifferentiated cells (which corresponds to the lack of explicit axons).

Incubation of cells in the presence of PG110 (20 ng/ml - 20 μg/ml) inhibited mediated NGF growth of axons, with apparent complete inhibition at 200 ng/ml, which suggests that PG110 is an effective inhibitor of the interaction between recombinant rat NGF and native receptors of neurotrophins in rats.

Example 4: the Stability in vivo of mutant antibodies against NGF PG110

In this example, the determined terminal half-life (T1/2) PG110 antibody in vivo in rats and cynomolgus macaques.

A. Studies in rats

Rats line sprag-douli was introduced through the 10-minute intravenous (IV) infusion PG110 antibody in a dosage of 3 mg/kg, 30 mg/kg or 100 mg/kg at 1 and day 56 of the study. Assessment of the toxicokinetic data was performed using the mean concentration in serum at time points of 6 animals in each point in each group. Nominal time points of blood sampling were: prior to the introduction of the drug and 0,25,1, 3, 6 and 24 h after drug administration at 1 and the bottom 56 of the study, as well as at points 8, 96, 168, 336, 504, 672, 840, 1008 and 1176 h after drug administration at 1 day of research. The bioassay of serum samples was performed in the analytical�certification laboratories Alta Analytical Laboratory with the use of a validated ELISA method. The pharmacokinetic analysis of data was performed with the use of SNBL USA Pharmacokinetics Analysis System 2.0 and software WinNonlin Professional, version 4.0 (Pharsight Corp.)

After IV injection of values of Tmax(the time to reach maximal concentration in serum) were in the range of from 0.25 to 1 h, which corresponded to the first two time points of sampling. All animals were observed two-phase distribution pattern with a terminal elimination half-lives of about 8 to 9 days. In particular, the values of T1/2specified in hours, for the three treatment groups are summarized below in table 4:

Table 4:
Terminal half-life of PG 110 in rats line sprag-douli
Level doses f mg/kg)Terminal half-life (h)
3217
30192
100207

Therefore, group mean values of elimination half-life in rats ranged from 192 to 217 hours (8-9 days).

B. the First study in rhesus

Javanese makaka introduced by RA�type, about 30-minute intravenous (IV) infusion PG110 antibody in a dosage of 3 mg/kg, 30 mg/kg or 100 mg/kg. the Animals were divided into males and females; each dose was tested on 2 males and 2 females (except test dose of 30 mg/kg only 1 female). Nominal time points of blood sampling to obtain data toxicokinetic analysis were: immediately after application (within 2 min after the infusion), 0,25,1, 3, 6,24,48, 96,168,336,504,672, 840,1008,1176, 1344,1512 and 1680 h after the infusion. The bioassay of serum samples was carried out in analytical laboratories Alta Analytical Laboratory with the use of a validated ELISA method. The pharmacokinetic analysis of data was performed with the use of SNBL USA Phannacokinetics Analysis System 2.0 and software WinNonlin Professional, version 4.0 (Pharsight Corp.)

After IV injection of values Tmah (time to reach maximal concentration in serum), measured from the start of infusion, were in the range from the end time of infusion up to about 1.6 h. all animals were observed two-phase character of the distribution (with the exception of one female that received a dose of 30 mg/kg, which saw a sharp decline in temporary point 504 h), with terminal half-lives from about 15 to 22 days. In particular, the values of T1/2specified in hours, for the six treatment groups are summarized in table 5 below:

Table 5:Terminal half-life of PG110 in cynomolgus macaquesThe dose level (mg/kg)PaulTerminal half-life(h)3Females (n=2)370Males (n=2)53130Females (n=1)471Males (n=2)450100Females (n=2)383Males (n=2)461

Therefore, group mean values of elimination half-life in cynomolgus macaques were in the range from 370 to 531 h (from 15 to 22 days). The mean values of elimination half-life were in General more males than females, although, given the small number of animals studied, it is not clear whether this is a statistically significant difference.

V. the Second study in rhesus

Javanese makaka did daily intravenous infusion of the antibody PG110 in those�ood period 4 nedir. Approximately 30-minute intravenous (IV) infusion of PG110 antibody in a dosage of 3 mg/kg, 30 mg/kg or 100 mg/kg did on 1, 8, 15 and 22 day. The animals were divided into males and females; each dose was tested on 3 males and 3 females. Serial blood samples were collected in the following nominal time points: before drug administration, immediately after application (within 2 min after the infusion), 0,25, 1, 3, 6,24, and 168 h after the infusion on day 1 and 22.

Additional samples were taken from all animals before drug administration on day 15 in animals that underwent a period of recovery, through 336, 504, 672, 840, 1008, 1176, 1344, 1512, 1680, 1848, 2016 and 2208 h after the last dose on day 22. The specified time in hours, starting 24 h after infusion, day pharmacokinetic analysis 1, 7,14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 92. All the actual time points of blood sampling converted into a format relative to the start of infusions.

The bioassay of serum samples on the concentration of PG110 was carried out in analytical laboratories Alta Analytical Laboratory with the use of a validated ELISA method. The pharmacokinetic analysis of data was performed with the use of SNBL USA Pharmacokinetics Analysis System 2.0 and software WinNonlin Professional, version 4.0 (Pharsight Corp.)

After introducing group IV average values of Tmax(the time to reach maximal concentration in serum), the measured�s from start of infusion, were in the range of from about 0.6 h to 2.6 h. all animals were observed two-phase distribution pattern with a terminal elimination half-lives of about 21-28 days (from 503 to 685 h). In particular, the values of T1/2specified in hours, for the six treatment groups are summarized in table 6 below:

Table 6.
Terminal half-life of PG110 in cynomolgus macaques
The dose level (mg/kg)PaulTerminal half-life (h)
3Females (n=3)503
Males (n=3)564
30Females (n=3)511
Males (n=3)619
100Females (n=3)532*
Males (n=3)685
* based on the values for n=2

Therefore, group mean values of the period p�luvyvedeniya in cynomolgus macaques were in the range of from about 21 to 28 days. The mean values of elimination half-life were in General more males than females, although, given the small number of animals studied, it is not clear whether this is a statistically significant difference.

Example 5: Efficacy of PG110 in comparison with the antibody of the predecessor rats

In this example, we have conducted additional experiments with the use of quantitative assessment of cell proliferation in TF-1 (as described in example 3 above) to compare the effectiveness of the PG110 antibody in the neutralization of NGF to that of his predecessor, αD11 rats. Antibody αD11 rats were supplied in two different series, one concentration of the original solution was to 0.73 mg/ml, and in another the concentration of the initial solution was to 0.63 mg/ml. PG110 Antibody αD11 and was evaluated by quantitative determination of cell proliferation, TF-1, mediated NGF human or rat NGF with application kit for the determination of cell proliferation using MTT (ATSS cat. No. 30-C).

Before applying for the analysis of TF1 cells were cultured for one week in RPMI-1640 (ATSS cat. No. 30-2001) containing 10% serum fruits of bovine (FBS, Cambrex DEI IF 4-80, lot 6SB0006) with 2 ng/ml GM-CSF (R&D Systems, cat. No. 215-GM-50). Cells were washed, were resuspended in RPMI-1640+10% FBS to a concentration of 300,000 cells/ml and re-seeded on 96-well plates (15,000 cells/well in 50 μl.

Not less than 60 min after the addition of 96-well plates for analysis of cells treated with recombinant NGF or person, or washed (10 ng/ml) in culture medium (10% FBS in RPMI-1640; 50 µl/well) containing an antibody against NGF. The medium containing NGF and the test antibody, prepared as 2-fold solution relative to the concentration in the test sample with at least 30 min prior to adding it to the pre-seeded cells. Subjects antibodies was used for analysis in the concentration range from 0.6 ng/ml to 24 mg/ml. In the analysis in all cases included control wells that contained either the medium or the cells TF-1 in the absence of NGF ("single cell test"). Each treatment was performed three times. After 40 h incubation period (37°C, 5% CO2), 10 µl of MTT reagent was added before further incubation for 4 h at 37°C. thereafter, the wells were incubated with a reagent containing a detergent (100 μl/well; gentle stirring) overnight at room temperature in the dark. Then registered the absorption at 570 nm. Final average OD values for the three repetitions of the measurement was calculated by subtracting the average values for the cell blank sample. The maximum inhibition was taken the mean value of the OD observed when cultured cells without NGF, in the absence of the test�th antibodies. As a zero level of inhibition was taken the mean value of the OD observed by culturing cells in the treatment of 10 ng/ml NGF, in the absence of the test antibody.

Inhibitory efficacy of antibodies against NGF was calculated as the value of IC50(i.e., the concentration of antibody required to reduce NGF-mediated proliferative response by 50%) using the software GraphPad Prism version 5.01. Curves of inhibition graphics were applied to each individually to obtain single values IC50for each of the test antibody in each experiment. Indicators of cell proliferation was normalized relative to the maximum OD values obtained by this quantitative definition, in the absence of added antibody test. After that, normalized quantitative indicators of response were applied to the schedule depending on the concentration of the antibody with a logarithmic scale, and display the values of IC50using the program GraphPad Prism nonlinear tracing nonlinear curve through points "lg(concentration of the inhibitor) relative to the response - variable slope".

Inhibitory effect of PG110 in comparison with its predecessor, the antibody αD11 rats, summarized below in table 7.

Tables� 7.
Summary of values of IC50(ng/ml) quantification of cell proliferation TF1
AntibodyNGF manNGF rats
Zh. average95% confidence intervalnZh. average95% confidence intervaln
PG11027,025,8 was 28.3226,213,6-50,42
αD11 rats (series 1)63,735,7-114344,831,4-64,13

αD11 rats (series 2)107154,91

The results show that the PG110 antibody has at�Erno 2 times higher efficiency in neutralizing NGF activity, compared to its predecessor, the antibody αD11 rats. Additionally, the values of the performance indicators obtained for αD11 rats, series 2, suggest that this series may have a lower efficiency than the material from series 1.

Example 6: Antibody against NGF PG110 does not cause the syndrome of a ricochet in the model system in animals

In this example, the applied model of skin damage in rats to evaluate the activity of monoclonal antibody PG110. Model of skin damage in rats was developed on the basis of observations that showed that in rats that received antibody against NGF, develops a response in the form of itching, a dose-dependent increase in the number of observed petting. The damage was not associated with a reduction in the innervation of the epidermis. Additionally, in rats, there was a rapid recovery after cessation of administration of the antibody. Activity that causes damage to the skin, observed in rodents, behavioural feature of which is grooming. Not limited to any one mechanism, the researchers hypothesized that the rats that received the antibody against NGF, continue to carry the grooming because of a violation of feedback needed to end the response in the form of scratching, which leads to skin damage. Further, damage to the skin can be quantified as an indicator of the activity of the� antibodies against NGF in rats.

In various clinical trials of antibody activity against NGF (such as a humanized antibody RN-642, further described in the publication of U.S. patent No. 20040237124 and work Abdiche, Y. N. et al. (2008) Protein Sci. 17:1326-1335) reported that the effectiveness of the antibody is reduced by a certain period of time post-dose (for example, in 14-21 days post-dose), followed by a recovery of activity of the antibody. There have been reports of increased pain and/or increase adverse events (such as abnormal sensitivity, range from the degree to a sense of pricking or tingling) in the period after the reference dose, when the activity of the antibody is reduced. This reduction in the activity of the antibody at a certain period of time after the dose is called in this document "the syndrome of a ricochet".

To evaluate the effect of PG110 antibody on grooming and scratching being in conscious rats male and female rats line sprag-douli injected monoclonal antibody PG110 (of 0.003, and 0.01, and 0.03, 0.3 or 3 mg/kg) as an intravenous loading dose or reference dose media. Rats received one dose weekly for four weeks. The criteria were: the number of episodes of grooming and scratching, body temperature, the latent period of licking of the paws, the latent period of the jump (trying to avoid) and the number and severity assessment p�damage to the skin over a period of time.

The results demonstrated that the observed skin lesions in rats, which were injected with the carrier, while in the groups of rats that were administered PG110, skin damage was observed in all animals at all tested doses of the antibody. Additionally, the number and severity of skin lesions increased over time and increased with increasing dosages of the antibody. The number of scratching episodes was also higher in animals that were administered the antibody, but the introduction of antibodies had no effect on grooming, body temperature, the latent period of licking of the paws or the latent period of the jump (attempted avoidance).

The severity of skin lesions was quantitatively evaluated by using estimates of damage, equal to the amount of damage multiplied by the area (in mm) damage. Assessment of damage over time to handle the PG110 antibody, in comparison with the carrier shown in the graph of figure 4. The results show that treated PG110 rats showed steady improvement in scores of injuries over time, in particular, at the highest tested doses (0.3 mg/kg and 3 mg/kg). That is, the antibody did not cause a significant syndrome of a ricochet for the time period of the experiment, which suggests the possibility of choosing the parameters of dosage and frequency �of reference for use in humans so to avoid the syndrome of a ricochet.

Thus, summarizing, the model of skin damage in rats demonstrates the advantage of antibodies against NGF PG110, which is that the PG110 antibody does not cause a marked syndrome of a ricochet, which was reported for other antibodies against NGF, and this suggests that PG110 demonstrates a more stable and long-lasting activity of t vivo. He limited to any one mechanism, it is believed that this ability PG110 to avoid the syndrome of a ricochet in vivo is associated with an increased terminal half-life observed in this antibody.

Example 7; Pharmacokinetics PG110 in humans

It is expected that PG110 will have a half-life in humans of approximately 10-30 days (range 10 to 40 days) with the multiphase nature of the distribution. On the basis of the target value Cmin~0,25 µg/ml (range from ~0.13 ág/ml to 0.40 µg/ml), it is expected that the person will be an effective intravenous dose of 10 mg (~0.15 mg/kg; range from ~0.1 mg/kg [5 mg] to ~0.2 mg/kg [15 mg]) every 4-12 weeks and subcutaneous doses of 20 mg (~0.3 mg/kg; range from -0.2 mg/kg [15 mg] up to ~0.4 mg/kg [30 mg]) every 4-12 weeks.

Predicted values of pharmacokinetic parameters were based on data on concentrations obtained for the two species (rat and monkey). Prediction of pharmacokinetics in humans was performed using sets with�osobov, which include scaling of pharmacokinetic parameters monkeys and rats, fixed allometric scaling, and methods based on preclinical and clinical data obtained for other monoclonal antibodies. Forecasting was performed for both mono-and biphasic distribution.

Method 1: Predicted values of pharmacokinetic parameters for humans were based on 2-component model corresponding to the plotted points on the profile curve, the concentration of PG110-time in two species (rat and monkey). Pharmacokinetic parameters for PG110 were predicted with the use of allometric scaling with fixed exponents of pharmacokinetic parameters for monkeys and rats.

For clearance:

For volume of distribution;

Table 8.
The prediction values of pharmacokinetic parameters for a person on the basis of allometric scaling with fixed exponents
V1ml/kgClearance CL ml/HR/kgV2ml/� CLDml/h/kg
Appropriate models pharmacokinetic parameters
Rat40,20,2236,52,23
Monkey29,30,10is 31.11,78
Predicted pharmacokinetic parameters for man
The predicted value for a person on the basis of data for rats40,20,03436,50,35
The predicted value for a person on the basis of data for monkeys29,30,042is 31.10,75
Average34,70,03833,80,55

Then calculated the half-life of PG110 in humans on the basis of PR�wskazany values of CL and V 1in humans, the predicted half-life of PG110 in humans was 26 days.

Method 2: Predicted values of pharmacokinetic parameters were based on data for two species (rat and monkey). The values of CL and V were projected by using the same method as in Method 1. The half-life PG110 for humans was predicted with the use of a modified allometric scaling with fixed exponent equal to 0.25:

td align="center"> 0,22
Table 9.
The prediction values of pharmacokinetic parameters for a person on the basis of the modified allometric scaling with fixed exponent
Clearance CL (ml/h/kg)Vss(ml/kg)t1/2(days)
RatMonkeyRatMonkeyRatMonkey
Observed for PG1100,09870608,619.1
Predicted values for man0,03430,0410706035,237
Predicted values for humans (average)0,03766536

Predicted using method 2 the half-life of PG110 in humans was 36 days.

Method 3: Prediction of pharmacokinetics in humans based on data on PG110 for monkeys and rats and preclinical and clinical pharmacokinetic parameters obtained for other monoclonal antibodies. Scaling was performed on the basis of the half-life of PG110 observed in rats and monkeys and the ratio rat/human and APE/man of the half-lives of other monoclonal antibodies. The calculated pharmacokinetic parameters (clearance, volume of distribution and half-life) other monoclonal antibodies in rats and monkeys were first compared with those obtained in to�incesti research. Differences between the rat, monkey and man, was calculated as the ratio of rat/human and APE/man. After that, we calculated pharmacokinetic parameters for PG110 person on the basis of its pharmacokinetic parameters in rats and monkeys, adjusted for the ratio rat/human and APE/person for other monoclonal antibodies. Predicted using method 3 the half-life of PG110 in person ranged from 11 to 29 days.

Table 10.
The prediction values of pharmacokinetic parameters for a person on the basis of previous experiments with other monoclonal antibodies
Clearance CL (ml/h/kg)Vss(ml/kg)t1/2(days)
R/HM/NR/HM/NR/HM/N
Antibody 1of 1.351.881,33 0,530,930,69

Antibody 23,571,43the 1.650,720,440,49
Antibody 31,581,46of 1.350,711,050,78
Average2,171.591,450,650,810,65
RatMonkeyRatMonkeyRatMonkey
Observed for PG1100,22 0,09870608,619,1
The predicted value for the person0,1010,06248,492,01129
R/H: the rat/man, M/N: monkey/man

Method 4: Predicted values of pharmacokinetic parameters for humans were based on 2-component model corresponding to the plotted points on the profile curve, the concentration of PG110-time in two species (rat and monkey). Pharmacokinetic parameters for PG110 were predicted with the use of allometric scaling with regression of pharmacokinetic parameters for monkeys and rats.

For clearance and volume of distribution:

Log (pharmacokinetic parameter)=a×Log (BW)+b

A linear regression was performed on the basis of pharmacokinetic parameters in rats and monkeys, and body weight (BW) for the estimation of the angular coefficient (a) and the point of intersection with the y-axis (b). After that, we calculated the pharmacokinetic parameters PG110 for a person using a typical body weight (BW) and calculated values of the angular coefficient and the point �of peresechenia with y axis.

Table 11.
The prediction values of pharmacokinetic parameters for a person on the basis of allometric scaling with regression
Body weight (BW)kgLnBW kgV1 mlClearance CL ml/hV2 mlCLDml/h
Rat0,25-1,3872,307-2,9072,210-0,585
Monkey51,6094.988-0,6805,0472,188
The calculated angular coefficient and the point of intersection with the y-axis based on the linear regression
Corner 0,89490,74320,9470,9255

factor
The intersection with the y axis3,5475-1,87663,5230,6982
Predicted pharmacokinetic parameters for man
People704,24815553,6001894103

Then calculated the half-life of PG110 in humans on the basis of predicted for human pharmacokinetic parameters. The predicted half-life of PG110 in humans was 12 days.

Based on the results obtained by these methods, it is expected that PG110 will have a half-life in humans of approximately 15-30 days (range 10 to 40 days) with the two-phase nature of the distribution (predicted pharmacokinetic parameters: V1 =2.5 l, CL=5.0 ml/h, V2=2.5 l, CLD=40 ml/h).

Example 8; Treatment of osteoarthritis in humans with the use of PG110

Started a clinical trial in humans to test the safety, tolerability and pharmacokinetics of PG110 in patients with pain, is characteristic of osteoarthritis of the knee. Study design and preliminary results are described below.

In this phase I, single-centre, placebo-controlled, double-blind, single ascending dose study evaluated six (6) levels: 0,003, 0,01, 0,03, 0,1, 0,3 and 1 mg/kg For each dose level cohort of 7 patients with pain, characteristic of osteoarthritis of the knee, (42 patients) randomly divided in the ratio of 6:1 to receive active treatment or placebo. Each patient was administered a single dose PG110 or placebo intravenously during the time period of 2 h in the morning on the day of the test (day 0) after a light Breakfast. Patients remained in the Department of clinical pharmacology (CPU) before the expiry of the period of about 24 hours after start of infusion (day 1) and made repeated visits at 4, 7,14,21, 28, 56 and 84 day study.

Blood samples for the quantitative determination of PG110 were taken on the first day of the study (day 0) (before drug administration and 1, 2, 3, 6 and 12 h) and 1,4, 7, 14, 21, 28, 56 and 84 days after administration of the drug. The concentration of PG110 in serum Oprah�elali with the use of validated method for quantitative analysis by ELISA. Also conducted quantitative determination of antibodies against PG110 samples taken on day 0 (before drug administration), 14, 28, 56 and 84.

This study evaluated the pharmacodynamic parameters, including the assessment of the pain patient questionnaire to determine the index of osteoarthritis of the Universities of Western Ontario and McMaster (WOMAC™), a questionnaire to determine the severity of your pain on a Mac-Gill, the test with the six-minute walk, ultrasound examination of the knee joint and the definition of high-sensitivity C-reactive protein.

Preliminary results on the evaluation of pain patients are summarized in table 12. Assessment of pain patients used to assess the severity of pain. Patients were asked to rate their answer on a scale of 0-100 mm VAS, where 0 mm corresponds to no pain and 100 mm represents the severe pain.

Table 12.
PG110 in patients with osteoarthritis: Evaluation of pain by the patient (VAS)
Assessment of pain by the patient (VAS, mm, change relative to

baseline) (mean ± standard rejected�(e)
Time (days)Placebo0.003 mg/kg0.01 mg/kg0.03 mg/kg0.1 mg/kg0.3 mg/kg
4-7,2±13,1-15,8±23,0-22,8±28,6is 16,5±17,5-23,0±17,6-31,7±18,2
7-10,2±12,1was 14.8±23,4-12,3±25,9-26,0±17,2-34,8±20,3-32,5±18,2
14-10,6±14,3-29,7±15,8-12,5±21,7occupies 15,5±22,7-24,3±28,30,5±17,2
21-11, 2 ±24,1-30,5±23,9-16,3±24,9-25,5±24,1-23,3±40,7to -20.0±18,5
28-9,6±11,9-28,3±25,9 is 16,5±22,8-30,7±22,5-45,2±22,4point is -35.3±20,5
560,5±15,0-24,3±25,8-11,8±19,8-24,0±19,8-48,8±23,7-52,8±19,3
84-7,8±16,4-25,0±20,79.2 per±18,2-21,0±23,2-29,3±37,1-34,0±26,0
Maximum pain assessment by the patient (VAS, mm)-22,4±13,0-41,0±14,5-30,2±25,3-37,2±17,7-48,8±23,7-55,3±13,7

Based on preliminary pharmacodynamic data obvious dose-effect was observed in the dose range of PG110 from 0 to 0.3 mg/kg.

The pharmacological half-life calculated based on the average amount of time to achieve the effect of the drug (the average holding time for the effect from the drug). It is calculated as the ratio of the area under the efficiency curve at first, scorretti�agreed in relation to the baseline (AUMEC) to the square dependence of accumulated adjusted relative to the baseline effect of the drug versus time (area under the curve of efficiency, AUEC):

Estimated pharmacological half-lives are summarized in table 13.

Table 13.
The pharmacological half-life of PG110
Dose PG110
0.003 mg/kg0.01 mg/kg0.03 mg/kg0.1 mg/kg0.3 mg/kg
The pharmacological half-life (mean ± standard deviation) (days)43,0±7,530,9±21,635,8±17,942,7±5,041,9±5,4

Calculate the pharmacological half-life was performed based on the data, stripped for the period up to 84 days post-dose PG110. As on day 84 was still observed long-term effect of PG110, especially at doses of 0.1 mg/kg and 0.3 mg/kg, estimated average Farmak�logical half-life PG110 is at least 5-7 weeks with a range of at least 4 to 8 weeks.

Preliminary pharmacodynamic data are consistent with the predicted therapeutic dose (from 0.10 to 0.3 mg/kg or 7-21 mg). The preliminary value of the pharmacological half-life suggests that it may be effective to introduce doses every 4 to 12 weeks.

Equivalents

Specialists in this field will be able to recognize or establish, using only standard methods of experimentation, numerous equivalents of specific examples of embodiments of the invention described in this document. It is assumed that such equivalents are included in the following claims. It is assumed that any combination of embodiments disclosed in the subordinate claims, is within the scope of the invention.

Inclusion as links

All publications, patents and patent applications pending, which are referenced in this document are included here as references.

1. Antibody against the nerve growth factor (NGF), comprising:
(i) variable region heavy chain containing CDR 1, 2 and 3 with amino acid sequences SEQ ID NO:3, 4 and 5, respectively,
(ii) variable region light chain contains the CDR 1, 2 and 3 with amino acid sequences SEQ ID NO:6, 7 and 8, respectively, and
(ii) a constant region of human IgG4, where the constant region is human IgG4 contains the amino acid sequence of SEQ ID NO:10.

2. The antibody according to claim 1, containing the variable region of the heavy chain containing the amino acid sequence of SEQ ID NO:1, and a variable region light chain containing the amino acid sequence of SEQ ID NO:2.

3. The antibody according to claim 1 comprising a heavy chain containing the amino acid sequence of SEQ ID NO:13 and a light chain containing the amino acid sequence of SEQ ID NO:16.

4. The antibody according to any one of claims.1-3, where the antibody is humanised.

5. Pharmaceutical composition for the relief of pain associated with the disease or condition in which the development or preservation of pain mediated NGF containing the antibody against NGF according to any one of claims.1-4 and a pharmaceutically acceptable carrier.

6. Set for treatment associated with NGF disease or condition that contains the antibody against NGF according to any one of claims.1-4 and the instructions for use.

7. Nucleic acid that encodes the heavy chain of the antibody according to any one of claims.1-4.

8. Nucleic acid that encodes the light chain of the antibody according to any one of claims.1-4.

9. The expression vector containing the nucleic acid according to claims.7 and 8.

10. The expression vector according to claim 9, which contains SEQ ID NO:11 and SEQ ID NO:14.

11. A host containing the expression vector according to claim 9 or 10, obtained for�I antibodies against NGF according to any one of claims.1-4.

12. Method of expression of antibodies against NGF according to claim 1, comprising cultivating a host cell according to claim 11.

13. The use of antibodies against NGF according to any one of claims.1-4 in a method of treating pain associated with a disease or condition in which the development or preservation of pain mediated NGF.

14. The use of antibodies against NGF according to any one of claims.1 to 4 to obtain drugs for the treatment of pain associated with the disease or condition in which the development or preservation of pain mediated NGF.

15. The use according to claim 13 or 14, where pain is the pain in osteoarthritis.

16. The use according to claim 13 or 14, where the pain is chronic pain of the lower back.



 

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32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of obtaining bivalent bispecific antibody, which includes transformation of host cell by vectors, containing molecules of nucleic acids, coding first light chain and first heavy chain of bivalent bispecific antibody, and vectors, containing molecules of nucleic acids, coding second light chain and second heavy chain of bivalent bispecific antibody, cultivation of host cell under conditions, providing synthesis of molecule of bivalent bispecific antibody from said culture. Said antibody contains first light chain and first heavy chain of antibody, specifically binding with first antigen, and second light chain and second heavy chain of antibody, specifically binding with second antigen, in which variable domains VL and VH of second light chain and second heavy chain are replaced by each other and constant domains CL and CH1 of second light chain and second heavy chain are replaced by each other.

EFFECT: invention makes it possible to increase output of correct bispecific antibody by increasing the level of correct heterodimerisation of heavy chains of wild type and modification of heavy chains resulting from crossing over.

2 cl, 31 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.

EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.

25 cl, 24 dwg, 8 tbl, 17 ex

Csf-1r antibody // 2547586

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.

EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.

23 cl, 18 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, in particular to peptydoglycane hydrolase biosynthesis, and represents a protein with the peptydoglycane hydrolase activity, a plasmid, containing a peptydoglycane hydrolase-coding fragment, a bacterium-producer, a method of microbiological peptydoglycane hydrolase synthesis, as well as a pharmaceutical composition, containing the obtained peptydoglycane hydrolase, for the therapy of diseases, caused by Gram-negative microflora.

EFFECT: elaborated method of microbiological synthesis makes it possible to obtain bacteriophage S-394 peptydoglycane hydrolase in an effective way.

22 cl, 2 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to chimeric proteins, nucleic acid, coding such a protein, an expression cassette, providing the expression of nucleic acid, a vector, including the expression cassette, a method of diagnostics and a set for diagnostics. The characterised chimeric Borrelia protein includes at least one sequence of an extracellular domain of the VlsE Borrelia protein of the first type, corresponding to a certain strain, and at least one sequence of IR6 area of the VlsE Borrelia protein of the second type or Borrelia of the first type, but corresponding to a strain, different from the strain of the first type, with Borrelia being selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

EFFECT: claimed inventions make it possible to carry out diagnostics of Lyme-borreliosis with an increased specificity and sensitivity.

15 cl, 8 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.

EFFECT: invention can find further application in diagnosing and treating cancer.

43 cl, 98 dwg, 20 tbl, 26 ex

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