HK1110513B - Anti-ngf antibodies and methods using same - Google Patents

Description

anti-NGF antibodies and methods of use thereof

Cross reference to related applications

The present application claims U.S. provisional patent application serial No. 60/436,905, filed on even 24/12/2002; U.S. provisional patent application serial No. 60/443,522 filed on 28/1/2003 and U.S. provisional patent application serial No. 60/510,006 filed on priority on 8/10/2003, all of which are hereby incorporated by reference in their entirety.

Background

The present invention relates to anti-NGF antibodies (e.g., anti-NGF antagonist antibodies). The invention further relates to the use of such antibodies in the treatment and/or prevention of pain, including post-operative pain, rheumatoid arthritis pain, and osteoarthritis pain.

Statement regarding federally sponsored research or development

Not applicable.

Background

Nerve Growth Factor (NGF) was the first neurotrophin identified and its role in the development and survival of peripheral and central neurons has been characterized. NGF has been shown to be a key survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons as well as in the development of basal forebrain cholinergic neurons. Smeyne et al, Nature 368: 246-249(1994) and Crowley et al, Cell 76: 1001-1011(1994). NGF upregulates neuropeptide expression in sensory neurons (Lindsay and Harmer, Nature 337: 362-364(1989)) and its activity binds to receptors through two distinct membranes: TrkA receptor and p75 common neurotrophin receptors (sometimes referred to as "high affinity" and "low affinity" NGF receptors, respectively). Chao et al, Science 232: 518-521(1986). For a review of NGF, see Huang et al, annu. rev. neurosci.24: 677-; bibel et al, Genes Dev.14: 2919-2937(2000). The crystal structures of NGF and complexes of NGF and trkA receptors have been determined. See Nature 254: 411 (1991); nature 401: 184-188(1996).

Nerve Growth Factor (NGF) was the first neurotrophin identified and its role in the development and survival of peripheral and central neurons has been characterized. NGF has been shown to be a key survival and maintenance factor in the development of peripheral sympathetic and embryonic sensory neurons as well as in the development of basal forebrain cholinergic neurons. (Smeyne et al, Nature 368: 246-. NGF upregulates neuropeptide expression in sensory neurons (Lindsay et al, Nature 337: 362-364(1989)) and its activity is mediated by two distinct membrane-bound receptors, the TrkA tyrosine kinase receptor and the p75 receptor, which is structurally related to other members of the tumor necrosis factor receptor family (Chao, et al, Science 232: 518-521 (1986)).

In addition to its role in the nervous system, NGF is increasingly shown to be involved in processes outside the nervous system. For example, NGF has been shown to enhance angiogenic permeability (Otten, et al, Eur JPharmacol.106: 199-201(1984)), enhance T-and B-cell immune responses (Otten, et al, Proc. Natl. Acad. Sci. USA 86: 10059-10063(1989)), induce lymphocyte differentiation and mast cell proliferation and cause the release of soluble biological signals from mast cells (Matsuda, et al, Proc. Natl. Acad. Sci. USA 85: 6508-6512 (1988); Pearce, et al, J. physiol.372: 379-393 (1986); Bischoff, et al, Blood 79: 2662-2669 (1992); Horigome, et al, J. biol. chem.268: 14881-14887 (1993)). Although it has been shown that exogenously added NGF can have all these effects, it is important to note that endogenous NGF is only rarely shown to be important in any of these processes in vivo (Torcia, et al, cell.85 (3): 345-56 (1996)). It is therefore unclear exactly how the effect of inhibiting the biological activity of endogenous NGF, if any, is.

NGF is produced by a number of Cell types including mast cells (Leon, et al, Proc. Natl. Acad. Sci. USA 91: 3739-3743(1994)), B lymphocytes (Torcia, et al, Cell 85: 345-356(1996), keratinocytes (Mardi, et al, J.biol. chem.268: 22838-22846), smooth muscle cells (Ueyama, et al, J Hypertens.11: 1061-1065(1993)), fibroblasts (Linoldhm, et al, Eur.J.Neurosci.2: 795-801(1990)), bronchial epithelial cells (Kassel, et al, Clin.Exp.Allergy 31: 1432-40(2001)), renal vascular membrane cells (Steiner, et al, amino.J.Physl.261: f792-798(1991)) and skeletal myotubes (Schwartz, et al, J Photochem. Photobiol. B66: 195-200 (2002)). NGF receptors are found on a variety of cell types outside the nervous system, for example, TrkA is found on human monocytes, T and B lymphocytes, as well as mast cells.

An association between increased NGF levels and various inflammatory diseases is observed in human patients and in some animal models. These include systemic lupus erythematosus (Bracci-Laudiero, et al, Neuroreport 4: 563-565(1993)), multiple sclerosis (Bracci-Laudiero, et al, neurosci.Lett.147: 9-12 (1992)), psoriasis (Raychaudhuri, et al, Acta Derm.l' enerol.78: 84-86(1998)), arthritis (Falcim, et al, Ann. Rheum. Dis.55: 745-.

Similarly, elevated NGF levels in peripheral tissues are associated with hyperalgesia and inflammation and are observed in many forms of arthritis. Synovium from patients with rheumatoid arthritis expresses high levels of NGF, whereas synovium from non-inflammatory patients is reported to have no detectable NGF (Aloe, et al, Arch. Rheum.35: 351-355 (1992)). Similar results were observed in rats that were experimentally induced for rheumatoid arthritis (Aloe, et al, Clin. Exp. Rheumatotol. 10: 203-204 (1992)). Increased NGF levels in transgenic arthritic mice have been reported to be accompanied by an increase in mast cell numbers (Aloe, et al, int. JTissue Reactions-exp. Clin. assays 15: 139-143 (1993)). PCT publication No. WO02/096458 discloses the use of anti-NGF antibodies of certain properties in the treatment of various NGF-related diseases, such as inflammatory diseases (e.g., rheumatoid arthritis). Injection of purified anti-NGF antibodies into arthritis transgenic mice carrying the human tumor necrosis factor alpha (TNF-alpha) gene has been reported to cause a decrease in mast cell number and decreased levels of histamine and substance P in the synovium of arthritic mice (Aloe et al, Rheumatotol. int.14: 249-252 (1995)). Exogenous administration of NGF antibody has been shown to reduce elevated TNF- α levels in arthritic mice (Manni et al, Rheumatotol. int.18: 97-102 (1998)).

Similarly, increased expression of NGF and a high affinity NGF receptor (TrkA) was observed in human osteoarthritic chondrocytes (Iannone et al, Rheumatology 41: 1413-.

Rodent anti-NGF antagonist antibodies have been reported. See, e.g., Hongo et al, Hybridoma (2000)19 (3): 215-227; ruberti et al, (1993) cell.molec.neurobiol.13 (5): 559-568. However, when rodent antibodies are therapeutically applied to humans, human anti-mouse antibody responses occur in many treated individuals. Furthermore, effector functions of mouse antibodies have been shown to be less efficient in humans. Thus, anti-NGF antagonist antibodies, including humanized anti-NGF antagonist antibodies, are highly desirable.

All references, publications and patent applications disclosed herein are hereby incorporated by reference in their entirety.

Summary of The Invention

The invention disclosed herein relates to nerve growth factor antibodies.

In another aspect, the invention is a humanized and affinity matured antibody E3, which specifically binds human and rodent nerve growth factor ("NGF"). The amino acid sequences of the E3 heavy and light chain variable regions are shown in FIGS. 1A (SEQ ID NO: 1) and 1B (SEQ ID NO: 2), respectively. The CDR portions of antibody E3 (including Chothia and Kabat CDRs) are depicted schematically in FIGS. 1A and 1B. The E3 heavy and light chains and the individual extended CDRs are also shown below (see, below, "antibody sequences").

In another aspect, the invention is an antibody comprising a fragment or region of antibody E3 (interchangeably referred to herein as "E3"). In one embodiment, the fragment is the light chain of antibody E3 as shown in figure 1B. In another embodiment, the fragment is the heavy chain of antibody E3 as shown in figure 1A. In another embodiment, the fragment comprises one or more variable regions from the light chain and/or heavy chain of antibody E3. In yet another embodiment, the fragment comprises one or more Complementarity Determining Regions (CDRs) from the light and/or heavy chain of antibody E3 as shown in fig. 1A and 1B.

In another aspect, the invention is an antibody comprising a light chain encoded by a polynucleotide produced by a host cell deposited with ATCC No. pta-4893 or ATCC No. pta-4894. In another aspect, the invention is an antibody comprising a heavy chain encoded by a polynucleotide produced by the host cell deposited under ATCC accession No. pta-4895. In another aspect, the invention is an antibody comprising (a) a light chain encoded by a polynucleotide produced by a host cell deposited under accession number ATCC No. PTA-4894 or ATCC No. PTA-4893 and (b) a heavy chain encoded by a polynucleotide produced by a host cell deposited under accession number ATCC No. PTA-4895 (for convenience herein, polynucleotides produced by deposited host cells are referred to as having accession numbers ATCC No. PTA-4894, PTA-4893 and PTA-4895). In another aspect, the invention is an antibody comprising a light chain variable region of a light chain encoded by a polynucleotide produced by a host cell deposited as ATCC No. pta-4894 or ATCC No. pta-4893. In another aspect, the invention is a heavy chain variable region antibody comprising a heavy chain encoded by the polynucleotide produced by the host cell deposited with ATCC No. pta-4895. In another aspect, the invention is an antibody comprising (a) the light chain variable region of a light chain encoded by a polynucleotide produced by a host cell deposited as ATCC No. pta-4894 or ATCC No. pta-4893 and (b) the heavy chain variable region of a heavy chain encoded by a polynucleotide produced by a host cell deposited as ATCC No. pta-4895. In yet another aspect, the invention is an antibody comprising one or more CDRs encoded by (a) the polynucleotide produced by the host cell deposited with ATCC No. pta-4894 and/or (b) the heavy chain encoded by the polynucleotide produced by the host cell deposited with ATCC No. pta-4895.

In some embodiments, the antibody comprises a human heavy chain IgG2a constant region. In some embodiments the antibody comprises a human light chain kappa constant region. In some embodiments, the antibody comprises a modified constant region, such as an immunologically inert constant region, e.g., that does not cause complement-mediated lysis, or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). In other embodiments, as described in eur.jimmunol, (1999) 29: 2613-2624; PCT application No. PCT/GB 99/01441; and/or modifications to the constant region as described in UK patent application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2a constant region comprising the following mutations: a330P331 to S330S331 (with reference to the amino acid numbering of the wild-type IgG2a sequence). Eur.j Immunol. (1999) 29: 2613-2624.

In another aspect, the invention provides a polypeptide (which may or may not be an antibody) comprising any one or more of: a) one or more CDRs of antibody E3 shown in fig. 1A and 1B; b) CDR H3 from the heavy chain of antibody E3 shown in fig. 1A; c) CDR L3 from the light chain of antibody E3 shown in fig. 1B; d) three CDRs from the light chain of antibody E3 shown in figure 1B; e) three CDRs from the heavy chain of antibody E3 shown in figure 1A; and f) three CDRs from the light chain and three CDRs from the heavy chain of antibody E3 shown in FIGS. 1A and 1B. The invention further provides a polypeptide (which may or may not be an antibody) comprising any one or more of: a) one or more (one, two, three, four, five or six) CDRs derived from antibody E3 shown in fig. 1A and 1B; b) CDRs derived from the CDR H3 of antibody E3 heavy chain shown in fig. 1A; and/or c) CDRs derived from the CDR L3 of the light chain of antibody E3 shown in FIG. 1B. In some embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (referred to herein as "extended" or "combined" CDRs). In some embodiments, a polypeptide (e.g., an antibody) binds NGF (e.g., human NGF). In some embodiments, the polypeptide comprises any of the CDF configurations (including combinations, variants, etc.) described herein.

In one aspect, the invention provides polypeptides (e.g., antibodies) comprising a polypeptide comprising SEQ ID NO: 9, wherein I34 is S, L, V, A or I; and N35 is replaced with N, T or S. For convenience herein, "substitution" or "being" in the context or in reference to an amino acid refers to the selection of an amino acid for a given position. Clearly, alternatives or alternatives may be the amino acids depicted in SEQ ID or figures.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S; and L63 is L or V.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and wherein Y110 is any amino acid.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and Y110 is any amino acid.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and Y96 is Y or R.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid; t93 is any amino acid and wherein Y96 is Y or R.

In one aspect, the invention provides polypeptides (e.g., antibodies) comprising a polypeptide comprising SEQ ID NO: 9, wherein I34 is S, L, V, A or I and N35 is N, T or S.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S and L63 is L or V.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and wherein Y110 is any amino acid.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and wherein Y110 is any amino acid.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y; and H32 is H, N or Q.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and wherein Y96 is Y or R.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid; t93 is any amino acid and wherein Y96 is Y or R.

In another aspect, the invention provides polypeptides (e.g., antibodies, including humanized antibodies) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, wherein I34 is S, L, V, A or I and N35 is N, T or S; SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S and L63 is L or V and SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G; wherein Y110 is Y, K, S, R or T. In some embodiments, the heavy chain variable region comprises SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; wherein Y110 is any amino acid. In other embodiments, the heavy chain variable region comprises SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and wherein Y110 is any amino acid. In some embodiments, the polypeptide (e.g., antibody) further comprises an antibody light chain variable region.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q; SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T; and SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and wherein Y96 is Y or R. In some embodiments, the light chain variable region comprises SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid, and T93 is any amino acid; wherein Y96 is Y or R. In some embodiments, the polypeptide (e.g., antibody) further comprises an antibody heavy chain.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, wherein I34 is S, L, V, A or I and N35 is N, T or S; SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S and L63 is L or V; and SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G; wherein Y110 is Y, K, S, R or T; said (b) is a light chain variable region comprising SEQ ID NO: 12 CDR1 region, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q; SEQ ID NO: 13, wherein I51 is I, T, V or a; s56 is S or T; SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and wherein Y96 is Y or R. In some embodiments, the light chain variable region comprises SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid. T93 is any amino acid and wherein Y96 is Y or R. In some embodiments, the heavy chain variable region comprises SEQ ID NO: 11, CDR3, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; wherein Y110 is any amino acid. In other embodiments, the heavy chain variable region comprises SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; and wherein Y110 is any amino acid. In some embodiments, the polypeptide further comprises an antibody light chain.

In another aspect, the invention provides polypeptides (e.g., antibodies, including humanized antibodies) comprising the amino acid sequence of SEQ ID NO: 9, wherein I34 is S, L, V A or I; and N35 is N, T or S; comprises the amino acid sequence of SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S and L63 is L or V; and a nucleic acid comprising SEQ id no: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G; wherein Y110 is Y, K, S, R or T. In some embodiments, the polypeptide comprises SEQ ID NO: 11, wherein Y100 is Y, L or R; and wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; and wherein Y110 is any amino acid. In other embodiments, the polypeptide comprises SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G and wherein Y110 is any amino acid. In some embodiments, the polypeptide (e.g., antibody) further comprises an antibody light chain variable region.

In another aspect, the invention provides polypeptides (e.g., antibodies, including humanized antibodies) comprising the amino acid sequence of SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y; and H32 is H, N or Q; comprises the amino acid sequence of SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T; and a polypeptide comprising SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and wherein Y96 is Y or R. In some embodiments, the polypeptide comprises SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid; t93 is any amino acid and wherein Y96 is Y or R. In some embodiments, the polypeptide (e.g., antibody) further comprises an antibody heavy chain variable region.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising (a) a polypeptide of SEQ ID NO: 9, wherein I34 is S, L, V A or I and N35 is N, T or S; SEQ ID NO: 10, wherein M50 is M, I, G, Q, S or L; a62 is A or S and L63 is L or V; and SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G; and wherein Y110 is Y, K, S, R or T; and (b) is SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q; SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T; and SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S; and wherein Y96 is Y or R. In some embodiments, the polypeptide comprises SEQ ID NO: 14, wherein S91 is S or E; k92 is any amino acid; t93 is any amino acid and wherein Y96 is Y or R. In some embodiments, the polypeptide comprises SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; wherein Y110 is any amino acid. In other embodiments, the polypeptide comprises SEQ ID NO: 11, wherein G98 is G, S, A, C, V, N, D or T; wherein G99 is G, S, A, C, V, N, D or T; wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T or G; and wherein Y110 is any amino acid. In some embodiments, the polypeptide further comprises an antibody light chain variable region.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a heavy chain variable region comprising: (a) SEQ ID NO: 9, wherein I34 is S, L, VA or I and N35 is replaced with N, T or S; (b) SEQ ID NO: 10, wherein M50 is I, G, Q, S or L; a62 is A or S and L63 is L or V; (c) SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G and wherein Y110 is Y, K, S, R or T; wherein the antibody binds NGF.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a light chain variable region comprising: (a) SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q; (b) SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T; and (c) SEQ ID NO: 14, wherein K92 is K, H, R or S and wherein Y96 is Y or R; wherein the antibody binds NGF.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising (a) a heavy chain variable region comprising: (i) SEQ ID NO: 9, wherein I34 is replaced with S, L, V A or I and N35 is replaced with N, T or S; (ii) SEQ ID NO: 10, wherein M50 is I, G, Q, S or L; a62 is A or S; and L63 is L or V; and (iii) SEQ ID NO: 11, wherein Y100 is Y, L or R; wherein Y101 is Y or W; wherein G103 is G, A or S; wherein T104 is T or S; wherein S105 is S, A or T; wherein Y106 is Y, R, T or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109 is D, N or G; wherein Y110 is Y, K, S, R or T; and (b) a light chain variable region comprising: (i) SEQ ID NO: 12, wherein S26 is S or F; d28 is D, S, A or Y and H32 is H, N or Q; (ii) SEQ ID NO: 13, wherein I51 is I, T, V or a and S56 is S or T; and (iii) SEQ ID NO: 14, wherein S91 is S or E; k92 is K, H, R or S and wherein Y96 is Y or R; wherein the antibody binds NGF.

Unless otherwise mentioned, the selection (e.g., substitution) of an amino acid in one position is independent of the selection of an amino acid in any other position.

In some embodiments, a polypeptide (e.g., an antibody) binds NGF (e.g., human NGF). In some embodiments, the polypeptide comprises any of the CDR configurations (including combinations, variants, etc.) described herein.

As is apparent from the description herein, the variable region numbering as applied herein is sequential numbering. One skilled in the art will readily understand that there are many antibody numbering systems (e.g., Kabat and Chothia numbering), and how to convert consecutive numbering to another numbering system, such as Kabat numbering or Chothia numbering.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 or 50 (e.g., a CDR3 sequence). In other embodiments, the polypeptide further comprises SEQ ID NO: 3. 4, 5, 6, 7 and 8. In other embodiments, the polypeptide further comprises SEQ ID NO: 9. 10, 11, 12, 13, 14 and 15, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides polypeptides (e.g., antibodies) comprising a sequence selected from (a) SEQ ID NOs: 28 and/or 29; (b) SEQ ID NO: 30 and/or 31; (c) SEQ ID NO: 32 and/or 33; (d) SEQ ID NO: 34 and/or 35; (e) SEQ ID NO: 36 and/or 37; (f) SEQ ID NO: 38 and/or 39 and (g) SEQ ID NO: 40 and 41 (e.g., a CDR region, such as a CDRH1 and/or a CDRH2 region). In some embodiments, the polypeptide comprises an amino acid sequence selected from SEQ id nos: 28. 30, 32, 34, 36, 38 and 40 (e.g., the CDR H1 region). In some embodiments, the polypeptide comprises a nucleic acid sequence selected from SEQ ID NOS: 29. 31, 33, 35, 37, 39 and 41 (e.g., CDR H2 region). In yet other embodiments, the polypeptide further comprises SEQ ID NO: 3. 4, 5, 6, 7 and 8. In yet other embodiments, the polypeptide further comprises SEQ ID NO: 9. 10, 11, 12, 13, 14 and 15, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising a sequence selected from (a) SEQ id nos: 18 and/or 19; (b) SEQ ID NOS: 20 and/or 21 and (c) SEQ ID NOS: 22 and/or 23 (e.g., CDR regions, such as CDRL1 and/or CDRL2 regions). In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 18. 20 and 22 (e.g., the CDR L1 region). In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 19. 21 and 23 (e.g., the CDR L2 region). In other embodiments, the polypeptide further comprises SEQ ID NO: 3. 4, 5, 6, 7, 8, or a pharmaceutically acceptable salt thereof. In other embodiments, the polypeptide further comprises SEQ ID NO: 9. 10, 11, 12, 13, 14 and 15, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides polypeptides (e.g., antibodies) comprising a sequence selected from (a) SEQ ID NOs: 51 and/or 52; (b) SEQ ID NO: 55 and/or 56; (c) SEQ ID NO: 57 and/or 58; (d) SEQ ID NO: 59 and/or 60; (e) SEQ ID NO: 61 and/or 62; (f) SEQ ID NO: 63 and/or 64 (e.g. a CDR region, such as a CDRL3 and/or a CDRH3 region). In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 51. 55, 57, 59, 61 and 63 (e.g., the CDR L3 region). In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 52. 56, 58, 60, 62 and 64 (e.g., the CDR H3 region). In yet other embodiments, the polypeptide further comprises SEQ ID NO: 18. 19, 30 and 31, or a pharmaceutically acceptable salt thereof. In other embodiments, the polypeptide further comprises SEQ ID NO: 3. 4, 5, 6, 7 and 8. In other embodiments, the polypeptide further comprises SEQ ID NO: 9. 10, 11, 12, 13, 14 and 15, or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a polypeptide (e.g., an antibody) comprising SEQ ID NO: 61. 63, 18, 19, 30 and 31 (e.g., CDR regions).

In one aspect, the invention provides anti-NGF antibodies (e.g., antagonist antibodies) that bind NGF (e.g., human NGF) with high affinity. In some embodiments, the high affinity is (a) with K_DLess than about 2nM (e.g., about any of 1nM, 800pM, 600pM, 400pM, 200pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, or less than 50 pM) and/or koff less than about 6X 10^-5s^-1Binds NGF; and/or (b) about 200pM, 150pM, 100pM, 80pM, 60pM, 40pM, 20pM, 10pM or any less than 10pM with an IC50 (NGF present at about 15 pM)One that inhibits (reduces and/or blocks) survival of a mouse E13.5 trigeminal neuron that is dependent on human NGF; and/or (c) inhibits (reduces and/or blocks) the survival of a human NGF-dependent mouse E13.5 trigeminal neuron with an IC50 (NGF present at about 1.5 pM) of any one of about 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, 2pM, 1pM, or 1pM or less; and/or (d) inhibits (reduces and/or blocks) the survival of mouse E13.5 trigeminal neurons that are dependent on rat NGF with an IC50 (about 15pM of NGF present) of any one of about 150pM, 125pM, 100pM, 80pM, 60pM, 40pM, 30pM, 20pM, 10pM, 5pM, or 5pM or less; and/or (E) inhibits (reduces and/or blocks) the survival of mouse E13.5 trigeminal neurons that are dependent on rat NGF with an IC50 (NGF is present at about 1.5 pM) of any one of about 30pM, 25pM, 20pM, 15pM, 10pM, 5pM, 4pM, 3pM, 2pM, 1pM, or 1pM or less; and/or (f) and/or binds NGF with a higher affinity than it binds the trkA receptor.

In another aspect, the invention provides polypeptides (e.g., antibodies) wherein polypeptide (a) is represented by K_DLess than about 2nM (e.g., about any of 1nM, 800pM, 600pM, 400pM, 200pM, 100pM, 90pM, 80pM, 70pM, 60pM, 50pM, or less than 50 pM) and/or koff less than about 6X 10^-1s^-1Binds NGF (e.g., human NGF); and/or (b) inhibits survival of a human NGF dependent mouse E13.5 trigeminal neuron with an IC50 (about 15pM of NGF present) of any one of about 200pM, 150pM, 100pM, 80pM, 60pM, 40pM, 20pM, 10pM, or less than 10 pM); and/or (c) inhibits survival of a human NGF dependent mouse E13.5 trigeminal neuron with an IC50 (NGF present at about 1.5 pM) of any one of about 50pM, 40pM, 30pM, 20pM, 10pM, 5pM, 2pM, 1pM, or 1pM or less; and/or bind NGF with a higher affinity than to trkA receptors. In some embodiments, polypeptide (a) binds NGF with a KD of about 2nM or less; and/or (b) inhibits survival of human NGF dependent mouse E13.5 trigeminal neurons with an IC50 of about 100pM or less, wherein IC50 is determined in the presence of about 15pM NGF; and/or (c) inhibits survival of human NGF dependent mouse E13.5 trigeminal neurons with an IC50 of about 10pM or less, wherein IC50 is determined in the presence of about 1.5pM NGF. In some embodiments, polypeptide (a) binds NGF with a KD of less than about 100 pM; and/or (b) inhibits the presence of human NGF-dependent mouse E13.5 trigeminal neurons with an IC50 of about 20pM or less Wherein IC50 is determined in the presence of about 15pM NGF; and/or (c) inhibits survival of human NGF dependent mouse E13.5 trigeminal neurons with an IC50 of about 2pM or less, wherein IC50 is determined in the presence of about 1.5pM NGF.

Specifically excluded from the present invention, as is apparent from the description herein, is a polypeptide embodiment consisting of an amino acid sequence identical to that of mouse monoclonal antibody 911. The extended CDR sequences of Mab 911 are shown in fig. 1A and 1B and in SEQ ID NO: 9-14.

In some embodiments, the invention provides any of the above polypeptides or antibodies, further wherein the polypeptide (e.g., antibody) is isolated. In some embodiments, the polypeptide (e.g., antibody) is substantially purified. In other embodiments, the polypeptide (e.g., antibody) is affinity matured. In other embodiments, the antibody is an antagonist antibody. In some embodiments, the polypeptide (e.g., an antibody) comprises a human framework sequence. In other embodiments, a polypeptide (e.g., an antibody) comprises one or more non-human framework residues. In some embodiments, the polypeptide (e.g., antibody) is represented by K_DNGF (e.g. human NGF) is bound at or below 2 nM. In some embodiments, the polypeptide comprises human amino acid substitutions relative to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more than 8) of a non-human amino acid sequence (e.g., a variable region, such as a CDR sequence, such as a framework sequence). In some embodiments, the polypeptide comprises at least 1, at least 2, or more than 2, such as at least 3, 4, 5, 6, or more than 6 amino acid substitutions relative to the parent polypeptide amino acid sequence (e.g., antibody 911 amino acid sequence, such as any one or more of SED ID NOs: 9-14). In some embodiments, the binding affinity of the antibody has been altered (in some embodiments, increased) relative to the affinity of the parent antibody (e.g., Mab 911). In other embodiments, the binding affinity of the antibody is lower than the binding affinity of trkA receptor for NGF (e.g., human NGF). In some embodiments, the polypeptide can be an antibody. In some embodiments, the antibody is a human antibody. In other embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a monoclonal antibody A diabody. In some embodiments, the antibody is an affinity matured antibody.

The present invention provides polynucleotides (including isolated polynucleotides) comprising polynucleotides encoding any of the above embodiments.

In another aspect, the invention provides an isolated polynucleotide comprising a polynucleotide encoding a fragment or region of antibody E3 (interchangeably referred to herein as "E3"). In some embodiments, the fragment is a light chain of antibody E3 as shown in figure 1B. In another embodiment, the fragment is the heavy chain of antibody E3 as shown in figure 1A. In other embodiments, the fragment comprises one or more variable regions from the light chain and/or heavy chain of antibody E3. In another embodiment, the fragment comprises one or more Complementarity Determining Regions (CDRs) from the light chain and/or heavy chain of antibody E3 as shown in fig. 1A and 1B.

In another aspect, the invention is an isolated polynucleotide comprising a polynucleotide encoding antibody E3. In some embodiments, the polynucleotide comprises one or both of the polynucleotides shown in figure 2 and figure 3.

In another aspect, the invention is an isolated polynucleotide encoding the light chain of E3 deposited as ATCC No. PTA-4893 or ATCC No. PTA-4894. In another aspect, the invention is an isolated polynucleotide encoding the heavy chain of E3 deposited under ATCC No. PTA-4895. In yet another aspect, the invention is an isolated polynucleotide comprising (a) the variable region encoded in the polynucleotide deposited under accession number ATCC No. PTA-4893 or PTA-4894 and (b) the variable region encoded in the polynucleotide deposited under accession number ATCC No. PTA-4895. In another aspect, the invention is an isolated polynucleotide comprising (a) one or more CDRs encoded in the polynucleotide deposited under ATCC accession No. PTA-4893 or PTA-4894; and/or (b) one or more CDRs encoded in the polynucleotide deposited as ATCC No. pta-4895.

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) or polypeptides described herein.

In another aspect, the invention provides vectors (including expression and cloning vectors) and host cells comprising any of the polynucleotides disclosed herein.

Specifically included in the present invention, as is apparent from the description herein, is a polynucleotide embodiment consisting of a polynucleotide identical to the polynucleotide sequence of mouse monoclonal antibody 911. The extended CDR sequences of Mab 911 are shown in fig. 1A and 1B and in SEQ ID NO: 9-14.

In another aspect, the invention is a host cell comprising a polynucleotide encoding a light chain of E3 and a polynucleotide encoding a heavy chain of E3, wherein the polynucleotide encoding the light chain of E3 has accession number ATCC No. pta-4893 and/or ATCC No. pta-4894 and the polynucleotide encoding the heavy chain of E3 has accession number ATCC No. pta-4895. In some embodiments, the host cell comprises the polynucleotides of (a) the variable region encoded by the polynucleotides of deposit No. ATCC No. PTA-4893 or PTA-4894 and/(b); said (b) is the variable region encoded in the polynucleotide deposited under ATCC No. PTA-4895. In some embodiments, the host cell comprises a polynucleotide encoding (a) one or more CDRs encoded by the polynucleotide of deposit No. ATCC No. PTA-4893 or PTA-4894 and/(b); said (b) is one or more CDRs encoded by the polynucleotide of deposit number ATCC No. PTA-4895. In some embodiments, the host cell is a mammalian cell.

In another aspect, the invention is an NGF complex to which antibody E3 binds. In another aspect, the complex is isolated. In another aspect, the complex is substantially pure.

In another aspect, the invention is an NGF complex to which any one of the antibodies or polypeptides described herein binds. In another aspect, the complex is isolated. In another aspect, the complex is substantially pure.

In another aspect, the invention is a pharmaceutical composition comprising any of the polypeptides (including antibodies such as antibody E3) or polynucleotides described herein, such as a pharmaceutical composition comprising antibody E3 or an antibody comprising a fragment of antibody E3, and a pharmaceutically acceptable excipient.

In another aspect, the invention is a method of producing antibody E3, comprising preparing a host cell comprising an expression vector encoding antibody E3; culturing the host cell or progeny thereof under conditions that allow for production of antibody E3; and purified antibody E3. In some embodiments, the expression vector comprises one or both of the polynucleotide sequences shown in figure 2 and figure 3.

In another aspect, the invention is a method of producing antibody E3, comprising expressing in a suitable cell a polynucleotide encoding the light chain of E3 and a polynucleotide encoding the heavy chain of E3, wherein the polynucleotide encoding the light chain of E3 has accession numbers ATCC No. pta-4893 and/or ATCC No. pta-4894, and the polynucleotide encoding the heavy chain of E3 has accession numbers ATCC No. pta-4895; the antibody is typically subsequently recovered and/or isolated.

In another aspect, the invention provides a method for producing any of the polypeptides described herein (e.g., an antibody) in a suitable cell by expressing one or more polynucleotides encoding the antibody (which may be expressed separately as a single light or heavy chain, or both light and heavy chains may be expressed from a vector), typically followed by recovering and/or isolating the antibody or polypeptide of interest.

In another aspect, the invention is a method of antagonizing NGF (e.g., human NGF) biological activity using any of the polypeptides disclosed herein, including antibodies such as antibody E3. In one embodiment, the method comprises contacting a human nerve growth factor with any of the polypeptides described herein (including antibody E3) to antagonize, reduce, block, or inhibit NGF activity (e.g., human nerve growth factor activity).

In another aspect, the invention is a method for detecting NGF using any of the polypeptides described herein (including antibodies, such as antibody E3). The presence of NGF is detected by detecting NGF complexes with any of the polypeptides described herein, such as antibody E3. The term "detecting" as used herein includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.

In another aspect, the invention is a method of treating pain by administering an effective amount of a composition comprising antibody E3 or any of the polypeptide (including antibody) or polynucleotide embodiments described herein. In some embodiments, the pain is post-surgical pain.

In another aspect, the invention is a method of preventing or treating rheumatoid arthritis pain in an individual by administering to the individual an effective amount of an anti-NGF antagonist antibody. anti-NGF antagonist antibodies have been shown, according to the invention, to inhibit or block pain associated with rheumatoid arthritis. In some embodiments, the pain is reduced within about 24 hours after administration of the anti-NGF antagonist antibody. In some embodiments, the pain is reduced about 4 days after administration of the anti-NGF antagonist antibody. In some embodiments, an improved indication of inflammatory disease in an individual is reduced in pain before or without the observation.

In another aspect, the invention provides a method of reducing the incidence, ameliorating, inhibiting, alleviating and/or delaying the onset, development or progression of rheumatoid arthritis pain in an individual comprising administering to the individual an effective amount of an anti-NGF antagonist antibody.

In another aspect, the invention is a method of preventing or treating osteoarthritis pain in an individual by administering to the individual an effective amount of an anti-NGF antagonist antibody.

In another aspect, the invention provides a method of treating inflammatory cachexia (weight loss) associated with rheumatoid arthritis in an individual comprising administering an effective amount of an anti-NGF antagonist antibody. In another aspect, the invention provides a method of reducing the incidence of, ameliorating, inhibiting, reducing and/or delaying the onset, development or progression of osteoarthritis pain in an individual comprising administering to the individual an effective amount of an anti-NGF antagonist antibody.

In another aspect, the invention provides kits and compositions comprising any one or more of the compositions described herein. These kits are generally used in any of the methods described herein in suitable packaging and provided with appropriate instructions.

The invention also provides any of the compositions and kits for any of the uses described herein, whether for use as a medicament and/or for the manufacture of a medicament.

Brief Description of Drawings

FIG. 1A: the amino acid sequence of the heavy chain variable region of the E3 antibody is shown (labeled "6" and "5 + affinity maturation H3). The Chothia CDR and Kabat CDR by underline form, bold and italic form description. FIG. 1A also shows the arrangement of the following amino acid sequences of the heavy chain variable region: (1) the CDRs H1(SEQ ID NO: 9), H2(SEQ ID NO: 10), and H3(SEQ ID NO: 11) of the mouse 911 antibody, (2) VH4-59 human embryonic receptor sequence (labeled "VH 4-59" or "2") (SEQ ID NO: 69); (3) the recipient sequence (labeled "CDR grafted" or "3") grafted with the extended CDR of mouse antibody 911 (SEQ ID NO: 70); (4) CDR-grafted recipient sequences (labeled "3 + one framework mutation" or "4") comprising a V71K substitution (SEQ ID NO: 71); (5) clones comprising the affinity matured CDRs H1 and H2 (labeled "5" or "4 + affinity matured H1, H2") (SEQ ID NO: 72); and antibody E3 (as described above) (SEQ ID NO: 1).

FIG. 1B: the amino acid sequence of the variable region of the E3 antibody light chain is shown (labeled "5" and "4 + affinity maturation L3). The Chothia CDR and Kabat CDR by underline form, bold and italic form description. FIG. 1B also shows the arrangement of the following light chain variable region amino acid sequences: (1) the CDRs L1(SEQ ID NO: 12), L2(SEQ ID NO: 13), and L3(SEQ ID NO: 14) of the mouse 911 antibody, (2) the O8 human germline acceptor sequence (labeled "O8" or "2") (SEQ ID NO: 73); (3) extension of the CDR-grafted recipient sequence (labeled "CDR-grafted" or "3") with mouse antibody 911 (SEQ ID NO: 74); (4) CDR-grafted recipient sequences (labeled "3 + affinity mutations L1, L2" or "4") (SEQ ID NO: 75); (5) clones containing the affinity matured CDRs L1 and L2 (labeled "5" or "4 + affinity matured L3"); and antibody E3 (as described above) (SEQ ID NO: 2).

FIG. 2: a polynucleotide comprising a polynucleotide sequence encoding the heavy chain variable region polynucleotide sequence of antibody E3 is shown (SEQ ID NO: 76).

FIG. 3: a polynucleotide comprising a polynucleotide sequence encoding the light chain variable region polynucleotide sequence of antibody E3 is shown (SEQ ID NO: 77).

FIG. 4: graph depicting NGF-dependent E13.5 neuronal survival in the presence of different concentrations of human and rat NGF. The X-axis corresponds to NGF concentration (ng/ml) and the Y-axis corresponds to counted neurons.

FIG. 5: graphs comparing NGF blocking by various Fab's in the presence of 0.04ng/ml human NGF (about 1.5 pM; shown in the lower panel) or 0.4ng/ml human NGF (about 15 pM; shown in the upper panel). Fab E3 at various concentrations on E13.5 mouse trigeminal neurons; murine 911 Fab; and Fab H19-L129 and Fab 8L2-6D 5. IC50 (expressed as pM) for each Fab at each NGF concentration was calculated and shown in figure 9. Fab E3 strongly blocked human NGF dependent trigeminal neuron survival with an IC50 of about 21pM in the presence of 15pM human NGF and an IC50 of about 1.2pM in the presence of 1.5pM human NGF. Fab 3C and H19-L129 also strongly blocked human NGF-dependent survival of trigeminal neurons. In both figures, the X-axis corresponds to antibody concentration (nM) and the Y-axis corresponds to counted neurons. IC50 was approximately 1.5pM NGF and 15pM represents the saturation concentration of NGF.

FIG. 6: graphs comparing NGF blocking by various Fab's in the presence of 0.04ng/ml rat NGF (about 1.5 pM; shown in the lower panel) or 0.4ng/ml rat NGF (about 15 pM; shown in the upper panel). Fab E3 at various concentrations on E13.5 mouse trigeminal neurons; murine Fab 911; and survival in FabH19-L129 and 8L2-6D5 were evaluated as described above. IC50 (expressed as pM) for each Fab at each NGF concentration was calculated and shown in figure 9. Fab E3 strongly blocked human NGF-dependent trigeminal neuron survival, with an IC50 of about 31.6pM in the presence of 15pM rat NGF and an IC50 of about 1.3pM in the presence of 1.5pM rat NGF. Fab 3C and H19-L129 also strongly blocked rat NGF-dependent trigeminal neuron survival. IC50 was approximately 1.5pM NGF and 15pM represents the saturation concentration of NGF. In both figures, the X-axis corresponds to antibody concentration (nM) and the Y-axis corresponds to counted neurons.

FIG. 7 is a graph depicting resting pain assessed 24 hours post-surgery and showing that treatment with 0.02mg/kg, 0.1mg/kg, 0.6mg/kg, or 1mg/kg anti-NGF antibody E3 reduced pain. "+" indicates statistically significant difference (p < 0.5) compared to negative control.

FIG. 8: to describe resting pain assessed 24 hours post-surgery and to show a graph of significant (p < 0.005) reduction in resting pain two hours post-surgery with an injection of 0.5mg/kg anti-NGF antibody E3.

FIG. 9: FIG. is a graph showing the results of BIAcore analysis of the binding affinity of human NGF to the mouse antibody 911 (Fab). Mouse 911 antibody with KD 3.7nM, K_off8.4×10^-5s^-1And K_on 2.2×10⁴ M^-1s^-1Binds NGF.

FIG. 10: FIG. is a graph showing the results of BIAcore analysis of the binding affinity of human NGF to antibody E3(Fab) (referred to as "3E Fab"). E3 with a KD of about 0.07nM (and a kon of about 6.0X 10⁵ M^-1s^-1And koff of about 4.2X 10^-5s^-1) Binds human NGF.

FIG. 11: graphs depicting antibody E3 blocking NGF interaction with its receptors trkA and p75, as assessed by the percent binding detected between NGF and trkA (shown as black circles) and NGF and p75 (shown as open squares), with the X-axis corresponding to the concentration of antibody 3E (fab) and the Y-axis corresponding to NGF binding (maximum RU percent). As shown by decreasing signal (as measured in RU), increased concentrations of Fab E3 blocked NGF interaction with both p75 and trkA. When antibody E3(Fab) concentration was the same as NGF concentration, no NGF binding was observed (as shown by zero signal).

FIG. 12: to depict the human NGF blocking ability of the intact antibodies E3 and Fab E3. Survival of E13.5 mouse trigeminal neurons in the presence of human NGF and various concentrations of Fab E3 and antibody E3 was assessed. The X-axis corresponds to NGF binding sites (nM) and the Y-axis corresponds to normalized counts of Trigeminal (TG) neurons. Intact antibody E3 and Fab 3E were shown to inhibit NGF-dependent survival of trigeminal neurons at similar levels when intact antibody and Fab concentrations were normalized to the number of NGF binding sites (one for Fab and two for Fab).

FIG. 13: to depict the inhibition of E13.5 trigeminal neuron dependent NGF survival by various concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 and 0.0nM) of antibody E3 (filled triangle; referred to as "3E"), antibody 911 (filled circle) and trkA receptor immunoadhesin (shaded square; referred to as "trkA-Fc") in the presence of 0.4ng/ml human NGF (at saturation). The X-axis corresponds to antibody concentration (nM) and the Y-concentration corresponds to counted neurons. These results demonstrate that antibody E3 blocks NGF significantly better than the mouse monoclonal anti-NGF antibody 911 or trkA immunoadhesin.

FIG. 14: to describe a graph in which the anti-NGF antagonist antibody E3 (referred to as "3E in the figure") or Fab 911 did not inhibit neuronal survival promoted by NT3, NT4/5 and MSP, even at antibody concentrations up to 200 nM. Data represent the average percentage of survival observed after 48 hours in culture relative to a positive control (100% survival of trigeminal neurons grown in the presence of saturated NGF concentration) in each experiment (mean + standard error, n-3 for each data point). Various concentrations (20nM, 2nM or 0.2nM) of E3 Fab (referred to in the figure as "3E") and the mouse antibody 911 Fab were used in the presence of non-added neurotrophins (referred to as "control"), 400pM NGF (referred to as "NGF-400 pM), 10 nNT 3 (referred to as" NT3-10nM) or 600pM MSP (referred to as "MSP-600 pM).

FIG. 15 is a graph depicting that anti-NGF antagonist antibody E3(Fab or intact antibody) (referred to as "3E in figure") or mouse antibody 911(Fab or intact antibody) did not inhibit neuronal survival promoted by NT3, NT4/5 and MSP even at antibody concentrations up to 200 nM. Various concentrations (200nM and 80nM) of E3 Fab, intact antibody, mouse antibody 911 intact antibody, and Fab were used in the absence of added neurotrophins (referred to as "no factor"), in the presence of 400pM NGF (referred to as "NGF-400 pM), 10nM NT3 (referred to as" NT3-10nM), or 600pM MSP (referred to as "MSP-600 pM).

FIG. 16: figure to describe that anti-NGF antagonist antibodies E3 or Fab E3 did not inhibit survival of E17 nodular neurons promoted by BDNF, NT4/5 or LIF. A mouse anti-NGF antagonist antibody 911 was also detected and similar results were observed. Various concentrations (200nM or 80nM) of intact antibody E3 (referred to as "3E" in the figure), Fab E3, intact antibody 911, or Fab911 were tested in the presence of 400pM BDNF (referred to as "BDNF-400 pM), 400pM NT4/5 (referred to as" NT4/5-400pM), or 2.5nM LIF (referred to as "LIF-2.5 nM) without added neurotrophins (referred to as" no factor ").

FIG. 17: figure to describe that anti-NGF antagonist antibodies E3 or Fab E3 did not inhibit E17 nodular neuron survival promoted by BDNF, NT4/5 or LIF. Various concentrations (200nM, 20nM, 2nM) of Fab E3 (referred to as "3E in the figure") or Fab911 were tested in the presence of non-added neurotrophins (referred to as "no control"), 400pM BDNF (referred to as "BDNF-400 pM), 400pM NT4/5 (referred to as" NT4/5-400pM) or 2.5nM LIF (referred to as "LIF-2.5 nM).

FIG. 18: graph demonstrating nociceptive responses in arthritic rats (rheumatoid arthritis model) following administration of anti-NGF antibodies (E3 and 911) to D14 and D19. E3 (intravenous 1mg/kg on days 14 and 19), 911 (intravenous 10mg/kg on days 14 and 19) or indomethacin (oral 3mg/kg indomethacin daily for 10 days) was administered to arthritic mice. The voicing strength values are expressed as mean ± standard error of mean in mV.

FIG. 19: graph demonstrating the effect of anti-NGF antibodies on body weight in rat arthritis (rheumatoid arthritis model) following administration of anti-NGF antibodies at D14 and D19. E3 (1 mg/kg intravenously on days 14 and 19), 911 (10 mg/kg intravenously on days 14 and 19), or indomethacin (3 mg/kg of indomethacin per day for 10 days) was administered to arthritic mice. Body weight values are expressed as mean ± standard error of mean in grams.

FIG. 20: graph demonstrating nociceptive responses in arthritic rats (rheumatoid arthritis model) following administration of different doses of anti-NGF antibody E3(0.003mg/kg, 0.03mg/kg, 0.3mg/kg and 5mg/kg) at D14 and D18. The voicing intensity is expressed as mean ± standard error of mean in mV.

FIG. 21: graph demonstrating the effect of anti-NGF antibody E3 on the percentage of body weight on day 14 (normalized to day 14) in arthritic rats (rheumatoid arthritis model) after administration of different doses of anti-NGF antibody E3(0.03mg/kg, 0.3mg/kg and 5mg/kg) at D14 and D18.

FIG. 22: graph demonstrating the effect of anti-NGF antibody E3 on weight loss in arthritic rats (rheumatoid arthritis model) following administration of different doses of anti-NGF antibody E3(0.03mg/kg, 0.3mg/kg and 5mg/kg) at D14 and D18. Body weight values were normalized to day 0.

Fig. 23 depicts the E3 heavy chain variable region amino acid sequence (fig. 23A) and the light chain variable region amino acid sequence (fig. 23B), as numbered with sequential numbering, Kabat numbering, and Chothia numbering.

Detailed Description

The invention disclosed herein provides anti-NGF antagonist antibodies that bind NGF (such as human NGF) with high affinity. The invention further provides antibodies and polypeptides derived from E3 that bind NGF, as well as methods of making and using these antibodies. In some embodiments, the invention provides humanized antibody E3 that binds nerve growth factor ("NGF"), and methods of making and using this antibody. The invention also provides E3 polypeptides (including antibodies) that bind NGF, as well as polynucleotides encoding E3 antibodies and/or polypeptides.

The invention disclosed herein also provides methods for preventing and/or treating rheumatoid arthritis pain in an individual by administering a therapeutically effective amount of an anti-NGF antagonist antibody.

The invention disclosed herein also provides methods for preventing and/or treating osteoarthritis pain in an individual by administering a therapeutically effective amount of an anti-NGF antagonist antibody.

The invention also provides methods for modulating antibody affinity and methods for characterizing CDR regions.

General techniques

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in the literature, such as Molecular Cloning: a Laboratory Manual, second edition, (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j.gait, eds., 1984); methods in molecular biology, Humana Press, supra; cell Biology: a Laboratory Notebook (J.E. Cellis, eds., 1998) Academic Press; animal Cell Culture (r.i. freshney, editors, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and tissue culture: laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, eds 1993-1998) J.Wiley and Sons; methods in Enzymology (academic Press, Inc.); handbook of Experimental Immunology (d.m.weir and c.c.blackwell, editions) in a book; gene Transfer Vectors for Mammaliancells (J.M.Miller and M.P.Calos, eds., 1987); current protocols Molecular Biology (F.M. Ausubel et al, eds., 1987) in the book; and (3) PCR: the Polymerase Chain Reaction, (Mullis et al, editors, 1994) in a book; current Protocols in Immunology (J.E.Coligan et al, eds., 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies: a practical proproach (D.Catty, eds., IRL Press, 1988-; monoclone antigens: a practicallappacach (p.shepherd and c.dean, editors, Oxford University Press, 2000) in one book; using antibodies: a Laboratory manual (E.Harlow and D.Lane (Cold spring Harbor Laboratory Press, 1999)), The antibiotics (M.Zantetti and J.D.Capra, eds., Harwood Academic Publishers, 1995), and The Cancer: Principles and Practice of Oncology (V.T.Devita et al, eds., J.B.Lippincott Company, 1993).

Definition of

An "antibody" is an immunoglobulin molecule that specifically binds a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only fully polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')₂Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and immunoglobulin molecules in any other modified configuration comprising an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain of an immunoglobulin, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Fv" is an antibody fragment that contains a complete antigen recognition and binding site. In the two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. In single chain Fv species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can be linked to a similar dimeric structure in a two chain Fv species. In this configuration the three CDRs of each variable domain interact to define the antigen binding specificity of the VH-VL dimer surface. However, even a single variable domain (or half of an Fv comprising only 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, but generally has less affinity than a complete binding site.

The Fab fragment also contains the light chain constant domain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

"monoclonal antibody" refers to a homogeneous population of antibodies, wherein the monoclonal antibodies comprise amino acids (naturally occurring or non-naturally occurring) that are involved in the selective binding of an antigen. The monoclonal antibody population is highly specific, being directed against a single antigenic site. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab') ₂Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site with the desired specificity and ability to bind an antigen. The source of the antibody or the manner in which the antibody is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animal, etc.) is not intended to be limiting.

As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or made by any technique known in the art or disclosed herein for making human antibodies. The definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library expressing human antibodies (Vaughan et al, 1996, Nature Biotechnology, 14: 309-. Human antibodies can also be prepared by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which endogenous immunoglobulin genes are partially or completely inactivated. This method is described in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425 and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce a target antigen (e.g., the B lymphocytes can be recovered from an individual or can be immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, page 77 (1985); boerner et al, 1991, J immunol, 147 (1): 86-95; and U.S. Pat. No. 5,750,373.

By "chimeric antibody" is meant an antibody in which a portion of the amino acid sequence of each of the heavy and light chains is homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to the corresponding sequence in another species. Generally, in these chimeric antibodies, the light and heavy chain variable regions mimic antibody variable regions from one species of mammal, while the constant portions are homologous to antibody sequences from another species. One clear advantage of such chimeric forms is that the B cell variable regions, e.g., using readily available hybridomas or from non-human host organisms, can be conveniently derived from presently known sources in combination with constant regions derived from, e.g., human cell preparations. The variable region has the advantages of easy preparation and no influence on the specificity of the variable region, the constant region is from human, and when the antibody is injected, the constant region from human is less likely to cause immune response to a human subject than the constant region from non-human source. However, the definition is not limited to this specific example.

A "functional Fc region" has at least one effector function of a native sequence Fc region. Example "effector functions" include C1q binding; complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and evaluated using a variety of assays known in the art for assessing such antibody effector functions.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that is different from a native sequence Fc region, but retains at least one effector function of the native sequence Fc region due to at least one amino acid modification. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example from about one to about ten amino acid substitutions, preferably from about one to about five amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% sequence identity, and most preferably at least about 90% sequence identity thereto, and more preferably at least about 95% sequence identity thereto, to the native sequence Fc region and/or to the Fc region of the parent polypeptide.

As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, as described in U.S. patent No. 5,500,362 or 5,821,337. Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and NK cells. Alternatively, or additionally, the ADCC activity of the molecule of interest may be in vivo, for example as described in Clynes et al, 1998, pnas (usa), 95: 652-656.

As used herein, "Fc receptor" and "FcR" describe receptors that bind the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, preferred fcrs are those which bind IgG antibodies (gamma receptors) and include Fc γ RI, Fc γ RII, and Fc γ RIII subclass receptors, including allelic variants of these receptors and alternatively spliced forms of such receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor") that have similar amino acid sequences but differ primarily in the cytoplasmic domain. FcR is used in ravatch and Kinet, 1991, ann.rev.immunol., 9: 457-92; capel et al, 1994, immunoassays, 4: 25-34; and de Haas et al, 1995, j.lab.clin.med., 126: 330-41. "FcR" also includes the neonatal receptor FcRn responsible for transfer of maternal IgG to the fetus (Guyer et al, 1976, J.Immunol., 117: 587; and Kim et al, 1994, J Immunol., 24: 249).

"complement dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that is complexed with a related antigen. To assess complement activation, for example, in Gazzano-Santoro et al, j.immunological Methods, 202: 163(1996), CDC assays were performed.

As used herein, the terms "E3", "3E", and "antibody E3" are used interchangeably to refer to antibodies comprising the heavy and light chain variable region amino acid sequences shown in FIGS. 1A (SEQ ID NO: 1) and 1B (SEQ ID NO: 2), respectively. The CDR portions of antibody E3 (including the Chothia and Kabat CDRs) are depicted graphically in FIGS. 1A and 1B. FIGS. 2 and 3 show polynucleotides encoding heavy and light chains, respectively, comprising the heavy and light chain variable regions shown in FIGS. 1A and 1B, respectively. The generation and characterization of E3 is described in the examples. Various biological functions associated with E3 include, but are not limited to, the ability to bind NGF and inhibit NGF biological activity and/or downstream pathways mediated by NGF signaling; and the ability to inhibit NGF-dependent survival of mouse E13.5 trigeminal neurons. As discussed herein, the antibodies of the invention can have any one or more of these characteristics. In some embodiments, the term "E3" refers to an immunoglobulin encoded by (a) a polynucleotide having the accession number ATCC No. pta-4893 or ATCC No. pta-4894 encoding E3 light chain; said (b) is the polynucleotide having deposit number ATCC No. pta-4895 encoding E3 heavy chain.

As used herein, "immunospecific" binding to an antibody refers to an antigen-specific binding interaction that occurs between the antigen-binding site of an antibody and the specific antigen recognized by the antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay, but does not react detectably with an unrelated protein).

An epitope of an antibody or polypeptide that "specifically binds" or "preferentially binds" (used interchangeably herein) is a term well known in the art, and methods of determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds more frequently, more rapidly, for a longer period of time, and/or with greater affinity than it reacts or binds to a candidate cell or substance. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or for longer periods of time than it binds to other substances. For example, an antibody that specifically or preferentially binds to an NGF epitope is one that binds with greater affinity, avidity, more readily, and/or for a longer period of time than to other NGF epitopes or non-NGF epitopes. It is also understood by reading this definition that, for example, an antibody that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second antibody. As such, "specific binding" or "preferential binding" does not necessarily (although it may be included) specific binding. Generally, but not necessarily, reference to binding indicates preferential binding.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The multimer may be linear or branched, it may comprise modified amino acids and it may be interrupted by non-amino acids. The term also encompasses naturally or indirectly modified amino acid polymers; the modification is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as attachment to a labeling component. Also included within this definition are, for example, polypeptides comprising one or more analogs of an amino acid (including, e.g., unnatural amino acids, etc.) as well as other modified polypeptides known in the art. It is understood that since the polypeptides of the invention are based on antibodies, the polypeptides may exist as single chains or as conjugated chains.

"Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be separated by non-nucleotide components. The polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", the replacement of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those having uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramides, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendent moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen (psoralen), etc.), modifications comprising chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications comprising alkylating agents, modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl groups typically present in the sugar may be substituted, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be attached to a solid support. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with amines or organic capping groups of from 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized to standard protecting groups. Polynucleotides may also comprise analogous forms of ribose or deoxyribose as are known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars, and the like A sugar such as arabinose, xylose or lyxose, pyranose, sedoheptulose, acyclic analogues and non-base nucleoside analogues such as methyl nucleosides. One or more phosphodiester linkages may be replaced by alternative linkers. Such alternative linking groups include, but are not limited to, those wherein the phosphate ester is comprised of P (O) S ("thioate"), P (S) S ("dithioate"), (O) NR₂("amidates"), P (O) R, P (O) OR', CO OR CH₂("formacetal") wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. It is not necessary that all linkages in a polynucleotide be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

The "variable region" of an antibody refers to the variable region of a light chain of the antibody or the variable region of a heavy chain of the antibody, either singly or in combination. Each heavy and light chain variable region consists of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs) also known as hypervariable regions. The CDRs of each chain are joined together in close proximity by the FRs, and are used with CDRs from other chains to form the antigen binding site of an antibody. There are at least two techniques for determining CDRs: (1) the interspecies variation-based approach (i.e., Kabat et al, sequential Proteins of Immunological Interest, (5 th edition, 1991, National institutes of Health, Bethesda MD)); and (2) crystallography studies based on antigen-antibody complexes (Chothia et Al, (1989) Nature 342: 877; Al-lazikani et Al, (1997) J.Molec.biol.273: 927-948)). As used herein, a CDR may refer to a CDR defined by one approach or a combination of both approaches.

The "constant region" of an antibody refers to either the antibody light chain constant region or the antibody heavy chain constant region, either singly or in combination.

As used herein, the terms "nerve growth factor" and "NGF" refer to nerve growth factor or variants of nerve growth factor that retain at least a portion of the biological activity of NGF. As used herein, NGF includes all mammalian species, including human, canine, feline, equine or bovine native sequence NGF.

"NGF receptor" refers to a polypeptide that is bound or activated by NGF. NGF receptors include the TrkA receptor and the p75 receptor of any mammalian species, including but not limited to human, canine, feline, equine, primate, or bovine.

As used herein, "anti-NGF antagonist antibody" (interchangeably referred to as "anti-NGF antibody") refers to an antibody that binds NGF and inhibits NGF biological activity and/or downstream pathways mediated by NGF signaling. anti-NGF antagonist antibodies comprise antibodies that block, antagonize, inhibit, or reduce (including significantly reduce) NGF biological activity, including downstream pathways mediated by NGF signaling, such as receptor binding and/or eliciting a response to NGF cells. For the purposes of the present invention, it is expressly understood that the term "anti-NGF antagonist antibody" encompasses all of the foregoing identified terms, titles and functional states and characteristics such that NGF itself, NGF biological activity (including but not limited to its ability to mediate any aspect of post-operative pain) or the consequences of biological activity are sufficiently nullified, reduced or neutralized to any meaningful degree. In some embodiments, an anti-NGF antagonist antibody binds NGF and prevents NGF dimerization and/or binding to an NGF receptor (e.g., p75 and/or trkA). In other embodiments, the anti-NGF antibody binds NGF and prevents trkA receptor dimerization and/or trkA autophosphorylation. Examples of anti-NGF antagonist antibodies are provided herein.

"biological activity" of NGF generally refers to the ability to bind to NGF receptors and/or activate NGF receptor signaling pathways. Without limitation, biological activity includes any one or more of the following: the ability to bind NGF receptors (such as p75 and/or trkA); the ability to promote trkA receptor dimerization and/or trkA autophosphorylation; the ability to activate NGF receptor signaling pathways; the ability to promote cellular differentiation, proliferation, survival, growth, and other changes in cellular physiology, including changes in neuronal morphology, synaptogenesis, synaptic function, neurotransmitter and/or neuropeptide release, and regeneration (for neurons, including peripheral and central neurons) following injury; the ability to promote survival of mouse E13.5 trigeminal neurons and the ability to mediate pain, including post-operative pain.

As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., contaminant free), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure.

"host cells" include individual cells or cell cultures that may be or have been used to incorporate a vector recipient for a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be identical (morphologically or complementarily to genomic DNA) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

As used herein, "treatment" is a method for obtaining a beneficial or intended clinical result. For the present invention, beneficial or aimed clinical results include, but are not limited to, one or more of the following: amelioration or reduction of any aspect of pain, including amelioration or reduction of acute, chronic, inflammatory, neuropathic, post-operative pain, rheumatoid arthritis pain, or osteoarthritis pain. For the present invention, beneficial or aimed clinical results include, but are not limited to, one or more of the following: including reduced severity, alleviation of one or more symptoms associated with pain, including any aspect of pain (e.g., reduction in pain duration, reduction in pain sensitivity or sensation).

An "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to elicit a beneficial or desired result, including a clinical result such as a reduction or reduction in the perception of pain. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to treat, alleviate, reduce the intensity of pain and/or prevent pain, including post-operative pain, rheumatoid arthritis pain and/or osteoarthritis pain. In some embodiments, an "effective amount" can reduce pain at rest (resting pain) or mechanically-induced pain (including post-exercise pain) or resting and mechanically-induced pain, and is administered before, during, or after incision, dissection, tearing, or injury and/or before, during, or after painful stimulation. As understood in clinical applications, an effective amount of a drug, compound or pharmaceutical composition may or may not be reached in combination with another drug, compound or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the single agent is combined with one or more other agents to achieve or achieve the desired result.

By "reduced incidence" of pain is meant any reduction in severity (which may include a reduction in the need and/or amount of other drugs and/or therapeutic agents typically used for the disease including, for example, opiates), duration and/or frequency (including, for example, delaying or increasing post-operative pain time in an individual). As understood by those skilled in the art, individuals may vary in their response to treatment, and as such, for example, "reducing the incidence of rheumatoid arthritis pain or osteoarthritis pain in an individual" reflects the administration of an anti-NGF antagonist antibody, based on the reasonable expectation that such administration may result in a reduction in such incidence in a particular individual.

"alleviating" pain (e.g., rheumatoid arthritis pain or osteoarthritis pain) or one or more symptoms of pain means reducing or ameliorating one or more symptoms of pain as compared to not administering an anti-NGF antagonist antibody. "alleviating" also includes shortening or reducing the duration of symptoms.

"alleviating" pain (e.g. rheumatoid arthritis pain or osteoarthritis pain) or one or more symptoms of pain means reducing the extent of one or more undesirable clinical manifestations of post-operative pain in an individual or population of individuals treated with an anti-NGF antagonist antibody according to the present invention.

As used herein, "delaying the progression of" a disease means delaying, impeding, slowing, arresting, stabilizing and/or delaying the progression of pain, such as post-operative pain, rheumatoid arthritis pain or osteoarthritis pain. The length of this delay may vary depending on the medical history and/or the individual undergoing treatment. As will be apparent to one skilled in the art, a sufficient or significant delay can be prophylactic so that the individual does not experience pain. A method of "delaying" the development of symptoms is to reduce the likelihood of developing symptoms over a given period of time and/or reduce the extent of symptoms over a given period of time as compared to not using this method. Such comparisons are generally based on clinical studies with a statistically significant number of subjects.

"pain" as used herein refers to any etiology, including acute and chronic pain, and any pain with an inflammatory component. Examples of pain include post-surgical pain, post-surgical pain (including toothache), migraine, headache and trigeminal neuralgia and pain associated with burns, wounds or kidney stones, pain associated with trauma (including traumatic head injury), neuropathic pain, pain associated with musculo-skeletal diseases such as rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid) arthropathies, non-articular rheumatological disorders and peri-articular disorders, and pain associated with cancer (including "breakthrough pain" and pain associated with advanced cancer), peripheral neuropathy and post-herpetic neuralgia. Examples of pain with an inflammatory component (in addition to some described above) include rheumatic pain, pain associated with mucositis and dysmenorrhea.

"post-operative pain" (interchangeably referred to as "post-incision pain" or "post-traumatic pain") refers to pain caused or caused by trauma, such as an incision, puncture, incision, tear, or trauma (including all surgical procedures, whether invasive or non-invasive). As used herein, post-operative pain does not include pain that occurs (is caused or caused) without external physical trauma. In some embodiments, post-surgical pain is internal or external (including peripheral) pain as well as trauma, incision, trauma, laceration or incision that occurs accidentally (e.g., traumatic trauma) or intentionally (e.g., surgical incision). As used herein, "pain" includes nociception and sensation of pain, and pain can be assessed objectively and subjectively using pain scores and other methods known in the art. As used herein, post-operative pain includes allodynia (i.e., an increased response to a normally non-noxious stimulus) and hyperalgesia (i.e., an increased response to a noxious or unpleasant stimulus), which can be thermal or mechanical (tactile) in nature. In some embodiments, the pain is characterized by heat sensitivity, mechanical sensitivity, and/or resting pain. In some embodiments, the post-surgical pain comprises mechanically induced pain or resting pain. In other embodiments, the post-surgical pain comprises resting pain. As is well known in the art, pain may be primary or secondary.

"biological samples" include a variety of sample types obtained from an individual and useful in diagnostic or monitoring assays. This definition includes blood or other liquid samples of biological origin, solid tissue samples such as biopsy samples or tissue cultures or cells of origin thereof, and progeny thereof. This definition also includes samples that are manipulated in any way after they have been obtained, for example by treating, solubilizing or concentrating certain components such as proteins or polynucleotides with reagents or embedding in semi-solid or solid matrices for preparing sections. The term "biological sample" encompasses clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

An "individual" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, and horses), primates, mice, and rats.

As used herein, "vector" means a construct capable of delivery in a host cell, and preferably expression of one or more genes or sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

As used herein, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter or enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity without reacting with the immune system of the subject. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or physiological (0.9%) saline. Compositions comprising such carriers are prepared by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, a. gennaro, Mack Publishing co., Easton, PA, 1990, and Remington, The Science and Practice of Pharmacy, 20 th edition, Mack Publishing, 2000).

The term "K" as used herein_off", is intended to refer to the off rate constant (off constant) for dissociation of the antibody from the antibody/antigen complex.

The term "K" as used herein_d", is intended to refer to the dissociation constant of the antibody-antigen interaction.

Antibody E3, E3-derived antibodies, compositions, and methods of use

E3 compositions, E3-derived compositions, and methods of making compositions

The present invention encompasses compositions comprising an antibody or polypeptide comprising E3; and pharmaceutical compositions comprising polynucleotides encoding E3 antibody or polypeptide sequences. As used herein, a composition comprises one or more antibodies or polypeptides (which may or may not be an antibody) that bind NGF and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind NGF. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art.

The invention also encompasses isolated antibody, polypeptide and polynucleotide embodiments. The invention also encompasses substantially pure antibody, polypeptide and polynucleotide embodiments.

The antibodies and polypeptides of the invention are characterized by any (one or more) of the following features: (a) ability to bind NGF; (b) the ability to decrease and/or inhibit NGF biological activity and/or downstream pathways mediated by NGF signaling; (c) reducing and/or inhibiting NGF dependent survival of mouse E13.5 trigeminal neurons; (d) lack any significant cross-reactivity with NT3, NT4/5, and/or BDNF; (e) the ability to treat and/or prevent pain (including post-operative pain); (f) ability to enhance NGF clearance; (g) the ability to reduce or inhibit activation of the trkA receptor, as measured, for example, by the kinase receptor activation assay (KIRA) (see U.S. Pat. No. 6,027,927).

The binding properties of antibody E3, which binds human NGF with high affinity and slow dissociation kinetics, compared to parent mouse anti-NGF monoclonal antibody 911, are outlined below. E3 binds human NGF with approximately 50-fold higher affinity than the parent mouse antibody 911.

Antibodies	KD	Koff	Kon
				911(Fab)	3.7nM	9×10-5s-1	2.2×104M-1s-1
E3	0.07nM	＜4×10-5s-1	6×105M-1s-1

The E3 antibody and related antibodies also showed strong ability to antagonize human NGF as assessed by in vitro assays (see examples 2 and 3). For example, antibody E3 antagonizes NGF-dependent survival of mouse E13 at about IC50 at about 21pM in the presence of 15pM human NGF and at about 1.2pM at IC50 in the presence of 1.5pM human NGF.

Thus, in another aspect, the antibodies or polypeptides of the invention are further identified and characterized as: (h) with low dissociation kinetics (in some embodiments, K_DLess than about 2nM and/or a koff of about 6X 10^-5 s^-1The following high affinity binds human NGF) and/or (i)15pM NGF (in some embodiments, human NGF) inhibits (blocks) mouse E13.5 trigeminal neuron-dependent NGF survival with an IC50 of about 100pM or less and/or about 1.5pM NGF with an IC50 of about 20pM or less.

In some embodiments, the antibody binds human NGF without significantly binding NGF from another vertebrate species (in some embodiments, a mammal). In some embodiments, the antibody binds human NGF and one or more NGF from another vertebrate species (in some embodiments, a mammal). In yet another embodiment, the antibody binds NGF without significant cross-reactivity with other neurotrophins (e.g., related neurotrophins, NT3, NT4/5, and/or BDNF). In some embodiments, the antibody binds NGF and at least one other neurotrophin. In some embodiments, the antibody binds NGF in a mammalian species, such as a horse or dog, but does not significantly bind NGF from other mammalian species.

In some embodiments, the invention is an antibody comprising a light chain encoded by a polynucleotide produced by a host cell deposited with ATCC No. pta-4893 or ATCC No. pta-4894. In another aspect, the invention is an antibody comprising a heavy chain encoded by a polynucleotide produced by the host cell deposited with ATCC No. pta-4895. The invention also encompasses various forms of E3 and equivalent antibody fragments (e.g., Fab ', F (ab')₂Fv, Fc, etc.), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified E3 configuration comprising a specific antigen of interest (NGF) recognition site. Equivalent antibodies to E3, including antibodies and polypeptide fragments (which may or may not be antibodies) to E3 and polypeptides comprising fragments of the E3 polypeptide, are identified and characterized by any standard(s) described above.

Accordingly, the present invention provides any of the following, or compositions (including pharmaceutical compositions) comprising any of the following: (a) antibody E3; (b) a fragment or region of antibody E3; (c) the light chain of antibody E3 as shown in figure 1B; (c) the heavy chain of the E3 antibody is shown in fig. 1A; (d) one or more variable regions from the light and/or heavy chain of antibody E3; (e) one or more CDRs (one, two, three, four, five, or six CDRs) of antibody E3 shown in fig. 1A and 1B; (f) the CDR H3 from the heavy chain of antibody E3 shown in fig. 1A; (g) CDR L3 from the light chain of antibody E3 is shown in fig. 1B; (h) three CDRs from the light chain of antibody E3 as shown in figure 1B; (i) three CDRs from the heavy chain of antibody E3 are shown in figure 1A; (j) three CDRs from the light chain and three CDRs from the heavy chain of antibody E3 are shown in fig. 1A and 1B; and (k) an antibody comprising any one of (b) to (i). As is apparent from the description herein, specifically excluded from the present invention are embodiments of polypeptides consisting of an amino acid sequence identical to the amino acid sequence of mouse monoclonal antibody 911. The extended CDR sequence of monoclonal antibody 911 is shown in fig. 1A and 1B and in SEQ id no: 9-14.

The CDR portions of antibody E3 (including Chothia and Kabat CDRs) are depicted diagrammatically in fig. 1A and 1B and consist of the following amino acid sequences: (a) heavy chain CDR1 ("CDR H1") GFSLIGYDLN (SEQ ID NO: 3); (b) heavy chain CDR2 ("CDR H2") IIWGDGTTDYNSAVKS (SEQ ID NO: 4); (c) heavy chain CDR3 ("CDR H3") GGYWYATSYYFDY (SEQ ID NO: 5); (d) light chain CDR1 ("CDR L1") RASQSISNNLN (SEQ ID NO: 6); (e) light chain CDR2 ("CDR L2") YTSRFHS (SEQ ID NO: 7); and (f) the light chain CDR3 ("CDR L3") QQEHTLPYT (SEQ ID NO: 8). Determination of CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs comprise Kabat CDRs. In other embodiments, the CDR is a Chothia CDR.

In some embodiments, the invention provides antibodies comprising at least one CDR that is substantially homologous to at least one CDR, at least two, at least three, at least four, at least 5 CDRs of E3 (or in some embodiments, substantially homologous to E3 or all 6 CDRs derived from E3). Other embodiments include antibodies having at least two, three, four, five, or six CDRs substantially homologous to at least two, three, four, five, or six CDRs of E3 or derived from E3. For the present invention, it is understood that while the degree of activity may vary (may be greater or less) compared to E3, binding specificity and/or overall activity is generally maintained (for treating and/or preventing pain or inhibiting NGF-dependent survival of trigeminal neurons in E13.5 mice).

The invention also provides polypeptides (which may or may not be antibodies) comprising an E3 amino acid sequence (shown in fig. 1A and 1B) having any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of the E3 sequence, wherein at least 3 amino acids are from the E3 variable region, are understood to be specifically excluded embodiments consisting of an amino acid sequence identical to the amino acid sequence of mouse monoclonal antibody 911. The extended CDR sequence of monoclonal antibody 911 is shown in fig. 1A and 1B, and in SEQ ID NO: 9-14. In one embodiment, the variable region is from an E3 light chain. In another embodiment, the variable region is from the E3 heavy chain. In another embodiment, 5 (or more than 5) consecutive amino acids are from the Complementarity Determining Regions (CDRs) of E3 shown in fig. 1A and 1B.

In another embodiment, the invention provides a polypeptide comprising an amino acid sequence having any one of: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of the E3 sequence, wherein the E3 sequence comprises any one or more of the following: amino acid residue L29 of CDRH1, I50 of CDRH2, W101 of CDRH3 and/or a103 of CDRH 3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91E of CDRL3, and/or H92 of CDRL3, it being understood that embodiments consisting of an amino acid sequence identical to the amino acid sequence of mouse monoclonal antibody 911 are specifically excluded.

As is apparent throughout this disclosure, a sequential amino acid numbering scheme is used to refer to the amino acid residues of the variable regions (i.e., the amino acid residues in each variable region are numbered sequentially). As is well known in the art, the Kabat and/or Chothia numbering systems are useful when comparing two antibodies or polypeptides, such as the E3 antibody and the E3 variant (or polypeptide suspected of being an E3 variant). It is well understood in the art how to convert sequential numbering to Chothia and/or Kabat numbering, as desired, for example for comparison between E3 and another polypeptide. Fig. 23 depicts the E3 variable regions numbered with sequential, Chothia, and Kabat numbering. Furthermore, for ease of comparison, it is generally understood that framework residues generally, but not often, have about the same number of residues. However, the CDR sizes may vary (i.e., insertions or deletions of one or more amino acid residues are possible). When comparing the E3 antibody and the candidate E3 variant (e.g., for CDR regions from the candidate sequence that are longer than the sequence of antibody E3 to which they are aligned), the following steps may be followed (although other methods are known in the art). The candidate antibody sequences were aligned with the heavy and light chain variable regions of the E3 antibody. The alignment may be performed manually or by computer using generally accepted computer programs. Alignment can be conveniently performed using a few amino acid residues commonly used for most Fab sequences. For example, the light and heavy chains each typically have two cysteines, which are often found at conserved positions. It is understood that the amino acid sequence of a candidate variant antibody may be longer (i.e., having inserted amino acid residues) or shorter (having deleted amino acid residues). Suffixes may be added to the number of residues to indicate an inserted additional residue, such as residue 34 abc. For example, for candidate sequences that are aligned, for example, with the E3 sequence, e.g., residues 33 and 35, but no residue is aligned with residue 35 between candidate sequences, residue 35 is simply not assigned to a residue. In another approach, when comparing CDRs of different lengths, it is generally known that comparisons can be made between structurally equivalent (e.g., the same position of an antigen-antibody complex) amino acids. For example, Chothia numbering (Al-Lazikani et Al, supra) places insertions and deletions in generally, but not in all cases, structurally correct positions. Structural equivalents may also be deduced or demonstrated by X-ray crystallography or double mutation cycle analysis (see Pons et al, (1999) prot. Sci.8: 958-968).

The binding affinity of an anti-NGF antibody to NGF (such as hNGF) can be about 0.10 to about 0.80nM, about 0.15 to about 0.75nM, and about 0.18 to about 0.72 nM. In some embodiments, the binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM, or greater than about 40 pM. In one embodiment, the binding affinity is between about 2pM and 22 pM. In other embodiments, the binding affinity is about 10nM or less, about 5nM, about 4nM, about 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10 pM. In some embodiments, the binding affinity is about 10 nM. In other embodiments, the binding affinity is about 10nM or less. In other embodiments, the binding affinity is about 0.1nM or about 0.07 nM. In other embodiments, the binding affinity is about 0.1nM or less or about 0.07nM or less. In other embodiments, the binding affinity is any of about 10nM, about 5nM, about 4nM, about 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10pM, to any of about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, or about 40 pM. In some embodiments, the binding affinity is any of about 10nM, about 5nM, about 4nM, about 3.5nM, about 3nM, about 2.5nM, about 2nM, about 1.5nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10 pM. In still other embodiments, the binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM, or greater than about 40 pM.

The binding affinity of an antibody to NGF can be determined by methods well known in the art. As described in the examples, one way to determine the binding affinity of an antibody to NGF is by determining the affinity of a monofunctional Fab fragment of the antibody. To obtain monofunctional Fab fragments, antibodies (e.g., IgG) can be cleaved with papain or expressed recombinantly. As described in the examples, the affinity of the anti-NGF Fab fragment of an antibody can be determined by surface plasmon resonance (BIAcore 3000)^TMSurface Plasmon Resonance (SPR) system, BIAcore, INC, Piscaway NJ). This method is useful for determining the binding affinity of an antibody to NGF of any species, including human NGF, NGF of another vertebrate (in some embodiments, a mammal) (e.g., mouse NGF, rat NGF, primate NGF), and for other neurotrophins, such as the related neurotrophins NT3, NT4/5, and/or BDNF.

In some embodiments, an antibody or polypeptide of the invention can inhibit (reduce and/or block) survival of mouse E13.5 trigeminal neuron dependent human NGF with any one of an IC50 (presence of about 15pM NGF) of about 200pM, 150pM, 100pM, 80pM, 60pM, 40pM, 20pM, 10pM, or less than 10 pM. In some embodiments, an antibody or polypeptide of the invention can inhibit (reduce and/or block) mouse E13.5 trigeminal neuron-dependent human NGF survival with an IC50 (presence of about 1.5pM NGF) of any one of about 50pM, 40pM, 30pM, 10pM, 20pM, 10pM, 5pM, 2pM, 1pM, or 1pM or less. In some embodiments, an antibody or polypeptide of the invention can inhibit (reduce and/or block) mouse E13.5 trigeminal neuron-dependent rat NGF survival with any one of an IC50 (presence of about 15pM NGF) of about 150pM, 125pM, 100pM, 80pM, 60pM, 40pM, 30pM, 20pM, 10pM, 5pM, or less than 5 pM. In some embodiments, an antibody or polypeptide of the invention can inhibit (reduce and/or block) mouse E13.5 trigeminal neuron-dependent rat NGF survival with an IC50 (presence of about 1.5pM NGF) of about 30pM, 25pM, 20pM, 15pM, 10pM, 5pM, 4pM, 3pM, 2pM, 1pM, or less than 1 pM. Methods for determining NGF-dependent survival of mouse E13 trigeminal neurons are known in the art and are described, for example, in example 2.

The invention also provides methods of making any such antibody or polypeptide. The antibodies of the invention can be prepared by procedures known in the art, some of which are set forth in the examples. The polypeptides may be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fusion polypeptides) or by chemical synthesis. Polypeptides of antibodies, particularly shorter polypeptides of up to about 50 amino acids, are generally prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, the E3 antibody can be produced by an automated polypeptide synthesizer using solid phase methods, see also U.S. patent nos. 5,807,715; 4,816,567 and 6,331,415. Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including methods using cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutaneimidate.

In another alternative, the antibody may be recombinantly produced using methods well known in the art. In one embodiment, a polynucleotide comprising sequences encoding the variable and light chain regions of antibody E3 (as shown in fig. 1A and 1B) is cloned into a vector for expression or propagation in a host cell (e.g., a CHO cell). In another embodiment, the polynucleotide sequences shown in FIGS. 2 and 3 are cloned into one or more vectors for expression or propagation. The sequences encoding the antibody of interest may be maintained in a vector for the host cell and the host cell then expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein. Methods for recombinant expression of antibodies in plants or milk are disclosed. See, e.g., Peeters et al, (2001) Vaccine 19: 2756; lonberg, n. and d.huskzar (1995) int.rev.immunol 13: 65; and Pollock et al, (1999) J Immunol Methods 231: 147. methods for preparing antibody derivatives, e.g., humanized antibodies, single chain antibodies, and the like, are known in the art.

The invention also encompasses single chain variable fragments ("ScFV") of antibodies of the invention, such as E3. Single chain variable region fragments can be prepared by linking the light and/or heavy chain variable regions using short linking peptides. Bird et al, (1988) Science 242: 423-426. An example of a linker peptide is (GGGGS)3(SEQ ID NO: 15) bridging between the carboxy terminus of one variable region and the amino terminus of the other variable region by about 3.5 nm. Linkers of other sequences have been referred to and used (Bird et al, (1988)). The linker may in turn be modified for other functions, such as linking a drug or linking a solid support. Single-stranded variants can be produced recombinantly or synthetically. For synthetic generation of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid comprising a polynucleotide encoding an scFv can be introduced into a suitable host cell, which is a eukaryotic cell, such as a yeast, plant, insect or mammalian cell, or a prokaryotic cell, such as E.coli. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures such as ligation of the polynucleotides. The generated scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair the two domains on the same chain, thus forcing the domains to pair with complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger, P., et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-.

The antibody can be a bispecific antibody, a monoclonal antibody, having at least two different antigen binding specificities. Bispecific antibodies can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al, 1986, Methods in Enzymology 121: 210). In general, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

According to the method for producing bispecific antibodies, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably to an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, there is a first heavy chain constant region (CH1) in at least one of the fusions that contains the site required for light chain binding. The DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into respective expression vectors and co-transfected into a suitable host organism. This provides in embodiments great flexibility in adjusting the ratio of the three polypeptide chains to each other, while using unequal ratios of the three polypeptide chains in the construct provides optimal yield. However, it is possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when expressing at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important.

In one approach, a bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure with immunoglobulin light chains in only half of the bispecific molecules helps the bispecific compound of interest to separate from unwanted immunoglobulin chain combinations. This method is described in PCT publication No. WO94/04690, published 3/3 of 1994.

Heterologous binding antibodies (heteroconjugate antibodies) comprising two covalently linked antibodies are also within the scope of the invention. Such antibodies are useful for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heterologous binding antibodies can be prepared by any conventional crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Pat. No. 4,676,980.

The antibody can be a humanized antibody, e.g., as known in the art and described herein.

Antibodies can be modified as described in PCT publication No. WO99/58572, published 11/18 1999. Such antibodies comprise, in addition to a binding domain for a target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of a human immunoglobulin heavy chain constant domain. Such antibodies are capable of binding to a target molecule without triggering significant complement-dependent lysis, or cell-mediated destruction of the target. Preferably, the effector domain is capable of specifically binding FcRn and/or fcyriib. These antibodies are generally based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are preferably used in chronic antibody therapy to avoid inflammatory and other adverse reactions to conventional antibody therapy.

The present invention encompasses modifications of antibody E3, including functionally equivalent antibodies that do not significantly affect their properties, as well as variants with enhanced or reduced activity. Modifications of the polypeptides are routine in the art and are further exemplified in the examples. Examples of modified polypeptides include polypeptides that are substituted with amino acid residues (including conservative substitutions), one or more amino acid deletions or additions that do not significantly detrimentally alter functional activity, or that use chemical analogs.

As used herein, a polypeptide "variant" is a polypeptide that differs from a native protein by one or more substitutions, deletions, additions and/or insertions, resulting in no substantial reduction in the immunoreactivity of the polypeptide. In other words, the ability of the variant to specifically bind an antigen may be enhanced or unchanged relative to the native protein, or may be reduced by less than 50%, and preferably less than 20%, relative to the native protein. Polypeptide variants preferably exhibit at least about 80%, more preferably at least about 90% and most preferably at least about 95% identity (as determined as described herein) to the identified polypeptide.

Amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes in the antibody DNA or by peptide synthesis. Such variants include, for example, the variants described herein as SEQ ID NOs: 1 or 2 amino acid sequence and/or deletion and/or insertion and/or substitution of residues. Any combination of any deletion, insertion and substitution is made to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of an antibody as preferred sites for mutagenesis or modification is known as "alanine scanning mutagenesis" and is described by Cunningham and Wells, 1989, Science, 244: 1081-1085. Residues or groups of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions that demonstrate sensitivity to the substitution function are then refined by introducing additional variants at or for the position of the substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or target region and the expressed antibody variants are screened for the desired activity. Library scanning mutagenesis, as described herein, can also be used to identify sites in an antibody suitable for mutagenesis or modification.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions that are one residue in length to polypeptides comprising one or more than one hundred residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with enzymes or polypeptides that increase the serum half-life of the antibody.

Substitutional variants have at least one amino residue removed from the antibody moiety and a different residue inserted at this position. The most interesting sites for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". If this substitution results in a change in biological activity, then what is referred to as an "exemplary substitution" in Table 1, or more substantial changes as described further below with respect to the amino acids, can be introduced and the product screened.

Table 1: amino acid substitutions

Residue of a proenzyme	Conservative substitutions	Example alternatives
			Alanine (A)	Valine	Valine; leucine; isoleucine
Arginine (R)	Lysine	Lysine; (ii) glutamine; asparagine
			Asparagine (N)	Glutamine	(ii) glutamine; (ii) histidine; aspartic acid; lysine; arginine
Aspartic acid (D)	Glutamic acid	Glutamic acid; asparagine
			Cysteine (C)	Serine	Serine; alanine
Glutamine (Q)	Asparagine	Asparagine; glutamic acid
			Glutamic acid (E)	Aspartic acid	Aspartic acid; glutamine
Glycine (G)	Alanine	Alanine
			Histidine (H)	Arginine	Asparagine; (ii) glutamine; lysine; arginine
Isoleucine (I)	Leucine	Leucine; valine; methionine; alanine; phenylalanine; norleucine
			Leucine (L)	Isoleucine	Norleucine; isoleucine; valine; methionine; alanine; phenylalanine
Lysine (K)	Arginine	Arginine; (ii) glutamine; asparagine
			Methionine (M)	Leucine	Leucine; phenylalanine; isoleucine
Phenylalanine (F)	Tyrosine	Leucine; valine; isoleucine; alanine; tyrosine
			Proline (P)	Alanine	Alanine
Serine (S)	Threonine	Threonine
			Threonine (T)	Serine	Serine
Tryptophan (W)	Tyrosine	Tyrosine; phenylalanine
			Tyrosine (Y)	Phenylalanine	Tryptophan; phenylalanine; threonine; serine
Valine (V)	Leucine	Isoleucine; leucine; methionine; phenylalanine; alanine; norleucine

The basic modification of antibody biological properties is accomplished by selecting substitutions that differ significantly in their role in maintaining: (a) the backbone structure of the polypeptide in the replacement region, e.g., a folded or helical configuration, (b) the charge or hydrophobicity of the target site molecule, or (c) the size of the side chain. Based on general side chain properties, naturally occurring residues are divided into:

(1) Hydrophobicity: norleucine, methionine, alanine, valine, leucine, isoleucine;

(2) neutral hydrophilicity: cysteine, serine, threonine;

(3) acidity: aspartic acid, glutamic acid;

(4) alkalinity: asparagine, glutamine, histidine, lysine, arginine;

(5) residues that influence chain orientation: glycine, proline; and

(6) aromaticity: tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions are made by exchanging one member of one of these classes for a member of another class.

Any cysteine residue not involved in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent mis-crosslinking. Conversely, particularly when the antibody is an antibody fragment such as an Fv fragment, a cysteine bond may be added to the antibody to improve its stability.

Amino acid modifications can range from changing or modifying one or more amino acids to completely redesigned regions, such as the variable regions. Changes in the variable region can alter binding affinity and/or specificity. In some embodiments, only one to five conservative amino acid substitutions are made in the CDR domains. In other embodiments, only one to three conservative amino acid substitutions are made in the CDR domains. In yet other embodiments, the CDR domains are CDRH3 and/or CDR L3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation, acetylation, and phosphorylation of different sugars. Antibodies are glycosylated at conserved sites in their constant regions (Jefferis and Lund, 1997, chem. Immunol.65: 111-128; Wright and Morrison, 1997, TibTECH 15: 26-32). The oligosaccharide side chains of immunoglobulins influence the function of the protein (Boyd et al, 1996, mol. Immunol.32: 1311-1318; Wittwe and Howard, 1990, biochem.29: 4175-4180) and the intramolecular interactions between glycoprotein moieties that can influence the conformation of the glycoprotein and the three-dimensional surface presented (Hefferis and Lund, supra, Wys and Wagner, 1996, Current Opin. Biotech.7: 409-416). Oligosaccharides may also target a given glycoprotein to certain molecules based on specific recognition structures. Glycosylation of antibodies has also been reported to affect Antibody Dependent Cellular Cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of glycosyltransferase- β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII) that catalyzes the formation of bisecting GlcNAc were reported to have improved ADCC activity (Umana et al, 1999, Mature Biotech.17: 176-180).

Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzyme to link a carbohydrate moiety to an asparagine residue. Thus, the presence of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it comprises one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Changes may also be made by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) to the original antibody sequence.

The glycosylation pattern of an antibody can also be altered without altering its underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell types used to express recombinant glycoproteins as potential therapeutic agents, such as antibodies, are rarely native cells, variations in antibody glycosylation patterns are predictable. (see, e.g., Hse et al, 1997, J.biol.chem.272: 9062-.

In addition to host cell selection, factors that influence glycosylation during recombinant production of antibodies include growth mode, medium composition, culture density, oxygenation, pH, purification protocols, and the like. Various methods have been proposed to alter glycosylation patterns in specific host organisms, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain classes of glycosylation, can be enzymatically removed from glycoproteins, for example, with endoglycosidase H (EndoH). In addition, recombinant host cells can be genetically engineered to be deficient in the processing of certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include coupling techniques known in the art, including but not limited to enzymatic methods, oxidative substitution, and chelation. For example, the modification may be used to bind a label of an immunoassay. Modified E3 polypeptides are prepared using methods well known in the art and can be screened using standard assays known in the art, some of which are described below and in the examples.

Other antibody modifications include antibodies modified as described in PCT publication No. WO99/58572, published 11/8 1999. In addition to the domain directed to the target molecule, the antibody comprises an effector domain having an amino acid sequence substantially homologous to all or part of the human immunoglobulin heavy chain constant domain. Such antibodies are capable of binding to a target molecule without inducing significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These antibodies are generally based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly useful in chronic antibody therapy to avoid inflammatory and other adverse reactions to conventional antibody therapy.

The invention also encompasses fusion proteins derived from one or more fragments or regions of an antibody (e.g., E3) or polypeptide of the invention. In one embodiment, fusion polypeptides are provided comprising at least 10 contiguous amino acids of the variable light chain region shown in figure 1B and/or at least 10 amino acids of the variable heavy chain region shown in figure 1A. In another embodiment, the fusion polypeptide comprises the light chain variable region and/or the heavy chain variable region of E3 as shown in fig. 1A and 1B. In another embodiment, the fusion polypeptide comprises one or more CDRs of E3. In yet another embodiment, the fusion polypeptide comprises the CDR H3 and/or CDR L3 of antibody E3. In another embodiment, the fusion polypeptide comprises any one or more of: amino acid residue L29 of CDRH1, I50 of CDRH2, W101 of CDRH3 and/or a103 of CDRH 3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91E of CDRL3, and/or H92 of CDRL 3. For the purposes of the present invention, an E3 fusion protein comprises one or more of the E3 antibody and another amino acid sequence that is not bound in the native molecule, e.g., a heterologous or homologous sequence to another region. Exemplary heterologous sequences include, but are not limited to, a "tag" such as a FLAG tag or a 6His tag. Labels are well known in the art.

The E3 fusion polypeptide can be prepared by methods known in the art, e.g., synthetically or recombinantly. Generally, the E3 fusion proteins of the invention are prepared by expressing polynucleotides encoding the fusion proteins prepared by recombinant methods described herein, although they may be prepared by other methods known in the art, including, for example, chemical synthesis.

The invention also provides compositions comprising an E3 antibody or polypeptide bound (e.g., linked) to a reagent that facilitates coupling to a solid support, such as biotin or avidin. For the sake of brevity, reference is generally made to E3 or antibodies as it is understood that such methods apply to any of the NGF binding embodiments described herein. Binding generally refers to linking the components described herein. Attachment (generally in close association with immobilization of these components at least for administration) can be accomplished in any manner. For example, when the reagent and the antibody each have a substituent capable of reacting with the other, a direct reaction between the reagent and the antibody is possible. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one of the reagent and antibody can react with a carbonyl-containing group, such as an anhydride or acid halide, on the other of the reagent and antibody, or with an alkyl group containing a good leaving group (e.g., halide).

The antibodies or polypeptides of the invention may be linked to a labeling agent (alternatively referred to as a "label"), such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. Labels which generally provide a (direct or indirect) signal are known in the art. Thus, the invention includes labeled antibodies and polypeptides.

The ability of the antibodies and polypeptides of the invention, e.g., to bind NGF; reducing or inhibiting NGF biological activity; reduction and/or blocking of NGF-induced survival of E13.5 mouse trigeminal neurons can be detected using methods known in the art, some of which are described in the examples.

The invention also provides compositions (including pharmaceutical compositions) and kits comprising antibody E3 and any or all of the antibodies and/or polypeptides described herein as explained in the disclosure.

Polynucleotides, vectors and host cells

The invention also provides isolated polynucleotides encoding the antibodies and polypeptides of the invention (including antibodies comprising the light and heavy chain variable region polypeptide sequences shown in FIGS. 1A and 1B), and vectors and host cells comprising the polynucleotides.

Accordingly, the present invention provides polynucleotides (or compositions, including pharmaceutical compositions) comprising a polynucleotide encoding any of: (a) antibody E3; (b) a fragment or region of antibody E3; (c) the light chain of antibody E3 as shown in figure 1B; (d) the heavy chain of antibody E3 is shown in fig. 1A; (e) one or more variable regions from the light and/or heavy chain of antibody E3; (f) one or more CDRs (one, two, three, four, five, or six CDRs) of antibody E3 are shown in fig. 1A and 1B; (g) CDR H3 from the heavy chain of antibody E3 shown in fig. 1A; (h) antibody E3 light chain CDR L3 is shown in fig. 1B; (i) three CDRs from the light chain of antibody E3 shown in figure 1B; (j) three CDRs from the heavy chain of antibody E3 are shown in figure 1A; (k) three CDRs from the light chain and three CDRs from the heavy chain of antibody E3 are shown in figures 1A and 1B; or (1) an antibody comprising any one of (b) to (k). In some embodiments, the polynucleotide comprises one or two of the polynucleotides shown in figure 2 and figure 3.

In another aspect, the invention is an isolated polynucleotide encoding the light chain of E3 deposited under ATCC No. PTA-4893 or ATCC NO. PTA-4894. In another aspect, the invention is an isolated polynucleotide encoding the heavy chain of E3 deposited as ATCC No. PTA-4895. In yet another aspect, the invention is an isolated polynucleotide comprising: (a) the variable region encoded in the polynucleotide deposited under ATCC accession number pta-4894 and (b) the variable region encoded in the polynucleotide deposited under ATCC accession number pta-4895. In another aspect, the invention is an isolated polynucleotide comprising: (a) one or more CDRs encoded in the polynucleotide deposited under ATCC No. pta-4894; and/or (b) one or more CDRs encoded in the polynucleotide deposited under ATCC No. pta-4895.

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein. Polynucleotides can be prepared by methods known in the art.

In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the compositions comprise an expression vector comprising a polynucleotide encoding an E3 antibody as described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In yet other embodiments, the composition comprises one or two polynucleotides shown in figure 2 and figure 3. The expression vector and the administration of the polynucleotide composition are further described herein.

In another aspect, the invention provides a method of making any of the polynucleotides described herein.

The invention also encompasses polynucleotides complementary to any such sequence. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, as well as mRNA molecules that do not contain introns. Other coding or non-coding sequences may, but need not, be present in the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or carrier materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. A polynucleotide variant comprises one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to a naturally-occurring immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to the polynucleotide sequence encoding the native antibody or portion thereof.

Two polynucleotide or polypeptide sequences are considered "identical" if the nucleotide or amino acid sequences in the two sequences are identical when aligned for maximum correspondence as described below. Comparison between two sequences is generally performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically 30 to about 75, 40 to about 50 positions, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences.

Optimal alignment of sequences for comparison can be performed using the Megalign program of bioinformatics software Lasergene suite (DNASTAR, Inc., Madison, Wis.) with default parameters. This program embodies several alignment schemes described in the following references: in Dayhoff, M.O. (1978) A model of evolution change in proteins-substrates for detecting differences relationships, in Dayhoff, M.O. (editor) Atlas of Protein sequences and structures, National biological Research Foundation, Washington DC Vol.5, Suppl.3, page 345 and 358; hein J, 1990, United approved approach Alignment and olefins, page 626-; higgins, d.g. and Sharp, p.m., 1989, cabaos 5: 151-153; myers, e.w. and Muller w., 1988, cabaos 4: 11-17; robinson, e.d., 1971, comb. 105; santou, n., Nes, m., 1987, mol.biol.evol.4: 406-425; sneath, p.h.a. and Sokal, r.r., 1973, Numerical taxomones and practice of Numerical taxomones, Freeman Press, San Francisco, CA; wilbur, w.j. and Lipman, d.j., 1983, proc.natl.acad.sci.usa 80: 726-730.

Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over at least a 20 position comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5 to 15%, or 10 to 12% as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of identical nucleic acid bases or amino acid residues present in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the result by 100 to yield the percentage of sequence identity.

Variants may also, or alternatively, be related to the native gene; or a portion or complement thereof is substantially homologous. Such polynucleotide variants are capable of hybridizing to naturally occurring DNA sequences (or complementary sequences) encoding a natural antibody under moderately stringent conditions.

Suitable "moderately stringent conditions" include prewashing in a solution of 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); 5 XSSC hybridization at 50 ℃ to 65 ℃ overnight; followed by two washes at 65 ℃ for 20 minutes each with 2X, 0.5X, and 0.2X SSC containing 0.1% SDS.

As used herein, "highly stringent conditions" or "highly stringent conditions" are: (1) washing with high ionic strength and high temperature, such as 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 deg.C; (2) during hybridization with a denaturing agent, such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at ph6.5 with 750mM sodium chloride, 75mM sodium citrate at 42 ℃; or (3) washing with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42 ℃, in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ and in 50% formamide at 55 ℃, followed by high stringency conditions consisting of 0.1 XSSC with EDTA at 55 ℃. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. (as needed) to adjust factors such as probe length, etc.

It will be appreciated by those of ordinary skill in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences which encode a polypeptide as described herein. Some of these polynucleotides have the lowest homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the invention. An allele is an endogenous gene that is altered as a result of one or more mutations, such as deletions, additions and/or substitutions, of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and/or database sequence comparison).

The polynucleotides of the invention may be obtained by chemical synthesis, recombinant methods or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. The DNA sequence of interest can be generated by one skilled in the art using the sequences provided herein and a commercially available DNA synthesizer.

For preparation of polynucleotides by recombinant means, as discussed further herein, a polynucleotide comprising a sequence of interest can be inserted into a suitable vector, and the vector can in turn be introduced into a suitable host cell for replication and amplification. The polynucleotide may be inserted into the host cell by any method known in the art. Cells are transformed by direct uptake, endocytosis, transfection, F-junction or electroporation into exogenous polynucleotide. Once introduced, the exogenous polynucleotide may be maintained in the cell as a non-integrating vector (e.g., a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al (1989).

Alternatively, PCR can replicate DNA sequences. PCR techniques are well known in the art and are described in U.S. Pat. nos. 4,683,195, 4,800,159, 4,754,065 and 4,83,202 and PCR: the polymerase Chain Reaction, edited by Mullis et al, Birkauswer Press, Boston (1994).

RNA can be obtained from the isolated DNA in a suitable vector and inserted into a suitable host cell. For example, as shown in Sambrook et al, (1989), when cells replicate and DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art.

Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector may vary depending on the host cell to be used, useful cloning vectors generally have the ability to replicate themselves, may have a single target of a particular restriction endonuclease, and/or may carry a marker gene for selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (e.g., pBS SK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs comprising a polynucleotide according to the invention. It is implied that the expression vector must be replicable in the host cell, either as an episome or as an integrated part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT publication No. WO 87/04462. Carrier components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also typically required.

Vectors comprising the polynucleotide of interest can be introduced into the host cell by any of a number of suitable methods, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment, lipofection, and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide to be introduced will often depend on the characteristics of the host cell.

The invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate a gene encoding an antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotic cells such as e.coli (e.coli) or bacillus subtilis (b.subtilis) and yeast such as saccharomyces cerevisiae (s.cerevisae), schizophylline (s.pombe) or kluyveromyces lactis (k.lactis). Preferably, the host cell expresses the cDNA at a level that is about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than the corresponding endogenous antibody or protein of interest in the host cell (if present). Screening for host cells that specifically bind NGF is achieved by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

Methods of derivatizing antibodies with E3 and E3

Antibody E3 that binds NGF can be used to identify or detect the presence or absence of NGF. For simplicity, it is understood that these methods apply to any of the NGF binding embodiments described herein (e.g., polypeptides), with general reference to E3 or antibodies. Detection generally involves contacting the biological sample with an antibody described herein that binds NGF and forms a complex between NGF and an antibody that specifically binds NGF (e.g., E3). The formation of such complexes may be in vitro or in vivo. The term "detecting" as used herein includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.

Any of a variety of known methods may be used for detection, including, but not limited to, immunoassays with antibodies that bind polypeptides, e.g., by enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), and the like; and functional assays for the encoded polypeptide, such as binding activity or enzyme assays. In some embodiments, the antibody is detectably labeled.

Diagnostic use of E3 and derivatives

The antibodies and polypeptides of the invention are useful for detecting, diagnosing, and monitoring diseases, disorders, or conditions associated with altered or abnormal NGF expression (in some embodiments, increased or decreased NGF expression) and/or inappropriate expression, such as the presence of expression in tissues and/or cells that normally lack NGF expression or the absence of NGF expression in tissues or cells that normally have NGF expression. The antibodies and polypeptides of the invention are further useful for detecting NGF expression in related diseases, e.g., altered or abnormal NGF sensitivity or reactivity. In some embodiments, NGF expression is detected in a sample from an individual suspected of having a disease, disorder, or disease associated with or characterized by an altered or abnormal sensitivity or reactivity to NGF expression (e.g., a cancer in which NGF promotes growth and/or metastasis).

Thus, in some embodiments, the invention provides methods of contacting a sample comprising an individual having altered or aberrant NGF expression with an antibody or polypeptide of the invention and methods of determining whether NGF levels are different from a control or comparative sample. In some embodiments, the individual suffers from arrhythmia, alzheimer's disease, and/or autonomic dysfunction.

In other embodiments, the invention provides methods comprising contacting a sample of an individual and determining the level of NGF expression. In some embodiments, the individual is suspected of having a disease, disorder characterized by or associated with altered or aberrant sensitivity or responsiveness to NGF expression. In some embodiments, the individual has small cell lung cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, hepatocellular cancer, or melanoma.

For diagnostic applications, antibodies are typically labeled with a detectable moiety, including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels. Methods of binding labels to antibodies are known in the art. In other embodiments of the invention, the antibodies of the invention do not require labeling, and the presence of the antibodies of the invention can be detected using a labeled antibody that binds to the antibodies of the invention.

The antibodies of the invention can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. In Zola, Monoclonal Antibodies: a Manual of Techniques, page 147-158 (CRC Press, Inc. 1987).

Antibodies may also be used in vivo diagnostic assays, such as in vivo imaging. In general, radionuclides for antibodies (e.g.¹¹¹In、⁹⁹Tc、¹⁴C、¹³1I、¹²⁵I or³H) The labeling allows the cells or tissues of interest to be located using immunoscintigraphy (immunoscintigraphy).

Antibodies may also be used as staining reagents in pathology according to techniques well known in the art.

Methods of using E3 and derivatives for therapeutic purposes

Antibody E3 is used to reduce and/or block the biological activity of NGF. This antagonistic activity is believed to be useful in the treatment of pathological conditions associated with endogenous NGF production, such as pain. Generally, an effective amount is administered to the individual in these embodiments. Thus, in one aspect, the invention provides methods of antagonizing human NGF biological activity using any of the polypeptides disclosed herein, including antibodies such as antibody E3. In one embodiment, the method comprises contacting human nerve growth factor with any of the polypeptides described herein (including antibody E3) such that human nerve growth factor activity is antagonized, reduced, blocked, or inhibited. In yet another embodiment, individuals suffering from pain (e.g., post-operative pain or rheumatoid pain) are treated with E3.

For the sake of brevity, it is understood that these methods apply to any of the E3 variant antibodies and polypeptides described herein, generally referred to as E3 or antibodies.

Various dosage forms of E3 or E3 fragments (e.g., Fab ', F (ab') 2, Fv, Fc, etc.) such as single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified E3 configuration comprising an antigenic NGF recognition site with the specificity of interest are useful for administration. In some embodiments, the E3 antibody or E3 multiple dosage forms thereof can be administered directly. In other embodiments, multiple dosage forms of E3 or its E3 (including any of the composition embodiments described herein) and pharmaceutically acceptable excipients may be administered in combination. Pharmaceutically acceptable excipients are well known in the art and are relatively inert substances that aid in the administration of a pharmacologically effective substance. For example, the excipient may form a shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for altering permeability, encapsulating agents, buffering agents and skin penetration enhancers. Excipients and dosage forms for parenteral and non-parenteral drug delivery are shown in Remington, The Science and practice of Pharmacy, 20 th edition, Mack Publishing (2000).

In some embodiments, these agents are prepared for injection administration (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.), although other modes of administration (e.g., oral, mucosal, by inhalation, sublingual, etc.) may also be used. Thus, the E3 antibody and equivalents thereof are preferably combined with a pharmaceutically acceptable carrier, such as saline, ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dosage, timing and repetition, depends on the particular individual and the medical history of that individual. In general, any of the following doses may be used: administering at least about 50mg/kg body weight; at least about 10mg/kg body weight; at least about 3mg/kg body weight; at least about 1mg/kg body weight; at least about 750 μ g/kg body weight; at least about 500 μ g/kg body weight; at least about 250 μ g/kg body weight; at least about 100 μ g/kg body weight; at least about 50 μ g/kg body weight; at least about 10 μ g/kg body weight; at least about 1 μ g/kg body weight or less than 1 μ g/kg body weight. For repeated administrations over several days or more, depending on the disease, continued treatment is carried out until the targeted suppression of disease symptoms occurs. An exemplary dosage regimen comprises administration of an initial dose of about 2mg/kg, followed by a weekly maintenance dose of about 1mg/kg of anti-NGF antibody or followed by a maintenance dose of about 1mg/kg every two weeks. However, other dosage regimens are useful depending on the mode of pharmacokinetic resolution that the practitioner wishes to achieve. Empirical considerations such as half-life generally aid in determining dosage. The progress of this treatment is readily monitored by conventional techniques and assays.

In some individuals, more than one dose may be required. The frequency of administration can be determined and adjusted during the treatment. For example, the frequency of administration can be determined or adjusted based on the type and severity of the pain being treated, whether the agent is being administered for prophylactic or therapeutic purposes, previous treatment, the clinical history of the patient and response to the agent, and the discretion of the attendant physician. anti-NGF antagonist antibodies (e.g., E3) are typically administered by the clinician until the dosage achieves the desired result. In some cases, a sustained release dosage form of the E3 antibody is appropriate. A variety of dosage forms and devices for accomplishing sustained release are known in the art.

In one embodiment, the dose of the E3 antibody (or polypeptide) may be in an individual administered one or more times^*And (4) empirically determining. An increased dose of E3 was administered to the individual. To assess the efficacy of E3 or other equivalent antibodies, markers of disease symptoms (e.g., pain) can be monitored.

For example, administration of an antibody (e.g., E3) or polypeptide according to the methods of the invention may be continuous or intermittent, depending on the physiological condition of the recipient, the purpose of administration being therapeutic or prophylactic, and other factors known to the skilled practitioner. Administration of the antibody can be substantially continuous during a preselected time period or can be a series of spaced doses, e.g., before, during, before and after, during and after, or before, during and after pain development. Can be administered before, during and/or after trauma, incision, trauma, surgery, and any event that may cause post-surgical pain.

Other dosage forms include suitable delivery forms known in the art, including, but not limited to, carriers such as liposomes. See, e.g., Mahato et al, (1997) pharm. res.14: 853-859. Liposome formulations include, but are not limited to, cytofectins, multilamellar liposomes, and unilamellar liposomes.

In some embodiments, more than one antibody or polypeptide may be present. The antibody may be monoclonal or polyclonal. Such compositions may comprise at least one, at least two, at least three, at least four, at least five different antibodies. Mixtures of antibodies, as they are often represented in the art, may be particularly useful in treating a wide range of populations of individuals.

Polynucleotides encoding any of the antibodies or polypeptides of the invention (e.g., antibody E3) can also be used to deliver and express any of the antibodies or polypeptides of the invention in a cell of interest. It is apparent that the expression vector can be used to directly express the E3 antibody or polypeptide. The expression vector can be administered by any method known in the art, such as intraperitoneal, intravenous, intramuscular, subcutaneous, intrathecal, intraventricular, oral, enteral, parenteral, intranasal, dermal, sublingual, or by inhalation. For example, administration of the expression vector includes local or systemic administration, including injection, oral administration, gene gun or transcatheter administration, and local administration. The person skilled in the art is familiar with the administration of expression vectors to obtain the expression of foreign proteins in vivo. See, for example, U.S. patent nos. 6,436,908; 6,413,942, and 6,376,471.

Targeted delivery can also be achieved using therapeutic compositions comprising polynucleotides encoding any of the antibodies or polypeptides described herein (e.g., antibody E3). Receptor-mediated DNA delivery techniques, such as those described in Findeis et al, Trends Biotechnol (1993) 11: 202; chiou et al, Gene Therapeutics: methods And Applications Of Direct Gene Transfer (J.A. Wolff, eds.) (1994) in one book; wu et al, J biol. chem. (1988) 263: 621 of the first and second substrates; wu et al, J biol. chem. (1994) 269: 542; zenke et al, proc.natl.acad.sci. (USA) (1990) 87: 3655; wu et al, J biol. chem. (1991) 266: 338. Therapeutic compositions comprising the polynucleotides are for topical administration in a gene therapy regimen in the range of about 100ng to about 200mg of DNA. Concentration ranges of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, and about 20 μ g to about 100 μ g of DNA may also be applied during the gene therapy regimen. The therapeutic polynucleotides and polypeptides of the invention may be delivered using gene delivery vectors. Gene delivery vectors can be of viral or non-viral origin (see, generally, Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185; and Kaplitt, Nature Genetics (1994) 6: 148). Expression of such coding sequences may be induced by endogenous mammalian or heterologous promoters. Expression of a coding sequence can be constitutive or regulatable.

Viral-based vectors for delivering a polynucleotide of interest and expressing it in a cell of interest are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. patent No. 5,219,740; 4,777,127; GB patent No. 2,200,651; and EP patent No. 0345242), alphavirus-based vectors (e.g., sindbis viral vectors, Semliki forest viruses (ATCC VR-67; ATCC VR-1247), Ross River viruses (Ross River viruses) (ATCC VR-373; ATCC CVR-1246) and Venezuelan equine encephalitis viruses (ATCC VR-923; ATCC VR-1250; CVR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649; WO 93/36 03769; WO 93/1912891; WO 3928462842/4684; WO 00676; WO 00655/4655; Cus; Cuss River viruses (see, e.g., PCT publication Nos. WO 3527/4655; WO), gene Ther (1992) 3: 147, administration of DNA linked to the killed adenovirus may also be used.

Non-viral delivery vectors and methods can also be used, including but not limited to polycationic concentrated DNA linked to killed adenovirus or not (see, e.g., Curiel, hum. Gene Ther. (1992) 3: 147); ligation of DNA via ligands (see, e.g., Wu, J.biol.chem. (1989) 264: 16985); eukaryotic cell delivery vector cells (see, e.g., U.S. Pat. No. 5,814,482; PCT publication No. WO 95/07994; WO 96/17072; WO95/30763 and WO97/42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA may also be used. Exemplary naked DNA introduction methods are described in PCT publication No. WO90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can be used as gene delivery vehicles are described in U.S. Pat. nos. 5,422,120; PCT patent numbers WO 95/13796; WO 94/23697; WO 91/14445; and EP patent No. 0524968. Other methods are described in Philip, mol.cell Biol. (1994) 14: 2411 and methods described in Woffendin, proc.natl.acad.sci. (1994) 91: 1581.

For all methods described herein, reference to anti-NGF antagonist antibodies also includes compositions comprising one or more of these agents. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients including buffers, which are well known in the art. The present invention may be used alone or in combination with other conventional therapeutic methods.

Methods of treating or preventing rheumatoid arthritis pain using anti-NGF antagonist antibodies

In some aspects, the invention provides methods for treating and/or preventing rheumatoid arthritis pain in an individual, including humans and non-human mammals. Thus, in one aspect, the invention provides a method of treating rheumatoid arthritis pain in an individual comprising administering an effective amount of an anti-NGF antagonist antibody. anti-NGF antagonist antibodies are well known in the art and described herein.

In another aspect, the invention provides a method for reducing, alleviating, inhibiting, reducing and/or delaying the incidence of the onset, development or progression of rheumatoid arthritis pain in an individual. Thus, in some embodiments, an anti-NGF antagonist antibody is administered prior to the development of pain or at the onset of pain in an individual with rheumatoid arthritis.

In another aspect, the invention provides a method for treating inflammatory cachexia (weight loss) associated with rheumatoid Arthritis in an individual comprising administering an effective amount of an anti-NGF antagonist antibody (Roubenoff et al, Arthritis Rheum.40 (3): 534-9 (1997); Roubenoff et al, J.Clin.invest.93 (6): 2379-86 (1994)).

The diagnosis or assessment of rheumatoid arthritis pain is well established in the art. The assessment is based on methods known in the art, such as patient pain characteristics using various pain criteria. See, e.g., Katz et al, Surg Clin North Am (1999)79 (2): 231-52; caraceni et al, JPain Symptom manager (2002)23 (3): 239-55, also commonly used criteria for determining disease status such as the American College of Rheumatology (ACR) (Felson, et al, Arthritis and Rheumatology (1993)36 (6): 729-. The anti-NGF antagonist antibody can be administered to the individual by any suitable route. Examples of different routes of administration are described herein.

The time course over which pain relief can be reduced is characterized. Thus, in some embodiments, a reduction in pain is observed about 24 hours after administration of the anti-NGF antagonist antibody. In other embodiments, a reduction in pain is observed about 36, 48, 60, 72 hours, or 4 days after administration of the anti-NGF antagonist antibody. In still other embodiments, a reduction in pain is observed before an improved sign of an inflammatory disease associated with rheumatoid arthritis is observed. In some embodiments, the frequency and/or intensity of pain is reduced, and/or the quality of life of the patient suffering from the disease is improved.

The preparation and use of anti-NGF antibodies for these methods is described in the following sections ("anti-NGF antagonist antibodies"; "identification of anti-NGF antagonist antibodies"; "administration of anti-NGF antagonist antibodies")

Methods of treating or preventing osteoarthritis pain with anti-NGF antagonist antibodies

In some aspects, the invention provides methods for treating and/or preventing osteoarthritis pain in an individual, including humans and non-human mammals. Accordingly, in one aspect, the invention provides a method of treating osteoarthritis pain in an individual comprising administering an effective amount of an anti-NGF antagonist antibody. anti-NGF antagonist antibodies are known in the art and described herein.

In another aspect, the invention provides a method for reducing, alleviating, inhibiting, reducing and/or delaying the incidence of development, progression or progression of osteoarthritis pain in an individual. Thus, in some embodiments, an anti-NGF antagonist antibody is administered prior to the development of pain or at the onset of pain in an individual with osteoarthritis.

The diagnosis or assessment of osteoarthritis pain is well established in the art. The assessment is based on methods known in the art, such as patient pain characteristics using various pain criteria. See, e.g., Katz et al, Surg Clin North Am (1999)79 (2): 231-52; caraceni et al, J PainSymptom manager (2002)23 (3): 239-55. For example, pain and response to treatment can be assessed using WOMAC ambulatory pain criteria (including pain, stiffness, and physical function) and 100mm visual simulation criteria (VAS).

The anti-NGF antagonist antibody can be administered to the individual by any suitable route. Examples of different routes of administration are described herein.

The time course over which pain relief can be achieved is characterized. Thus, in some embodiments, a reduction in pain is observed about 24 hours after administration of the anti-NGF antagonist antibody. In other embodiments, a reduction in pain is observed about 36, 48, 60, 72 hours, or 4 days after administration of the anti-NGF antagonist antibody. In some embodiments, the frequency and/or intensity of pain is reduced, and/or the quality of life of the patient suffering from the disease is improved.

The preparation and use of anti-NGF antibodies for use in these methods is described in the following sections ("anti-NGF antagonist antibodies"; "identification of anti-NGF antagonist antibodies"; "administration of anti-NGF antagonist antibodies").

anti-NGF antagonist antibodies

The methods described herein (in relation to rheumatoid arthritis pain and osteoarthritis pain) employ anti-NGF antagonist antibodies, which refer to any antibody molecule that blocks, inhibits or reduces (including significantly reduces) NGF biological activity, including through NGF signaling-mediated downstream pathways, such as receptor binding and/or eliciting a response to NGF cells.

anti-NGF antagonist antibodies should exhibit one or more of the following characteristics: (a) bind NGF and inhibit NGF biological activity or downstream pathways mediated by NGF signaling function; (b) any aspect of preventing, alleviating or treating rheumatoid arthritis pain or osteoarthritis pain; (c) blocking or reducing NGF receptor activity (including TrkA receptor dimerization and/or autophosphorylation); (d) increased clearance of NGF; (e) inhibit (decrease) NGF synthesis, production or release. anti-NGF antagonist antibodies are known in the art, see, e.g., PCT publication nos. WO01/78698, WO01/64247, U.S. patent nos. 5,844,092, 5,877,016, and 6,153,189; hongo et al, Hybridoma, 19: 215-227 (2000); cell.molec.biol.13: 559-568 (1993); gene bank (GenBank) accession numbers U39608, U39609, L17078 or L17077.

For the present invention, antibodies react with NGF in a manner that inhibits NGF and/or inhibits downstream pathways mediated by NGF signaling function. In some embodiments, the anti-NGF antagonist antibody recognizes human NGF. In yet other embodiments, the anti-NGF antagonist antibody specifically binds human NGF. In some embodiments, the anti-NGF antagonist antibody does not significantly bind to a related neurotrophin, such as NT-3, NT4/5, and/or BDNF. In still other embodiments, the anti-NGF antibody binds NGF and is effective to inhibit NGF binding in vivo to its TrkA and/or p75 receptor and/or is effective to inhibit NGF activation of its TrkA and/or p75 receptor. In yet another embodiment, the anti-NGF antagonist antibody is a monoclonal antibody. In yet other embodiments, the anti-NGF antibody is a humanized antibody (as described herein for antibody E3). In some embodiments, the anti-NGF antibody is human. In one embodiment, the antibody is a human antibody that recognizes one or more epitopes on human NGF. In another embodiment, the antibody is a mouse or rat antibody that recognizes one or more epitopes on human NGF. In another embodiment, the antibody recognizes one or more epitopes on NGF selected from primate, canine, feline, equine and bovine. In yet a further embodiment, the anti-NGF antagonist antibody binds substantially the same NGF epitope as one or more antibodies selected from the group consisting of the MAb 911, MAb 912 and MAb 938 antibodies (see, Hongo, et al, Hybridoma 19: 215-227 (2000)). In other embodiments, the antibody binds to the same epitope as Mab 911. In other embodiments, the antibody comprises a constant region that is immunologically inactive (e.g., does not elicit complement-mediated lysis or relies on antibody cell-mediated cytotoxicity (ADCC)). ADCC activity can be assessed as disclosed in U.S. patent No. 5,500,362. In some embodiments, the constant region is as described in eur.j.immunol. (1999) 29: 2613-2624; PCT publication No. PCT/GB 99/01441; and/or modified as described in UK patent publication No. 9809951.8.

In some embodiments, the anti-NGF antagonist antibody is a humanized mouse anti-NGF monoclonal antibody designated antibody "E3", any of the E3 related antibodies described herein, or a fragment thereof, of an NGF antagonist.

Antibodies useful in the invention can comprise monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab ', F (ab') 2, Fv, Fc, etc.), chimeric antibodies, diabodies, heteroconjugate antibodies, single chain antibodies (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of an immunoglobulin molecule comprising a specific antigen recognition site of interest, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. The antibody may be murine, human, or any other source (including chimeric or humanized antibodies).

The binding affinity of an anti-NGF antagonist antibody to NGF (such as hNGF) can be about 0.10 to about 0.80nM, about 0.15 to about 0.75nM, and about 0.18 to about 0.72 nM. In one embodiment, the binding affinity is between about 2pM and 22 pM. In some embodiments, the binding affinity is about 10 nM. In other embodiments, the binding affinity is about 10nM or less. In other embodiments, the binding affinity is about 0.1nM or about 0.07 nM. In other embodiments, the binding affinity is about 0.1nM or less or about 0.07nM or less. In other embodiments, the binding affinity is any one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50pM or less to any one of about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, or about 40 pM. In some embodiments, the binding affinity is any of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50pM or less. In some embodiments, the binding affinity is less than any of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50pM or less. In still other embodiments, the binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM, or greater than about 40 pM.

One way to determine the binding affinity of an antibody to NGF is by measuring the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, an antibody (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of anti-NGF Fab fragments of antibodies can be determined by surface plasmon resonance (BIAcore 3000)^TMSurface Plasmon Resonance (SPR) system, BIAcore, INC, Piscaway NJ). CM5 chips were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Human NGF (or any other NGF) can be diluted with 10mM sodium acetate pH4.0 and injected into the activated chip at a concentration of 0.005 mg/mL. With variable flow times through a single chip channel, two ranges of antigen densities can be obtained: 100-200 Reaction Units (RU) were used for detailed kinetic studies and 500-600 RU for screening assays. The chip can be blocked with ethanolamine. Regeneration studies showed that in more than 200 injections, a mixture of Pierce elution buffer (product No. 21004, Pierce Biotechnology, Rockford IL) and 4M NaCl (2: 1) was effective in removing bound Fab while retaining on-chip hNGF activity. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15 NaCl, 3mM EDTA, 0.005% surfactant P29) was used as the running buffer for the BIAcore assay. Serial dilutions (0.1-10x estimate K) _D) The purified Fab sample was injected at 100 uL/min for 1 min and allowed to dissociate for 2 hours. The concentration of the Fab protein is determined by ELISA and/or SDS-PAGE electrophoresis using known concentrations of Fab (as determined by amino acid analysis) as a standard. Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data to a 1: 1 langmuir binding model (Karlsson, r.roos, h.fagerstat, l.petersson, B. (1994) Methods Enzymology 6: 99-110) using the BIA evaluation program. Equilibrium dissociation constant (KD) values are calculated as koff/kon. This method is useful for determining the binding affinity of an antibody to any NGF, including human NGF, other vertebrate NGF (in some embodiments, mammalian), such as mouse NGF, rat NGF, primate NGF, and for other neurotrophins, such as the related neurotrophins NT3, NT4/5, and/or BDNF.

In some embodiments, the antibody binds human NGF without significant binding to NGF from another vertebrate species (in some embodiments, a mammal). In some embodiments, the antibody binds human NGF as well as one or more NGF from other vertebrate species (in some embodiments, mammals). In some embodiments, the antibody binds human NGF as well as one or more NGF from other vertebrate species (in some embodiments, mammals). In yet other embodiments, the antibody binds NGF without significant cross-reactivity with other neurotrophins (e.g., related neurotrophins, NT3, NT4/5, and/or BDNF). In some embodiments, the antibody binds NGF and at least one other neurotrophin. In some embodiments, the antibody binds NGF of a mammalian species, such as a horse or dog, but does not significantly bind NGF from other mammalian species.

Epitopes may be continuous or discontinuous. In one embodiment, the antibody substantially binds to a peptide selected from the group consisting of antibodies, and antibodies, e.g., as described in Hongo et al, Hybridoma, 19: antibodies to MAb 911, MAb 912 and MAb938 described in 215-. In another embodiment, the antibody binds essentially the same hNGF epitope as MAb 911. In yet another embodiment, the antibody binds essentially to the same epitope as MAb 909. Hongo et al, supra. For example, an epitope may comprise one or more of: residues K32, K34 and E35 in hNGF variable region 1 (amino acids 23-35); residues F79 and T81 in hNGF variable region 4 (amino acids 81-88); residues H84 and K88 in variable region 4; r103 residues between hNGF variable region 5 (amino acids 94-98) and hNGFC terminal (amino acids 111-118); residues E11 in hNGF pre-variable region 1 (amino acids 10-23); residues Y52 between hNGF variable region 2 (amino acids 40-49) and hNGF variable region 3 (amino acids 59-66); residues L112 and S113 in hNGF C-terminus; r59 and R69 residues in hNGF variable region 3; or residues V18, V20 and G23 in hNGF pre-variable region. Furthermore, an epitope may comprise one or more of variable region 1, variable region 3, variable region 4, variable region 5, N-terminal region and/or C-terminal region of hNGF. In yet another embodiment, the antibody significantly reduces the solvent proximity of the R103 residue of hNGF. It will be appreciated that although the epitopes described above are related to human NGF, the skilled person can align the structure of human NGF with NGF from other species and identify possible counterparts to these epitopes.

In one aspect, antibodies that inhibit NGF (e.g., human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies) can be prepared using immunogens that express full-length or partial NGF sequences. Alternatively, an immunogen comprising cells that overexpress NGF may be used. Another example of an immunogen that may be used is NGF protein comprising full-length NGF or a partial NGF protein.

anti-NGF antagonist antibodies can be prepared by any method known in the art. The immunization routes and protocols of the host animal as further described herein are generally consistent with established and conventional techniques for antibody stimulation and production. General techniques for generating human and mouse antibodies are known in the art and described herein.

It is contemplated that any mammalian subject, including humans or antibody producing cells thereof, may be manipulated as a basis for the generation of mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated with an amount of immunogen including the immunogen as described herein intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally.

Hybridomas may be obtained using Kohler, b, and Milstein, c. (1975) Nature 256: 495-497 or as determined by Buck, d.w. et al, In Vitro, 18: 377-381(1982) improved somatic hybridization techniques were prepared from lymphocytes and immortalized myeloma cells. Myeloma cell lines available for use in the hybridization include, but are not limited to, X63-Ag8.653 and myeloma cell lines from the cell distribution center of the California, san Diego, Salk research, USA. In general, the technique involves fusing myeloma cells and lymphoid cells with a fusing agent such as polyethylene glycol or by electrical methods well known to those skilled in the art. After fusion, the cells are isolated from the fusion medium and grown in a selective growth medium, such as inosine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Any of the media described herein, with or without serum added, can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to cell fusion techniques, EBV immortalized B cells can be used to produce anti-NGF monoclonal antibodies according to the invention. The hybridomas are expanded and passaged and the supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., transmission immunoassay, enzyme immunoassay, or fluorescent immunoassay) as desired.

Hybridomas that may be used as a source of antibodies include all derivatives, progeny cells, of the parent hybridoma that produce an NGF-specific monoclonal antibody or portion thereof.

Hybridomas producing such antibodies can be grown in vitro or in vivo using well-known procedures. If desired, the monoclonal antibodies can be isolated from the culture medium or body fluids by conventional immunoglobulin purification steps such as ammonium sulfate, gel electrophoresis, dialysis, chromatography, ultrafiltration. If an undesired activity is present, for example by flowing the preparation over an adsorbent prepared with the immunogen attached to a solid phase and eluting or releasing the antibody of interest from the immunogen. Immunization of a host animal with human NGF or a fragment comprising the target amino acid sequence linked to a protein that is immunogenic to a species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional agent or derivative such as maleimidobenzoyl sulfosuccinimidyl ester (bound through a cysteine residue), N-hydroxysuccinimide (bound through a lysine residue), glutaraldehyde, succinic anhydride, SOC12, or R1N ═ C ═ NR (where R and R1 are different alkyl groups), can produce a number of antibodies (e.g., monoclonal antibodies).

If desired, the anti-NGF antagonist antibody of interest (monoclonal or polyclonal) can be sequenced and the polynucleotide sequence then cloned into a vector for expression or propagation. The sequences encoding the antibody of interest may be maintained in a vector for the host cell and the host cell may then be propagated and frozen for later use. In the alternative, the polynucleotide sequences may be used in genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant regions are engineered to more closely resemble human constant regions to avoid immune responses in humans if the antibodies are used in clinical trials and treatments. It is desirable to genetically manipulate antibody sequences to obtain greater affinity for NGF and greater efficacy in inhibiting NGF. It will be apparent to those skilled in the art that one or more polynucleotide changes may be made to an anti-NGF antagonist antibody while still retaining its ability to bind NGF.

Humanized monoclonal antibodies generally have four steps: they are: (1) determination of the nucleotides of the starting antibody light and heavy chain variable domains and prediction of the amino acid sequence (2) design of humanized antibodies, i.e., determination of the antibody framework regions used in the humanization process (3) current humanization methodologies/techniques and (4) transfection and expression of the humanized antibody. See, for example, U.S. Pat. nos. 4,816,567; 5,807,715, respectively; 5,866,692, respectively; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

Many "humanized" antibody molecules comprising antigen binding sites derived from non-human immunoglobulins include chimeric antibodies having rodent or modified rodent V regions and their associated Complementarity Determining Regions (CDRs) fused to human constant domains. See, for example, Winter et al, Nature 349: 293-: 4220-4224 (1989), Shaw et al, J Immunol.138: 4534-4538(1987) and Brown et al, Cancer Res.47: 3577-3583(1987). Other references describe rodent CDRs grafted onto human support Framework Regions (FRs) prior to fusion with appropriate human antibody constant regions. See, e.g., Riechmann et al, Nature 332: 323-327(1988), Verhoeyen et al, Science 239: 1534-1536(1988) and Jones et al, Nature 321: 522-525(1986). Other references describe rodent CDRs supported by recombinant modification of rodent framework regions. See, for example, european patent No. 0519596. These "humanized" molecules are designed to reduce unintended immunological reactions against the rodent anti-human antibody portion that limit the duration and efficacy of therapeutic applications of these molecules in human recipients. For example, the antibody constant region can be engineered such that it is not immunologically active (e.g., does not initiate complement lysis). See, e.g., PCT publication Nos. PCT/GB 99/01441; british patent No. 9809951.8. Other methods that can also be used to humanize antibodies are described by Daugherty et al, nucleic acids res.19: 2471-2476(1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867, respectively; 5,866,692, respectively; 6,210,671 and 6,350,861; and PCT patent No. WO 01/27160.

In yet another alternative, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulins. Transgenic animals designed to produce more desirable (e.g., fully human antibodies) or more potent immune responses may also be used to produce humanized or human antibodies.An example of such a technique is Xenomouse from Abgenix, Inc. (Fremont, CA)^TMAnd HuMAb-Mouse from Medarex, Inc (Princeton, NJ)And TC Mouse^TM。

In the alternative, the antibody may be recombinantly produced and expressed using any method known in the art. In another alternative, the antibody may be recombinantly produced by phage display technology. See, for example, U.S. Pat. nos. 5,565,332; 5,580,717; 5,733,743, respectively; and 6,265,150; and Winter et al, annu, rev, immunol.12: 433-455(1994). Alternatively, phage display techniques (McCafferty et al, Nature 348: 552-553(1990)) can be used to generate human antibodies or antibody fragments in vitro from the gene library of the variable (V) domain of an unimmunized donor immunoglobulin. According to this technique, antibody V domain genes are cloned in-frame into the major or minor envelope protein genes of filamentous phages such as M13 or fd and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for a review see, e.g., Johnson, Kevin s. and Chiswell, David j., Current Opinion in structural biology 3: 564-571(1993). Several sources of V gene fragments are available for phage display. Clackson et al, Nature 352: 624-628(1991) different permutations of anti-oxazolone antibodies were isolated from small random combinatorial libraries of splenic V genes from immunized mice. A V gene bank from an unimmunized human donor can be constructed and constructed essentially according to Mark et al, j.mol.biol.222: 581-597(1991) or Griffith et al, EMBO J.12: 725-734(1993) isolate antibodies against different antigens, including self-antigens. In the innate immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes confer higher affinity, and B cells displaying high affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be performed by using a nucleic acid sequence known as "chain shuffling" Marks, et al, Bio/technol.10: 779 technical analogy to 783 (1992). In this approach, the affinity of "original" human antibodies obtained by phage display can be improved by replacing the heavy and light chain V region genes with naturally occurring series of variants (libraries) obtained from the non-immune donor V domain genes. This technique produces antibodies and antibody fragments with affinities in the pM-nM range. Waterhouse et al, Nucl. acids Res.21: 2265-2266(1993) describes a strategy for the preparation of very large phage antibody libraries (also known as "master-of-the-world libraries"). Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity to the starting rodent antibody. According to this method, also known as "epitope imprinting", the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a human V domain gene bank, resulting in rodent-human chimeras. The selection of the antigen results in the isolation of human variable regions that restore a functional antigen binding site, i.e. the selection of the epitope dominant (imprinting) partner (partner). When this process is repeated in order to replace the remaining rodent V domains, human antibodies are obtained (see PCT patent No. WO93/06213 published on month 1, 4, 1993). Unlike conventional humanization of rodent antibodies by CDR grafting, this technique provides fully humanized antibodies without rodent-derived framework or CDR residues.

Although the above discussion relates to humanized antibodies, it is clear that the general principles discussed apply to the preparation of antibodies according to this principle for use in, for example, dogs, cats, primates, horses and cattle. One or more aspects of the humanized antibodies described herein may be combined, e.g., CDR grafting, framework mutations and CDR mutations are also apparent.

Antibodies can be prepared recombinantly by first isolating the antibody and antibody-producing cells from the host animal, obtaining the gene sequence and expressing the antibody recombinantly in host cells (e.g., CHO cells) with the gene sequence. Other methods that may be used are expression of antibody sequences in plants (e.g., tobacco) or transgenic milk. Methods for recombinant expression of antibodies in plants or milk have been disclosed. See, e.g., Peeters, et al, Vaccine 19: 2756 (2001); lonberg, n, and d.huskzar int.rev.immunol 13: 65 (1995); and Pollock, et al, J Immunol Methods 231: 147(1999). Methods for preparing antibody derivatives, such as humanized antibodies, single chain antibodies, and the like, are known in the art.

Immunoassays and flow cytometric sorting techniques such as Fluorescence Activated Cell Sorting (FACS) can also be used to isolate antibodies specific for NGF.

Antibodies can be bound to many different carriers. The carrier may be active and/or inactive. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the present invention, the nature of the carrier may be soluble or insoluble. Other suitable carriers that are or can be determined to be useful for binding the antibody are known to those of skill in the art using routine experimentation. In some embodiments, the vector comprises a moiety that targets the myocardium.

DNA encoding the monoclonal antibody can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector (such as that disclosed in PCT publication No. WO 87/04462) which is then transfected into a host cell such as an E.coli cell, a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulin protein to obtain synthesis of monoclonal antibodies in the recombinant host cell. See, for example, PCT patent No. WO 87/04462. DNA can also be modified, for example, by replacing the homologous murine sequences with the coding sequences for the human heavy and light chain constant domains, Morrison et al, proc.nat.acad.sci.81: 6851(1984) or by covalently linking all or part of the sequence encoding the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In that manner, a "chimeric" or "hybrid" antibody having binding specificity for an anti-NGF monoclonal antibody herein is prepared.

anti-NGF antagonist antibodies can be characterized by methods well known in the art. For example, one approach is to identify the epitope to which the antibody binds, or "epitope mapping". There are many methods available in the art for mapping and characterizing epitope positions on proteins, including, for example, solving the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays described in chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, new york, 1999. In other examples, epitope mapping can be used to determine the sequence to which an anti-NGF antagonist antibody binds. Epitope mapping is commercially available from a variety of sources, such as the Pepscan system (Edelhertweg 15, 8219 PH Lelystad, holland). The epitope may be a linear epitope, i.e. a conformational epitope comprising a stretch of amino acids, or not necessarily comprised in a stretch of amino acids, but formed by three-dimensional interactions of amino acids. Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinant) and used in binding assays with anti-NGF antagonist antibodies. In another example, the epitope to which an anti-NGF antagonist antibody binds can be determined in a systematic screen using overlapping peptides derived from NGF sequences and determining binding by the anti-NGF antagonist antibody. According to gene fragment expression assays, open reading frames encoding NGF are fragmented randomly or by specific genetic constructs and the reactivity of the expressed fragment of NGF with pre-test antibodies is determined. For example, a gene fragment can be generated by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled NGF fragment is then determined by immunoprecipitation or gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a library of defined overlapping peptide fragments can detect binding to the test antibody in a simple binding assay. In other examples, antigen binding domain mutagenesis, domain switching experiments, and alanine scanning mutagenesis can be performed to identify sufficient and/or essential residues for epitope binding. For example, a domain switching assay can be performed using mutant NGF in which various fragments of an NGF polypeptide are replaced (switched) with sequences from a closely related, but antigenically distinct protein, such as another member of the neurotrophin family. By assessing the binding of an antibody to mutant NGF, the importance of a particular NGF fragment to antibody binding can be assessed.

Another method that can be used to characterize anti-NGF antagonist antibodies is to perform a competition assay with other antibodies known to bind to the same antigen, i.e., various fragments on NGF, to determine whether the anti-NGF antagonist antibody binds to the same epitope as the other antibodies. Competitive assays are well known to those skilled in the art. As in Hongo et al, Hybridoma 19: 215- (2000), examples of antibodies useful in the competition assay of the invention include monoclonal antibodies 911, 912, 938.

The expression vectors can be used to directly express anti-NGF antagonist antibodies. Those skilled in the art are familiar with the administration of expression vectors to obtain expression of foreign proteins in vivo. See, for example, U.S. patent nos. 6,436,908; 6,413,942, respectively; and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, gene gun or catheterized administration, and local administration. In another embodiment, the expression vector is administered directly into the sympathetic trunk or ganglia, or directly into the coronary arteries, atria, ventricles, or pericardium.

Targeted delivery can also be achieved with therapeutic compositions comprising expression vectors or subgenomic polynucleotides. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al, Trends Biotechnol (1993) 11: 202; chiou et al, Gene Therapeutics: methods And Applications of direct Gene Transfer (J.A. Wolff, eds.) (1994); wu et al, J.biol.chem. (1988) 263: 621 of the first and second substrates; wu et al, j.biol.chem. (1994) 269: 542; zenke et al, proc.natl.acad.sci.usa (1990) 87: 3655; wu et al, j.biol.chem. (1991) 266: 338. In gene therapy protocols, the topical administration of therapeutic compositions containing polynucleotides ranges from about 100ng to about 200mg of DNA. Concentration ranges of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, and about 20 μ g to about 100 μ g may also be used in gene therapy protocols. Gene delivery vehicles can be used to deliver therapeutic polynucleotides and polypeptides of the invention. Gene delivery tools can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1: 51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) 1: 185 and Kaplitt, Nature genetics (1994) 6: 148). Expression of such coding sequences is induced using endogenous mammalian promoters or heterologous promoters. Expression of the coding sequence may be constitutive or regulated.

Viral-based vectors for delivery and expression of polynucleotides of interest in cells of interest are well known in the art. Representative virus-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO 91/02805; U.S. Pat. Nos. 5,219,740, 4,777127; GB patent No. 2,200,651 and EP0345242), alphavirus-based vectors (e.g., sindbis viral vectors, Simplerian forest viruses (ATCC VR-67, ATCC VR-1247), Ross River viruses (ATCC VR-373, ATCC VR-1246) and Venezuelan equine encephalitis viruses (ATCC VR-737, ATCC VR-1250, ATCC VR-1249 ATCC VR-532), and adeno-associated (AAV) vectors (see, e.g., PCT publication Nos. WO94/12649, WO 93/1918, WO 6/91, WO 3528938, WO 362827/11984 and WO 11955/00655), gene Ther (1992) 3: 147 into inactivated adenovirus.

Non-viral delivery tools and methods can also be utilized, including, but not limited to, polycation-concentrated DNA linked or unlinked to an inactivated adenovirus (see, e.g., Curiel, hum. Gene Ther. (1992) 3: 147), ligand-linked DNA (see, e.g., Wu, J.biol. chem. (1989) 264: 16985), eukaryotic cell delivery tool cells (see, e.g., U.S. Pat. No. 5,814,482, PCT publication Nos. WO95/07994, WO96/17072, WO95/30763, and WO97/42338), and nuclear charge neutralization or fusion to the cell membrane. Naked DNA may also be used. Representative naked DNA introduction methods are described in PCT publication No. WO90/11092 and U.S. Pat. No. 5,580,859. Liposomes that are useful as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, PCT publication Nos. WO95/13796, WO94/23697, WO91/14445, and EP 0524968. Additional methods are described in Philip, mol.cell Biol. (1994) 14: 2411 and Woffendin, proc.natl.acad.sci. (1994) 91: 1581.

Identification of anti-NGF antagonist antibodies

anti-NGF antagonist antibodies can be identified or characterized by methods known in the art, whereby a reduction, alleviation or neutralization of NGF biological activity is detected and/or assayed. For example, the kinase receptor activation (KIRA) assay described in U.S. patent nos. 5,766,863 and 5,891,650 can be used to identify anti-NGF agents. This ELISA-type assay is suitable for qualitative or quantitative determination of kinase activity and for identifying and characterizing potential antagonists of selected rptks, such as TrkA, by measuring the autophosphorylation of the kinase domain of a receptor protein tyrosine kinase (hereinafter "rPTK"), such as the TrkA receptor. The first phase of the assay involves phosphorylation of a kinase domain of a kinase receptor, such as the TrkA receptor, wherein the receptor is present in the cell membrane of a eukaryotic cell. The receptor may be an endogenous receptor or a nucleic acid encoding a receptor, or a receptor construct that can be transformed into a cell. Typically, the first solid phase (e.g., the wells of the first assay plate) is coated with a substantially homogeneous population of such cells (typically a mammalian cell line) to adhere the cells to the solid phase. Often the cells are adherent cells and thus naturally adhere to the first solid phase. If a "receptor construct" is used, it will generally comprise a fusion of the kinase receptor and a marker polypeptide. The marker polypeptide is recognized by a capture agent (typically a capture antibody in the ELISA portion of the assay). An analyte, such as a candidate anti-NGF antagonist antibody, is then added to the well with adherent cells along with NGF, exposing (or contacting) the tyrosine kinase receptor (e.g., TrkA receptor) with NGF and the analyte. This assay enables the identification of antibodies that inhibit TrkA activation by their ligand NGF. Following exposure to NGF and analyte, adherent cells are lysed with lysis buffer (with detergent dissolved therein) and gently agitated so that the released cell lysate can be used directly in the ELISA portion of the assay without concentration or clarification.

The cell lysate thus prepared is then ready for use in the ELISA phase of the assay. As a first step of the ELISA stage, the second solid phase (typically the wells of an ELISA microplate) is coated with a capture agent (often a capture antibody) that specifically binds to a tyrosine kinase receptor, or, for receptor constructs, to a marker polypeptide. The coating of the second solid phase is performed such that the capture reagent adheres to the second solid phase. The capture agent is typically a monoclonal antibody, but polyclonal antibodies may also be used, as described in the examples herein. The resulting cell lysate is then exposed to or contacted with an attached capture agent to cause the receptor or receptor construct to adhere to (or be captured on) the second solid phase. A rinsing step is then performed to remove unbound cell lysate, leaving the captured receptor or receptor construct. The adhesion or capture receptor or receptor construct is then exposed to or contacted with an anti-phosphotyrosine antibody that identifies phosphorylated tyrosine residues in tyrosine kinase receptors. In one embodiment, the anti-phosphotyrosine antibody is conjugated (directly or indirectly) to an enzyme that catalyzes a color change of the non-radioactive chromogenic reagent. Thus, phosphorylation of the receptor can be measured by a subsequent color change of the reagent. The enzyme may be conjugated directly to the anti-phosphotyrosine antibody, or a conjugation molecule (e.g., biotin) may be conjugated to the anti-phosphotyrosine antibody and the enzyme may then be conjugated to the anti-phosphotyrosine antibody via the conjugation molecule. Finally, the conjugation of the anti-phosphotyrosine antibody to the capture receptor or receptor construct is determined, for example, by a color change of a chromogenic reagent.

anti-NGF antagonist antibodies can also be identified by incubating a candidate agent with NGF and monitoring any one or more of the following characteristics: (a) bind NGF and inhibit NGF biological activity or downstream pathways mediated by NGF signaling function; (b) inhibit, block or reduce NGF receptor activation (including TrkA dimerization and/or autophosphorylation); (c) increased clearance of NGF; (d) any aspect of treating or preventing rheumatoid arthritis pain or osteoarthritis pain; (e) inhibit (decrease) NGF synthesis, production or release. In some embodiments, anti-NGF antagonist antibodies are identified by incubating a candidate agent with NGF and monitoring binding and/or concomitant reduction or neutralization of NGF biological activity. Binding assays may be performed with purified NGF polypeptides or with cells that are naturally expressed or transfected to express NGF polypeptides. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete with known anti-NGF antagonists for binding to NGF is assessed. This assay can be performed in a variety of formats, including ELISA formats. In other embodiments, anti-NGF antagonist antibodies are identified by incubating a candidate agent with NGF and monitoring binding and concomitant inhibition of trkA receptor dimerization and/or autophosphorylation.

After initial identification, the activity of candidate anti-NGF antagonist antibodies can be further confirmed and refined by known bioassays that detect the biological activity of the target. Alternatively, the candidate may be screened directly using a bioassay. For example, NGF promotes a number of morphologically recognizable changes in responding cells. These include, but are not limited to, promoting differentiation of PC12 cells and enhancing neurite outgrowth from these cells (Greene et al, Proc Natl Acad Sci usa.73 (7): 2424-8, 1976), promoting outgrowth of neurite-responsive sensory and sympathetic ganglion explants (Levi-Montalcini, R. and Angeletti, P.Nerve growth factor. physiol.Rev.48: 534-569, 1968) and promoting the survival of NGF-dependent neurons such as embryonic dorsal root ganglion, trigeminal ganglion or sympathetic ganglion neurons (e.g., Chun & Patterson, Dev.biol.75: 705-711 (1977); buchman & Davies, Development 118: 989-, assays for inhibition of NGF biological activity require culturing NGF responsive cells with NGF and an analyte, such as a candidate anti-NGF antagonist antibody, after an appropriate time, the cellular response (cell differentiation, neurite outgrowth or cell survival) is assayed.

The ability of a candidate anti-NGF antagonist antibody to block or neutralize NGF biological activity can also be monitored by monitoring the activity of a candidate agent in Hongo et al, Hybridoma 19: 215-227(2000) was evaluated for inhibition of NGF-mediated survival in the embryonic rat dorsal root ganglion survival assay.

Administration of anti-NGF antagonist antibodies

The anti-NGF antagonist antibody can be administered to the individual (for rheumatoid arthritis and osteoarthritis individuals) by any suitable route. The examples described herein are not intended to be limiting but rather illustrative of the available technologies, as will be apparent to those of skill in the art. In some embodiments, the anti-NGF antagonist antibody is administered to the individual according to known methods such as intravenously, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, sublingual, intrasynovial, by inhalation, intrathecal, oral, inhalation, or topical route. Administration may be systemic, e.g., intravenous, or may be topical. Commercially available nebulizers are used for liquid dosage forms, including jet nebulizers and ultrasonic nebulizers for administration. Liquid dosage forms can be directly nebulized, while lyophilized powders can be nebulized after reconstitution. Alternatively, anti-NGF antagonist antibodies can be aerosolized using fluorocarbon formulations and pressurized metered dose aerosols, or inhaled as lyophilized and pulverized powders.

In one embodiment, the anti-NGF antagonist antibody is administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of anti-NGF antagonist antibodies or local delivery catheters such as infusion catheters, indwelling catheters or trocars, synthetic grafts, adventitial sleeves, shunts and stents or other implantable devices, site-specific vectors, direct injection or direct application. See, for example, PCT publication No. WO00/53211 and U.S. Pat. No. 5,981,568.

Various dosage forms of anti-NGF antagonist antibodies are available for administration. In some embodiments, an NGF antagonist antibody can be administered systemically. In some embodiments, the anti-NGF antagonist antibody and pharmaceutically acceptable excipients can be in a variety of dosage forms. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that aid in the administration of a pharmaceutically effective substance. For example, excipients may impart shape or viscosity, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying permeabilities, capsules, buffers, and skin penetration enhancers. Excipients and dosage forms for parenteral and non-parenteral drug delivery are shown in Remington, The Science and Practice of pharmacy, 20 th edition, Mack Publishing (2000).

In some embodiments, these formulations are formulated for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). Thus, these formulations may be combined with a pharmaceutically acceptable carrier such as raw saline, ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dosage, time, and repetition, depends on the particular individual and the individual's medical history.

anti-NGF antibodies can be administered by any suitable method, including by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). anti-NGF antibodies can also be administered by inhalation as described herein. In general, for administration of anti-NGF antibodies, the initial candidate dose may be about 2 mg/kg. For the present invention, typical daily dosages may range from about 1 to 3 to 30 to 300 to 3 to 30 to 100mg/kg or more, depending on the factors set forth above. For example, an anti-NGF antibody may be administered at about 1. mu.g/kg, about 10. mu.g/kg, about 20. mu.g/kg, about 50. mu.g/kg, about 100. mu.g/kg, about 200. mu.g/kg, about 500. mu.g/kg, about 1mg/kg or about 2 mg/kg. Repeated administration for several or more days will depend on the disease, and treatment will continue until the desired suppression of the presence of symptoms is achieved or until a therapeutic level sufficient to reduce pain is achieved. An exemplary dosage regimen comprises administration of an initial dose of about 2mg/kg, followed by a weekly maintenance of about 1mg/kg of anti-NGF antibody, or followed by a maintenance of about 1mg/kg every two weeks. However, other dosage regimens are useful depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, in some embodiments, dosages of one to four times per week are contemplated. The progress of this treatment is readily monitored by conventional techniques and assays. The dosage regimen, including the NGF antagonist employed, may vary over time.

For the purposes of the present invention, the appropriate dosage of anti-NGF antagonist antibody will depend on the anti-NGF antagonist (or combination thereof) employed, the type and severity of pain to be treated, whether the formulation is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the formulation, and the discretion of the attending physician. Typically, the clinician administers an anti-NGF antagonist antibody until a dosage is reached that achieves the desired result. The dosage and/or frequency may vary with the course of treatment.

Empirical considerations such as half-life generally aid in determining dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, pain-based treatment and/or inhibition and/or alleviation and/or delay. Alternatively, a sustained continuous release dosage form of the anti-NGF antagonist antibody may be appropriate. A variety of dosage forms and devices for achieving sustained release are known in the art.

In one embodiment, the dosage for the anti-NGF antagonist antibody can be empirically determined in an individual administered one or more administrations of the anti-NGF antagonist antibody. The individual is administered an increasing dose of an anti-NGF antagonist antibody. To assess the efficacy of anti-NGF antagonist antibodies, one can assess this indicator of pain.

For example, administration of an anti-NGF antagonist antibody according to the methods of the invention can be continuous or intermittent, depending on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to practitioners. Administration of anti-NGF antagonist antibody can be substantially continuous over a preselected period of time or can be a series of intermittent doses, e.g., before, during, or after pain development; before; during the period; before and after; during and after; before and during; or before, during and after the pain develops.

In some embodiments, more than one anti-NGF antagonist antibody may be present. At least one, at least two, at least three, at least four, at least five different, or more than five anti-NGF antagonist antibodies can be present. In general, these anti-NGF antagonist antibodies have complementary activities that do not adversely affect each other.

Therapeutic dosage forms of anti-NGF antagonist antibodies for use according to The invention are prepared for storage in lyophilized form or as a liquid solution by mixing The antibody of The desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington, The Science and Practice of Pharmacy, 20 th edition, mack publishing (2000)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexane diamine chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant such as TWEENTM, PLURONICSTM, or polyethylene glycol (PEG).

By methods known in the art, as described in Epstein et al, proc.natl.acad.sci.usa 82: 3688 (1985); hwang et al, proc.natl acad.sci.usa 77: 4030 (1980); and the methods described in U.S. Pat. nos. 4,485,045 and 4,544,545 to prepare liposomes containing anti-NGF antagonist antibodies. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes of the desired diameter.

The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in microemulsions, for example, by agglomeration techniques or by interfacial polymerization. Such techniques are disclosed in Remington, The Science and practice of Pharmacy, 20 th edition, Mack Publishing (2000).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamic acid, non-degradable acetyl ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D- (-) -3-hydroxybutyric acid.

Dosage forms for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic anti-NGF antagonist antibody compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or a vial having a hypodermic needle that can pierce the stopper.

The compositions according to the invention may be administered in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for oral, parenteral or rectal administration, or by inhalation or insufflation.

For the preparation of solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g., conventional tablet ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a intimately admixed compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into the unit dosage forms described above containing from 0.1 to about 500mg of the active ingredient of the present invention. The tablets or pills of the novel composition may be coated or mixed to provide a dosage form that provides extended action benefits. For example, a tablet or pill may comprise an inner dose and an outer dose component which is an envelope over the inner dose component. The two components may be separated by the intestinal layer which acts in the stomach as a barrier to decomposition and allows the inner component to pass intact into the duodenum or delayed release. A variety of materials may be used for such enteric or coating layers, such materials including, for example, a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include, inter alia, nonionic agents such as polyoxyethylene sorbitol (e.g., TweenTM 20, 40, 60, 80, or 85) and other sorbitan (e.g., span 20, 40, 60, 80, or 85). Compositions with surfactants conveniently comprise between 0.05 and 5% surfactant and may be between 0.1 and 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipitTM, Liposony, IndonitroTM, Lipofundin and Lipiphysan. The active ingredient may be dissolved in the pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and formed into an emulsion with a phospholipid (e.g., lecithin, soybean phospholipid or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically comprise up to 20%, for example between 5 and 20% oil. The fat emulsion may comprise fat droplets of between 0.1 and 1.0 μm, especially between 0.1 and 0.5 μm, and has a pH in the range of 5.5 to 8.0.

The emulsion composition may be a composition prepared by mixing the nerve growth factor antibody with IntralipidTM or its components (soybean oil, lecithin, glycerin and water).

Compositions for inhalation or insufflation include solutions and suspensions or mixtures thereof in pharmaceutically acceptable water or organic solvents, and powders. The liquid or solid composition may comprise suitable pharmaceutically acceptable excipients as indicated above. In some embodiments, the composition may be administered by the oral or nasal respiratory route for local or systemic effects. The composition in a preferably sterile pharmaceutically acceptable solvent can be sprayed by the application of a gas. The nebulized solution may be breathed directly in the nebulizing device or the nebulizing device may be connected to a face mask, tent, or intermittent positive pressure ventilator. The solution, suspension or powder composition may be administered, preferably orally or nasally, from a device that delivers the dosage form in an appropriate manner.

Treatment efficacy can be assessed by methods well known in the art.

Kit comprising the antibodies and polynucleotides of the invention

The invention also provides kits comprising antibodies or polynucleotides for detection and/or treatment. Thus, in some embodiments, the kit comprises antibody E3. In some embodiments, the kit comprises any of the antibodies or polypeptides described herein.

In another aspect, the kit can be used in any of the methods described herein, including, for example, treating an individual for pain (including post-operative pain, rheumatoid arthritis pain, and osteoarthritis pain). The kits of the invention are suitably packaged and may optionally provide additional components such as buffers and instructions for use of the antibodies in any of the methods described herein. In some embodiments, the kit comprises instructions for treating pain. In some embodiments, a kit comprises an anti-NGF antagonist antibody described herein and instructions for treating and/or preventing rheumatoid arthritis pain in an individual. In other embodiments, the kit comprises an anti-NGF antagonist antibody described herein and instructions for treating and/or preventing osteoarthritis pain in an individual. In some embodiments, the anti-NGF antagonist antibody is antibody E3.

In another aspect, the invention provides a kit comprising a polynucleotide encoding an E3 polypeptide as described herein. In some embodiments, the kit further comprises instructions for using the polynucleotide in any of the methods described herein.

Methods for modulating antibody affinity and methods for characterizing CDRs

We have developed a new method for characterising CDRs of an antibody and/or altering (e.g. improving) the binding affinity of a polypeptide, such as an antibody, known as "library scanning mutagenesis". In general, library scanning mutagenesis functions as follows. One or more amino acid positions in a CDR are substituted with two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using methods known in the art. This results in a small clone library (in some embodiments, one for each amino acid position analyzed, each library having a complexity of two or more members (if there are two or more amino acid substitutions at each position.) typically, libraries also include clones that contain the native (unsubstituted) amino acid, for small numbers of clones from each library, for example, about 20-80 clones (depending on the complexity of the library) on the target polypeptide binding affinity screening and increased, the same, reduced or no binding candidates were identified. The BIAcore surface plasmon resonance analysis is used to detect about 2-fold or more than 2-fold differences in binding affinity. Binding affinity of (4). When the starting antibody is used with relatively high affinity, e.g. K_DBIAcore is particularly useful when bound to about 10nM or less than 10 nM. Screening using BIAcore surface plasmon resonance is described in the examples herein.

In other embodiments, binding affinity is determined using Kinexa Biocensor, scintillation proximity assay, ELISA, ORIGEN Immunoassay (IGEN), fluorescence quenching, fluorescence migration, and/or yeast display. In other embodiments, the binding affinity is screened using a suitable bioassay.

In some embodiments, all 20 natural amino acids are substituted at each amino acid position in the CDRs (in some embodiments, one amino acid at a time) using mutagenesis methods known in the art (some of which are described herein). This results in miniclone libraries (in some embodiments, one for each amino acid position analyzed), each library having a complexity of 20 members (if all 20 amino acids were used for substitution at each position).

In some embodiments, the library to be screened comprises substitutions at two or more positions in the same CDR or two or more CDRs. Thus, in some embodiments, the library comprises substitutions at two or more positions in one CDR. In another embodiment, the library comprises substitutions at two or more positions in two or more CDRs. In yet other embodiments, the library comprises substitutions at 3, 4, 5, or 5 or more positions, wherein the positions are found in two, three, four, five, or six CDRs. In some embodiments, the substitutions are made with low redundant codons. See, e.g., Balint et al, (1993) Gene 137 (1): 109-18.

In some embodiments, the CDRs are CDRH3 and/or CDRL 3. In other embodiments, the CDR is one or more CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH 3. In some embodiments, the CDRs are Kabat CDRs, Chothia CDRs, or extended CDRs.

Candidates with improved binding can be sequenced, thus identifying CDR substitution mutations that result in improved affinity (also referred to as "improved" substitutions). For example, as demonstrated in example 1, with this approach allows identification of a single substitution that improves binding even when 18 other substitutions estimated at the same amino acid position result in no binding (i.e., loss of antibody function). The bound candidates can also be sequenced, thus identifying CDR substitutions that retain binding.

In some embodiments, multiple rounds of screening are performed. For example, candidates with improved binding (each candidate comprising an amino acid substitution at one or more positions of one or more CDRs) are also used to design a second library comprising at least the original and replacement amino acids at each improved CDR position (i.e., at the amino acid position of a CDR where the substitution mutant shows improved binding). The preparation and screening or selection of such libraries is discussed further below.

Library scanning mutagenesis also provides a means for characterizing CDRs, as well as information about the importance of each amino acid position on antibody-antigen complex stability, as regards the frequency of clones with improved binding, identical binding, reduced binding or no binding. For example, if a CDR position remains bound when changed to all 20 amino acids, this position is identified as a position that may not be desirable for antigen binding. Conversely, if a CDR position remains bound for only a small percentage of substitutions, this position serves as a position important to the CDR function. Thus, library scanning mutagenesis methods yield information about positions in the CDR that can be changed to many different amino acids (including all 20 amino acids), as well as positions in the CDR that cannot be changed or can be changed to only a few amino acids. This aspect is discussed and exemplified in example 1.

In some embodiments, candidates with improved affinity are combined in a second library that may further include additional substitutions depending on the complexity of the library of interest or the screening or selection method of interest permitted to be used, including the improved amino acid, the original amino acid at that position. Further, adjacent amino acid positions can be randomized to at least two or more amino acids, as desired. Randomization of adjacent amino acids may allow additional conformational flexibility in the mutated CDRs, which in turn may allow or facilitate the introduction of a larger number of improved mutations. In some embodiments, the library also comprises position substitutions that do not show improved affinity in the first round of screening.

Screening or selection of a second library of library members having improved and/or altered binding affinity using methods known in the art, including BIAcore plasmon resonance analysis, and using any method known in the art for selection, including phage display, yeast display, and ribosome display selection.

Methods for modulating antibody affinity and characterizing CDRs

These methods are used to prescreen CDR amino acid positions to identify amino acid substitutions that improve binding or maintain binding. The pre-identification of important residues, substitutions that improve binding, and/or substitutions that preserve antibody function allow for efficient design and screening of affinity maturation libraries.

The method is also used to characterize the CDRs and provide comprehensive information on the importance of each amino acid position in the CDRs for binding to antigen. The method can also be used to identify alternatives that improve binding.

Each position may be the use of a small library at random (in some embodiments, one at a time), allowing for selection of substitution mutants with sensitive methods such as BIAcore, which provides detailed kinetic information. When screening larger libraries, screening methods are often impractical. Instead, screening methods such as phage display, yeast display, and ribosome display are typically used to identify clones that retain binding. Phage display and ELISA assays may depend primarily on the concentration of protein samples prepared from the clones, and thus tend to be primarily biased toward clones with increased expression, increased stability, or reduced toxicity, rather than identifying clones with increased binding affinity. Furthermore, differences in clonal expression levels may mask small improvements in binding affinity. This disadvantage is particularly evident when antibodies with high affinity are used as starting material, since very low levels of antigen must be used in order to make the screening sufficiently stringent.

In contrast, the methods described herein, such as randomization of each position (in some embodiments, one position at a time), allow the introduction and characterization of the effects of, for example, all 20 amino acid substitutions at a given position. This analysis provides information on how many substitutions are tolerated (i.e., antibody binding is maintained) at a given position, which in turn provides information about the importance of each amino acid to the function of the antibody. Further, even where many or most substitutions at a given location produce non-functional (non-binding) antibodies, substitutions that produce improved binding can be identified. In contrast, alanine scanning mutagenesis, which is commonly used to identify important CDR positions, provides information as to whether alanine substitution allows or prevents binding. Typically, alanine substitution positions that prevent binding are removed from the affinity maturation library. However, in many cases, alanine may be a weak surrogate at the CDR positions.

The invention also allows the identification and characterization of the effects of single CDR mutations. In contrast, methods such as phage display introduce and select for many mutations simultaneously, and thus potentially increase the risk that positive mutations will be masked by the presence of deleterious mutations in a particular clone.

The present method is also useful for improving affinity while maintaining the binding specificity of the original (starting) antibody, and within the scope of the present method allows the identification of small numbers of mutations (e.g., 1, 2, 3, 4, or 5 mutations in a single CDR) that result in improved binding affinity. In contrast, methods such as phage display generally improve binding affinity with simultaneous multiple mutations, which can result in altered antibody specificity and/or increased unwanted cross-reactivity.

The following examples are provided to illustrate, but not to limit, the present invention.

Examples

Example 1: humanization and affinity maturation of mouse antagonist anti-NGF antibody 911

A. General procedure

The following general method is applied in this example.

Library generation

As in Kay et al, (1996), Phage display of peptides and proteins: the libraries were generated by PCR cassette mutagenesis using degenerate oligonucleotides as described in the section of the analytical Manual, San Diego, Academic Press (see page 277-291). A doped codon (doping codon) NNK was used to randomize one amino acid position to include 20 possible amino acids. To randomize an amino acid position to include only one set of amino acids with specific properties, as in Balint et al, (1993) Gene 137 (1): 109-18 with doped codons. As in Innis et al, (1990) PCR protocols: a guide to methods and applications (see pages 177-183) using recombinant PCR for site-directed mutagenesis.

Small-Scale Fab preparation

Small scale expression was optimized in 96-well culture plates for screening Fab libraries. Starting from the transformation of E.coli with the Fab library, colonies were picked and inoculated both on master plates (LB agar + ampicillin (50. mu.g/mL) + 2% glucose) and on working plates (2 mL/well, 96 well/plate containing 1.5mL LB + ampicillin (50. mu.g/mL) + 2% glucose). Both plates were grown at 30 ℃ for 8-12 hours. The main plate was stored at 4 ℃ and cells from the working plate were pelleted by centrifugation at 5000 rpm and resuspended with 1mL LB + ampicillin (50. mu.g/mL) +1mM IPTG to induce Fab expression. Cells were harvested by centrifugation after 5 hours of expression at 30 ℃ and resuspended in 500. mu.L of HBS-EP buffer (100mM HEPES buffer pH7.4, 150mM NaCl, 0.005% P20, 3mM EDTA). Lysis of HBS-EP resuspension cells was obtained by one round of freezing (-80 ℃) followed by thawing at 37 ℃. The cell lysate was centrifuged at 5000 rpm for 30 minutes to separate the cell debris from the Fab-containing supernatant. The supernatant was then injected into the BIAcore cytoplasmic resonator to obtain affinity information for each Fab. Fab-expressing clones were obtained from the master culture plate for DNA sequencing and for large-scale Fab production and detailed features are described below.

Large Scale Fab preparation

To obtain detailed kinetic parameters, fabs were expressed and purified from bulk cultures. Erlenmeyer flasks containing 200mL LB + ampicillin (50. mu.g/mL) + 2% glucose were inoculated with 5mL overnight cultures from selected Fab expressing E.coli clones. Clones were incubated at 30 ℃ until OD_550nmReached 1.0 and then induced by replacing the medium with 200ml + LB ampicillin (50. mu.g/ml) +1mM IPTG. After 5 hours of expression at 30 ℃, cells were pelleted by centrifugation and then resuspended in 10mL PBS (pH 8). Lysis of the cells was obtained by two rounds of freeze/thaw (-80 ℃ and 37 ℃ respectively). Cell lysate supernatant was loaded onto a Ni-NTA superfluidic agarose (Qiagen, valencia. ca) column equilibrated with PBS, pH8, then washed with 5 column volumes of PBS, pH 8. Different fabs were eluted in different fractions with PBS (pH8) +300mM imidazole. Fab-containing fractions were collected and dialyzed against PBS and then quantified by ELISA prior to affinity characterization.

Whole antibody preparation

For expression of the intact antibody, the heavy and light chain variable regions were cloned into 2 mammalian expression vectors (Eb.911. E3 or Eb.pur.911.3E for the light chain and Db.911.3E for the heavy chain; described herein) and transfected into HEK 293 cells with lipofectamine (lipofectamine) for transient expression. The antibody was purified by standard methods using protein a.

Biacore assay

Affinity of anti-NGF Fab and monoclonal antibody Using BIAcore3000^TMSurface Plasmon Resonance (SPR) system (BIAcore, INC, Piscaway NJ). According to the supplier's instructions, CM5 chip with N-ethyl-N' - (3-two methyl ammonia)Cyclopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Human NGF was diluted in 10mM sodium acetate, pH4.0 and injected at a concentration of 0.005mg/mL onto activated chips. Two ranges of antigen density were obtained with variable flow times through the respective chip channels: 100-200 Reaction Units (RU) for detailed kinetic studies and 500-600 RU for screening assays. The chip is blocked by ethanolamine. Regeneration studies showed that Pierce elution buffer (product No. 21004, Pierce Biotechnology, Rockford, IL) in mixture with 4M NaCl (2: 1) was effective in removing bound Fab while maintaining hNGF activity on the chip for more than 200 injections. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15 NaCl, 3mM EDTA, 0.005% surfactant P29) was used as the running buffer for all BIAcore assays.

Screening assays

Screening BIAcore assays were optimized to determine affinity from library Fab clones. Supernatants from small culture lysates were injected at 50. mu.l/min for 2 min. Dissociation times of 10 to 15 minutes were used to determine single index dissociation rates (k) using BIA evaluation software _off). Injection display k_offSamples with rates in the same range as the templates used to generate the library (clone 8L2-6D5, k)_off 1×10^-3s^-1) For determination and dissociation time up to 45 min to allow better k_offThe value is obtained. Show improved (slower) k_offCloning of values for expression on Large Scale and determination of the complete kinetic parameter k on purified proteins_onAnd k_off. This assay can detect differences in affinity by about 2-fold or more than 2-fold.

Affinity determination assays

Serial dilutions (0.1-10X estimated K)_D) The purified Fab sample was injected at 100 μ L/min for 1 min and the dissociation time was allowed to reach 2 hours. The concentration of the Fab protein was determined by ELISA and/or SDS-PAGE electrophoresis using known concentrations of standard Fab (determined by amino acid analysis). Data were fitted to a 1: 1 Langmuir binding model (Karlsson, R.Roos, H.Fagerst) by using the BIA evaluation programam, L.Petersson, B. (1994) Methods Enzymology 6: 99-110) while obtaining a kinetic binding rate (k)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) Value passing k_off/k_onAnd (4) calculating.

B. Humanization and affinity maturation of mouse antagonist anti-NGF antibody 911

The mouse antagonist anti-NGF antibody 911 (see Hongo et al, (2000) Hybridoma19 (3): 215-227) was selected for humanization and affinity maturation. Monoclonal antibody 911 binds human and rat NGF with high affinity and shows no significant cross-reactivity with neurotrophins NT3, NT4/5 or BDNF. See Hongo, supra. Papain-cleaved Fab fragments of mouse monoclonal antibody 911 were determined using BIAcore analysis as described above. Papain cleavage of Fab fragment by murine monoclonal antibody 911 with K _DAbout 10nM binds human NGF.

Humanization and affinity maturation were performed in several steps as follows:

(1) preparation of CDR-grafting template the light chain extended CDRs (i.e., including both Kabat and Chothia CDR regions) of antibody 911 were grafted to human germline recipient sequence 08 with JK2, and the heavy chain extended CDRs of antibody 911 were grafted to human germline recipient sequence VH4-59 with JH 4. The amino acid sequences of the human germline recipient sequences are shown in FIGS. 1A and 1B. The amino acid numbering is sequential. Using the protein framework described above, DNA sequences were designed for encoding synthetic genes with murine CDR human frameworks. These humanized heavy and light chain domains are referred to as hVH and hVL, respectively. Codons were optimized for e.coli and hamster applications. Essentially as described in Prodromou et al, (1992) Protein Eng 5 (8): 827-9 by extension of several overlapping oligonucleotides (69-90 bases long) of full length hVL and hVH, respectively, with two short flanking primers for each strand. The resulting DNA fragment of the correct length was gel purified and then cloned into an E.coli bicistronic expression plasmid (ampicillin resistance). Expression of the antibody was under the control of the IPTG-inducible lacZ promoter, as compared to the expression of the antibody in Barbas (2001) Phage display: a similar description is given in a Laboratory, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press (see vector pComb3X, page 2.10). However, modifications include the addition and expression of additional domains: human kappa light chain constant domain (see GenBank accession number CAA09181) and IgG2a human immunoglobulin CHI constant domain (GenBank accession number P01859).

The amino acid sequence of the variable region of the CDR-grafted antibody (also referred to as the "template") is designated 8L2-4D5 and is also shown in FIGS. 1A and 1B. The affinity of 8L2-4D5 was determined by BIAcore analysis as described above. 8L2-4D5 with K_DAbout 38nM binds human NGF.

(2) Introduction of point mutations in the framework sequence the substitution of V71K was introduced into the CDR-grafted heavy chain using recombinant PCR site-directed mutagenesis as described in Innis et al, (1995) PCR strands, San Diego, Academic Press. This substitution replaces the human framework residues with the corresponding mouse framework residues. The antibody produced was designated 8L2-6D5, and the amino acid sequence of the 8L2-6D5 heavy chain variable region is shown in FIG. 1A. The affinity of 8L2-6D5 was determined using BIAcore analysis as described above. Fab fragments of 8L2-6D5 bind human NGF with a Kd of about 15 nM. 8L2-6D5 was chosen as template for affinity maturation.

(3) Humanization and affinity maturation of the CDRs L1, L2, H1 and H2 the CDRs L1, L2, H1 and H2 were humanized and affinity matured. Amino acid positions in CDRs L1, L2, H1 and H2 that are not essential to the CDR structure were identified for Chothia extreme (canonical) based structures (see Al-Lazikani et Al (1997) J.mol.biol.273 (4): 927-48) and randomized as follows. Two libraries containing light chain mutations or heavy chain mutations and grafted CDR L3 or CDR H3, respectively, as shown in table 2 were purified using, for example, Kay et al, (1996) Phage display of peptides and polypeptides: PCR cassette mutagenesis of degenerate oligonucleotides as described in the book by laboratory Manual, San Diego, Academic Press, by using a DNA sequence as described in Balint et al, (1993) Gene137 (1): 109-18, doped (doping) codons. In general, antibodies based on sequences with human germline antibodies 9 11, the light and heavy chain amino acid sequences are arranged to change the amino acid residues to those more common in human antibodies. In addition to the CDR H2 at residue 50 being methionine, wild type (unsubstituted) amino acid residues are also represented in the library, with wild type methionine not represented in the library. Methionine residues are susceptible to oxidation and, therefore, substitution of this residue is expected to improve the stability of the resulting antibody. The Fab library was cloned into the vector pComb3X plus human CH1 and C as described above_κIn the region.

Table 2:

1. heavy chain H1/H2 library

CDR-H1

Changing I34 to F, L, V, S, P, T, A or I

N35 changed to N, T, S or Y

CDR-H2

Changing M50 into all 20 natural amino acids

A62 changed to A or S

L63 changed to L or V

2. Light chain L1/L2 library

CDR-L1

Changing S26 to S, A, V or F

Changing D28 to D, A, S or Y

Changing H32 to H, N, K, D, E, Q or Y

CDR-L2

Changing Y50 to Y, D, A or S

Changing I51 to I, T, A or V

Changing F54 to F or L

Changing S56 to S or T

For affinity screening experiments, eachThe library was further paired with the corresponding CDR-grafted light or heavy chain (e.g., H1/H2 library paired with CDR-grafted light chain), antibodies were expressed, and each clone was screened for human NGF affinity using the BIOCORE Surface Plasmon Resonance (SPR) system (BIAcore, inc. piscataway, NJ) according to the supplier's instructions and as described above. Determining k _off、k_onAnd k_D. Since the majority of the changes in affinity are generally at k_offIn rate, and further due to k_offThe rate is independent of antibody concentration, so antibody clones are based on k_offAnd (4) rate arrangement.

The sequences of the binding clones were determined and the binding clone sequences are shown in table 3.

Table 3: l1 and L2 amino acid sequences, H1 and H2 amino acid sequences, and kinetic data for binding clones after screening for affinity of H1/H2 or L1/L2 library clones

CDR1-2 mutant kinetic data
					Light chain library clones paired with 8L2 heavy chain	CDRL1 amino acid sequence	CDRL2 amino acid sequence	koff (s-1)	*KD (nM)
8L2-6D5 (control)	RASQDISNHLN(SEQ ID NO：12)	YISRFHS(SEQ ID NO：13)	**1e-3	25
					L129	RASQSISNNLN	YTSRFHS	4.5e-4	11

	(SEQ ID NO：18)	(SEQ ID NO：19)
					L208	RASQYISNHLN (SEQID NO：20)	YTSRFHS (SEQ ID NO：21)	4.6e-4	11
L97	RASQSISNQLN (SEQ ID NO：22)	YVSRFHS (SEQ ID NO：23)	5.6e-4	14
					L81	RAFQAISNQLN (SEQ ID NO：24)	YISRFHT (SEQ ID NO：25)	7.4e-4	18
L6	RAFQSISNQLN (SEQ ID NO：26)	YASRFHS (SEQ ID NO：27)	8.2e-4	20
					Heavy chain library clones paired with 6D5 light chain	CDRH1 amino acid sequence	CDRH2 amino acid sequence	koff (s-1)	*KD (nM)
8L2-6D5 (control)	GFSLIGYDIN (SEQ ID NO：9)	MIWGDGTTDYNSAL (SEQ ID NO：10)	1e-3	25
					H109	GFSLIGYDSN (SEQ ID NO：28)	IIWGDGTTDYNSAL (SEQ ID NO：29)	1.6e-4	4
H19	GFSLIGYDLN (SEQ ID NO：30)	IIWGDGTTDYNSAV (SEQ ID NO：31)	2.4e-4	6
					H222	GFSLIGYDVT (SEQ ID NO：32)	GIWGDGTTDYNSAV (SEQ ID NO：33)	3.8e-4	9.5
H225	GFSLIGYDVT (SEQ ID NO：34)	GIWGDGTTDYNSSV (SEQ ID NO：35)	3.8e-4	9.5
					H18	GFSLIGYDAT (SEQ ID NO：36)	GIWGDGTTDYNSAV (SEQ ID NO：37)	4.2e-4	10.5
H9	GFSLIGYDVS (SEQ ID NO：38)	IIWGDGTTDYNSSV (SEQ ID NO：39)	4.1e-4	10.2
					H227	GFSLIGYDIS	QIWGDGTTDYNSSV	5.4e-4	13.5

	(SEQ ID NO：40)	(SEQ ID NO：41)
					H17	GFSLIGYDAS(SEQ ID NO：42)	GIWGDGTTDYNSSV(SEQ ID NO：43)	6.1e-4	15.2
H28	GFSLIGYDST(SEQ ID NO：44)	SIWGDGTTDYNSAL(SEQ ID NO：45)	7.5e-4	18.7

The bold amino acids as indicated above are randomized.

^*K for KD_on4e4M^-1s^-1And (4) calculating.

^**For convenience, "e" as used herein means "x 10". Thus 4e4 interchangeably represents 4 × 10⁴。

CDRs comprising the following substitutions remain bound:

CDR-H1

i34: s, L, V, I and A, in combination.

N35: n, T and S, in combination.

CDR-H2

M50: m, I, G, Q, S, L, and combining.

A62: a and S, combined.

L63: l and V, combined.

CDR-L1

S26: s and F, combining.

D28: D. s, A, Y, and combining.

H32: H. n, Q, and combining.

CDR-L2

Y50: and Y, combining.

I51: I. t, V, A, and combining.

F54: and F, combining.

S56: s and T, combining.

CDRs containing the following substitutions were generally selected based on binding affinity and combined with a single clone designated H19-L129:

CDR-H1: I34L; N35N (unchanged)

CDR-H2: M50I; a62A (no change); L63V

CDR-L1: S26S (no change); D28S; H32N

CDR-L2: Y50Y (no change); I51T; F54F (no change); S56S (unchanged)

These mutations were combined (amplification of the H and L chains by PCR, cleavage of the PCR product with restriction enzymes and vector (pRN8) and ligation of 3 fragments) into a single clone designated H19-L129, which also included the grafted H3 and L3 CDRs. The sequences of the H19-L129 heavy and light chain variable regions are shown in FIGS. 1A and 1B, and Table 4 shows the amino acid sequences of CDRs L1, L2, H1, and H2. H19-L129 bound NGF with a KD of approximately 1nM, as determined by BIAcore analysis described herein.

Table 4: amino acid sequences of CDRs H1, H2, L1 and L2 and kinetic data of the combination clone H19-L129

Combined cloning: mutations in CDRH1, H2, L1 and L2	CDRL1CDRH1 amino acid sequence	CDRL2CDRH2 amino acid sequence	Koff(s-1)	*KD(nM)
					H19-L129	CDR-L1：RASQSISNNLN(SEQ ID NO：18)CDR H1：GFSLIGYDLN(SEQ ID NO：30)	CDRL2：YTSRFHS(SEQ ID NO：19)CDR-H12IIWGDGTTDYNSAV(SEQ ID NO：31)	1.1e-4	3.5

^*K for KD_on4e4M^-1s^-1And (4) calculating.

(4) Affinity maturation of H3 and L3 CDRs: affinity maturation of the H3 and L3 CDRs was performed in two steps. First, in a process called "library scanning mutagenesis", each amino acid residue in H3 and L3 was individually pre-screened to identify amino acid positions where mutations result in increased binding affinity for human NGF. Based on the results of library scanning mutagenesis (also referred to as "small library random analysis"), a set of amino acid positions in H3 and L3 were screened for the preparation of affinity maturation libraries, and the affinity of the affinity maturation libraries for human NGF were screened as described in BIAcore analysis. It should be understood that these techniques may be applied generally.

(a) Library scanning mutagenesis

Each amino acid position in the H3 and L3 CDRs was individually pre-screened to identify substitutions that resulted in increased binding affinity for human NGF. The frequency of amino acid substitutions at any given position that result in improved binding, identical binding, poor binding or no binding provides information concerning positions in the CDR that can be changed to many different amino acids, including all 20 amino acids, as well as concerning positions in the CDR that cannot be changed or can be changed to only a few amino acids. Amino acid substitutions that result in increased affinity were also identified. Based on this screening result, a set of amino acid positions in CDR H3 and L3 were screened for the preparation of affinity maturation libraries.

Separate Fab libraries were prepared in which each amino acid in the L3 and H3 CDRs was randomized into all 20 amino acids at a time, resulting in a few (5 libraries for the light chain and 13 libraries for the heavy chain) small libraries of complexity with a probability of 20 amino acids at each amino acid position. In all cases, the library is represented by natural (i.e., unchanged) amino acids. As in Kay et al (1996), Phage displayo Peptides and Proteins: a laboratory Manual, San Diego, Academic Press, randomize one amino acid position with the doped codon NNK to include 20 possible amino acids, and prepare libraries by PCR cassette mutagenesis with degenerate oligonucleotides. Since the lower affinity of the CDR-grafted antibody allows easy detection of differences in affinity in the H3 and L3 mutations during screening, 8L2-6D5 (CDR-grafted antibody, with framework mutation V71K) was used as template for library construction. Thus, each member of the library contained a CDR3(H3 or L3) with one amino acid substitution and 5 grafted CDRs.

From 20-80 clones from each small library were screened using BIAcore analysis as described herein. The binding affinity of the samples to NGF in one channel of the BIAcore chip and the simultaneous analysis of the samples by BIAcore by binding five histidine-tagged antibodies in the other channel of the sensor chip to detect the heavy chain C-terminal histidine tag. Clones expressing the protein were classified as having the same affinity, poor affinity, better affinity or no binding using the koff classification: the results of this analysis are shown in table 5.

Table 5: based on koff, clones expressing the protein were classified as having the same affinity, poor affinity, better affinity or no binding:

all cloned sequences with improved affinity were determined, showing the frequency and identity of amino acid substitutions resulting in increased affinity. In addition, several clones that retain similar affinity to the 8L2-6D5 clone were screened from each library to determine the amino acid substitutions allowed at a given position, although substitutions do not necessarily increase binding affinity. The results of this analysis are summarized in table 6.

TABLE 6

CDR H3 mutation (8L2-6D5 template, including antibody 911 CDR-H3 amino acid sequence: GGYYYGTSYYFDY (SEQ ID NO: 11)	Koff(s-1) 1E-3	KD(nM) 25
			Y100L	1.2E-3	30
Y100R	1.1E-3	27
			Y101W	5.6E-4	14
G103A	1.6E-4	4
			T104S	2.2E-3	55
S105A	5.1E-4	13
			S105T	6.4E-4	16
Y106R	1.6E-3	40
			Y106T	2.0E-3	50
Y106M	2.7E-3	67
			Y107F	1.4E-3	35
F108W	1.22E-3	30
			D109N	1.5E-3	37
D109G	1E-3	25
			Y110K	1.4E-3	35
Y110S	1.5E-3	37
			Y110R	1.6E-3	40
Y110T	1.7E-3	42
			CDR L3 mutations (8L2-6D5 template, including wild-type (unsubstituted) CDR-L3 amino acid sequence: QQSKTLPYT (SEQ ID NO: 14)	Koff(s-1) 1E-3	KD * (nM) 25
S91E	2.5E-4	6
			Y96R	1.7E-3	42

^*K for KD_on4e4M^-1s^-1And (4) calculating.

Several mutations resulted in an increase in binding affinity compared to the 8L2-6D5 template, at least the following mutations resulted in a significant increase in binding affinity: (H _ Y101W (CDR sequence GGYWYGTSYYFDY (SEQ ID NO: 46)); H _ S105A (CDR sequence GGYYYGTAYYFDY (SEQ ID NO: 47)); H _ S105T (CDR sequence GGYYYGTTYYFDY (SEQ ID NO: 48)); H _ G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO: 49); and L _ S91E (CDR sequence QQEKTLPYT (SEQ ID NO: 50)).

The results of this experiment were used to guide the selection of amino acid positions for generating affinity maturation libraries.

This experiment also provides information on the frequency of amino acid substitutions that resulted in improved binding, identical binding, poor binding, or no binding at any given position, as shown in table 5. This information allows identification of amino acid positions in the CDRs that can be changed to many different amino acids, including all 20 amino acids, as well as amino acid positions in the CDRs that can be changed to several amino acids or very few amino acids (in some embodiments, no amino acid changes). These results also demonstrate amino acid substitutions that increase binding affinity.

(b) Affinity maturation

Next, the results of the small library random analysis (above) were used to select residues of H3 and L3 libraries that produced H3 and L3 CDR affinity maturation. Y101 and G103 residues of CDR H3 and S91 and K92 residues of CDR L3 were selected for generating H3 and L3 libraries for CDR affinity maturation of H3 and L3.

This library combines mutations of H3 and L3 simultaneously in CDR-grafted clone 8L2-6D5 and in the H19-L129 background, respectively, and has a diversity of 80 different clones. Table 7 shows the amino acid residues selected for substitution as well as the amino acids substituted at each position.

Amino acid residues selected for substitution in Table 7, H3 and L3 and amino acids substituted at each position

CDR-H3：

Y101 was changed to Y and W, C (note that C was included, since codon C was also generated with the codon TRS in one degenerate oligonucleotide).

Change G103 to A, P, S

CDR-L3：

Change S91 to E.

K92 was changed to all twenty amino acids. A. R, K and H, in combination.

Each polypeptide was expressed as Fab and affinity of human NGF was screened for 96 individual clones of each library using BIACORE analysis according to the manufacturer's instructions and described above. The results of this analysis are shown in table 8.

Table 8.

CDR L3H 3 combination mutation (8L2-6D5 template)	Koff(s-1)1E-3	KD *(nM) 25
			L _ S91E; l _ K92A (CDR sequence QQEATLPYT (SEQ ID NO: 51)) H _ Y101W; h _ G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO: 52))	5.5E-4	13
L _ S91E; l _ K92R (CDR sequence QQERTLPYT (SEQ ID NO: 53)) H _ Y101W; h _ G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO: 54)))	1.0E-4	25
			CDR L3H 3 combinatorial mutations (H19-L129 template, mature H1H2L1L 2)	Koff(s-1)1.1e-4	KD *(nM)
L _ S91E; l _ K92H (CDR sequence QQEHTLPYT (SEQ ID NO: 55)) H _ Y101W; h _ G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO: 56)) (clone E3)	1.2E-5	0.3
			L _ S91E; l _ K92S (CDR sequence QQESTLPYT (SEQ ID NO: 57)) H _ Y101W; h _ G103S (CDR sequence GGYWYSTSYYFDY (SEQ ID NO: 58))	4.7E-5	1.1
L _ S91E; l _ K92K (CDR sequence QQEKTLPYT (SEQ ID NO: 59)) H _ Y101W; h _ G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO: 60))	2E-5	0.5
			L _ S91E; l _ K92R (CDR sequence QQERTLPYT (SEQ ID NO: 61)) H _ Y101W; h _ G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO: 62)) (clone 3C)	1.4E-5	0.35
L _ S91E; l _ K92R (CDR sequence QQERTLPYT (SEQ ID NO: 63)) H _ Y101Y; h _ G103A (CDR sequence GGYYYATSYYFDY (SEQ ID NO: 64))	1.5E-5	0.37

^*K for KD_on4e4M^-1s^-1And (4) calculating.

Based on binding affinity, the best clones E3 (interchangeably referred to as "3E") and 3C were selected for further characterization. E3 contains the following CDR substitutions: CDR-H3: Y101W, G103A; and CDR-L3: S91E, K92H, which can be combined into a single clone that also includes the following L1, L2, H1 and H2 mutations:

CDR-H1：I34L；

CDR-H2：M50I；L63V；

CDR-L1：D28S；H32N；

CDR-L2：I51T。

the sequences of the E3 heavy and light chain variable regions are shown in fig. 1A and 1B. 3C comprises the following CDR substitutions: CDR-L3: S91E; K92R; CDRH 3: Y101W; G103A, combined into a single clone that also included the L1, L2, H1 and H2 mutations describing clone 3E.

The 3E and 3C sequences were cloned into mammalian expression vectors for Fab and whole antibody production, and expressed in HEK293 cells and purified by Ni-NTA or protein a affinity chromatography. The purified protein was accurately quantified by amino acid analysis.

In addition to the use of 100 RU NGF on a chip to prevent re-binding, the binding affinity of Fabs E3 and 3C to human NGF was determined using BIAcore analysis according to the manufacturer's instructions and as described above. Briefly, several concentrations of antibody (Fab) were injected onto CM5 chips with 100 RU of immobilized human NGF for 2 minutes and allowed to dissociate for 1800 seconds. The mouse antibody 911(Fab) was analyzed as a control. The data were analyzed using BIA evaluation software according to the manufacturer's instructions. Analytical node for antibodies E3 and 911The results are shown in fig. 9 and 10. E3 was at KD of about 0.07nM (and at k)_onAbout 6.0e5M-Is-1 and k_offAbout 4.2e-5s-1) binds human NGF. 3C with KD of about 0.35nM (k)_offAbout 1.4E-5) binds human NGF. In contrast, the mouse antibody 911 was shown to have a KD of 3.7nM, k_off 8.4×10^-5s^-1And k is_on 2.2×10⁴Ms^-1Binds NGF.

Based on the high binding affinity, antibody E3 (interchangeably referred to as 3E) was selected for further analysis. To test the ability of E3 to prevent NGF interaction with NGF receptors trkA and p75, 2.5nM human NGF was premixed with 0 to 50nM antibody E3(Fab) and incubated for one hour. After incubation, samples were injected at 10 μ l/min onto BIAcore CM5 chips containing 260 RU p75 (channel 2) and 600 RUtrkA (channel 3) and percent binding was determined. The results of this analysis are shown in FIG. 11. As shown by the decrease in signal (measured in RU), the increased concentration of Fab E3 blocked NGF interaction with p75 and trkA, indicating that Fab E3 blocked human NGF interaction with trkA and p 75. When antibody E3(Fab) concentration was equal to NGF concentration (about 2.5nMNGF concentration), no NGF binding was observed (as shown by zero signal). The fact that zero percent NGF receptor binding occurs when the NGF concentration is the same as that of antibody 3E indicates that 2.5nM NGF is at least ten times higher than the kD of E3 for NGF and is in equilibrium.

Example 2: evaluation of the ability of anti-NGF antibodies to interfere with NGF Using a mouse E13.5 trigeminal neuron survival assay

The ability of Fab E3 or whole antibody E3 to block NGF activity was assessed by determining in vitro the ability of the antibody to inhibit the survival of NGF dependent mouse E13.5 trigeminal neurons. The trigeminal ganglion contains cutaneous sensory neurons innervated by the facial region. Since NGF is required to support the survival of trigeminal neurons, the survival of mouse E13.5 trigeminal neurons is a sensitive assay to evaluate the blocking of NGF activity by anti-NGF antagonist antibodies. For example, at saturation concentrations of NGF, survival by 48 hours of culture was close to 100%. In contrast, less than 5% of neurons survived 48 hours in the absence of NGF.

The survival assay was performed as follows: passage of CO to pregnant Swiss Webster female mice₂Inhalation was euthanized. The uterine horns were removed and the embryonic stage E13.5 embryos were extracted and the embryo heads were removed. The trigeminal ganglia were dissected using an electrolytic sharp tungsten needle, then the ganglion was trypsinized, mechanically dissociated and cultured in poly-L-ornithine and laminin coated 96-well plates at a density of 200-300 cells per well in well-defined serum-free medium.

Blocking Activity of anti-NGF Fab or antibody blocking activity was determined by adding different doses of anti-NGF antibodies Mab 911(Fab), 8L2-6D5 to trigeminal neurons; H19-L129; e3 and 3C; and concentrations of 0.4ng/ml (. about.15 pM; this concentration represents the saturation concentration of NGF for survival) and 0.04ng/ml (. about.1.5 pM; this concentration is around IC50 were assessed using human or rat NGF at home. After 48 hours of culture, cells were subjected to automated immunocytochemistry on a Biomek FX liquid handling workstation (Beckman Coulter) as follows: fixing with 4% formaldehyde, 5% sucrose and PBS; permeabilization with PBS containing 0.3% Triton X-100; blocking non-specific binding sites with 5% normal goat serum, 0.11% BSA in PBS; and sequentially incubated with primary and secondary antibodies to detect neurons. The primary antibody is an established neuronal phenotypic marker, a rabbit polyclonal antibody (PGP9.5, Chemicon) directed against protein gene product 89.5. The secondary antibody was an Alexa Fluor 488 goat anti-rabbit (Molecular Probes) together with the nuclear dye Hoechst 33342(Molecular Probes) that labels all cells present in culture. Image acquisition and image analysis were performed on a Discovery-I/GenII imager (Universal Imaging Corporation). Images were automatically obtained at two wavelengths for Alexa Fluor 488 and Hoechst 33342, and since nuclear staining was present in all wells, nuclear staining used as a reference point was used for the image-based autofocus system of the imager. The appropriate number of targets and imaging sites for each well is selected to cover the entire surface of each well. Based on their specific staining with anti-PGP 9.5 antibody, automated image analysis was set up to count the number of neurons present per well after 48 hours of culture. A selective filter applied based on careful onset of the image and morphology and fluorescence intensity yields an accurate count of neurons per well.

The results of this experiment demonstrate that Fab E3 blocks NGF activity with high affinity. The results are shown in FIGS. 4 to 6 and Table 9.

FIG. 4 is a graph showing NGF-dependent E13.5 neuronal survival in the presence of different concentrations of human and rat NGF.

FIG. 5 is a graph comparing the effects of different Fab's on blocking NFG in the presence of 0.04ng/ml human NGF (about 1.5 pM; shown in the lower panel of FIG. 5) or 0.4ng/ml human NGF (about 15 pM; shown in the upper panel of FIG. 5). 1.5pM NGF was around the EC50 for NGF-promoted survival, while 15pM represents the saturating concentration of NGF. Fab E3 at different concentrations; murine 911 Fab; and H19-L129 Fab and 8L2-6D5 Fab the survival of E13.5 mouse trigeminal neurons was assessed as above. At each NGF concentration, each Fab IC50 (expressed as pM) was calculated and shown in table 9. Fab E3 strongly blocked human NGF-dependent survival of trigeminal neurons with an IC50 of about 21pM in the presence of 15pM human NGF and an IC50 of about 1.2pM in the presence of 1.5pM human NGF. Fab 3C and H19-L129 also strongly blocked human NGF-dependent survival of trigeminal neurons.

FIG. 6 is a graph comparing NGF blocking effects of various Fab's in the presence of 0.04ng/ml rat NGF (about 1.5 pM; shown in the lower panel of FIG. 6) or 0.4ng/ml rat NGF (about 15 pM; shown in the upper panel of FIG. 6). 1.5pM NGF was around EC50, while 15pM represents the saturation concentration of NGF. At various concentrations Fab E3; murine 911 Fab; and H19-L129 Fab and 8L2-6D5 Fab the survival of E13.5 mouse trigeminal neurons was evaluated as described above. At each NGF concentration, EC50 (expressed as pM) was calculated for each Fab and shown in table 9. Fab E3 strongly blocked rat NGF dependent survival of trigeminal neurons with an IC50 of about 31.6pM in the presence of 15pM rat NGF and an IC50 of about 1.3pM in the presence of 1.5pM rat NGF.

Table 9:

human NGF	IC50 (15 pM NGF present)	IC50 (presence of 1.5pM NGF)
	pM	pM
			8L2-6D5 Fab	1580.5	461.8
H19-L129 Fab	60.1	9.6
			3E Fab	＜21.0	＜1.2
3C Fab	80.9	5.6
			911 Fab	322.3	63.5
			Rat NGF	IC50(15pM NGF)	IC50(1.5pM NGF)
				pM	pM
8L2-6D5 Fab	730.3	169.4
			H19-L129 Fab	31.0	6.0
3E Fab	＜8.3	＜1.3
			3C Fab	31.6	6.0
911 Fab	161.0	34.6

In different experiments, we compared the inhibition of NGF-dependent E13.5 neuronal survival by the intact antibody E3 and Fab 3E in the presence of 0.4ng/ml (saturating concentration) of human NGF. The results of the analysis are shown in fig. 12. When the concentrations of intact antibody and Fab were normalized to NGF binding site numbers (Fab with one binding site and intact antibody with two binding sites), it was shown that intact antibodies E3 and Fab 3E had similar levels of inhibition of NGF-dependent survival. These results demonstrate no affinity due to binding of intact antibody to NGF dimer.

In another experiment, we compared the ability of antibody E3, antibody 911 and trkA receptor immunoadhesin (consisting of the NGF receptor trkA extracellular domain fused to the immunoglobulin Fc domain CH2-CH 3) to inhibit NGF-dependent E13.5 neuronal survival at various concentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128 and 0.0nM) in the presence of 0.4ng/ml (saturation). These results are shown in fig. 13. These results demonstrate that antibody E3 blocks NGF better than antibody 911 or trkA immunoadhesin.

Example 3: evaluation of specificity of anti-NGF antibody E3 Using mouse trigeminal and ganglion neuron survival assays

The ability of antibody E3 to specifically block NGF activity was assessed by determining the ability of the antibody to inhibit the survival of mouse E17/18 trigeminal neurons in vitro in the presence of saturating concentrations of NGF, NGF-related neurotrophin NT3 or NGF-independent neurotrophic factor, Macrophage Stimulating Protein (MSP). Since NGF is required to maintain the survival of these neurons at a higher concentration than the NGF level required to maintain the survival of E13.5TG neurons, survival of mouse E17/18 trigeminal neurons is a sensitive assay to assess NGF blocking activity of anti-NGF antagonist antibodies, which are also maintained by NT3 or MSP; thus, the survival of these neurons is also a sensitive assay to assess whether anti-NGF antagonist antibodies also block NT3 or MSP.

The ability of antibody E3 to specifically block NGF activity was also assessed by determining the ability of the antibody to inhibit the survival of the mouse nodule E17 ganglion in the presence of saturating concentrations BDNF or NT 4/5. Survival of nodal neurons is maintained by BDNF or NT 4/5; thus, the survival of these neurons is a sensitive assay to evaluate the BDNF or NT4/5 blocking ability of anti-NGF antagonist antibodies.

The survival assay was performed as follows: passage of CO to pregnant Swiss Webster female mice ₂Inhalation was euthanized. The uterine horns were removed and the embryos (day 17 or 18 of embryonic stage) were removed and the embryo heads were removed. The trigeminal and ganglion ganglia were dissected and washed. The ganglion was then trypsinized, mechanically dissociated and cultured at a density of 200-300 cells per well in defined serum-free medium in poly-L-ornithine and laminin coated 4-well culture plates (Greiner).

E17/18 trigeminal neurons were either without neurotrophic factor (negative control) or in the presence of saturating concentrations of human NGF (400pM and 15pM) (positive control); NT3(400 pM); or in MSP (600 pM). Duplicate cultures were established including different concentrations of E3 and 911 Fab as well as intact antibodies. The concentrations of Fab and intact antibody are indicated with each binding site (e.g., intact antibody contains two binding sites, while Fab contains one binding site).

E17 nodose neurons were grown in the absence of added neurotrophic factors (negative control) or with saturating concentrations of BDNF (400pM) (positive control) or NT4/5(400pM) or NGF-independent growth factor ILF (interleukin inhibitor). Since the purpose of this experiment was to detect the specificity of the antibody, a high concentration of neurotrophins was used. Duplicate cultures were established including addition or non-addition of antibodies E3 and 911. After 48 hours of culture, the total number of neurons surviving in each well under each condition was determined by manual counting with a phase contrast microscope.

The results of these experiments demonstrate that the E3 and 911 antibodies completely block the survival promoting effect of NGF on E18 trigeminal neurons. In contrast, the E3 and 911 antibodies had no effect on the survival of trigeminal neurons promoted by NT3 or MSP, or on ganglion neurons promoted by BDNF or NT4/5 or LIF. These results demonstrate that antibody E3 has selective specificity for NGF, and no interaction was detected between these antibodies and other NGF-related neurotrophins (NT3, NT4/5, BDNF) at concentrations 1000-fold to 10,000-fold higher than the effective concentration for NGF blockade. Furthermore, these results demonstrate that the NGF-dependent neuronal death observed with the addition of antibody or Fab E3 in NGF-supplemented cultures was due to specific interactions between such antibodies and NGF and not to general toxic effects. A mouse anti-NGF antagonist antibody 911 was also tested and similar results were observed. Note that due to the use of high concentrations of neurotrophins, the titration conditions for both antibodies E3 and 911 were very close and that they were also expected to bind NGF at similar levels since the differences in the binding affinity of these antibodies to NGF were less pronounced under such conditions.

The results of these experiments are shown in fig. 14, 15, 16 and 17. Data for each experiment is shown as the mean percent survival (± mean standard error, n ═ 3 for each data point) after 48 hours of culture relative to the survival observed in positive controls (e.g., 100% survival of trigeminal neurons grown in the presence of saturating NGF concentrations, and 100% survival of growing ganglion neurons in the presence of saturating BDNF concentrations, respectively). Fig. 14-15 are graphs showing that anti-NGF antagonist antibody E3 or Fab E3 did not inhibit survival promoted by NT3 and MSP even at antibody concentrations up to 200 nM. In contrast, the 20nM antibody E3 or Fab 3E and Fab 911 completely blocked NGF-induced survival. Mouse anti-NGF antagonist antibody 911 was also tested and similar results were observed. In particular, FIG. 14 is a graph showing a comparison of the survival effect of different concentrations (20nM, 2nM or 0.2nM) of E3 Fab (referred to in the figure as "3E") and the mouse antibody 911 Fab on E18 trigeminal neurons in the presence of no neurotrophin (referred to as "control"), 400pM NGF (referred to as "NGF-400 pM), 10nM NT3 (referred to as" NT3-10nM) or 600pM MSP (referred to as "MSP-600 pM). FIG. 15 is a graph depicting the effect of comparing the different concentrations (200nM and 80nM) of E3 Fab and whole antibody and mouse antibody 911 whole antibody and Fab on E17 trigeminal neuron survival in the presence of either no neurotrophin (referred to as "no factor"), 400pM NGF (referred to as "NGF-400 pM), 10nM NT3 (referred to as" NT3-10nM) or 600pM MSP (referred to as "MSP-600 pM).

FIGS. 16-17 are graphs showing that anti-NGF antagonist antibodies E3 or Fab E3 do not inhibit E17 nodal neurons promoted by BDNF, NT4/5, or LIF. Mouse anti-NGF antagonist antibody 911 was also detected and similar results were observed. In particular, FIG. 16 is a graph showing a comparison of the survival effect of the intact antibody E3 (referred to as "3E" in the graph), Fab E3, intact antibody 911, or Fab 911 on E17 nodal neurons at different concentrations (200nM or 80nM) in the absence of added neurotrophins (referred to as "no factor"), 400pM BDNF (referred to as "BDNF-400 pM), 400pMNT4/5 (referred to as" NT4/5-400pM), or 2.5nM LIF (referred to as "LIP-2.5 nM). FIG. 17 is a graph depicting the comparative effect of different concentrations (200nM, 20nM, 2nM) of Fab E3 (referred to as "3E in the graph"), or Fab 911 on E17 nodal neuron survival, in the presence of 400pM BDNF (referred to as "BDNF-400 pM), 400pM NT4/5 (referred to as" NT4/5-400pM), or 2.5nM LIF (referred to as "LIP-2.5 nM) without added neurotrophin (referred to as" control ").

Example 4: preparation of mammalian expression vector for antibody E3 and expression in mammalian cells

Three mammalian expression vectors were designed and constructed for expression of antibody E3 in mammalian cells.

The vector db.911.3e is an expression vector comprising the heavy chain variable region of the E3 antibody and the constant region of human IgG2a, and is suitable for transient or stable expression of the heavy chain, and consists of a nucleotide sequence corresponding to the region db.911.3e, the nucleotides of which are: the mouse cytomegalovirus promoter region (nucleotides 1-612); synthesizing an intron (nucleotide 619-1507); the DHFR coding region (nucleotides 707-1267); human growth hormone signal peptide (nt 1525-1602); the heavy chain variable region of antibody 3E (nucleotide 1603-1965); a human heavy chain IgG2a constant region comprising the following mutations: the mutations were A330P331 to S330S331 (amino acid numbering refers to wild-type IgG2a sequence; see Eur. J. Immunol. (1999) 29: 2613-2624); SV40 late polyadenylation signal (nucleotide 2974-3217); the SV40 enhancer region (nucleotides 3218-3463); a phage fl region (nucleotide 3551-4006) and a beta lactamase (AmpR) coding region (nucleotide 4443-5300). Db.911.3E was deposited with the ATCC on 8.1.2003 and assigned ATCC accession number PTA-4895.

The vector Eb.911.3E is an expression vector comprising the variable region of the light chain of the E3 antibody and the constant region of the human K chain, and is suitable for transient expression of the light chain. Eb.911.3e consists of a nucleotide sequence corresponding to the following region: the mouse cytomegalovirus promoter region (nucleotides 1-612); the EF-1 intron (nucleotide 619-1142); human growth hormone signal peptide (nucleotide 1173-1150); the variable region of the light chain of the antibody E3 (nucleotides 1251-1571); the human kappa chain constant region (nucleotides 1572-1892); the SV40 late polyadenylation signal (nucleotide 1910-2153), the SV40 enhancer region (nucleotide 2154-2399); a phage fl region (nucleotides 2487-. Eb.911.3E was deposited at the ATCC on 8.1.2003 and assigned ATCC accession number PTA-4893.

The vector eb.pur.911.3e is an expression vector comprising the light chain variable region of the E3 antibody and the human kappa constant region, and is suitable for stable expression of the light chain. Eb.pur.911.3e consists of a nucleotide sequence corresponding to the following region: the mouse cytomegalovirus promoter region (nucleotides 1-612); the human EF-1 intron (nucleotide 619-1758); the coding region of the pac gene (puromycin R) (nucleotide 739-1235); the region of the human hsp 705' UTR (nucleotide 1771-1973); human growth hormone signal peptide (nucleotide 1985-2062); the variable region of the light chain of antibody E3 (nucleotide 2063-2383); the human kappa chain constant region (nucleotides 2384 and 2704); SV40 late polyadenylation signal (nucleotide 2722-2965); the SV40 enhancer region (nucleotide 2966-3211); a phage f1 region (nucleotides 3299-3654) and a beta lactamase (AmpR) coding region (nucleotides 4191-5048). Eb.pur.911.e3 was deposited with the ATCC on 8/1/2003 and assigned ATCC accession No. PTA-4894.

Transient cell expression was performed as follows: CHO and HEK293T cells were transiently co-transfected with 25 μ g of each plasmid (i.e., one plasmid containing the heavy chain and one plasmid containing the light chain) in 150mm dishes. The DNA was mixed with 100. mu.l lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. The DNA-lipid complexes were contacted with the cells in serum-free or antibiotic-free DMEM/F12 for 5 hours. After this incubation, the medium was changed to Opti-MEM (Invitrogen) without any additives for two days for expression. The cell supernatants containing the antibodies were harvested up to four consecutive times with subsequent medium replacement. The supernatant was purified by affinity chromatography using a MapSelect protein A resin (Amershambiosciences 17-5199-02). The antibody was bound to protein A resin in 0.3M glycine, 0.6M NaCl buffer pH 8, and then eluted with 0.1M citrate buffer pH 3. The fractions containing the antibody were immediately neutralized with 1M Tris buffer pH 8.0, and then the antibody fractions were dialyzed in PBS and concentrated.

Example 5: anti-NGF antibody E3 is effective in treating post-operative pain

We assessed the efficacy of treatment with antibody E3 using a pain model that mimics post-operative pain. Antibody E3 contained the following mutated human heavy chain IgG2a constant regions: A330P331 to S330S331 (amino acid numbering refers to wild type IgG2a sequence; see Eur. J Immunol. (1999) 29: 2613-2624); a human light chain kappa constant region; and the heavy and light chain variable regions shown in tables 1A and 1B.

Male Sprague Dawley rats weighing between 220 and 240 grams were purchased from Harlan (Wisconsin) and acclimated in animal facilities for one week prior to surgery.

Surgical procedures are based on techniques such as Brennan, et al, Pain 64: 493 501 (1996). Animals were anesthetized with 2% isoflurane (isonurane) in an air mixture and maintained during surgery through a nose cone. The right hind plantar aspect was padded with povidone iodine starting 0.5cm from the heel edge and extending to the toes and was incised 1-cm longitudinally through the center of the skin and fascia. The foot is measured with a ruler in the flexed position. The metatarsal muscles were lifted with curved forceps and cut longitudinally. The muscle is cut through the full depth of the origin and insertion point. Bleeding was controlled by applying mesh pad pressure throughout the procedure. The wound was closed with two mattress sutures (5-0 ethilon black monofilament). The sewing is performed by 5-6 knots, and the first knot is loose. The wound site was swabbed with bacitracin solution. Animals recovered and rested in a clean cage for two or more hours before the onset of behavioral testing.

Assessment of resting pain cumulative pain scores were used to assess pain associated with weight bearing. Animals were placed in plastic mesh (grid: 8 mm) of a clean plastic cage²) Above, the plastic cage was mounted on a platform (h: 18 ") to facilitate inspection of their underfoot. After a 20 minute acclimation period, the degree of weight bearing was evaluated in 0 to 2 minutes. If the foot is whitish or pressed against the mesh for 0 minutes, full weight bearing is indicated. If the foot retracts into the skin upon just touching the mesh, the skin is given no blush or dent for 1 point. Giving a score of 2 if the foot is completely out of the mesh. The paw withdrawal was considered to be 2 points if the rat was still at rest. Each animal was observed every 5 minutes for 1 minute, for 30 minutes. A total of 6 scores (0-12) obtained during 1/2 hours were used to assess pain in the incised foot. A 2-score frequency was also calculated and used to assess the incidence of severe pain or total protection in the animal's feet. Each animal was tested 24 hours before surgery (baseline), and 2 hours, 24 hours, 48 hours, and 72 hours after surgery. The results of this experiment are shown in FIG. 1, where FIG. 1 depicts the cumulative resting pain score observed in animals treated with 35mg/kg anti-NGF mouse antibody 911. These results demonstrate that treatment with NGF antibodies significantly reduced post-operative resting pain. Weight bearing correlates well with how well the animal is willing to use the limb and is therefore an effective means of reducing pain.

E3 antibody was injected intraperitoneally (i.p.) at various antibody concentrations (0.004, 0.01, 0.02, 0.1, 0.6, and 1mg per kg animal body weight) 15 hours prior to incision. The negative control group was not injected with antibody but was injected intraperitoneally with saline solution. Fentanyl was injected intraperitoneally at 0.01mg/kg 30 minutes prior to detection 24 hours post-surgery as a positive control. In each trial 8 animals were included for each case (n-8 per group) and 56 animals were included in the control group. Surgery was performed as above and cumulative pain scores were determined. Resting pain was assessed 24 hours after surgery.

As shown in FIG. 7, post-operative humanized anti-NGF antibody E3 significantly reduced resting pain (p < 0.05) when administered at doses from 0.02mg/kg to 1 mg/kg. "+" indicates significant difference from control (p < 0.05). Treatment with 0.02mg/kg was at least as effective as treatment with 0.01mg/kg fentanyl in reducing pain behavior. The fentanyl dose is 10 times the normal human dose of the potent opioid.

In another experiment, the efficacy of the E3 antibody in reducing post-operative pain was tested when administered post-operatively. Antibody E3(0.5mg/kg) was injected intravenously (i.v.) two hours after surgery. The control group was not administered with antibody but was injected intravenously with saline solution. Surgery was performed and resting pain, expressed as cumulative pain scores, was assessed 24 hours after surgery. As shown in figure 8, treatment with anti-NGF antibody twenty-four hours post-incision significantly reduced resting pain (p < 0.05) when the antibody was administered 2 hours post-incision. These results demonstrate that the E3 antibody is effective at reducing post-operative pain when administered post-operatively.

Example 6: evaluation of analgesic Effect of anti-NGF antagonist antibody 911 in rat model of rheumatoid arthritis

The analgesic effect of anti-NGF antibody 911 inducing chronic arthritis in rats by Complete Freund's Adjuvant (CFA) was investigated in the vocalization test in comparison with indomethacin as a reference substance (see, Hongo et al, Hybridoma 19 (3): 215- (2000)).

Fifty (50) male Lewis rats (LEWIS LEW/Crl Ico) (Charles River Belgium) weighing 150g to 220g at the beginning of the experimental period were included in this study. All animals were acclimatized for at least 5 days prior to the start of the experiment and kept in controlled room conditions of temperature (19.5-24.5 ℃), relative humidity (45-65%) and 12 hours light/dark cycle in all studies and were fed free filtered tap water and standard laboratory pellet feed (u.a.r, france). Animals were individually identified on the tail.

On day 0 (D0), arthritis was induced in rats by intradermally injecting 0.05ml of mineral oil (10mg/ml) of a suspension of mycobacterium casei (Difco, usa) into the tail. Day 14 (D14), Arthritis rats were included in the study based on their ability to vocalize when the hind feet were gently flexed and the Arthritis index assessed using inflammation scores for each hind and forefoot (see Kuzuna et al, chem. pharm. Bull. (Tokyo) 23: 1184-1191 (1975); Pearson et al, Arthritis Rheum.2: 440-459 (1959)). Animals were scored based on the following criteria: 0 minute: the appearance is normal; 1 minute: erythema; and 2, dividing: erythema with mild edema; and 3, dividing: intense inflammation without stiffness; and 4, dividing: stiffness. Only animals that vocalized on gentle bending and appeared to be 2 or 3 points were included in the study.

Four groups of 10 rats were included in the study. For group 1 (vehicle), day 14 (D14), after selection, rats were administered vehicle (saline) intravenously. On day 18 (D18), pain intensity was assessed by gently bending hind legs and the vocal level intensity was recorded for each animal. For group 2 (4 days), on day 14, after selection, rats were administered 911(10mg/kg) intravenously. On day 18 (D18), pain intensity was assessed after 24 hours by gently bending hind legs and the vocal level intensity was recorded for each animal. For group 3 (24 hours), rats were administered 911(10mg/kg) intravenously 17 days after CFA injection. The intensity of the hind paw pain was assessed by gentle bending and the intensity of the vocalization level was recorded for each animal. For group 4 (indomethacin), on day 18 (D18), pain intensity was assessed by gently bending hind paw one hour after oral administration of indomethacin (10mg/kg), and the intensity of vocalization level was also recorded for each animal. The substance to be tested is administered in an unknown and random manner by the intravenous route in a volume of 5ml/kg, while indomethacin is administered by the oral route in a volume of 10 ml/kg.

The analgesic effect of anti-NGF antibody 911 is shown in table 10. Results for each group were expressed as the assessed pain intensity and recorded vocalization level in mV (mean ± SEM) for each animal and the percentage change in pain intensity calculated from the vehicle treated group mean. Statistical significance between the treated and vehicle groups was determined by the residual variable Dunnett test (Dunnett's test) after one-way anova (P < 0.05).

TABLE 10 analgesia of 911 in complete Freund's adjuvant-induced chronic arthritis in rats

Substance (day of dose administration)	Carrier (D14)	911(D14)	911(D17)	Indometacin (D18)
					Dosage (mg/kg)		10	10	10
Intensity of pain (mV)	971.0±116.2	234.7±34.4*	247.2±41.8*	145.8±29.9*
					% of the variables	-	-76	-75	-85

Results expressed as mean. + -. sem

Each group of n-10 rats

Day 0 (D0): induction of chronic arthritis by administration of CFA

Carrier: salt water

911(10mg/kg) was administered intravenously at D14 or D17 and a pain assay at D18 was performed.

Indomethacin (10mg/kg) was administered orally at D18 and pain measurements were performed one hour after dose administration.

Dannett test: it shows a significant difference P < 0.05 from the vehicle-treated group

As shown in table 10, anti-NGF antibody 911 significantly reduced pain in a rheumatoid rat model 24 hours or 4 days after a single administration of the antibody.

Example 7: pharmacological Effect of anti-NGF antagonist antibodies E3 and 911 in rat model of rheumatoid arthritis

The pharmacological effects (anti-inflammatory and analgesic effects) of the anti-NGF antagonist antibodies E3 and 911 were studied in rats in a chronic arthritis model induced by Complete Freund's Adjuvant (CFA) in comparison with indomethacin, which was used as an internal positive control substance. The analgesic effects of E3 and 911 were evaluated by measuring nociceptive responses. Anti-inflammatory effects were assessed by foot volume, arthritis index (inflammation score), body weight and hindfoot weight. At the end of the experiment, podocyte cytokine levels (IL-6, IL-1. beta., TNF-. alpha., and TGF-. beta.1), serum circulating TGF-. beta.1, E3, and 911 plasma concentrations, biological parameters, and X-ray photography were performed.

Experimental methods

1. Design of research

80 male Lewis rats (LEWIS Lew/Ico) of 5 weeks of age were included in this study (Charles river Laboratories, Belgium). All animals were acclimatized for at least 5 days prior to the start of the experiment and were kept in controlled conditioned rooms at temperature (19.5-24.5 ℃), relative humidity (45-65%) and 12 hours light/dark cycle in all studies, and were fed free filtered tap water and standard laboratory pellet feed (SAFE, France). Once in the animal facility, they were housed 5 animals per cage and observed for a 10 day acclimation period prior to any testing. Animals were individually identified on the tail.

Five groups were included in the study, 10 animals per group (5 week old male Lewis rat from Charles River Laboratories-Belgium-Lewis Lew/Ico): group 1. Non-arthritic rats/saline (vehicle), bolus intravenous administration, n-10; group 2: arthritic rats/saline (vehicle), bolus intravenous administration, n-10; group 3: arthritis rats/indomethacin 3mg/kg, orally administered daily for 10 days, n-10; group 4: arthritic rat/E3, 1mg/kg, bolus intravenous administration, n-10; group 5: arthritis rats/911, 10mg/kg, bolus i.v. administration, n-10. The dose is expressed as free active substance (mg/kg). E3 and 911 were prepared from stock solutions temporarily to the desired concentration in saline. E31 mg/kg: 3.41mL stock solution (0.88mg/mL) q.s.p.15mL saline. 91110 mg/kg: 12mL of stock solution (2.5mg/mL) q.s.p.15mL of brine. All diluted solutions (before intravenous injection) were sterilized with a 0.20 μm sterile filter membrane; the diluted solution pH and osmolarity values were determined prior to each intravenous injection. Before the first intravenous administration, the osmolarity (mosm/L) of saline, E3, and 911 were 278, 269, and 308, respectively; the pH of brine, E3, and 911 were 5.93, 6.76, and 6.71, respectively. Before the second intravenous administration, the osmolarity (mosm/L) of saline, E3, and 911 were 280, 270, and 309, respectively; the pH of brine, E3, and 911 were 5.86, 6.72, and 6.59, respectively.

On days 14 and 19 post-arthritis induction, intravenous injections were administered by bolus injection with 5mL/kg volumes of E3 or 911 or saline in a numbered random order. The non-arthritic group was administered by bolus intravenous injection with 5mL/kg volume of saline on days 14 and 19. Indomethacin was extemporaneously prepared in 1% methylcellulose. Indomethacin was administered once daily by oral route (p.o.) in the numbered randomized order for 10 days from day 14 to day 23 post-arthritis induction in a volume of 10 mL/kg.

2. Induction of arthritis

On day 0 (D0), arthritis was induced in 70 rats by tail intradermal injection of 0.05ml of mycobacterium casei suspension. A group of 10 rats did not receive any intradermal injection (non-arthritic rats). On day 14 (D14), arthritic rats were included in this study as the following criteria: all included rats showed an increase in mean paw volume (mean of left and right paw volumes) of at least 0.30ml compared to the mean paw volume (mean of left and right paw volumes) of the non-arthritic group (paw volume determination as described below); all included rats showed vocalization in mild bending (nociceptive response assays as follows); and all included rats showed an arthritis index score of 2-3 for each hind paw (animals scored 0, 1 or 4 were discarded).

3. Body weight

Animals were weighed once daily from day 0 to day 24 (except before treatment: days D1, D2, D8, D9, D10 weekend). All assays were performed between 9:00 and 12:00 am, except for D14 (7: 30-9:00am) and D24 (7: 30-8:00 am).

3. Determination of foot volume

The volume of the right hind paw and left hind paw of each rat (arthritic and non-arthritic) was measured with a plethysmometer. The measurements were performed at the following times (after induction of arthritis): day 14 (bolus intravenous or before oral administration); and day 24 (5 days after the last bolus intravenous injection or 24 hours after the last oral administration). All measurements were performed at 9:00 and 12:00 am. All data was collected and saved by WinDas software.

4. Index of arthritis

The arthritis index was evaluated with the inflammation score for each hind and forefoot (arthritic rats): 0 minute: the appearance is normal; 1 minute: erythema; and 2, dividing: erythema with mild edema; and 3, dividing: acute inflammation without stiffness; and 4, dividing: stiffness. This assessment was performed at the following times (after induction of arthritis): day 14 (bolus intravenous or before oral administration); and day 24 (5 days after the last bolus intravenous injection or 24 hours after the last oral administration). All measurements were performed between 2:00 and 3:00 pm (D14), 8:00 to 9:00am (D24) in the afternoon. All data was collected and saved by WinDas software.

5. Determination of nociceptive response (Sound test)

Nociceptive responses were assessed by repeating 2 gentle bends of the right and left hind paw (arthritic rats) with the operator's fingers at 4 to 5 second intervals. The level of vocalization was recorded for each hind paw (2 times: right hind paw: s1 and s 3; 2 times: left hind paw: s2 and s4) for each animal. This assessment was performed at the following times (after induction of arthritis): day 14 (bolus intravenous or before oral administration); day 18 (1 hour before or after the second bolus intravenous injection); and day 24 (5 days after one bolus intravenous injection or 24 hours after the last oral administration). All measurements were taken between 8:00 and 9:00am in the morning, except at D14 (7: 30-9:00 in the morning) and D24 (7: 30-9:00 in the morning).

6. Blood collection for determination of E3 or 911 concentrations and circulating TOP-1 and blood parameters

On day 24 (after paw volume and arthritic index determination and sound test), blood samples (approximately 800-.

Measurement of E3 or 911 concentrations (groups 2, 4 and 5): a portion of the blood sample was collected in a tube containing Li-heparin (maintained on ice) and centrifuged at 2500-. Plasma samples (at least 100 μ L) were obtained, frozen in liquid nitrogen, and stored at-80 ℃. One sample was slightly hemolyzed (vehicle-treated arthritic rat # 36).

Determination of circulating TFG-. beta. (groups 1-2-3-4-5): a portion of the blood sample was collected in a microtube for serum preparation at room temperature. After sample collection, the blood was mixed and coagulated for 30 minutes before centrifugation. The tube was centrifuged at about 6000g for 3 minutes. Each serum sample (at least 100. mu.L, except for rats #52 and # 53) was aliquoted and stored at-20 ℃ until sample activation for TFG-beta analysis. These aliquots (50 tubes) were stored for 6 months from the end of the study. Some samples were slightly hemolyzed (vehicle-treated non-arthritic rats: #2, #5, #9, # 10; vehicle-treated arthritic rats: #53, # 63; E3-treated arthritic rats #31, # 51; 911-treated arthritic rats #52, #62 and # 64). TFG-. beta.levels were determined using a human TFG-. beta.1 ELISA kit (reference DB100, batch Nos. 212258 and 213610, R & D System-France).

Blood collection for blood parameters (group 1-2-3-4-5: 50 tubes): blood samples were collected in tubes containing K3-EDTA (at least 100. mu.L). The determination of the parameters was performed on the day of collection and no samples were stored. Blood parameters include red blood cells, white blood cells, platelets, hemoglobin, hematocrit as measured by a hemocytometer (D24). Some blood parameters were not determined due to clotting of the samples (vehicle-treated non-arthritic rats: # 10; E3-treated arthritic rats: #59, # 67; 911-treated arthritic rats: # 16).

7. Podocyte factor levels

On day 24 (5 days after the last bolus i.v. injection or 24 hours after the last oral administration) (post X-ray), the hind feet (arthritic and non-arthritic rats) of each animal were weighed and collected in labeled polyethylene tubes. Tissue samples were frozen in liquid nitrogen and stored at-80 ℃.

Preparation of joint homogenate: and (5) preparing the frozen feet into powder by using a biological pulverizer. The powdered hind paw was then placed in a 50ml conical centrifuge tube containing 3ml PBS supplemented with 50 μ l of an anti-protease cocktail and homogenized on ice using an Ultra-Turrax homogenizer (50% max). The homogenate was then centrifuged at 2000Xg for 15 minutes at 4 ℃ and the supernatant filtered through a 0.2 μ M sartorius filter, aliquoted and stored at-80 ℃ until use.

Cytokine level determination: the cytokine levels were determined in duplicate for TFG-alpha (rat TFG-alpha ELISA kit, reference RTA00, batch 213718, R & D System-France), IL-1 beta (rat IL-1 beta ELISA kit, reference RLB00, batch 212435, R & D System-France), IL-6 (rat IL-6 ELISA kit, reference R6000, batch 211773, 214008 and 214362, R & D System, France) and TFG-beta 1 (human TFG-beta 1 ELISA kit, reference DB100, batch 212258 and 213610, R & D System, France) according to the manufacturer's protocol. The split-filled hind paw homogenate is stored at-80 ℃.

X-ray analysis

On day 24, after blood collection, animals were sacrificed and X-ray pictures (hind legs) were obtained for assessment of joint damage. The X-ray analysis focused on joint erosion, joint space, periosteal abnormalities of the two hind feet. All radiographs were analyzed by looking at seven different items: the seven items are soft tissue injury, malformation, demineralization, joint space, erosion, osteogenesis and periosteal reaction. For each animal, the first six items were analyzed separately by looking at the worst hind foot condition. Periosteal responses were analyzed by tail viewing. For each item, scores ranged from 0 (normal) to 4 (maximum injury). Thus the total score is from 0 to 28. The same reader who does not know the animal (treated or untreated) explains the radiography.

9. Observation of

For unknown reasons, one animal (#65) died D23 after indomethacin administration (before D23 administration).

10. Analysis and expression of results

At each time point, all results were reported as mean ± mean standard error in 10 rats in each group. Paw volumes are expressed in ml calculated from the mean of the right and left paw volumes. The arthritis index was calculated from the total score obtained for each 4 feet. Nociceptive responses were assessed by recording the intensity of vocalization levels in mV for each animal (mean of 4 values: 2/foot). Percentage of nociceptive response inhibition was determined from the mean of vehicle-treated arthritic group [ (mean of vehicle-treated arthritic group-mean of treated arthritic group/mean of vehicle-treated arthritic group) ^*100]And (6) performing calculation. Body weight is expressed in grams. Hind paw (left and right paw) weights are expressed in grams. Cytokine levels (IL-6, IL-I β, TNF- α and TGF- β 1) were expressed in pg/ml for each hindpaw. Circulating levels of TGF-. beta.1 are expressed in pg/ml. The radiation index and the total radiation index (total score) for each parameter (demineralization, erosion, periosteal reaction, soft tissue injury, joint space, bone formation, deformity) were calculated from the total score obtained for each parameter. When the equality variable or normality test failed, the intra-group significance of the difference between the values of the vehicle treated group (arthritic rats) and the vehicle treated group (non-arthritic rats) was assessed by the student test or the Mann-Whitney rank sum test. The in-group significance of the dispersion between the vehicle treated group (arthritic rats) and the E3 and 911 and indomethacin treated groups was assessed by variable analysis of the one-way variation number analysis followed by the unpaired dunnett t test. The probability of P ≦ 0.05 was considered significant. All statistical analyses were by Sigmastat^TMAnd (4) carrying out software.

Results

1. Nociceptive response (Sound test)

As shown in table 11 and fig. 8, D14, nociceptive responses in the arthritis treated groups with vehicle, indomethacin, E3, and 911 were 4147 ± 331, 4386 ± 235, 4644 ± 367, and 4468 ± 143, respectively. Compared with the arthritis treatment group treated by the carrier (D18: 1511 + -398 to 5279 + -326 mV; D24: 1552 + -508 to 5905 + -345 mV), the indomethacin 3mg/kg (10 days) orally administered at D18 and D24 each day has strong and obviously reduced nociceptive response about-3768 mV (inhibition%: 71%) and-4353 mV (inhibition%: 74%), respectively. E3 (1 mg/kg administered intravenously in D14 and D19) strongly reduced nociceptive responses by about-4167 mV (% inhibition: 79%) and-5905 mV (% inhibition: 100%) in D18 and D24 compared to vehicle-treated arthritis groups (D18: 1112 + -401 vs 5279 + -326 mV; D24: 0 + -00 vs 5905 + -345 mV). Compared with the arthritis group treated by the carrier (D18: 1347 + -492 to 5279 + -326 mV; D24: 547 + -307 to 5905 + -345 mV) in D18 and D24, 911 (10 mg/kg intravenously applied on days D14 and D192) the nociceptive response was strongly reduced to about-3932 (% inhibition: 74%) and-5358 mV (% inhibition: 91%), respectively.

TABLE 11 Effect of intravenous injection of E3 and 911(2 days: D14-D19) on nociceptive responses in rat rheumatoid arthritis

Values expressed as mean. + -. standard error of mean in mV

Except that D24 was used for indomethacin (n-9), each group was n-10

Dannett t test:^*p is less than or equal to 0.05 for rats with arthritis treated by carrier

2. Body weight

As shown in table 12 and fig. 19, a significant decrease in body weight gain was observed in arthritic rats from D0 to D14 due to the establishment of arthritis. D14 (selected days) showed a significant reduction in body weight (289. + -.2 vs 217. + -.4 g) in arthritic rats compared to non-arthritic rats (Student t-test P < 0.05). However, no significant difference in weight (D14) was detected in all arthritis groups. The body weight was moderately and significantly increased in the indomethacin-treated group (3 mg/kg daily for 10 days) compared to the vehicle-treated arthritic group (261 + -5 vs. 218 + -3 g) from D17 to D24, with a maximum increase of about 43g at D24. From D17 to D24, body weight increased moderately and significantly after E3 treatment (1 mg/kg administered intravenously at D14 and D19) with a maximum increase of about 46g at D24 compared to the vehicle treated arthritis group (264 ± 5g vs 218 ± 3 g). From D18 to D24, body weight increased moderately and significantly after 911 treatment (10 mg/kg administered intravenously at D14 and D19) compared to vehicle treated arthritis (265 ± 7 vs 218 ± 3g), with a maximum increase of about 47g at D24.

TABLE 12 Effect of E3 and 911 intravenous injection (2 days: D14-D19) on body weight in rat rheumatoid arthritis

Values are expressed in grams as mean ± standard error of mean. Except for the animals for indomethacin (n-9) for D23 and D24, each group of n-10 animals

Dannett t test:^*p is less than or equal to 0.05 for rats with arthritis treated by carrier

3. Foot volume

At D14, randomization was performed to obtain a homogeneous set of foot volumes. As shown in table 13, at D14, the hindfoot volume (mean of right and left foot volumes) was significantly higher in the arthritic group than in the non-arthritic group (2.10 ± 0.05 versus 1.44 ± 0.02mL (Student t-test P < 0.05)). Indomethacin (3 mg/kg per day orally for 10 days) significantly reduced foot volume by about 0.75mL (D24) (1.59 ± 0.03mL versus 2.34 ± 0.08mL) compared to the vehicle treated arthritis group. E3 (1 mg/kg administered intravenously at D14 and D19) slightly and significantly increased the foot volume by about 0.37mL (2.71 ± 0.09mL vs 2.34 ± 0.08mL) compared to the vehicle-treated arthritis group. 911 (10 mg/kg administered intravenously at D14 and D19) slightly and significantly increased the foot volume by about 0.36mL (2.70 ± 0.11mL vs 2.34 ± 0.08mL) compared to the vehicle-treated arthritis group.

TABLE 13 Effect of E3 and 911 intravenous injection (2 days: D14-D19) on foot volume in rat rheumatoid arthritis

Values are expressed in mL as mean ± mean standard error.

Except for D24 (n-9) for indomethacin, each group of n-10 animals

Dannett t test:^*p is less than or equal to 0.05 for rats with arthritis treated by carrier

4. Index of arthritis

As shown in Table 14, the arthritis indexes at D14 in the arthritis groups treated with vehicle, indomethacin, E3 and 911 were 10.1. + -. 0.8, 8.7. + -. 0.6, 10.2. + -. 0.4 and 9.4. + -. 0.7, respectively. Compared with the group treated with vehicle for arthritis, indomethacin strongly and significantly reduced the arthritis index (2.7 ± 0.7 vs. 10.7 ± 0.6) with a maximum reduction of about 8.0 after daily oral administration of 3mg/kg (for 10 days). E3 (1 mg/kg administered intravenously at D14 and D19) did not affect the arthritis index (11.4. + -. 0.4 vs 10.7. + -. 0.6) compared to the vehicle-treated arthritis group. 911 (10.9. + -. 0.7 vs. 10.7. + -. 0.6 intravenous administration at D14 and D19) did not affect the phase of the arthritis index compared to the vehicle treated arthritis group.

TABLE 14 Effect of E3 and 911 intravenous injection (2 days: D14-D19) on the arthritis index in rats rheumatoid arthritis

Values are expressed as mean ± mean standard error (score)

With the exception of indomethacin (n-9), each group contained 10 animals

Dannett t test: ^*P is less than or equal to 0.05 for rats with arthritis treated by carrier

5. Podocyte factor levels

As shown in Table 15, at D24, cytokine levels in the left and right paw were increased by up to approximately 3.5(IL-1 β), 4 (TNF-. alpha.), and 1.8 (TGF-. beta.1) fold in the vehicle treated group of arthritis compared to the non-arthritis vehicle treated group. No significant differences between the two groups were observed for IL-6 levels in the right and left feet. Cytokine levels in the arthritis group were similar in the left and right paw: for IL-6, IL-1 beta, TNF-alpha and TGF-beta 1, 259.7 + -38.5 to 219.2 + -32.4, 4802.8 + -365.5 to 4007.1 + -380.4, 17.8 + -1.6 to 18.6 + -1.9 and 9735.0 + -1219.8 to 9161.4 + -846.1 pg/ml respectively. Indometacin slightly but significantly reduced TGF- β 1 levels by about 1.3 fold (7057.4 + -335.6 vs 9161.4 + -846.1) after daily oral administration of 3mg/kg (for 10 days) in the right foot compared to vehicle treated groups, without altering IL-6, TNF- α or IL-1 β levels. In the left foot, a similar but not significant effect was observed. In both feet, E3 (1 mg/kg administered intravenously at D14 and D19) did not affect IL-6, IL-1 β, TNF- α or TGF- β 1 levels compared to the vehicle treated group. 911 (10 mg/kg administered intravenously at D14 and D19) increased IL-1 β levels (6215.3 ± 666.7 vs 4007.1 ± 380.4) in the right foot compared to the vehicle treated group. In both feet, there was no effect on other cytokine levels.

TABLE 15 Effect of E3 and 911 on podocyte cytokine levels after intravenous injection (D14 and D19, 2 days) in Rheumatoid arthritis rats

Left paw cytokine levels

Right paw cytokine levels

Values expressed as mean. + -. standard error of mean in pg/ml

Except for the non-arthritis/vehicle group (right foot), the arthritis/vehicle group (left foot) and indomethacin (n-9), each group of n-10 animals

Dannett t test:^*p of rats treated with arthritis by the carrier is less than or equal to 0.05

6. Determination of circulating TGF-. beta.1

As shown in table 16, serum TGF- β 1 levels were increased in the arthritis vehicle treated group (81715.7 ± 1984.1 versus 60269.9 ± 2142.8) compared to the non-arthritis vehicle treated group at D24. Compared with the group treated with vehicle for arthritis, indomethacin significantly reduced serum TGF-beta 1 levels by about 1.5 times (57222.2 + -3194.1 vs 81715.7 + -1984.1) after daily oral administration of 3mg/kg (for 10 days). E3 (1 mg/kg administered intravenously at D14 and D19) and 911 (10 mg/kg administered intravenously at D14 and D19) significantly reduced serum TGF- β 1 levels so that cytokine levels in the E3 and 911 treated groups were comparable to those in the vehicle treated non-arthritic group (69408.8 + -3926.7 and 67214.5 + -3649.4, respectively, vs 60269.9 + -2142.8).

TABLE 16 Effect of E3 and 911 intravenous injection (at D14 and D19, 2 days) on serum TGF-. beta.1 levels in rheumatoid arthritis rats

Values expressed as mean. + -. standard error of mean in pg/ml

Except for the non-arthritis/vehicle group (right foot), the arthritis/vehicle group (left foot) and indomethacin (n-9), each group of n-10 animals

Dannett t test:^*the P of the rats with arthritis treated by the carrier is less than or equal to 0.05

7. Hematological parameters

As shown in table 17, hematological parameters such as white blood cells and platelets were higher in vehicle-treated arthritic rats compared to vehicle-treated non-arthritic rats (Student t-test P < 0.05). While red blood cells, hemoglobin and hematocrit were unchanged (Student's t-test P > 0.05). Compared to the vehicle-treated arthritis group, the indomethacin did not affect the blood parameters after daily oral administration of 3mg/kg indomethacin (for 10 days). E3 (1 mg/kg administered intravenously at Dl4 and D19) did not affect blood parameters compared to the vehicle treated arthritic group. 911 (10 mg/kg administered intravenously at D14 and D19) did not affect blood parameters compared to the vehicle treated arthritic group.

TABLE 17 Effect of E3 and 911 intravenous injection (at D14 and D19, 2 days) on blood parameters (measured at D24) in rat rheumatoid arthritis

Values are expressed as mean. + -. standard error of mean

Variance analysis: for rats with arthritis treated by the carrier, P is more than 0.05

7. Weight of hind foot

As shown in Table 18, the weight of the left and right hind paw in vehicle-treated arthritic rats was greater than the weight of the left and right hind paw in vehicle-treated non-arthritic rats (3.43 + -0.11 vs. 1.98 + -0.01 and 3.32 + -0.12 vs. 1.99 + -0.02 g, respectively) (Student's t-test or Mann-Withney P < 0.05). Indometacin significantly reduced hindfoot weight after daily oral administration of 3mg/kg (for 10 days) compared to vehicle treated arthritic group (left hindfoot: 2.23 + -0.04 vs. 3.43 + -0.11 g; right hindfoot: 2.20 + -0.05 vs. 3.32 + -0.12 g). E3 (1 mg/kg administered intravenously at D14 and D19) only significantly reduced left hind paw weight (left hind paw: 3.86. + -. 0.14 vs 3.43. + -. 0.11 g; right hind paw: 3.72. + -. 0.13 vs 3.32. + -. 0.12g) compared to the vehicle treated arthritic group. 911 (10 mg/kg administered intravenously at D14 and D19) significantly increased the weight of only the right hind paw (left hind paw: 3.73. + -. 0.12 vs 3.43. + -. 0.11 g; right hind paw: 3.83. + -. 0.15 vs 3.32. + -. 0.12g) compared to the vehicle-treated arthritic group.

TABLE 18 Effect of E3 and 911 on hind paw weight (measured at D24) following intravenous injection (at days D14 and D192) in rat rheumatoid arthritis

Values expressed in grams as mean ± mean standard error

Except for indomethacin (n-9), each group of n-10 animals

Dannett t test:^*the P of the rats with arthritis treated by the carrier is less than or equal to 0.05

X-ray analysis

As shown in table 19, a total score of 0.0 ± 0.0 was observed in vehicle-treated non-arthritic rats. Rats with arthritis treated by the carrier have a total score of 15.1 + -1.3, demineralization (2.4 + -0.3), erosion (2.7 + -0.3), soft tissue injury (3.1 + -0.2) and joint space (3.3 + -0.2); moderate scores for periosteal response (1.0. + -. 0.3), bone formation (0.8. + -. 0.2) and malformations (1.8. + -. 0.2). Indomethacin (administered orally at 3mg/kg daily for 10 days) strongly and significantly reduced the overall score by about 10.7(4.4 ± 0.9 versus 15.1 ± 1.3) compared to the vehicle-treated arthritic group. E3 (1 mg/kg administered intravenously at D14 and D19) did not affect the overall score (14.2. + -. 1.3 vs 15.1. + -. 1.3) compared to the vehicle treated arthritic group. 911 (10 mg/kg administered intravenously at D14 and D19) did not affect the overall score (15.4. + -. 1.0 vs 15.1. + -. 1.3) compared to the vehicle-treated arthritic group.

TABLE 19 Effect of E3 and 911 on X-ray parameters (measured at D24) following intravenous injection (at days D14 and D192) in rat rheumatoid arthritis

Values are expressed as mean. + -. standard error of mean

Except for indomethacin (n-9), each group of n-10 animals

Dannett t test:^*the P of the rats with arthritis treated by the carrier is less than or equal to 0.05

Conclusion

Under the experimental conditions described above, E3 (intravenously administered at 1mg/kg for 2 days: D14-D19) and 911 (intravenously administered at 10mg/kg for 2 days: D14-D19) showed strong analgesic effects, but did not show significant anti-inflammatory effects in this arthritis model.

Example 8: effect of varying doses of anti-NGF antibody E3 in rat model of rheumatoid arthritis

The ability of E3 to produce reduced pain was further investigated by studying the dose-response relationship between administration of E3 and pain reduction in arthritic rats. Rats were treated with adjuvant to induce arthritis as described above. Ten rats injected without adjuvant were used as non-arthritic controls. Fourteen days after adjuvant injection, animals were used qualitatively in the study based on the criteria set forth above, ten rats were randomly added to eight groups and their intensity of vocalization was examined. They were then administered saline or 0.003mg/kg, 0.01mg/kg, 0.03mg/kg, 0.1mg/kg, 0.3mg/kg, 1mg/kg or 5mg/kg of E3 antibody on day 14 as described above. The animals were tested for vocalization on days 16, 18, 20 and 24. On day 18 after the vocalization test, the animals were re-administered with saline or the same dose of E3. Animals were weighed daily starting on day 14. Thus, animals were administered twice with the given dose of antibody or saline on days 14 and 18, and pain was assessed five times on days 14, 16, 18, 20 and 24. The data are shown in tables 20-22 and in FIGS. 20-22.

TABLE 20 Effect of different doses of E3 on nociceptive response (intensity of vocalization) in rheumatoid arthritis rats. Mean average standard error expression of voicing intensity values in mV

Effect on pain-induced vocalization with different doses of anti-NGF antibody E3 (data shown in table 20) results obtained by statistical analysis using two-way rank and variance analysis to compare the pairings between arthritic animals treated with vehicle and with given doses of antibody E3. There was a highly significant effect (p < 0.0001) on all levels of the tested E3. Even at the lowest dose tested (0.003mg/kg E3), the difference in vocalization was significant (p < 0.0001).

As shown in table 20 and fig. 20, treatment with 1mg/kg antibody E3 showed rapid and strong relief of pain as in the above experiment. Within two days (the earliest time point of detection), the sound production intensity decreased by 90%. Treatment with lower concentrations of E3 also strongly reduced pain, although pain reduction at lower doses required longer time to manifest. On day 24, the significant reduction in efficacy, except for the highest dose tested, was probably due to the reduction in the true level of plasma E3 in the test rats that occurred after the immune response. Doses as low as 0.003mg/kg were evident in this model to provide at least partial pain relief.

TABLE 21 Effect of different doses of E3 on body weight in rheumatoid arthritis rats (corrected to day 14)

20	107.36	1.78	111.26	0.77	113.57	0.83	115.32	1.11
									21	108.50	2.01	113.31	0.87	116.71	0.92	119.11	1.21
23	109.25	2.15	115.59	1.38	123.35	1.13	126.36	1.94
									24	108.77	2.08	115.58	1.43	124.41	1.00	127.25	1.79

TABLE 22 Effect of different doses of E3 on body weight in rheumatoid arthritis rats (corrected to day 0)

The effect on body weight of animals treated with different doses of anti-NGF antibody E3 was analyzed by a two-way variation analysis to compare the results obtained for the pairing between arthritic animals treated with the vehicle and those with the given dose of antibody E3. With the weight data corrected to day 14 (Table 21), the 0.03mg/kg E3 dose resulted in a significant change in body weight (p < 0.005). The difference between treated and untreated arthritic animals was significant in all higher doses of E3 (p ≦ 0.0001). With the weight data corrected to day 0 (table 22), the 0.03mg/kg E3 dose resulted in a significant change in body weight (p < 0.002). The difference between treated and untreated arthritic animals was significant in all higher doses of E3 (p < 0.0001).

Again, consistent with earlier studies, rats treated with E3 showed less weight loss than saline-treated arthritic rats (table 22 and figure 22). Indeed, treatment of rats with high dose antibody E3 restored earlier weight loss and actually increased faster than their non-arthritic counterparts (table 21 and figure 21).

Preservation of biological materials

The following biological materials were deposited in the united states, virginia, Manassas, 10801 university boulevard, American Type Culture Collection (ATCC):

deposit date of material ATCC

Eb.911.3E 3 light chain PTA-48932003 year, 1 month 8 days

Eb.pur.911.3E 3 light chain PTA-48942003 year 1, 8 days

Db.911.3E 3 heavy chain PTA-48952003 year 1 month 8 days

The vector Eb.911.3E is a polynucleotide encoding the light chain variable region of E3; vector eb.pur.911.3e is a polynucleotide encoding the light chain variable region of E3, and vector db.911.3e is a polynucleotide encoding the heavy chain variable region of E3.

This deposit was prepared under the provisions of the budapest treaty on the international recognition of the deposit of microorganisms (budapest treaty) for the patent procedure and its procedures. This ensured that viable cultures were maintained for 30 years from the date of storage. This deposit was obtained under the Budapest treaty by ATCC and under the protocol between Rinat neurosciences and ATCC. The protocol ensures that progeny of the deposited culture are permanently and unrestrictedly available to the public at the time of publication of the relevant U.S. patent or at the time of publication of any U.S. or foreign patent application, whichever is first published, and that progeny are available to persons identified by the U.S. patent and trademark office as determined by 35 USC section 122 and rules associated therewith, including section 1.14 of 37 CFR, referenced specifically as 886 OG 638.

The assignee of the present application agreed that if a culture of the deposited material died or was lost or destroyed when cultured under appropriate conditions, the replacement of the material with another identical culture was immediately notified. The availability of the deposited material does not constitute an admission that the invention is being practiced by any government authority in accordance with the authority of its patent laws.

Antibody sequences

Heavy chain variable region (Kabat CDR underlined; Chothia CDR in bold and italics)

QVQLQESGPGLVKPSETLSLTCTVS GFSLIGYDLNWIRQPPGKGLEWIG IIWGDGTTD

YNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCAR GGYWYATSYYFDYWGQG

TLVTVS(SEQ ID NO：1)

Light chain variable region (Kabat CDR underlined; Chothia CDR in bold and italics)

DIQMTQSPSSLSASVGDRVTITC RASQSISNNLNWYQQKPGKAPKLLIY YTSRFHSG

VPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQEHTLPYTFGQGTKLEIKRT (SEQIDNO：2)

E3 heavy chain extension CDR

CDRH1：GFSLIGYDLN(SEQ ID NO：3)

CDRH2：IIWGDGTTDYNSAVKS(SEQ ID NO：4)

CDRH3：GGYWYATSYYFDY(SEQ ID NO：5)

E3 light chain extension CDR

CDRL1：RASQSISNNLN(SEQ ID NO：6)

CDRL2：YTSRFHS(SEQ ID NO：7)

CDRL3：QQEHTLPYT(SEQ ID NO：8)

Mouse monoclonal antibody 911 extended CDR

911 heavy chain extension CDR

CDRH1：GFSLIGYDIN(SEQ ID NO：9)

CDRH2：MIWGDGTTDYNSALKS(SEQ ID NO：10)

CDRH3：GGYYYGTSYYFDY(SEQ ID NO：11)

911 light chain extension CDR

CDRL1：RASQDISNHLN(SEQID NO：12)

CDRL2：YISRFHS(SEQ ID NO：13)

CDRL3：QQSKTLPYT(SEQ ID NO：14)

E3 heavy chain amino acid sequence (complete)

QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTT

DYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYW

GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC

CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV

DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEK

TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

GK(SEQ ID NO：16)

3E light chain amino acid sequence (complete antibody)

DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSG

VPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSV

FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHXVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：17)

3E heavy chain nucleotide sequence (complete antibody)

CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCT

GTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGG

ATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTG

ATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGA

CACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACAC

GGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTT

TGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGG

CCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCC

GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGG

AACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCC

TCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGC

ACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGAC

AAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCA

GTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATG

ATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGA

CCCAGAGGTGCAGTTAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCA

AGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTG

CTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGT

GTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGG

ACAGCCAAGAGAGCCACAGGTGTATACCCTGCCACCATCCAGAGAGGAGATGA

CCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACA

TCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACC

CCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGG

ACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAG

GCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAA(

SEQ ID NO：65)

3E heavy chain variable domain nucleotide sequence

CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCT

GTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGG

ATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTG

ATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGA

CACCTCCAAGAACCAGTTCTCCCTGAAGCTGACGCTCTGTGACCGCCGCGGACAC

GGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTT

TGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA(SEQ ID NO：66)

3E light chain nucleotide sequence (complete antibody)

GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGC

GTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATC

AGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCA

CTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTC

ACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAG

CATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGCACTG

TGGCTGCACCATCTGTCTTCATCTTTCCTCCATCTGATGAGCAGTTGAAATCCGG

AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGT

ACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCA

CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGA

GCAAAGCAGACTACGAGAAACACMAAGTCTACGCCTGCGAAGTCACCCATCAG

GGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA(SEQ ID

NO：67)

3E light chain variable domain nucleotide sequence

GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGC

GTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTAT

CAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTT

CCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCAC

CTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACA

GGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAAC

GC(SEQ ID NO：68)

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims (30)

1. An anti-Nerve Growth Factor (NGF) antibody comprising:

(a) a heavy chain variable region comprising:

(i) a CDR1 region, wherein the sequence of the CDR1 region is SEQ ID NO: 3;

(ii) a CDR2 region, wherein the sequence of the CDR2 region is SEQ ID NO: 4;

(iii) a CDR3 region, wherein the sequence of the CDR3 region is selected from SEQ ID NOs: 5. 58 and 60; and

(b) a light chain variable region comprising:

(i) a CDR1 region, wherein the sequence of the CDR1 region is SEQ ID NO: 6;

(ii) a CDR2 region, wherein the sequence of the CDR2 region is SEQ ID NO: 7;

(iii) a CDR3 region, wherein the sequence of the CDR3 region is selected from SEQ ID NOs: 8. 57, 59 and 61.

2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region, wherein the antibody binds NGF, wherein the heavy chain variable region comprises:

(c) a CDR1 region, wherein the sequence of the CDR1 region is SEQ ID NO: 3;

(d) a CDR2 region, wherein the sequence of the CDR2 region is SEQ ID NO: 4; and

(e) a CDR3 region, wherein the sequence of the CDR3 region is SEQ ID NO: 5.

3. the antibody of claim 1 or 2, wherein the antibody comprises a light chain variable region, wherein the antibody binds NGF, the light chain variable region comprising:

(f) a CDR1 region, wherein the sequence of the CDR1 region is SEQ ID NO: 6;

(g) A CDR2 region, wherein the sequence of the CDR2 region is SEQ ID NO: 7; and

(h) a CDR3 region, wherein the sequence of the CDR3 region is SEQ ID NO: 8.

4. the antibody of claim 3, wherein the antibody further comprises a heavy chain variable region comprising:

(i) a CDR1 region, wherein the sequence of the CDR1 region is SEQ ID NO: 3;

(j) a CDR2 region, wherein the sequence of the CDR2 region is SEQ ID NO: 4; and

(k) a CDR3 region, wherein the sequence of the CDR3 region is SEQ ID NO: 5.

5. the antibody of claim 1, wherein the sequence of the heavy chain variable region is SEQ ID NO: 1.

6. the antibody of claim 1, wherein the sequence of the light chain variable region is SEQ ID NO: 2.

7. the antibody of claim 1, wherein the amino acid sequence of the heavy chain is SEQ ID NO: 16.

8. the antibody of claim 1, wherein the amino acid sequence of the light chain is SEQ ID NO: 17.

9. the antibody of claim 1, wherein the antibody binds human NGF with a KD of 100pM or less.

10. The antibody of claim 9, wherein the antibody also binds rodent NGF.

11. An anti-NGF antibody comprising

(a) A heavy chain variable region, wherein the sequence of the heavy chain variable region is SEQ ID NO: 1; and

(b) A light chain variable region, wherein the sequence of the light chain variable region is SEQ ID NO: 2.

12. an anti-NGF antibody comprising

(a) A heavy chain, wherein the amino acid sequence of said heavy chain is SEQ ID NO: 16; and

(b) a light chain, wherein the amino acid sequence of the light chain is SEQ ID NO: 17.

13. a pharmaceutical composition comprising (a) an antibody according to any preceding claim, and (b) a pharmaceutically acceptable excipient.

14. A kit comprising the antibody of any one of claims 1-12.

15. A method of making the antibody of any one of claims 1-12, the method comprising expressing a polynucleotide encoding the antibody of any one of claims 1-12 in vitro in a non-human host cell.

16. Use of an effective amount of an antibody according to any one of claims 1-12 in the manufacture of a medicament for treating rheumatoid arthritis pain in an individual.

17. The use of claim 16, wherein the individual has reduced pain within 24 hours of administration of the anti-NGF antibody.

18. The use of claim 16, wherein the individual has reduced pain within 4 days of administration of the anti-NGF antibody.

19. Use of an effective amount of an antibody according to any one of claims 1-12 in the manufacture of a medicament for treating weight loss associated with rheumatoid arthritis in an individual.

20. The use of claim 16 or 19, wherein the anti-NGF antibody specifically binds human NGF.

21. The use of claim 16 or 19, wherein the anti-NGF antibody is a monoclonal antibody comprising the amino acid sequence of SEQ id no: 1 and 2.

22. The use of claim 16 or 19, wherein the subject is a human.

23. An isolated polynucleotide comprising a nucleotide sequence encoding the antibody of any one of claims 1-12.

24. The polynucleotide of claim 23, wherein the nucleotide sequence encoding the heavy chain of the antibody is seq id NO: 65.

25. the polynucleotide of claim 23, wherein the nucleotide sequence encoding the antibody heavy chain variable domain is SEQ ID NO: 66.

26. the polynucleotide of claim 23, wherein the nucleotide sequence encoding the light chain of the antibody is seq id NO: 67.

27. the polynucleotide of claim 23, wherein the nucleotide sequence encoding the antibody light chain variable domain is SEQ ID NO: 68.

28. the polynucleotide of claim 23, wherein the nucleotide sequence encoding the antibody heavy chain variable domain is SEQ ID NO: 66 and the nucleotide sequence encoding the antibody light chain variable domain is seq id NO: 68.

29. a vector comprising a polynucleotide, wherein said polynucleotide is a nucleotide sequence encoding an antibody of any one of claims 1-12.

30. An isolated host cell comprising a polynucleotide, wherein said polynucleotide is a nucleotide sequence encoding the antibody of any one of claims 1-12.